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Fifty Years: Reflections Since the First Successful Heart Transplant
JLC Swanevelder, PC Gordon, JG Brink, JT Gutsche, RA Dyer and JG Augoustides
J Cardiothorac Vasc Anesth (2018) 32(1): 14-18

Synthetic extracellular matrix mimic hydrogel improves efficacy of mesenchymal stromal cell therapy for ischemic cardiomyopathy
MC Ciuffreda, G Malpasso, C Chokoza, D Bezuidenhout, KP Goetsch, M Mura, F Pisano, NH Davies and M Gnecchi
Acta Biomater (2018)

BACKGROUND: Mesenchymal stromal cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. Low cell engraftment limits the release of soluble paracrine factors (SF) over time and, consequently, MSC efficacy. We tested whether a synthetic extracellular matrix mimic, a hydrogel containing heparin (H-HG), could ameliorate MSC engraftment and binding/release of SF, thus improving MSC therapy efficacy. METHODS AND RESULTS: In vitro, rat bone-marrow MSC (rBM-MSC) were seeded and grown into H-HG. Under normoxia, the hydrogel did not affect cell survival (rBM-MSC survival >90% at each time point tested); vice versa, under hypoxia the biomaterial resulted to be protective for the cells (p<.001 vs rBM-MSC alone). H-HG or control PEG hydrogels (HG) were incubated with VEGF or bFGF for binding/release quantification. Data showed significantly higher amount of VEGF and bFGF bound by H-HG compared with HG (p<.05) and a constant release over time. In vivo, myocardial infarction (MI) was induced in female Sprague Dawley rats by permanent coronary ligation. One week later, saline, rBM-MSC, H-HG or rBM-MSC/H-HG were injected in the infarct zone. The co-injection of rBM-MSC/H-HG into infarcted hearts significantly increased cardiac function. Importantly, we observed a significant gain in MSC engraftment, reduction of ventricular remodeling and stimulation of neo-vasculogenesis. We also documented higher amounts of several pro-angiogenic factors in hearts treated with rBM-MSC/H-HG. CONCLUSIONS: Our data show that H-HG increases MSC engraftment, efficiently fine tunes the paracrine MSC actions and improves cardiac function in infarcted rat hearts. STATEMENT OF SIGNIFICANCE: Transplantation of MSC is a promising treatment for ischemic heart disease, but low cell engraftment has so far limited its efficacy. The enzymatically degradable H-HG that we developed is able to increase MSC retention/engraftment and, at the same time, to fine-tune the paracrine effects mediated by the cells. Most importantly, the co-transplantation of MSC and H-HG in a rat model of ischemic cardiomyopathy improved heart function through a significant reduction in ventricular remodeling/scarring and amelioration in neo-vasculogenesis/endogenous cardiac regeneration. These beneficial effects are comparable to those obtained by others using a much greater number of cells, strengthening the efficacy of the biomaterial used in increasing the therapeutic effects of MSC. Given its efficacy and safety, documented by the absence of immunoreaction, our strategy appears readily translatable to clinical scenarios.

Transmural capillary ingrowth is essential for confluent vascular graft healing
T Pennel, D Bezuidenhout, J Koehne, NH Davies and P Zilla
Acta Biomater (2018) 65: 237-247

Spontaneous endothelialization of synthetic vascular grafts may occur via three independent or concurrent modalities: transanastomotic (TA) outgrowth, transmural (TM) ingrowth or fallout (FO) from the blood. The limited TA and FO endothelialization, which occurs in humans, results in poor long-term patency in the small diameter position, where TM ingrowth may offer a clinically relevant alternative. To achieve sequential analysis of each mode of healing, loop grafts comprising anastomotically isolated angiopermissive polyurethane control grafts were abluminally sealed using either ePTFE wraps or solid polyurethane skins and implanted in the rat infrarenal aortic loop model for twelve weeks. Positive control grafts showed improved endothelialization and patency compared to the abluminally isolated mid-grafts. Furthermore, the mid-graft healing was accelerated with surface heparin and heparin-growth factor (VEGF, PDGF) modification in a three-week sub-study. We are thus able to distinguish between the three vascular graft endothelialization modes, and conclude that fallout plays a secondary role to TM healing. The increased endothelialisation for growth factor presenting grafts indicates the promise of this simple approach but further optimization is required. STATEMENT OF SIGNIFICANCE: In addition to the full elucidation of, and differentiation between, the three healing/endothelialisation modes of vascular grafts, the significance of the work relates to the near-complete lack of endothelialisation of small diameter vascular grafts in humans (1-2cm transanastomotic outgrowth on a graft that may be 60cm long) even after decades of implantation. The concomitant retained midgraft thrombogenicity leads, together with anastomotic hyperplastic responses, to poor long-term outcomes. The large impact of successful translation of the current research to the achievement of full endothelialisation of long peripheral grafts in humans via transmural ingrowth (half a millimetre distance; thickness of the graft wall), is evident, and supported by the large improvements in clinical patencies achievable in by pre-seeding of ePTFE grafts with confluent endothelia.

Sinus of Valsalva-right atrial tunnel causing heart failure in a 38-year-old
SL Dellis, T Pennel, Q Said-Hartley and P Zilla
J Thorac Cardiovasc Surg (2018) 155(1): e51-e53

High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization
M McLuckie, CA Schmidt, A Oosthuysen, N Sanchez-Macedo, H Merker, D Bezuidenhout, SP Hoerstrup and N Lindenblatt
J Biomed Mater Res A (2017) 105(9): 2543-2550

Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 microm thickness, and interconnected pore size of 150 microm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017.

Group A Streptococcus, Acute Rheumatic Fever and Rheumatic Heart Disease: Epidemiology and Clinical Considerations
LJ Zuhlke, A Beaton, ME Engel, CT Hugo-Hamman, G Karthikeyan, JM Katzenellenbogen, N Ntusi, AP Ralph, A Saxena, PR Smeesters, D Watkins, P Zilla and J Carapetis
Curr Treat Options Cardiovasc Med (2017) 19(2): 15

OPINION STATEMENT: Early recognition of group A streptococcal pharyngitis and appropriate management with benzathine penicillin using local clinical prediction rules together with validated rapi-strep testing when available should be incorporated in primary health care. A directed approach to the differential diagnosis of acute rheumatic fever now includes the concept of low-risk versus medium-to-high risk populations. Initiation of secondary prophylaxis and the establishment of early medium to long-term care plans is a key aspect of the management of ARF. It is a requirement to identify high-risk individuals with RHD such as those with heart failure, pregnant women, and those with severe disease and multiple valve involvement. As penicillin is the mainstay of primary and secondary prevention, further research into penicillin supply chains, alternate preparations and modes of delivery is required.

Dual electrospinning with sacrificial fibers for engineered porosity and enhancement of tissue ingrowth
J Voorneveld, A Oosthuysen, T Franz, P Zilla and D Bezuidenhout
J Biomed Mater Res B Appl Biomater (2017) 105(6): 1559-1572

Porosity, pore size and pore interconnectivity are critical factors for cellular infiltration into electrospun scaffolds. This study utilized dual electrospinning with sacrificial fiber extraction to produce scaffolds with engineered porosity and mechanical properties. Subsequently, scaffolds were covalently grafted with heparin, a known anti-coagulant with growth-factor binding properties. We hypothesized that the tissue ingrowth would correlate positively with the porosity of the scaffolds. Pellethane(R) (PU) was spun simultaneously with poly(ethylene oxide) (PEO, subsequently extracted). Low, medium and high porosity scaffolds and heparinized versions of each were characterized and implanted in vivo for evaluation of cellular infiltration and inflammation subcutaneously in male Wistar rats (7,14 and 28 days, n = 6). Average pore-size for low (76 +/- 0.2%), medium (83 +/- 0.5%) and high (90 +/- 1.0%) porosity scaffolds was 4.0 +/- 2.3 microm, 9.9 +/- 4.2 microm and 11.1 +/- 5.5 microm (p < 0.0001). Heparinization resulted in increased fiber diameter (3.6 +/- 1.1 microm vs. 1.8 +/- 0.8 microm, p < 0.0001) but influenced neither pore-size (p = 0.67) nor porosity (p = 0.27). Cellular infiltration for low, medium and high porosity scaffolds reached 33 +/- 7%, 77 +/- 20% and 98 +/- 1% of scaffold width, respectively, by day 28 of implantation (p < 0001); heparinization did not affect infiltration (p = 0.89). The ultimate tensile strength (UTS) and Young's modulus (Ey ) of the constructs increased linearly with increasing PU fiber fraction (UTS: r(2) = 0.97, p < 0.0001, Ey : r(2) = 0.76, p < 0.0001) and heparinization resulted in decreased strength but increased stiffness compared to non-heparinized scaffolds. Increased PEO to PU fraction in the scaffold resulted in predictable losses to mechanical strength and improvements to cellular infiltration, which could make PEO to PU fraction a useful optimization parameter for small diameter vascular grafts. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1559-1572, 2017.

50th Anniversary of the first Human Heart Transplant-How is it seen today?
K Sliwa and P Zilla
Eur Heart J (2017) 38(46): 3402-3404

Improved vascularization of porous scaffolds through growth factor delivery from heparinized polyethylene glycol hydrogels
A Janse van Rensburg, NH Davies, A Oosthuysen, C Chokoza, P Zilla and D Bezuidenhout
Acta Biomater (2017) 49: 89-100

Surface modification with heparin has previously been shown to increase vascularization of porous scaffolds. In order to determine its efficacy with sustained release, heparin (Hep) was covalently incorporated into degradable (Type D) and non-degradable (Type N) polyethylene glycol (PEG) hydrogels. After in vitro characterization of their physicochemical properties, growth factor (GF) loaded, heparinised Type D gels were formed within the pores of porous polyurethane disks, which were then implanted and evaluated in a subcutaneous model. Type N gels formed faster (3.1+/-0.1 vs. 7.2+/-0.2min), were stiffer (10.0+/-0.5kPa vs. 7.1+/-1.2kPa) and more stable than degradable gels (>6month stability vs. disintegration 22d in vitro; all p<0.001). Sustained release of covalently incorporated (CI) heparin from Type N (56days; first order kinetics) and Type D (21days; zero order kinetics) was achieved, as opposed to non-covalently incorporated (NI) heparin that eluted in a burst release within the first 2days. While Type D gels initially impeded tissue ingrowth into the porous scaffolds, they were completely degraded and replaced by ingrown tissue after 28days in vivo. At the latter timepoint disks containing gels without Hep or with non-covalently incorporated Hep were less vascularized than empty (no gel) controls. In contrast, the incorporation of covalently heparinized (no GF) and GF containing gels (no Hep) resulted in a 50% and 42% (p<0.05) improvement in vascularization, while an increase of 119% (p<0.001) was achieved with a combination of covalently attached Hep and GF. These gels thus provide a sustained release system for heparin and GF that extends the duration of their action to local tissue ingrowth. STATEMENT OF SIGNIFICANCE: The paper describes the modification and covalent incorporation of heparin into degradable and non-degradable polyethylene glycol hydrogels in a way that provides for the hydrolytic cleavage of the linker for the release of the heparin in original and active form, and in an extended (21-56d) controlled (zero and first order respectively) manner. The successful use of these gels as growth-factor containing and releasing matrices for the improvement of in vivo vascularization holds promise for many potential uses in tissue engineering and regenerative medicine applications, such as vascular grafts and myocardial infarction therapy, where the antithrombotic and/or growth factor binding/potentiating properties are required.

The world's first human-to-human heart transplant at Groote Schuur Hospital: 50 years later
J Brink, T Pennel, K Seele and P Zilla
S Afr Med J (2017) 107(12): 1035-1036

SASCI/SCTSSA joint consensus statement and guidelines on transcatheter aortic valve implantation (TAVI) in South Africa
J Scherman and H Weich
Cardiovasc J Afr (2016) 27(6): 399-400

Management of valvular disease in pregnancy: a global perspective
K Sliwa, MR Johnson, P Zilla and JW Roos-Hesselink
Eur Heart J (2015) 36(18): 1078-89

Valvular heart disease (VHD) in pregnant women, whether due to congenital or acquired aetiologies, poses a challenge to clinicians and their patients. Significant valve disease, which can affect a single valve or several valves, increases the risk of pregnancy to the mother and foetus and requires a careful preconception risk assessment and, subsequently during pregnancy, specialized care to minimize maternal and foetal morbidity and mortality. The goal of this paper is to provide a guide to risk assessment and to give an overview of the optimal cardiac and obstetric management, including surgical intervention, taking into consideration the resources available in higher and lower-to-middle income countries. This manuscript provides a practical approach and is not replacing comprehensive guidelines on the management of VHD or cardiovascular disease in pregnancy. It focuses on common valvular diseases and does not cover the large variety of aortic disease with and without valve disease or complex congenital heart disease in detail.

Micro-structurally detailed model of a therapeutic hydrogel injectate in a rat biventricular cardiac geometry for computational simulations
MS Sirry, NH Davies, K Kadner, L Dubuis, MG Saleh, EM Meintjes, BS Spottiswoode, P Zilla and T Franz
Comput Methods Biomech Biomed Engin (2015) 18(3): 325-31

Biomaterial injection-based therapies have showed cautious success in restoration of cardiac function and prevention of adverse remodelling into heart failure after myocardial infarction (MI). However, the underlying mechanisms are not well understood. Computational studies utilised simplified representations of the therapeutic myocardial injectates. Wistar rats underwent experimental infarction followed by immediate injection of polyethylene glycol hydrogel in the infarct region. Hearts were explanted, cryo-sectioned and the region with the injectate histologically analysed. Histological micrographs were used to reconstruct the dispersed hydrogel injectate. Cardiac magnetic resonance imaging data from a healthy rat were used to obtain an end-diastolic biventricular geometry which was subsequently adjusted and combined with the injectate model. The computational geometry of the injectate exhibited microscopic structural details found the in situ. The combination of injectate and cardiac geometry provides realistic geometries for multiscale computational studies of intra-myocardial injectate therapies for the rat model that has been widely used for MI research.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats
S Sharp, M Poglitsch, P Zilla, NH Davies and ED Sturrock
J Renin Angiotensin Aldosterone Syst (2015) 16(4): 1149-58

INTRODUCTION: The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. METHODS: The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. RESULTS: Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. CONCLUSION: LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions.

Personalised computational cardiology: Patient-specific modelling in cardiac mechanics and biomaterial injection therapies for myocardial infarction
KL Sack, NH Davies, JM Guccione and T Franz
Heart Fail Rev (2016) 21(6): 815-826

Predictive computational modelling in biomedical research offers the potential to integrate diverse data, uncover biological mechanisms that are not easily accessible through experimental methods and expose gaps in knowledge requiring further research. Recent developments in computing and diagnostic technologies have initiated the advancement of computational models in terms of complexity and specificity. Consequently, computational modelling can increasingly be utilised as enabling and complementing modality in the clinic-with medical decisions and interventions being personalised. Myocardial infarction and heart failure are amongst the leading causes of death globally despite optimal modern treatment. The development of novel MI therapies is challenging and may be greatly facilitated through predictive modelling. Here, we review the advances in patient-specific modelling of cardiac mechanics, distinguishing specificity in cardiac geometry, myofibre architecture and mechanical tissue properties. Thereafter, the focus narrows to the mechanics of the infarcted heart and treatment of myocardial infarction with particular attention on intramyocardial biomaterial delivery.

Infarcted rat myocardium: Data from biaxial tensile and uniaxial compressive testing and analysis of collagen fibre orientation
MS Sirry, JR Butler, SS Patnaik, B Brazile, R Bertucci, A Claude, R McLaughlin, NH Davies, J Liao and T Franz
Data Brief (2016) 8: 1338-43

Myocardial infarction was experimentally induced in rat hearts and harvested immediately, 7, 14 and 28 days after the infarction induction. Anterior wall infarct samples underwent biaxial tensile and uniaxial compressive testing. Orientation of collagen fibres was analysed following mechanical testing. In this paper, we present the tensile and compressive stress-strain raw data, the calculated tensile and compressive moduli and the measured angles of collagen orientation. The presented data is associated with the research article titled "Characterisation of the mechanical properties of infarcted myocardium in the rat under biaxial tension and uniaxial compression" (Sirry et al., 2016) [1].

Cast Tube Assay: A 3-D in vitro assay for visualization and quantification of horizontal chemotaxis and cellular invasion
BC Whitehead, D Bezuidenhout, C Chokoza, NH Davies and KP Goetsch
Biotechniques (2016) 61(2): 66-72

Directed cell motility, as controlled by soluble factors, is crucial for many biological processes, including development, cancer progression, and wound healing. The use of directed cell motility also shows promise for applications in regenerative medicine such as therapeutic angiogenesis. Unfortunately, current in vitro 3-D migration and invasion models limit our understanding and application of these processes. Here, we present a novel and cost-effective 3-D chemotaxis assay for assessing the invasive response of cells to a chemoattractant extracellular matrix (ECM). Our system takes advantage of a custom-casting chamber to set two gels in contact with each other along a defined front, one containing a suitable chemoattractant and the other the cells. Rotation of the chamber allows easy visualization of invasion across the interface. The effectiveness of the assay was demonstrated by studying the invasion of both human dermal fibroblasts (FBs) and smooth muscle cells (SMCs) into a polyethylene glycol (PEG) hydrogel containing basic fibroblast growth factor (bFGF). Incorporation of bFGF resulted in significantly increased and directional invasion for both cell groups.

Characterisation of the mechanical properties of infarcted myocardium in the rat under biaxial tension and uniaxial compression
MS Sirry, JR Butler, SS Patnaik, B Brazile, R Bertucci, A Claude, R McLaughlin, NH Davies, J Liao and T Franz
J Mech Behav Biomed Mater (2016) 63: 252-264

Understanding the passive mechanical properties of infarcted tissue at different healing stages is essential to explore the emerging biomaterial injection-based therapy for myocardial infarction (MI). Although rats have been widely used as animal models in such investigations, the data in literature that quantify the passive mechanical properties of rat heart infarcts is very limited. MI was induced in rats and hearts were harvested immediately (0 day), 7, 14 and 28 days after infarction onset. Left ventricle anterioapical samples were cut and underwent equibiaxial and non equibiaxial tension followed by uniaxial compression mechanical tests. Histological analysis was conducted to confirm MI and to quantify the size of the induced infarcts. Infarcts maintained anisotropy and the nonlinear biaxial and compressive mechanical behaviour throughout the healing phases with the circumferential direction being stiffer than the longitudinal direction. Mechanical coupling was observed between the two axes in all infarct groups. The 0, 7, 14 and 28 days infarcts showed 438, 693, 1048 and 1218kPa circumferential tensile moduli. The 28 day infarct group showed a significantly higher compressive modulus compared to the other infarct groups (p=0.0060, 0.0293, and 0.0268 for 0, 7 and 14 days groups). Collagen fibres were found to align in a preferred direction for all infarct groups supporting the observed mechanical anisotropy. The presented data are useful for developing material models for healing infarcts and for setting a baseline for future assessment of emerging mechanical-based MI therapies.

Excessive volume of hydrogel injectates may compromise the efficacy for the treatment of acute myocardial infarction
P Wise, NH Davies, MS Sirry, J Kortsmit, L Dubuis, CK Chai, FP Baaijens and T Franz
Int J Numer Method Biomed Eng (2016) 32(12)

Biomaterial injectates are promising as a therapy for myocardial infarction to inhibit the adverse ventricular remodeling. The current study explored interrelated effects of injectate volume and infarct size on treatment efficacy. A finite element model of a rat heart was utilized to represent ischemic infarcts of 10%, 20%, and 38% of left ventricular wall volume and polyethylene glycol hydrogel injectates of 25%, 50%, and 75% of the infarct volume. Ejection fraction was 49.7% in the healthy left ventricle and 44.9%, 46.4%, 47.4%, and 47.3% in the untreated 10% infarct and treated with 25%, 50%, and 75% injectate, respectively. Maximum end-systolic infarct fiber stress was 41.6, 53.4, 44.7, 44.0, and 45.3 kPa in the healthy heart, the untreated 10% infarct, and when treated with the three injectate volumes, respectively. Treating the 10% and 38% infarcts with the 25% injectate volume reduced the maximum end-systolic fiber stress by 16.3% and 34.7% and the associated strain by 30.2% and 9.8%, respectively. The results indicate the existence of a threshold for injectate volume above which efficacy does not further increase but may decrease. The efficacy of an injectate in reducing infarct stress and strain changes with infarct size. Copyright (c) 2016 John Wiley & Sons, Ltd.

Regulation of tissue ingrowth into proteolytically degradable hydrogels
KP Goetsch, M Bracher, D Bezuidenhout, P Zilla and NH Davies
Acta Biomater (2015) 24: 44-52

UNLABELLED: Regulation of the rate of cell ingrowth into and within a matrix is desirable for efficient tissue regeneration. Polyethylene glycol hydrogels crosslinked with matrix metalloproteinase (MMP) susceptible peptide sequences permit cell-controlled invasion. In this study, hydrogels of the same stiffness polymerised using different ratios of a readily degradable MMP peptide sequence (PAN-MMP) and a MMP peptide with a limited degradation capacity (MMP-9) were assessed both in vitro and in vivo for cellular invasion. The degree of invasion into the various hydrogels was found to be tightly linked to the relative proportion of each peptide both in vitro and in vivo. Furthermore a good correlation between in vitro and in vivo ingrowth was observed. These findings demonstrate a highly tunable model for regulating cellular invasion which is readily translatable to in vivo models. This finding may allow for further optimisation of aspects of regenerative scaffolds such as tissue invasion, growth factor release and cellular encapsulation. STATEMENT OF SIGNIFICANCE: Degradable hydrogels are used in a wide range of tissue regeneration approaches. A particularly advantageous variant of these hydrogels is where due to peptide based crosslinking of the polymeric hydrogels, cell invasion rate is dependent on cellular enzymatic activity. This present study demonstrates a further refinement whereby both cellular and tissue invasion rates are finely regulated through the polymerisation of a hydrogel with varying combinations of enzymatically degradable peptides. Importantly this allows for invasion rates to be controlled without altering the biomechanical properties of the hydrogel such as stiffness. The latter can further influence cellular behaviour thus potentially interfering with the desired outcome.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats
S Sharp, M Poglitsch, P Zilla, NH Davies and ED Sturrock
J Renin Angiotensin Aldosterone Syst (2015) 16(4): 1149-58

INTRODUCTION: The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. METHODS: The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. RESULTS: Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. CONCLUSION: LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions.


Assessment of the immunogenicity of mechanically induced interferon aggregates in a transgenic mouse model
Paul Human, Helen Ilsley, Cynthia Roberson, Eric Grovender, Bill Van Antwerp, Eric Fogt and Peter Zilla
​Journal of Pharmaceutical Sciences (2015) 104(2): 722-730

Background: Pump delivery of human Interferon alpha-2B (IFNα2b) has the potential for inducing immunogenic drug aggregates.  We therefore evaluated the immunogenicity of mechanically induced IFNα2b aggregates to assess this risk. Methods: Transgenic human-IFNα2b (TG) and wild-type (WT) FVB/N mice (n=8 and n=9/group respectively) were administered mechanically agitated drug (45Hz for 6h [LLA] or 24h [HLA]), chemically modified drug (low pH [pH 4.0] or metal oxidized [OXD]) or unstressed drug [Native].  Mice received IFNα2b (50µg; 100µg/ml; s.c.) formulations on days 0, 7, 14 and 21.  Drug-binding and neutralizing antibody titres were determined after 28d. Results: Aggregate concentrations were highest in OXD and HLA formulations but OXD had more dimers/trimers.  Geometric mean titres were 1:131; 1:728; 1:1,573; 1:871 and 1:10,240 for WT mice (n=9) and 1:207; 1:587; 1:1,810; 1:571 and 1:2,153  for TG mice (n=8) for Native, LLA, HLA, pH4 and OXD groups respectively. Conclusion: Mechanical agitation of IFNα2b induced equivalent titres of immunoglobulin to that of metal oxidation, both capable of binding to or neutralizing the drug in WT and TG mice. Thus, by limiting metal contamination and by inclusion of a stabilizing agent to mitigate drug aggregation the risk of anti-drug immunoglobulin may be reduced in a pump delivery scenario.

Management of valvular disease in pregnancy; a global perspective
Karen Sliwa, MR Johnson, Peter Zilla, JW Roos-Hesselink
Eur Heart J (2015) 36(18):1078-1089

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensis system in myocardial infarcted rats
Sarah Sharp, M Poglitsch, Peter Zilla, Neil H Davies, ED Sturrock

Journal of the Renin-Angiotensin-Aldosterone System (2015) Published on-line DOI: 10.1177/1470320314568438

Polymeric heart valves for surgical implantation, catheter-based technologies and heart assist devices (Review)
Deon Bezuidenhout, David F Williams, Peter Zilla
Biomaterials (2015) 36:6-25

The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulator models
Tim Pennel, G Fercana, Deon Bezuidenhout, Agneta Simionescu, TH Chuan, Peter Zilla, Dan Simionescu
Biomaterials (2014) 35:6311-6322

Massive hemoptysis 18 months after a stabbing attack
Jacques Scherman, TD Nguyen, Peter Zilla, MY Emmert
Annals of Thoracic Surgery (2014) 98(2):728

Nitric oxide release from polydimethylsiloxane-based polyurethanes
EB Nguyen, Peter Zilla, Deon Bezuidenhout
J Appl Biomater Funct Mater (2014) 12(3): 172-182

Constrictive pericarditis requiring pericardiectomy at Groote Schuur Hospital, Cape Town, South Africa: Causes and perioperative outcomes in the HIV era (1990-2012)
Arthur Mutyaba, Sarvesh Balkaran, Robert Cloete, Naude du Plessis, Motasim Badri, Johan Brink and Bongani Mayosi
J Thorac Cardiovasc Surg 2014;148:3058-65

Objective: The causes of constrictive pericarditis and predictors of perioperative outcome after pericardiectomy have not been clearly elucidated, especially in Africa, where the disease characteristics differ from those in developed countries. Furthermore, the effect of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) on pericardial constriction and outcomes after surgery is unknown. We investigated the causes of constrictive pericarditis, outcomes after pericardiectomy, and predictors of mortality in Cape Town, South Africa, during a 22-year period of high HIV/AIDS prevalence.
Methods: A retrospective review of the medical records of all patients who had undergone pericardiectomy for constrictive pericarditis at Groote Schuur Hospital from January 1, 1990 to December 31, 2012 was performed.
Results: Of 121 patients, 36 (29.8%) had proven tuberculosis, 74 (61.2%) had presumed tuberculosis, 6 (5%) had idiopathic causes, and 5 (4%) had miscellaneous causes of constrictive pericarditis. Seventeen patients (14%) died perioperatively with low cardiac output syndrome the main cause of mortality. On multivariable analysis, serum sodium (hazard ratio, 0.88; 95% confidence interval, 0.80-0.97; P=0.009) and preoperative New York Heart Association class IV (hazard ratio, 3.42; 95% confidence interval, 1.29-9.08; P=0.014; vs combined class I-III) were independent predictors of early mortality. Of the 121 patients, 14 (11.6%) were HIV positive, with a mean CD4 cell count of 284±133 cells/µL. No early deaths occurred in the HIV-positive patients.
Conclusions: Tuberculosis is the main cause of constrictive pericarditis in South Africa. Despite its efficacy at relieving the symptoms of heart failure, pericardiectomy is associated with high perioperative mortality that was not influenced by HIV status. New York Heart Association functional class IV and hyponatremia predict for early mortality after pericardiectomy.

Huge left-ventricular pseudoaneurysm compressing coronary artery 10 weeks after stabbing attack
Jacques Scherman, Thi Dan Linh Nguyen, Peter Zilla, Maximilian Emmert.
European Heart Journal (2014) 35(6): 385

Covalent incorporation and controlled release of active dexamethasone from injectable polyethylene glycol hydrogels
Deon Bezuidenhout, Anel Oosthuysen, Neil Davies, Lage Ahrenstedt, Stephan Dobner, Peter Roberts, Peter Zilla.
Journal of Biomedical Materials Research (A) (2013) 101A(5): 1311-1318

Dexamethasone (Dex) is used in a wide range of applications, but may have undesirable systemic side effects. A number of techniques have thus been developed to deliver the substance locally. In this study, dexamethasone was acrylated, pegylated, and tethered to hydrolytically degradable (acrylate based) and nondegradable (vinyl sulfone based) polyethylene glycol hydrogels by nucleophilic addition. Hydrogel swelling, drug elution and drug activity were followed over an extended period in vitro. Nondegradable gels were stable for more than a year, while degradable gels showed increasing swelling ratios due to degradation that resulted in disintegration after  12 days. Near-linear (zero order) release could be achieved in some cases with the degradable gels, while release from the nondegradable gels approximated first order initial release kinetics. Significantly delayed release was observed in all cases where the Dex was linked to the gels, when compared with controls where the drug was merely physically incorporated. Eluates from the gels containing the tethered drug showed high levels of activity for extended time periods, while the activity of the eluates from gels containing nonbound dexamethasone decreased rapidly within the first few days. Dexamethasone can thus be incorporated using nucleophilic addition chemistry to produce gels that are capable of sustained release of the active drug. The methodology is applicable to a variety of drugs that contain hydroxyl groups.

Micro-structurally detailed model of a therapeutic hydrogel injectate in a rat biventricular cardiac geometry for computational simulations
Mazin Sirry, Neil Davies, Karen Kadner, Laura DuBuis, Muhammad Saleh, Ernesta Meintjies, Bruce Spottiswoode, Peter Zilla, Thomas Franz.
Computer Methods in Biomechanics and Biomedical Engineering (2015)18(3):325-331

Protective constriction of coronary vein grafts with knitted nitinol
Lovendran Moodley, Thomas Franz, Paul Human, Michael Wolf, Deon Bezuidenhout, Jacques Scherman, Peter Zilla.
European Journal of Cardio-Thoracic Surgery (2013) 44(1): 64-71

Outcomes of myocardial infarction hydrogel injection therapy in the human left ventricle dependent on injectate distribution
Renee Miller, Neil Davies, Jeroen Kortsmit, Peter Zilla, Thomas Franz.
International Journal for Numerical Methods in Biomedical Engineering (2013) 29(8)

Myocardial infarction therapies involving biomaterial injections have shown benefits in inhibiting progression towards heart failure. However, the underlying mechanisms remain unclear. A finite element model of the human left ventricle was developed from magnetic resonance images. An anteroapical infarct was represented at acute (AI) and fibrotic (FI) stage. Hydrogel injections in the infarct region were modelled with layered (L) and bulk (B) distribution. In the FI, injectates reduced end-systolic myofibre stresses from 291.6% to 117.6% (FI-L) and 115.3% (FI-B) of the healthy value, whereas all AI models exhibited sub-healthy stress levels (AI: 90.9%, AI-L: 20.9%, AI-B: 30.5%). Reduction in end-diastolic infarct stress were less pronounced for both FI (FI: 294.1%, FI-L: 176.5%, FI-B: 188.2%) and AI (AI: 94.1%, AI-L: 35.3%, AI-B: 41.2%). In the border zone, injectates reduced end-systolic fibre stress by 8–10% and strain from positive (AI) and zero (FI) to negative. Layered and bulk injectates increased ejection fraction by 7.4% and 8.4% in AI and 14.1% and 13.7% in FI. The layered injectate had a greater impact on infarct stress and strain at acute stage, whereas the bulk injectate exhibited greater benefits at FI stage. These findings were confirmed by our previous in vivo results.

Differentiating transmural from transanastomotic prosthetic graft endothelialization through an isolation loop-graft model
Timothy Pennel, Peter Zilla, Deon Bezuidenhout.
Journal of Vascular Surgery (2013) 58(4): 1053-1061

A slow-release fibrin matrix increases adeno-associated virus transduction of wound repair cells in vivo
Christian Schmidt, Deon Bezuidenhout, Peter Zilla, Neil Davies.
Journal of Biomaterials Applications (2013)

Virus-mediated gene therapy is a promising strategy for numerous tissue engineering applications. Fibrin-based scaffolds have been previously used as vehicles for localised delivery of adenovirus to wound sites. However, their utility in the delivery of adeno-associated viruses to wound repair cells has not yet been determined. The influence of fibrin concentration on efficacy of delivery of AAV-2 to wound tissue was assessed in this study. Fibrin scaffolds containing recombinant AAV-2 encoding for  -galactosidase were polymerised in porous polyurethane discs and implanted subcutaneously in rats. A fibrin scaffold with a concentration of 50 mg/ml showed significantly elevated levels of  -galactosidase activity within explanted discs at 10 days compared to 10 mg/ml and 25 mg/ml fibrin. These findings inform efforts to optimise biodegradable scaffolds for the localised delivery of AAV in tissue engineering.

The use of finite element methods and genetic algorithms in search of an optimal fabric reinforced porous graft system
Mark S Yeoman, BD Reddy, Helmut C Bowles, Peter Zilla, Deon Bezuidenhout, Thomas Franz
Annals of Biomedical Engineering 2013;37(11):2266-2287

Recommendations for the treatment of hypertension in the elderly and very elderly – a scotoma within international guidelines
Hans-Hendrik Schafer, Jacob de Villiers, Isabella Sudano, Sandra Dischinger, Glan-Reto Theus, Peter Zilla, Thomas Dieterle.
Swiss Medical Weekly (2012) 142: 13574

The recommendations of international scientific societies for the treatment of hypertension in the geriatric population are different. Lack of outcome trials, non-standardised terminology as well as differing levels of evidence contribute to the inconsistencies in the guidelines. This review article compares six international guidelines (ESH-ESC 2007/2009, SHG 2009, DHL 2008, CHEP 2010, NICE 2011 and JNC7 2003) as well as the consensus document of the ACCF/AHA 2011 in terms of their recommendations of drug classes, target blood pressure values and the use of combination therapy. Generally, antihypertensive therapy appears to be clinically beneficial in geriatric patients. Target blood pressure values of <140–150/90 mm Hg and <140/90 mm Hg can be used as a general guideline for octogenarians (80–89 yrs) and septuagenarians (70–79 yrs) respectively. While angiotensin-II converting enzyme inhibitors and diuretics appear to be advantageous in treating combined systolic-diastolic hypertension, calcium-channel blockers and diuretics are to be recommended in the management of isolated systolic hypertension. Combination therapy often increases the efficacy of the treatment as well as patient medication adherence. Furthermore, by making the most of drug combination synergy, lower doses may be used resulting in fewer side-effects.

The beneficial effects of deferred delivery on the efficiency of hydrogel therapy post myocardial infarction
Karen Kadner, Stephan Dobner, Thomas Franz, Deon Bezuidenhout, Mazin Sirry, Peter Zilla, Neil Davies.
Biomaterials (2012) 33(7): 2060-2066

Biomaterials are increasingly being investigated as a means of reducing stress within the ventricular wall of infarcted hearts and thus attenuating pathological remodelling and loss of function. In this context, we have examined the influence of timing of delivery on the efficacy of a polyethylene glycol hydrogel polymerised with an enzymatically degradable peptide sequence. Delivery of the hydrogel immediately after infarct induction resulted in no observable improvements, but a delay of one week in delivery resulted in significant increases in scar thickness and fractional shortening, as well as reduction in end-systolic diameter against saline controls and immediately injected hydrogel at both 2 and 4 weeks post-infarction (p < 0.05). Hydrogels injected at one week were degraded significantly slower than those injected immediately and this may have played a role in the differing outcomes. The hydrogel assumed markedly different morphologies at the two time points having either a fibrillar or bulky appearance after injection immediately or one week post-infarction respectively. We argue that the different morphologies result from infarction induced changes in the cardiac structure and influence the degradability of the injectates. The results indicate that timing of delivery is important and that very early time points may not be beneficial.

Rheumatic heart disease: The tip of the iceberg
Karen Sliwa, Peter Zilla.
Circulation (2012) 125: 3060-3062

Blood pressure target attainment in the background of guidelines: the very elderly in Swiss primary care
Hans-Hendrik Schafer, Isabella Sudano, Gian-Reto Theus, Peter Zilla, Georg Noll, Michael Burnier.
Family Practice (2012) 29(5): 511-520

Off-the-shelf human decellularized tissue-engineered heart valves in a non-human primate model
Benedikt Weber, Petra Dijkman, Jacques Scherman, Bart Sanders, Maximilian Emmert, Jurg Grunenfelder, Renier Verbeek, Mona Bracher, Melanie Black, Thomas Franz.
Biomaterials (2013) 34: 7269-7280

Heart valve tissue engineering based on decellularized xenogenic or allogenic starter matrices has shown promising first clinical results. However, the availability of healthy homologous donor valves is limited and xenogenic materials are associated with infectious and immunologic risks. To address such limitations, biodegradable synthetic materials have been successfully used for the creation of living autologous tissue- engineered heart valves (TEHVs) in vitro. Since these classical tissue engineering technologies necessitate substantial infrastructure and logistics, we recently introduced decellularized TEHVs (dTEHVs), based on biodegradable synthetic materials and vascular-derived cells, and successfully created a potential off-the- shelf starter matrix for guided tissue regeneration. Here, we investigate the host repopulation capacity of such dTEHVs in a non-human primate model with up to 8 weeks follow-up. After minimally invasive de- livery into the orthotopic pulmonary position, dTEHVs revealed mobile and thin leaflets after 8 weeks of follow-up. Furthermore, mild-moderate valvular insufficiency and relative leaflet shortening were detected. However, in comparison to the decellularized human native heart valve control e representing currently used homografts e dTEHVs showed remarkable rapid cellular repopulation. Given this sub- stantial in situ remodeling capacity, these results suggest that human cell-derived bioengineered decel- lularized materials represent a promising and clinically relevant starter matrix for heart valve tissue engineering. These biomaterials may ultimately overcome the limitations of currently used valve re- placements by providing homologous, non-immunogenic, off-the-shelf replacement constructs.

Cell specific ingrowth hydrogels
Mona Bracher, Deon Bezuidenhout, Matthias P Lutolf, Thomas Franz, Michelle Sun, Peter Zilla, Neil Davies.
Biomaterials (2013) 34: 6797-6803

Extracellular mimetic hydrogels formed from peptide crosslinkers and polyethylene glycol monomers permit cell-controlled invasion. The use of matrix metalloproteinase specific peptides might further allow for selective control of different cell-type invasion. In this study, the invasion of fibroblasts and vascular smooth muscle cells (VSMC) into hydrogels polymerised with either a peptide generally permissive for matrix metalloproteinase (MMP) degradation or peptides preferentially cleaved by MMP- 14 or MMP-9 enzymes were compared. The two cell-types invaded the MMP permissive hydrogel equally. However, invasion of VSMC into MMP-14 selective peptide crosslinked hydrogels was diamet- rically opposite in nature to that of fibroblasts whereby VSMC showed a two-fold increase into these hydrogels relative to that observed in permissive hydrogels whilst fibroblasts had a relative two-fold decrease (p < 0.01). These findings are suggestive that invasion and growth of different cell-types in engineered synthetic extracellular matrix mimics may be controlled selectively by the choice of protease specific peptide crosslinker and this could have general utility in tissue regenerative and engineering approaches.

Pulmonary artery banding:  Still a valuable option in developing countries?
Andre Brooks, Agnetha Geldenhuys, Liesl Zuhlke, Paul Human, Peter Zilla.
European Journal of Cardio-Thoracic Surgery (2012) 41: 272-276

OBJECTIVE: We examined whether the socio-economic circumstances of a developing country justify pulmonary artery banding (PAB) for the deferral of perceived high-risk patients requiring biventricular repair.
METHODS: A retrospective cohort analysis was done on 143 consecutive patients with ventricular anatomy suitable for a biventricular repair, who had a pulmonary artery band applied between 1 January 2002 and 31 December 2007 as they were considered too high a risk to undergo corrective surgery. The goal in all patients was to lower their risk of definitive surgery by improving their clinical condition. The minimum follow-up period was 2 years with the closing date for data collection being 31 January 2010. The mean weight and age at PAB was 5.34 ± 2.94 kg and 9.9 ± 17.3 months. The end points of the study were mortality, interval hospital readmission, growth pattern post-banding, whether or not definitive correction was achieved, and the current follow-up status of uncorrected patients.
RESULTS: The hospital mortality was 8% (n = 12), the inter-stage mortality 21% (n = 30), and the total mortality 29% (n = 42). Positive growth was not shown in 50% following the banding procedure. The mean number of inter-current hospital admissions was 1.5 ± 2 times per patient. At the termination of data collection, after a mean interval of 24.5 ± 14.3 months, debanding and full correction was achieved in 43% (n = 62). In addition to the 29% (n = 42) that were confirmed to be dead, an additional 28% (n = 39) were not corrected and of these almost half were regarded as lost to follow-up. Thus, of the entire cohort of patients, 57% (n = 81) have not achieved definitive correction at the termination of data collection.
CONCLUSION: A strategy of deferring biventricular repair by the application of a pulmonary artery band is ineffective under Third World conditions largely due to lack of patient compliance. This study shows that the overall mortality in the inter-stage period following PAB is high prior to definitive correction. Less than half of patients will eventually be repaired in a reasonable time frame and patient follow-up is unreliable. We conclude that consideration should be given to early definitive repair even in perceived high-risk cases.

Remodeling leads to distinctly more intimal hyperplasia in coronary than in infra-inguinal vein grafts.
Peter Zilla, Lovendran Moodley, Jacques Scherman, Hugo Krynauw, Jeroen Kortsmit, Paul Human, Michael Wolf, Thomas Franz.
Journal of Vascular Surgery (2012) 55(6): 1734-1741

Flow patterns and shear forces in native coronary arteries are more protective against neo-intimal hyperplasia than those in femoral arteries. Yet, the calibre mismatch with their target arteries makes coronary artery bypass grafts (CABGs) more likely to encounter intimal hyperplasia than their infra-inguinal counterparts due to the resultant slow flow velocity and increased wall stress. In order to allow a site-specific, flow-related comparison of remodeling behavior, saphenous vein bypass grafts were simultaneously implanted in femoral and coronary position.
Saphenous vein grafts were concomitantly implanted as coronary and femoral bypass grafts using a senescent non-human primate model. Duplex ultrasound-based blood flow velocity profiles as well as vein graft and target artery dimensions were correlated with dimensional and histo-morphological graft remodeling in large, senescent Chacma baboons (n=8; 28.1±4.9kg) over a 24 week period. 
At implantation, the cross sectional quotient between target arteries and vein grafts was Qc=0.62±0.10 for femoral grafts versus 0.17±0.06 for coronary grafts and as such the dimensional graft-to-artery mismatch was 3.6 times higher (p<0.0001) in coronary grafts. Together with different velocity profiles, these site-specific dimensional discrepancies resulted in a 57.9±19.4% lower maximum flow velocity (p=0.0048); 48.1±23.6% lower maximal cycling wall shear stress (p=0.012) and 62.2±21.2% lower mean velocity (p=0.007) in coronary grafts. After 24 weeks, the luminal diameter of all coronary grafts had contracted by 63% (from ID 4.49±0.60 to 1.68±0.63mm; p<0.0001) [sub-intimal diameter: -41.5%, p=0.002] while 57% of the femoral interposition grafts had dilated by 31% (from ID 4.21±0.25 to 5.53±1.30mm; p=0.020). Neo-intimal tissue was 2.3 times thicker in coronary than in femoral grafts (561±73µm versus 240±149µm ; p=0.001). Overall, the luminal area of coronary grafts was, on average, 4.1 times smaller than that of femoral grafts.
Although coronary and infra-inguinal bypass surgery uses saphenous veins as conduits they undergo significantly different remodeling processes in these two anatomical positions.

Deferred delivery augments the efficiency of hydrogel therapy post myocardial infarction.
Karen Kadner, Stephan Dobner, Deon Bezuidenhout, Thomas Franz, Mazin Sirry, Peter Zilla, Neil Davies.
Biomaterials  (2012) 33(7): 2060-2066

Biomaterials are increasingly being investigated as a means of reducing stress within the ventricular wall of infarcted hearts and thus attenuating pathological remodelling and loss of function. In this context, we have examined the influence of timing of delivery on the efficacy of a polyethylene glycol hydrogel polymerised with an enzymatically degradable peptide sequence. Delivery of the hydrogel immediately after infarct induction resulted in no observable improvements, but a delay of one week in delivery resulted in significant increases in scar thickness and fractional shortening, as well as reduction in end-systolic diameter against saline controls and immediately injected hydrogel at both 2 and 4 weeks post-infarction (p < 0.05). Hydrogels injected at one week were degraded significantly slower than those injected immediately and this may have played a role in the differing outcomes. The hydrogel assumed markedly different morphologies at the two time points having either a fibrillar or bulky appearance after injection immediately or one week post-infarction respectively. We argue that the different morphologies result from infarction induced changes in the cardiac structure and influence the degradability of the injectates. The results indicate that timing of delivery is important and that very early time points may not be beneficial.

Sustaining neovascularisation of a scaffold through staged release of VEGF-A and PDGF-BB.
Neil Davies, Christian Schmidt, Deon Bezuidenhout, Peter Zilla.
Tissue Engineering (A) (2012) 18

Tissue regeneration into a three-dimensional scaffold requires the stimulation of blood vessel ingrowth. We have employed a freely interconnecting porous scaffold developed by us to determine the utility of a covalently bound heparin surface coating for the delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) in vivo. The heparin surface was shown to release VEGF far more rapidly than PDGF-BB in vitro (VEGF: 75 ng/h for 24 h; PDGF-BB: 86 pg/h for >7 days). In rat subcutaneous implants, at 10 days the heparin surface alone increased vessel ingrowth substantially (p<0.05 vs. unmodified scaffold), release of VEGF resulted in a further increase (p<0.05 vs. heparinized scaffold), whereas PDGF-BB had no additional effect. The increase induced by the combination of growth factors was similar to VEGF alone. After 2 months, PDGF-BB, but not VEGF delivery, resulted in a substantial increase in vascularization above that induced by heparin (p<0.05). At the longer time point the combination of growth factors was similar to PDGF-BB. However, only the combination of growth factors significantly elevated the number of ingrowing arterioles (p<0.05 vs. heparinized scaffold). Thus, the covalent modification of a porous scaffold with heparin allows for the differential release of VEGF and PDGF-BB that results in both a rapid and sustained increase in scaffold vascularization.

Patient-specific prediction of intrinsic mechanical loadings on sub-muscular pectoral pacemaker implants based on an inter-species transfer function.
Hamman De Vaal, James Neville, Micah Litow, Jacques Scherman, Peter Zilla, Thomas Franz.
Journal of Biomechanics (2011) 44: 2525-2531

With the steady technological development enabling reduced device dimensions and new patient populations, detailed data on mechanical in vivo loads become increasingly important to ensure reliability of implantable medical devices. Based on an intra-species correlation of in-line and transverse force of the Pectoralis major established previously for the Chacma baboon (de Vaal et al., 2010a), a simplified physiological model and a mechanical equivalent model were developed for a sub-muscular pectoral device implant considering Pectoralis major, Pectoralis minor and rib cage. By assessing the morphometric and mechanical parameters of these musculo-skeletal structures and the associated model parameters, the intra-species correlation was shown to exhibit (a) robustness for a larger intra-species subject population and (b) linear scale variance allowing application for humans under consideration of the inter-species difference of the attachment angles of Pectoralis major. The transfer function provides a basis for the prediction of patient-specific maximum mechanical loadings on a sub-muscular pectoral cardiac pacemaker implant through non- or minimal invasive measurements on the patient.

Knitted nitinol represents a new generation of constrictive external vein graft meshes.
Peter Zilla, Lovendran Moodley, Michael Wolf, Deon Bezuidenhout, Mazin Sirry, Nasser Rafiee, Wilhelm Lichtenberg, Melanie Black, Thomas Franz.
Journal of Vascular Surgery (2011) 54: 1439-1450

OBJECTIVE: Constriction of vein grafts with braided external nitinol meshes had previously led to the successful elimination of neointimal tissue formation. We investigated whether pulse compliance, smaller kink-free bending radius, and milder medial atrophy can be achieved by knitting the meshes rather than braiding, without losing the suppressive effect on intimal hyperplasia. METHODS: Pulse compliance, bending stiffness, and bending radius, as well as longitudinal-radial deformation-coupling and radial compression, were compared in braided and knitted nitinol meshes. Identical to previous studies with braided mesh grafts, a senescent nonhuman primate model (Chacma baboons; bilateral femoral interposition grafts/6 months) mimicking the clinical size mismatch between vein grafts and runoff arteries was used to examine the effect of knitted external meshes on vein grafts: nitinol mesh-constricted (group 1); nitinol mesh-constricted and fibrin sealant (FS) spray-coated for mesh attachment (group 2); untreated control veins (group 3), and FS spray-coated control veins (group 4). RESULTS: Compared with braided meshes, knitted meshes had 3.8-times higher pulse compliance (3.43 +/- 0.53 vs 0.94 +/- 0.12%/100 mm Hg; P = .00002); 30-times lower bending stiffness (0.015 +/- 0.002 vs 0.462 +/- 0.077 Nmm(2); P = .0006); 9.2-times narrower kink-free bending radius (15.3 +/- 0.4 vs 140.8 +/- 22.4 mm; P = .0006), and 4.3-times lower radial narrowing caused by axial distension (18.0% +/- 1.0% vs 77.0% +/- 3.7%; P = .00001). Compared with mesh-supported grafts, neointimal tissue was 8.5-times thicker in group I (195 +/- 45 mum) vs group III (23.0 +/- 21.0 mum; P < .001) corresponding with a 14.3-times larger neointimal area in group I (4330 +/- 957 x 103 mum(2)) vs group III (303 +/- 221x 103 mum(2); P < .00004). FS had no significant influence. Medial muscle mass remained at 43.4% in knitted meshes vs the 28.1% previously observed in braided meshes. CONCLUSION: Combining the suppression of intimal hyperplasia with a more physiologic remodeling process of the media, manifold higher kink-resistance, and lower fraying than in braided meshes makes knitted nitinol an attractive concept in external vein graft protection. CLINICAL RELEVANCE: A main reason for vein graft failure is neointimal hyperplasia and its associated pathology. Low shear stress and high circumferential wall stress play a key role in the development of flow-limiting neointimal tissue. By reducing the diameter and concomitantly increasing the flow velocity, radially constrictive external meshes were shown to nearly eliminate intimal hyperplasia. The introduction of a knitted mesh consisting of superelastic nitinol led to a more artery-like remodeling. Together with a higher kink resistance and less fraying, it holds high promise for the acute as well as the medium-term to long-term fate of vein grafts.

A mathematical method for constraint-based cluster analysis towards optimized constrictive diameter smoothing of saphenous vein grafts.
Thomas Franz, Daya Reddy, Paul Human, Peter Zilla.
Medical & Biological Engineering & Computing (2011) 48(6): 519-529

This study was concerned with the cluster analysis of saphenous vein graft data to determine a minimum number of diameters, and their values, for the constrictive smoothing of diameter irregularities of a cohort of veins. Mathematical algorithms were developed for data selection, transformation and clustering. Constrictive diameter values were identified with interactive pattern evaluation and subsequently facilitated in decision-tree algorithms for the data clustering. The novel method proved feasible for the analysis of data of 118 veins grafts, identifying the minimum of two diameter classes. The results were compared to outcome of a statistical recursive partitioning analysis of the data set. The method can easily be implemented in computer-based intelligent systems for the analysis of larger data sets using the diameter classes identified as initial cluster structure.

Induced chronic hypoxia negates the pro-angiogenic effect of surface immobilized heparin in a polyurethane porous scaffold.
Christian Schmidt, Deon Bezuidenhout, Lawrence Higham, Peter Zilla, Neil Davies.
Journal of Biomedical Materials Research (A) (2011) 98(4): 621-628

Porous scaffolds are frequently utilized in tissue regeneration. We have developed a polyurethane (PU) scaffold with a freely interconnecting porosity that can be modified with a covalently linked heparinized surface. The ability of this surface functionality to stimulate vessel and cellular growth into the PU scaffold has been evaluated by subcutaneous implantation of discs in the rat under normoxia and chronic hypoxia (hypobaric chamber) for 10 days. The heparinized surface alone was able to significantly increase vascularization and cellularization under normoxia (p < 0.05), but this response was negated by hypoxia. Addition of vascular endothelial growth factor to heparinized discs resulted in increased vascularity and cellularization under both conditions (p < 0.05). This suggests that endogenous growth factor production was limiting under chronic hypoxia but that an angiogenic response could still occur with exogenous delivery of factors.

Injectable living marrow stromal cell-based autologous tissue engineered heart valves: first experiences with a one-step intervention in primates.
Benedikt Weber, Jacques Scherman, Maximilian Emmert, Juerg Guenenfelder, Renier Verbeek, Mona Bracher, Melanie Black, Jeroen Kortsmit, Thomas Franz, Roman Schoenauer.
European Heart Journal (2011) 32(22): 2830-2840

Aims A living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates.
Methods and results Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n = 6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118 ± 17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodelling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress–strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue.
Conclusion Here, we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.

A numerical tool for the coupled mechanical assessment of anastomoses of PTFE arterio-venous access grafts.
M Ngoepe, B Reddy, Del Kahn, C Meyer, Peter Zilla, Thomas Franz.
Cardiovascular Engineering and Technology (2011) 2: 160-172

The anastomotic angle is assumed to affect the performance of arterio-venous (AV) access grafts by altering wall shear stress (WSS) and wall tension. The objective of this study was to develop a coupled numerical tool to assess fluid and structural anastomotic mechanics of a straight upper arm access graft. 3D computational fluid dynamics (CFD) and finite element (FE) models were developed for arterial and venous anastomoses with different graft attachment angles. The fluid simulations were executed using flow velocity profiles for anastomotic inlets obtained from a whole-graft CFD model. A mesh adaptation algorithm was developed to couple CFD and FE meshes and capture fluid structure interactions. The coupling algorithm enabled transfer of blood pressure (BP) and WSS predicted with the CFD models to the FE models as loadings. The deformations induced in the FE models were used to update the CFD geometries after which BP and WSS were recalculated and the process repeated until equilibrium between fluid and solid models. Maximum BP in the vein was 181 mmHg. WSS peaked at 2.3 and 0.7 Pa and the structural wall stress reached 3.38 and 3.36 kPa in arterial and venous anastomosis. Since flow-induced wall tension has been identified as a contributor to access graft failure along with WSS, the computational tool will be useful in studying the coupled mechanics in these grafts. Initial investigations of arterial and venous anastomotic end-to-side configuration indicated a slightly better performance of the 90° configuration over 135° arterial and 45° venous configurations.

Degradation-induced change in mechanical properties of an electro-spun polyester-urethane scaffold for vascular tissue regeneration.
Hugo Krynauw, Lucy Bruchmuller, Deon Bezuidenhout, Peter Zilla, Thomas Franz.
Journal of Biomedical Materials Research (B) (2011) 99B: 359-368

Tailored sizes of constrictive external vein meshes for coronary artery bypass surgery.
Thomas Franz, Paul Human, Stephan Dobner, Daya Reddy, Melanie Black, Helen Ilsley, Michael Wolf, Deon Bezuidenhout, Lovendran Moodley, Peter Zilla.
Biomaterials (2010) 31(35): 9301-9309

Background: External mesh-constriction of vein grafts was shown to mitigate intimal hyperplasia by lowering circumferential wall stress and increasing fluid shear stress. As under-constriction leaves vein segments unsupported and thus prone to neointimal proliferation while over-constriction may cause wall folding optimal mesh sizing holds a key to clinical success.
Methods: Diameter fluctuations and the occurrence of wall folding as a consequence of external constriction with knitted Nitinol meshes were assessed in saphenous vein grafts from 100 consecutive coronary artery bypass (CABG) patients. Subsequently, mesh dimensions were identified that resulted in the lowest number of mesh sizes for all patients either guaranteeing tight continual mesh contact along the entire graft length (stipulation A) or preventing wall folding (stipulation B). A mathematical data classification analysis and a statistical single-stage partitioning approach were independently applied alternatively prioritizing stipulation A or B.
Results: Although the risk of folding linearly increased when constriction exceeded 24.6% (Chi squared test p=0.0004) the actual incidence of folding (8.6% of veins) as well as the degree of lumenal encroachment (6.2±2.1%) were low. Folds were always single, narrow longitudinal formations (height: 23.3±4.0% of inner diameter / base: 16.6±18.1% of luminal circumference). Both analytical methods provided an optimum number of 4 mesh sizes beyond which no further advantage was seen. While the size ranges recommended by both methods assured continual tight mesh contact with the vein the narrower range suggested by the mathematical data classification analysis (3.0 to 3.7mm) put 20.6±12.5% of length in 69% of veins at risk of folding as opposed to 21.3±25.9% being at risk in the wider size range (3.0 to 4.2mm) suggested by the statistical partitioning approach.
Four mesh sizes would provide uninterrupted mesh contact in 98% of vein grafts in CABG procedures with only 26% of their length being at risk of relatively mild wall folding.

Modification, crosslinking and reactive electrospinning of a thermoplastic medical polyurethane for vascular graft applications.
Jaco Theron, Hansie Knoetze, Ron Sanderson, Roger Hunter, Kibrit Mequanint, Thomas Franz, Peter Zilla, Deon Bezuidenhout.
Acta Biomaterialia (2010) 6(7): 2434-2447

Thermoplastic polyurethanes are used in a variety of medical devices and experimental tissue engineering scaffolds. Despite advances in polymer composition to improve their stability, the correct balance between chemical and mechanical properties is not always achieved. A model compound (MC) simulating the structure of a widely used medical polyurethane (Pellethane®) was synthesized and reacted with aliphatic and olefinic acyl chlorides to study the reaction site and conditions. After adopting the conditions to the olefinic modification of Pellethane®, processing into flat sheets, and crosslinking by thermal initiation or ultraviolet radiation, mechanical properties were determined. The modified polyurethane was additionally electrospun under ultraviolet light to produce a crosslinked tubular vascular graft prototype. Model compound studies showed reaction at the carbamide nitrogen, and the modification of Pellethane with pentenoyl chloride could be accurately controlled to up to 20% (correlation: ? = 0.99). Successful crosslinking was confirmed by insolubility of the materials. Initiator concentrations were optimized and the crosslink densities shown to increase with increasing modification. Crosslinking of Pellethane containing an increasing number of pentenoyl groups resulted in decreases (up to 42%, p < 0.01) in the hysteresis and 44% in creep (p < 0.05), and in a significant improvement in degradation resistance in vitro. Modified Pellethane was successfully electrospun into tubular grafts and crosslinked using UV irradiation during and after spinning to render them insoluble. Prototype grafts had sufficient burst pressure (>550 mmHg), and compliances of 12.1 ± 0.8 and 6.2 ± 0.3%/100 mmHg for uncrosslinked and crosslinked samples, respectively. It is concluded that the viscoelastic properties of a standard thermoplastic polyurethane can be improved by modification and subsequent crosslinking, and that the modified material may be electrospun and initiated to yield crosslinked scaffolds. Such materials hold promise for the production of vascular and other porous scaffolds, where decreased hysteresis and creep may be required to prevent aneurismal dilation.

A constitutive model for the warp-weft coupled non-linear behavior of knitted biomedical textiles.
Mark Yeoman, Daya Reddy, Helmut Bowles, Deon Bezuidenhout, Peter Zilla, Thomas Franz.
Biomaterials  (2010) 31(32): 8484-8493

Knitted textiles have been used in medical applications due to their high flexibility and low tendency to fray. Their mechanics have, however, received limited attention. A constitutive model for soft tissue using a strain energy function was extended, by including shear and increasing the number and order of coefficients, to represent the non-linear warp-weft coupled mechanics of coarse textile knits under uniaxial tension. The constitutive relationship was implemented in a commercial finite element package. The model and its implementation were verified and validated for uniaxial tension and simple shear using patch tests and physical test data of uniaxial tensile tests of four very different knitted fabric structures. A genetic algorithm with step-wise increase in resolution and linear reduction in range of the search space was developed for the optimization of the fabric model coefficients. The numerically predicted stress–strain curves exhibited non-linear stiffening characteristic for fabrics. For three fabrics, the predicted mechanics correlated well with physical data, at least in one principal direction (warp or weft), and moderately in the other direction. The model exhibited limitations in approximating the linear elastic behavior of the fourth fabric. With proposals to address this limitation and to incorporate time-dependent changes in the fabric mechanics associated with tissue ingrowth, the constitutive model offers a tool for the design of tissue regenerative knit textile implants.

Vascular Surgery: Biomaterials.
Deon Bezuidenhout, Peter Zilla.
Encyclopedia of Materials: Science and Technology (2010): 9513-9518

This is a review paper and does not include an abstract.

A computational study of structural designs for a small-diameter composite vascular graft promoting tissue regeneration.
Mazin Sirry, Peter Zilla, Thomas Franz.
Cardiovascular Engineering and Technology (2010) 1(4): 269-281

The structural integrity and arterial mechanics are important aspects for tissue regenerative vascular grafts with ingrowth permissible porous scaffolds. This paper presents a computational study of structural designs for a small-diameter vascular graft comprising porous polyurethane scaffold and knitted reinforcement mesh using Nitinol and polyurethane wire, respectively. Finite element models of the porous scaffold with the knitted mesh as embedded or external reinforcement were generated using validated constitutive models for porous polyurethane and Nitinol. Simulating a luminal pressure of up to 200 mmHg, deformations and stresses were recorded in porous scaffold and knitted mesh. The models predicted compliance between 1.2 and 15.7%/100 mmHg for the reinforced grafts and 65.1 and 106.4%/100 mmHg for the non-reinforced grafts. For the reinforced grafts, maximum stress was 97.0, 28.2, and 0.055 MPa in Nitinol wire, polyurethane wire, and porous polyurethane scaffold, respectively, at 120 mmHg. The corresponding maximum strain was 0.27, 5.0, and 22.5%. Stress and strain remained safe in the Nitinol mesh and the porous polyurethane but became critical in the polyurethane mesh between 120 and 200 mmHg. Despite compression due to luminal pressure load, the porous scaffold remained ingrowth permissible for cells, capillaries, and arterioles up to 200 mmHg. The outcomes of this study provided preliminary concepts for the structural designs for a tissue regenerative composite vascular graft toward improved mechanical performance and structural integrity. The implemented modeling approach can be used in the further development and optimization of small-diameter tissue-regenerating vascular grafts.

The use of finite element models and generic algorithms in search of an optimal fabric reinforced porous graft system.
Mark Yeoman, Daya Reddy, Helmut Bowles, Peter Zilla, Deon Bezuidenhout, Thomas Franz.
Annals of Biomedical Engineering (2009) 37(11): 2266-87

The mechanics of arteries result from the properties of the soft tissue constituents and the interaction of the wall layers, predominantly media and adventitia. This concept was adopted in this study for the design of a tissue regenerative vascular graft. To achieve the desired structural properties of the graft, most importantly a diametric compliance of 6%/100 mmHg, finite element methods and genetic algorithms were used in an integrated approach to identify the mechanical properties of an adventitial fabric layer that were required to optimally complement an intimal/medial polyurethane layer with interconnected porosity of three different size classes. The models predicted a compliance of 16.0, 19.2, and 31.5%/100 mmHg for the non-reinforced grafts and 5.3, 5.5, and 6.0%/100 mmHg for the fabric-reinforced grafts. The latter, featuring fabrics manufactured according to the required non-linear mechanical characteristics numerically predicted, exhibited an in vitro compliance of 2.1 +/- 0.8, 3.0 +/- 2.4, and 4.0 +/- 0.7% /100 mmHg. The combination of finite element methods and genetic algorithms was shown to be able to successfully optimize the mechanical design of the composite graft. The method offers potential for the application to alternative concepts of modular vascular grafts and the incorporation of tissue ingrowth and biodegradation.

Dimensional analysis of human saphenous vein grafts: Implications for external mesh support.
Paul Human, Thomas Franz, Jacques Scherman, Lovendran Moodley, Peter Zilla.
Journal of Thoracic and Cardiovascular Surgery (2009) 137(5): 1101-1108

Objective: Diameter mismatch, luminal irregularities and side branches are main triggers of vein graft failure through neointimal hyperplasia. Constrictive external mesh-support was shown to mitigate this process. Optimal mitigation depends on detailed knowledge of the fluid dynamic imperfections of the saphenous vein anatomy.
Methods: In 200 consecutive patients undergoing coronary artery bypass grafting all harvested saphenous veins (length 34.4±10.8cm) were analyzed regarding diameter irregularities, side branch distribution and micro-structure.
Results: The mean outer diameter (OD) of surgically distended saphenous veins was 4.2±0.6mm (males 4.3±0.6mm vs females 3.9±0.5mm; p<0.0001). While the OD significantly decreased over the initial 18cm (-7.6%; p<0.0001) the overall increase between malleolus and thigh was non-significant (+11.2%;N.S.). Smaller diameter veins (<3.5mm) had more pronounced diameter fluctuations than larger ones (31.8±11.0% vs 21.2±8.8%; p<0.0001) with more than 71% of all veins showing caliber changes of more than 20%. There was one side branch every 5.4±4.3cm with a significantly higher incidence between 20 and 32cm from the malleolus (p<0.0001 to distal / p<0.0004 to proximal).  Generally, females had more side branches than males (0.30±0.15cm-1 versus 0.25±0.12cm-1; p=0.0190). Thick-walled veins (565.7±138.4µm) had a significantly higher number of large side branches (p<0.0001) while thin-walled veins (398.7±123.2µm) had significantly more small side branches (p<0.0001). Pronounced intimal thickening ('cushions') was found in 28% of vessels (119.8±28.0µm vs 40.1±18.2µm; p<0.0001).
Conclusion: While the preferential location of side branches may be addressed by the deliberate discarding of infra-genicular vein segments, a diameter constriction of 27% would eliminate diameter irregularities in 98% of vein grafts.

Utilization of shape memory in external vein-graft meshes allows extreme diameter constriction for suppressing intimal hyperplasia: A non-human primate study.
Peter Zilla, Michael Wolf, Nasser Rafiee, Lovendran Moodley, Deon Bezuidenhout, Melanie Black, Paul Human, Thomas Franz.
Journal of Vascular Surgery (2009) 49(6): 1532-42

Constrictive external Nitinol meshes have been shown to suppress neo-intimal tissue formation and preserve endothelial integrity in vein grafts. As this mitigating effect increased with the degree of constriction we investigated whether extreme constriction was possible without leading to detrimental luminal encroachment.
A senescent non-human primate model (Chacma baboons / bilateral femoral interposition grafts) mimicking the clinical size-mismatch between vein grafts and run-off arteries was used. Control grafts were either untreated (group 1) or spray-coated with fibrin glue (group 2). Nitinol meshes constricting the lumen by ≤80% (group 3) were compared with longitudinally pleated meshes of identical circumference that constricted the lumen by >90% (group 4). Anastomotic size mismatch at implantation was expressed as quotient of cross-sectional area of run-off artery to vein graft (QC).
At 6 months all vein grafts without mesh-support showed thick, eccentric layers of neo-intimal tissue (group 1: 348±130µm [QC median at implant 0.19]; group 2: 318±142µm [QC median at implant 0.17])). Fibrin glue-spraying had no effect. In contrast, neo-intimal tissue was absent in all mesh-supported grafts (p<0.007 in all cases) both at 6 weeks / 6 months (group 3: 7.5±8.8µm and 2.5±4.4µm [QC median at implant 1.47]); group 4: 1.3±0.6µm and 3.8±5.6µm [QC median at implant 3.09]). Except for mild tissue buckling (fold hight<356µm) in one group 3 graft none of the mesh-constricted grafts showed wall folds. Endothelial coverage was only complete in the mesh-supported groups (100% in group 3 and 4 versus 85±14% (p<0.023) in group 1. Fibrin glue alone (52±48%) did not preserve endothelialization of control grafts (p<0.38).
Extreme vein graft constriction using external Nitinol meshes is possible without detrimental tissue buckling. Although moderate constriction was found to be sufficient for mitigating diffuse intimal hyperplasia and endothelial detachment, extremer constriction may occasionally be required to eliminate luminal irregularities.

The first human heart transplant and further advances in cardiac transplantation at Groote Schuur Hospital and the University of Cape Town.
Johan Brink, Joannis Hassoulas.
Cardiovascular Journal of Africa (2009) 20(1): 31-35

Christiaan (Chris) Barnard was born in South Africa in 1922 and qualified in medicine at the University of Cape Town in 1946. Following surgical training in South Africa and the USA, Barnard established a successful open-heart surgery program at Groote Schuur Hospital and the University of Cape Town in 1958.  In 1967, he led the team that performed the world's first human-to-human heart transplant. The article describing this remarkable achievement was published just 3 weeks after the event in the South African Medical Journal and is one of the most cited articles in the Cardiovascular field. In the lay media as well, this first transplant also remains the most publicized event in world medical history,
Although the first heart transplant patient survived only 18 days, four of Groote Schuur Hospital's first 10 patients survived for more than one year, two living for 13 and 23 years, respectively. This relative success amid many failures worldwide did much to generate guarded optimism that heart transplantation would eventually become a viable therapeutic option.
This first heart transplant and subsequent ongoing research in cardiac transplantation at the University of Cape Town and in a few other dedicated centres over the subsequent 15 years laid the foundation for heart transplantation to become a well-established form of therapy for end-stage cardiac disease.  During this period from 1968 to 1983, Chris Barnard and his team continued to make major contributions to organ transplantation, notably the development of the heterotopic (“piggy-back”) heart transplant; advancing the concept of brain death, organ donation and other related ethical issues; better preservation and protection of the donor heart (including hypothermic perfusion storage of the heart; studies on the haemodynamic and metabolic effects of brain death; and even early attempts at xenotransplantation.

The effects of cross-link density and chemistry on the calcification potential of diamine-extended glutaraldehyde-fixed bioprosthetic heart-valve materials.
Deon Bezuidenhout, Anel Oosthuysen, Paul Human, Christoph Weissenstein, Peter Zilla.
Biotechnol Appl Biochem (2009) 54(3): 133-40

Despite indications that GA (glutaraldehyde)-crosslinked tissues remain prone to long-term degradation and calcification, it is still the reagent of choice in the fixation of bioprosthetic heart valves. We have shown previously that increased GA concentrations and diamine extension of cross-links with lysine incorporation lead to mitigated in vivo calcification, mainly of porcine aortic-wall tissue. The present study was performed to assess the correlation between the cross-link density of all three commonly used tissue types [PW (porcine aortic wall), PL (porcine aortic leaflet) and BP (bovine pericardium)] and tissue calcification in the subcutaneous rat model after GA treatment with or without lysine. The effect of lysine enhancement, and increased GA concentration in the presence of lysine, resulted in significant increases in tissue cross-linking in all three tissue types. Although increased GA concentration on its own resulted in decreased calcification without an increase in cross-link density, overall positive correlations were found between denaturation temperature and RPD (resistance towards protease degradation) [correlation coefficient (rho) values: rhoPW =0.922, rhoPL =0.783 and rhoBP =0.955], whereas negative correlations existed between RPD and calcification (rhoPW=-0.836, rhoPL=-0.929 and rhoBP=-0.579). The combination of lysine enhancement and an increase in GA concentration from 0.2 to 3% resulted in 79, 44 and 56% decreases in calcification in PW, PL and BP. In the case of BP, a decrease in calcification of 81% could be achieved merely by adding lysine extension to low-concentration (0.2 %) GA cross-linking. Thus it is concluded that the increase in cross-link density achieved by lysine incorporation, and by increased GA concentration in the presence of lysine, results in significant and marked decreases in calcification of all three types of tissues commonly used in bioprosthetic heart valves.

Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography.
Christian Schmidt, Deon Bezuidenhout, Mike Beck, Elizabeth van der Merwe, Peter Zilla, Neil Davies.
Biomaterials (2009) 30(30): 5959-68

Microcomputed tomography (micro-CT) is increasingly being used to analyze the three-dimensional structure and architecture of microvascular networks. Therefore we have evaluated a micro-CT analysis of VEGF-induced vessel ingrowth into a porous polyurethane scaffold through comparison with analyses by CD31 immunohistochemistry, vascular perfusion by intravital Lycopersicon esculentum lectin perfusion and vascular corrosion casting. Micro-CT scanning found a similar level of vascularisation within the VEGF treated scaffolds to that determined by the other analytical methods. However, although the relative increase in vascularisation (17 fold above PBS controls p<0.05) induced by VEGF determined by micro-CT was similar to the perfusion based analyses (20.1 and 10.4 fold for lectin perfusion and vascular corrosion respectively p<0.05), it differed substantially from that determined by CD31 immunohistochemistry (3.2 fold p<0.05). This difference was due to a large proportion of unperfused vessels in the PBS control that were not present in the VEGF group. The increase in perfusion probably resulted in part from an increase in average vessel diameter. Though this increase was detected by micro-CT, the actual diameters were overestimated by 60-90% most likely as a consequence of a merging effect for juxtaposed vessels. Thus whilst micro-CT gives an accurate three-dimensional quantification of the VEGF-induced increase in perfused vessels, resolution needs to be maximized for accurate sizing of a microvascular network's components.

Aortic valve leaflet mechanical properties facilitate diastolic valve function.
Thorsten Koch, Daya Reddy, Peter Zilla, Thomas Franz.
Computational Methods in Biomechanical and Biomedical Engineering (2009) 1

This work was concerned with the numerical simulation of the behaviour of aortic valves whose material can be modelled as non-linear elastic anisotropic. Linear elastic models for the valve leaflets with parameters used in previous studies were compared with hyperelastic models, incorporating leaflet anisotropy with pronounced stiffness in the circumferential direction through a transverse isotropic model. The parameters for the hyperelastic models were obtained from fits to results of orthogonal uniaxial tensile tests on porcine aortic valve leaflets. The computational results indicated the significant impact of transverse isotropy and hyperelastic effects on leaflet mechanics; in particular, increased coaptation with peak values of stress and strain in the elastic limit. The alignment of maximum principal stresses in all models follows approximately the coarse collagen fibre distribution found in aortic valve leaflets. The non-linear elastic leaflets also demonstrated more evenly distributed stress and strain which appears relevant to long-term scaffold stability and mechanotransduction.

A synthetic non-degradable polyethylene glycol hydrogel retards adverse post-infarct left ventricular remodeling.
Stephan Dobner, Deon Bezuidenhout, Padmini Govender, Peter Zilla, Neil Davies.
Journal of Cardiac Failure (2009) 15(7): 629-636
Left ventricular remodeling after myocardial infarction is a key component of heart failure and it has long been postulated that it may result from increased wall stress. It has recently been suggested that an injectable, non-degradable polymer may limit pathological remodeling in a manner analogous to that of cardiac support devices. We have tested a non-degradable polyethylene glycol (PEG) gel in a rat infarction model. METHODS AND RESULTS: After permanent ligation of the left anterior descending artery in male Wistar rats, PEG gel reagents were injected into the infarcted region and polymerized in situ. At 4 weeks, fractional shortening and infarct volume were unchanged relative to a saline injected control, but the infarct-induced left ventricular end-diastolic diameter (LVEDD) increase was substantially reduced (43%, P < .05) and wall thinning was completely prevented. At 13 weeks, the LVEDD were similar for both saline- and PEG-injected hearts. The non-degradable PEG gels did elicit a macrophage-based inflammatory reaction. CONCLUSIONS: The injection of non-degradable synthetic gel was effective in ameliorating pathological remodeling in the immediate postinfarction healing phase, but was unable to prevent the dilation that occurred at later stages in the healed heart.

Association of Ang-2 with integrin beta 2 controls Ang-2/PDGF-BB-dependent upregulation of human peripheral blood monocyte fibrinolysis.
Louise Bezuidenhout, Peter Zilla, Neil Davies.
Inflammation (2009) 32(6): 393-401

Angiopoietin-2 (Ang-2), an angiogenic factor that is generally considered an autocrine factor for endothelial cells was shown in a previous study to upregulate peripheral blood monocyte fibrinolysis in concert with platelet-derived growth factor-BB (PDGF-BB). This upregulation of fibrinolysis was demonstrated to be due to upregulation of elements of the matrix metalloproteinase and serine protease fibrinolytic pathways. The manner in which Ang-2 interacts with monocytes was not elucidated though no expression of the angiopoietin receptor tyrosine kinase Tie-2 was found for monocytes. In this study Ang-2 was found to bind to integrin beta(2), and functional inhibition of integrin beta(2) eliminated Ang-2/PDGF-BB-mediated upregulation of monocyte fibrin invasion. Additionally, integrin beta(2) blockade significantly inhibited the Ang-2/PDGF-BB based increase in matrix metalloproteinase-9 (MMP-9) and membrane type-1-MMP (MT1-MMP). Furthermore, Ang-2/PDGF-BB-upregulated urokinase plasminogen-activator receptor (uPAR) was shown to be associated in complexes with integrin beta(2). In addition, Ang-2 was shown to upregulate PDGFR-beta expression in monocytes. Therefore several components of the mechanism via which the novel interaction of Ang-2 and PDGF-BB with monocytes occurs have been identified.

Constrictive external nitinol meshes inhibit vein graft intimal hyperplasia in non-human primates.
Peter Zilla, Paul Human, Michael Wolf, Wilhelm Lichtenberg, Nasser Rafiee, Deon Bezuidenhout, Nazlia Samodien, Christian Schmidt, Thomas Franz.
Journal of Thoracic and Cardiovascular Surgery (2008) 136(3): 717-25

Objective: External mesh-support of vein grafts has been shown to mitigate the formation of intimal hyperplasia (IH). The aim of the present study was to address the issue of optimal mesh size in 
a non-human primate model that mimics the dimensional mismatch typically encountered between clinical vein grafts and their target arteries. 
Methods: The effect of mesh-size on intimal hyperplasia and endothelial preservation was assessed in bilateral femoral interposition grafts in Chacma baboons (n?=32/n=8 per mesh size). No mesh-suppoport (Group I) was compared with external Nitinol meshes at three different sizes: loose-fitting (Group II); 25% diameter-constricting (Group III) and 50% diameter-constricting 
(Group IV). Mesh-sizes were not only seen in isolation but also against the back ground of anastomotic size mismatch at implantation, expressed as quotient of cross sectional area of host 
artery to vein graft [QC].  
Results: Significant amounts of intimal hyperplasia were found in Group I [QCmedian 0.20;  IH6weeks = 1.63±0.34mm2; IH12weeks = 1.73±0.5mm2] and Group II [QCmedian 0.25; IH6week s= 1.96±1.64mm2; IH12weeks = 2.88±1.69mm2]. In contrast, Group III [QCmedian 0.45; IH6weeks = 0.08±0.13mm2; IH12weeks = 0.18±0.32mm2] and IV (QCmedian 1.16; IH6weeks = 0.02±0.03mm2; IH12weeks = 0.11±0.10mm2] showed dramatically suppressed IH (p<0.01) at both time points. Endothelial integrity was only preserved in group IV (p<0.05). There were no significant differences in vascularization and inflammation in either inter-layer or inter-group comparisons. 
Conclusion: By using an animal model that addressed the clinical phenomenon of diameter discrepancy between vein graft and bypassed artery, we could demonstrate that suppression of intimal hyperplasia required constrictive mesh sizes.

A computational study of knitted nitinol structures for their prospective use as external vein reinforcement.
Helena van der Merwe, Daya Reddy, Peter Zilla, Deon Bezuidenhout, Thomas Franz.
Journal of Biomechanics (2008) 41(6): 1302-1309

External reinforcement has been suggested for autologous vein grafts to address the mismatch of mechanical properties and fluid dynamics of graft and host vessel, a main factor for graft failure. A finite-element tool was developed to investigate the mechanical behaviour, in particular radial compliance, of knitted Nitinol meshes (internal diameter: 3.34 mm) with two different knit designs (even versus uneven circumferential loops) and three different wire thicknesses (0.05, 0.0635 and 0.075 mm) under physiological conditions. The Nitinol material parameters were obtained from experimental testing. The compliance predicted for the 80-120 mmHg physiological blood pressure range was 2.5, 0.9 and 0.6%/100 mmHg for the even loop design and 1.2, 0.5 and 0.5%/100 mmHg for the uneven loop design, for wire thicknesses of 0.05, 0.0635 and 0.075 mm. The highest stress, at 120 mmHg, was found in the even loop mesh with the thinnest wire to be 268 MPa, remaining 44.5% below the stress initiating stress-induced phase transformation. The maximum stress decreased to 132 and 91 MPa with increasing wire thickness of the same loop design. The uneven loop design exhibited maximum stress levels of 65.3%, 63.6% and 87.9% of the even loop values at 0.05, 0.0635 and 0.075 mm wire thickness. The maximum strain of 0.7%, at 120 mmHg, remained un-critical considering a typical high-cycle recoverable strain of 2%. It was demonstrated that the numerical approach developed was feasible of effectively evaluating design variations of knitted Nitinol meshes towards vein graft behaviour equivalent to arterial mechanics.

Genetic polymorphisms of alcohol metabolising enzymes: their role in susceptibility to oesophageal cancer.
DP Li, C Dandara, Gabi Walther, MI Parker.
Clin Chem Lab Med (2008) 46: 323-328

Abstract Background: Alcohol is a major risk factor for susceptibility to oesophageal cancer in the South African population. The role of polymorphisms in alcohol metabolising enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) in predisposition of this
population to oesophageal cancer is unknown. Alcohol metabolising enzymes exhibit polymorphisms that result in variant alleles with either increased or decreased activity. Methods: The role of these polymorphisms in increased risk of oesophageal cancer was investigated in 238 patients and 268 controls from Black and Mixed Ancestry South Africans, using the PCR/RFLP technique. Results: The ADH3*2/*2 genotype was significantly associated with increased risk for oesophageal cancer amongst Black subjects (odds ratio, 2.19; p=0.004).
The low activity ALDH2*2 allele was significantly associated with increased risk for oesophageal cancer amongst the Black subjects (odds ratio, 2.35; p=0.0084).
Conclusions: It was observed that ADH variants, ADH2*1 and ADH3*2, were associated with increased risk for oesophageal cancer, possibly due to the tolerance of the carriers of these alleles to alcohol consumption compared to those with high
activity alleles (ADH2*2 and ADH2*3) which are associated with higher production of the unpleasant acetaldehyde intermediate.

Prosthetic heart valves: catering for the few.
Peter Zilla, Johan Brink, Paul Human, Deon Bezuidenhout.
Biomaterials  (2008) 29(4): 385-406

Prosthetic heart valves epitomize both the triumphant advance of cardiac surgery in its early days and its stagnation into a retrospective, exclusive first world discipline of late. Fifty-two years after the first diseased heart valve was replaced in a patient, prostheses largely represent the concepts of the 1960s with many of their design-inherent complications. While the sophisticated medical systems of the developed world may be able to cope with sub-optimal replacements, these valves are poorly suited to the developing world (where the overwhelming majority of potential valve recipients reside), due to differences in age profiles and socio-economic circumstances. Therefore, it is the latter group which suffered most from the sluggish pace of developments. While it previously took less than 7 years for mechanical heart valves to develop from the first commercially available ball-in-cage valve to the tilting pyrolytic-carbon disc valve, and another 10 years to arrive at the all-carbon bi-leaflet design, only small incremental improvements have been achieved since 1977. Similarly, bioprosthetic valves saw their last major break-through development in the late 1960s when formalin fixation was replaced by glutaraldehyde cross linking. Since then, poorly understood so-called 'anti-calcification' treatments were added and the homograft concept rediscovered under the catch-phrase 'stentless'. Still, tissue valves continue to degenerate fast in younger patients, making them unsuitable for developing countries. Yet, catheter-delivered prostheses almost exclusively use bioprosthetic tissue, thereby reducing one of the most promising developments for patients of the developing world into a fringe product for the few first world recipients. With tissue-engineered valves aiming at the narrow niche of congenital malformations and synthetic flexible leaflet valves being in their fifth decade of low-key development, heart valve prostheses seem to be destined to remain an unsatisfying and exclusive first world solution for a long time to come.

Forty years on: The anesthetic for the world’s first human-to-human heart transplant remembered.
Peter Gordon, Johan Brink.
Journal of Cardiothoracic and Vascular Anesthesia (2008) 22(1): 133-138

The first human-to-human heart transplant was performed by a team led by Christiaan Barnard in Cape Town, South Africa, on December 3, 1967. The event, performed at the geographically isolated tip of Africa, stunned the world. The anesthesiologist was Dr Joseph Ozinsky. The anesthetic technique and monitoring used differed greatly from current practice. Anesthesia was maintained with nitrous oxide, oxygen, and halothane. No nondepolarizing agents were used, and no opioids were administered before weaning from cardiopulmonary bypass. The operation caused enormous worldwide interest in cardiac transplantation and stimulated the growth of bioethics and issues related to brain death. By the end of 1967, a number of centers had the skill and knowledge to perform a heart transplant in man. The basic technique of orthotopic heart transplantation (OHT) was developed from laboratory animal experimentation. Carrel and Guthrie had unsuccessfully attempted experimental heart transplantation in 1905. Between 1946 and 1955, Demikhov performed hundreds of transplant operations in dogs without the benefit of hypothermia or cardiopulmonary bypass. He developed techniques to transplant hearts intrathoracically, initially heterotopically and later orthotopically. The first successful orthotopic heart transplants in dogs were described by Lower and Shumway in 1960. Their technique, modified slightly by Barnard in 1968, became the standard approach for OHT. The first heart transplant in man was performed by James Hardy who in 1964 transplanted a chimpanzee heart into a patient in cardiogenic shock after a myocardial infarction. The operation was unsuccessful, and the patient died on the operating table because the heart was too small to bear the load.
In 1956, Barnard, then a promising young surgeon at Groote Schuur Hospital (GSH), accepted a 2-year scholarship to work in Owen Wangensteen’s Department of Surgery at the University of Minnesota. There he was exposed to the newly developing field of cardiac surgery under the mentorship of Walter Lillehei. He returned to Cape Town in 1958 with a de Wall-Lillehei heart lung machine and bubble oxygenator donated by the US government, set up a cardiac surgery center at GSH, and built up a formidable team, with particular expertise in the correction of congenital heart defects and valvular surgery. In 1963, he developed an interest in transplantation and performed his first heart transplant operation in a dog. In 1967, he returned to the US, spending 3 months studying immunosuppressive therapy with pioneer kidney transplant surgeon David Hume in Richmond, VA, and 2 weeks with Thomas Starzl in Denver, CO. On returning to Cape Town, he performed his only kidney transplant operation on a patient who survived 20 years. By the end of 1967, Barnard, now 45 years old, had performed 48 animal heart transplants, and the team was ready to perform a human-to-human heart transplant if a suitable recipient and donor could be found.

The dosage dependence of VEGF stimulation on scaffold neovascularisation.
Neil Davies, Stephan Dobner, Deon Bezuidenhout, Christian Schmidt, Mike Beck, Andreas Zisch, Peter Zilla.
Biomaterials  (2008) 29(26): 3531-8

Growth factors are often used in tissue regeneration to stimulate vascularisation of polymeric scaffolds, with vascular endothelial growth factor (VEGF) having been extensively studied for short-term vessel ingrowth. We have therefore evaluated the effect of different concentrations of VEGF on the vascularisation of a porous scaffold in the short-, intermediate- and long-term, by delivering 15, 150 and 1500ng VEGF/day to polyurethane scaffolds by osmotic pumps for up to 6 weeks. An increased vascularisation months after termination of VEGF delivery was only achieved with 150ng/day (46%, p<0.05). This dosage consistently showed elevated levels of vascularisation (144, 125, 160 and 60% above PBS controls at 10, 20, 30 and 42 days, respectively, p<0.05), whilst the vessels induced by the highest dosage, though initially maximally elevated (265 and 270% at 10 and 20 days, p<0.05) tended to regress after 20 days of VEGF delivery. Pericyte coverage was decreased at 20 days for the highest dosage (30%, p<0.05). Lectin perfusion demonstrated that vessels within the scaffold were connected to the host vasculature at all time points and perfusion was substantially raised by VEGF delivery at day 20. These results suggest concentration of VEGF plays a critical role in the nature and persistence of vasculature formed in a tissue regenerative scaffold.

Ang-2 and PDGF-BB cooperatively stimulate human peripheral blood monocyte fibrinolysis.
Louise Bezuidenhout, Mona Bracher, G Davison, Peter Zilla, Neil Davies.
J Leukoc Biol (2007) 81(6): 1496-1503

Angiopoietin-2 (Ang-2) is a growth factor, which was identified originally as playing a critical role in vessel remodeling during angiogenesis. More recent evidence has indicated additional involvement in vascular homeostatic responses such as coagulation and inflammation, which are central to wound healing. We therefore determined whether a relationship existed between Ang-2 and monocytes, one of the initial cell types to be recruited to a wound, in the context of fibrin clot invasion. Ang-2 significantly increased monocyte invasion of fibrin in the presence of serum. In the absence of serum, it required a combination of Ang-2 and platelet-derived growth factor BB (PDGF-BB) to increase invasion by threefold. Furthermore, it was shown that the heightened invasion was dependent on serine proteases and matrix metalloproteinases (MMPs) and that the combination of Ang-2 and PDGF-BB increased urokinase plasminogen-activator receptor expression, as well as MMP-9 and membrane type 1 MMP expression. These data give further credence to the concept of Ang-2 as a key regulator of several essential phases of wound healing.

Prosthetic vascular grafts: Wrong models, wrong questions and no healing.
Peter Zilla, Deon Bezuidenhout, Paul Human.
Biomaterials  (2007) 28(34): 5009-5027

In humans, prosthetic vascular grafts remain largely without an endothelium, even after decades of implantation. While this shortcoming does not affect the clinical performance of large bore prostheses in aortic or iliac position, it contributes significantly to the high failure rate of small- to medium-sized grafts (SMGs). For decades intensive but largely futile research efforts have been under way to address this issue. In spite of the abundance of previous studies, a broad analysis of biological events dominating the incorporation of vascular grafts was hitherto lacking. By focusing on the three main contemporary graft types, expanded polytetrafluoroethylene (ePTFE), Dacron and Polyurethane (PU), accumulated clinical and experimental experience of almost half a century was available. The main outcome of this broad analysis-supported by our own experience in a senescent non-human primate model-was twofold: Firstly, inappropriate animal models, which addressed scientific questions that missed the point of clinical relevance, were largely used. This led to a situation where the vast majority of investigators unintentionally studied transanastomotic rather than transmural or blood-borne endothelialization. Given the fact that in patients transanastomotic endothelialization (TAE) covers only the immediate perianastomotic region of sometimes very long prostheses, TAE is rather irrelevant in the clinical context. Secondly, transmural endothelialization seems to have a time window of opportunity before a build-up of an adverse microenvironment. In selecting animal models that prematurely terminate this build-up through the early presence of an endothelium, the most significant 'impairment factor' for physiological tissue regeneration in vascular grafts remained ignored. By providing insight into mechanisms and experimental designs which obscured the purpose and scope of several decades of vascular graft studies, future research may better address clinical relevance.

Reduction of calcification of carbodiimide-processed heart valve tissue by prior blocking of amine groups with monoaldehydes.
Frank Everaerts, M Gillissen, Mark Torrianni, Peter Zilla, Paul Human, Marc Hendriks, Johan Feijen.
Journal of Heart Valve Disease (2006) 15(2): 269-277

Failure of implanted bioprostheses due to calcification is a commonly occurring phenomenon. In order to prevent calcification, many alternative cross-linking methods to glutaraldehyde (GA) have been developed and evaluated. METHODS: In a novel approach an improved carbodiimide (EDC) cross-linking method that comprises a two-step process was developed. First, the available amine groups in (tissue) collagen were blocked with a monoaldehyde, followed by an EDC-activated cross-linking reaction of the carboxyl groups in the tissue with a poly (propylene glycol) bis 2-(amino-propyl) ether (Jeffamine). RESULTS: Samples processed via this method have shown a significantly reduced calcification in a subdermal juvenile rat model compared to samples with standard GA treatment. In the present study, heart valve tissue was blocked with various monoaldehydes, followed by reaction with Jeffamine using carbodiimide cross-linking chemistry. Leaflet calcification was almost eliminated using different aldehydes, whereas wall calcification was maximally 95% reduced when propionaldehyde was used as blocking agent, as compared to a carbodiimide cross-linked control without Jeffamine and blocked amine groups. CONCLUSION: Amine blocking and cross-linking technology appears promising in the design of the next generation of tissue valves. Calcification was significantly reduced compared to GA cross-linking. The mechanistic insight of decreased wall calcification is still unknown, and research investigations are ongoing.

Bioprosthetic tissue preservation by filling with a poly(acrylamide) hydrogel.
Anel Oosthuysen, Peter Zilla, Paul Human, Christian Schmidt, Deon Bezuidenhout.
Biomaterials  (2006) 27: 2123-2130

Glutaraldehyde (GA) fixation has been used for more than 40 years as the preferred treatment to suppress immunogenicity and increase durability of bioprosthetic tissues (BPT) used in heart valve prostheses.  This fixative and its reaction products have, however, been implicated in the calcific degeneration and long-term failure of these devices. The current study investigates stabilization of bioprosthetic tissue and the mitigation of calcification by filling aortic wall samples with a synthetic hydrogel (acrylamide), with and without pre-treatment with GA. Histological and gravimetric analysis showed full penetration of the acrylamide (AAm) into the fresh tissue, while only partial filling could be achieved with GA prefixed tissue.  The observed decrease in amino-group content (0.156±0.011 to 0.123±0.010 mmol/mg, p<0.03) and corresponding increase in shrinkage temperature (67.2±0.4 to 78.1±0.9°C, p<0.0001) when fresh tissue was filled, indicate the participation of tissue-amines in a process that leads to BPT crosslinking.  These effects were much less pronounced when the tissue was pre-fixed with GA.  Filling increased the tensile stiffness of fresh tissue (to levels half that of 0.2% GA fixed tissue), but decreased the stiffness of GA pre-fixed tissue.  When compared to standard 0.2% GA fixed samples, fresh tissue filled with AAm showed 87% (p<0.0035) less calcification while exhibiting similar resistance toward degradation by protease.  Filling did not result in significant decreases in calcification when the tissue was pre-fixed with GA.

Comparison of Processing and Sectioning Methodologies for Arteries Containing Metallic
Peter Rippstein, Melanie Black, Marie Boivin, John P Veinot, Xiaoli Ma, Yong-Xiang Chen, Paul Human, Peter Zilla, Edward R O’Brien.
Journal of Histochemistry & Cytochemistry (2006) 54: 673-681

The histological study of arteries with implanted metallic scaffolding devices, known as stents,
remains a technical challenge. Given that the arterial response to stent implantation can
sometimes lead to adverse outcomes, including the re-accumulation of tissue mass within the stent
(or in-stent restenosis), overcoming these technical challenges is a priority for the advancement of
research and development in this important clinical field. Essentially, the task is to section the
stent-tissue interface with the least amount of disruption of tissue and cellular morphology.
Although many methacrylate resin methodologies are successfully applied towards the study of
endovascular stents by a variety of research laboratories, the exact formulations, as well as
subsequent processing and sectioning methodology remain largely coveted. In this paper we
describe in detail a methyl methacrylate resin embedding methodology that can successfully be
employed towards tungsten carbide blade, as well as saw and grinding sectioning methods and
transmission electron microscopy. In addition, we present a comparison of the two sectioning
methodologies in terms of their effectiveness with regards to morphological, histochemical and
immunohistochemical analyses.

Gene-environment interaction: the role of SULT1A1 and CYP3A5 polymorphisms as risk modifiers for squamous cell carcinoma of the oesophagus.
C Dandara, DP Li, Gabi Walther, MI Parker.
Carcinogenesis (2006) 27(4): 791-797

An imbalance in the activities of enzymes involved in the metabolism, conjugation and transport of xenobiotics may account for the variability in susceptibility to the development of complex diseases such as cancer between different population groups. In this study we investigated a functional polymorphism in the SULT1A1 gene in 245 patients and 288 controls. Previous studies have shown that the 638G-->A polymorphism that results in the substitution of arginine by histidine at codon 213 (SULT1A1*2) results in decreased SULT1A1 activity. The same group of samples used in this study had been previously genotyped for CYP3A5 genetic polymorphisms. Among Black subjects the burning of wood or charcoal for cooking and keeping warm was significantly associated with increased risk for oesophageal cancer (OC) (AOR, 15.2; P=0.001) as was the consumption of home-brewed beer (AOR, 6.97; P=0.0001). Among the Mixed Ancestry group, tobacco smoking combined with alcohol consumption were significantly associated with higher risk for OC (AOR, 5.18; P=0.0005). In both Blacks and Mixed Ancestry subjects, starting to smoke below the age of 20 years was associated with significantly increased risk for OC (AOR, 3.5 among the Blacks and AOR, 12 among the Mixed Ancestry). The homozygous SULT1A1*2/*2 genotype was associated with increased risk for OC among smokers. The SULT1A1*2/*2 genotype in combination with the CYP3A5 heterozygous genotypes was associated with significantly increased risk for OC (AOR, 3.60; P=0.001) with the risk being even higher among smokers compared with non-smokers. The above findings confirm the association between alcohol consumption and tobacco smoking with increased risk for OC. The genotype results show that SULT1A1*2/*2 genotype is associated with increased risk for OC among subjects exposed to tobacco-smoke-related carcinogens.

Diamine-extended glutaraldehyde and carbodiimide crosslinks act synergistically in mitigating bioprosthetic aortic wall calcification.
Peter Zilla, Deon Bezuidenhout, Mark Torrianni, Marc Hendriks, Paul Human.
Journal of Heart Valve Disease (2005) 14: 538-545

Objective: The extension of glutaraldehyde (GA) cross-links with diamine bridges was previously shown to significantly reduce bioprosthetic heart valve calcification. The present study investigated whether the additional cross-linking of functional carboxyl groups could augment this anticalcific effect at the low glutaraldehyde concentrations typically used in commercial heart valve production.
Methods: Entire aortic roots of medium sized pigs were fixed after 48 hours of cold storage. Cross-linking of amino-functional groups was either achieved by GA fixation alone (0.2% or 0.7%) or with an interim treatment with the diamine L-Lysine (25mM, 50mM or 100mM; 37°C; 2 days). Carboxyl groups were activated with carbodiimide (N’-{3-dimethylaminopropyl}-N-ethyl carbodiimide hydrochloride/EDC 240mM) and cross-linked with an oligomeric diamine (Polypropylene glycol-bis-aminopropyl ether / Jeffamine™/ 60mM 230D). By permutation of treatments and combinations thereof, a total of 17 groups were compared. Aortic wall discs (12mm diameter) were implanted subcutaneously into seven-week-old Long Evans rats for 60 days. Tissue calcification was determined by histology and atomic absorption spectrophotometry.
Results: There was no significant difference in tissue calcification if either glutaraldehyde or carbodiimide fixation was used on its own. Equally, the combined cross-linking with GA and EDC/Jeffamine™ did not achieve a mitigation of tissue calcification below levels seen in at least one of the two treatments alone. When commercial GA fixation was mildly diamine-enhanced with l-Lysine (25mM), additional EDC/Jeffamine™ cross-linking of carboxyl groups resulted in a distinct additive effect in both 0.2% (-31%; p<0.0002) and 0.7% (-36%; p=0.0073) GA-fixed tissue. Relative to conventional GA fixation, this combination mitigated aortic wall calcification by 43% (p<0.0001) and 34% (p=0.0014) in 0.2% and 0.7% GA-fixed tissue respectively. An increase in L-Lysine concentration to 100mM further reduced calcification of 0.7% GA-fixed tissue (18.5%; p=0.016), but had no additional effect on 0.2% GA-fixed tissue (0.6%; p=0.463).
Conclusion: A distinct reduction of bioprosthetic aortic wall calcification can be achieved by combining diamine-extended conventional GA fixation with a diamine-extended carbodiimide based cross-linking step.

Carbodiimide treatment dramatically potentiates the anti-calcific effect of alpha-amino oleic acid on glutaraldehyde fixed aortic wall tissue.
Peter Zilla, Deon Bezuidenhout, Paul Human.
Annals of Thoracic Surgery (2005) 79: 905-910

Objective: Bi-functional amines were previously found to act as bridging molecules between the terminal ends of incomplete glutaraldehyde cross-links. The additional cross-links thus formed between  -NH2 groups of tissue were seen to significantly inhibit bioprosthetic calcification. In the current study, the potential ability of  -amino oleic acid to act as a bridging molecule between  -NH2- and COOH- dependent cross-links was hypothesised to similarly augment the anti-calcification effect of the  -amino oleic acid molecule.
Methods: Porcine aortic wall tissue from Medtronic Freestyle™ valve bioprostheses incorporating the AOA™ anti-calcification process additionally underwent carboxyl-group cross-linking with Jeffamine™ using carbodiimide (EDC). Tissue was subdermally implanted into 5-week old Long Evans rats for 60 days. Standard 0.2%GA-fixed tissue served as a control. To further assess the impact of storage solution on AOA tissue, samples were either stored in GA (0.2%GA) or EDC (25mM Carbodiimide) before implantation. Tissue calcification was assessed by atomic absorption spectroscopy and histochemical staining.
Results: Aldehyde end-capping with  -amino oleic acid achieved only a modest reduction of calcification in GA-treated aortic wall tissue (-20.0%; p<0.05). Replacing GA with EDC as a storage solution led to a further 32.4% (p<0.01) mitigation of calcification in Freestyle tissue. Incorporating an intermediate EDC/Jeffamine cross-linking step achieved a distinct additional reduction of calcification by 40.4% (p<0.05). Overall, aortic wall calcification was 59.7% (p<<0.0001) lower if commercial Freestyle tissue underwent an additional EDC/Jeffamine cross-linking step and subsequent storage in EDC. Relative to control GA-fixed tissue, this represented a 67.8% (p<<0.0001) reduction. Incorporation of  -amino oleic acid was essential for the beneficial effect of the additional EDC/Jeffamine cross-linking step.
Conclusion: Potentially utilizing both the amino- and the carboxyl moieties of  -amino oleic acid for tissue binding dramatically reduces aortic wall calcification of glutaraldehyde fixed tissue.

The selective modulation of endothelial cell mobility on RGD peptide-containing surfaces by YIGSR peptides.
Matthias Fittkau, Peter Zilla, Deon Bezuidenhout, Matthias P Lutolf, Paul Human, Jeffery Hubbel, Neil Davies.
Biomaterials  (2005) 26(2): 167-74

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.

Missed Stabbed Heart in an Infant.
A.B. van As, Johan Brink.
Injury Extra (2005) 36: 445-446

Case report: An 11-month-old infant was admitted to a local hospital after an alleged assault with a knife. The child was crying but haemodynamically stable. On examination a 2 cm long laceration was noted over the left shoulder, through the deltoid muscle. The wound was sutured with nylon 3.0, after which the child was discharged. However, the child was brought back to the hospital by its mother after 2 days with the history that the left arm was underutilized and the child distressed when the arm was passively moved. Anteroposterior and lateral radiographs of the thorax revealed a large knife blade retained in the left chest. The blade was situated subcutaneously under the sutured wound on the left shoulder with the tip directed towards the cardiac region. A left sided chest drain was inserted and the patient was transferred to our trauma unit. The child was prepared for theatre, intravenous antibiotics were administered and an emergency left thoracotomy was performed. The blade was identified within the chest and the whole tract visualized before it was removed. The tip of the blade penetrated the pericardium and was located in the myocardium. The pericardium was opened widely and there was little intra-pericardial blood present and no bleeding from the wound in the left ventriclular myocardium, which had been penetrated tangentially to a maximum depth of about 1 cm. No bleeding occurred when the blade was carefully pulled from the myocardium in a reverse direction and removed from the chest via the thoracotomy. The pericardium was partially reapproximated and an intrathoracic drain was positioned and the chest was closed in layers. To complete the procedure, the shoulder joint was washed out with normal saline. The postoperative course was uncomplicated.

Heart transplantation: the contributions of Christiaan Barnard and the University of Cape Town/Groote Schuur Hospital.
Johan Brink, David Cooper.
World Journal of Surgery (2005) 29(8): 953-961

Christiaan (Chris) Neethling Barnard was born in South Africa and qualified in medicine at the University of Cape Town in 1946. Following surgical training in South Africa and the USA, Barnard established a successful open-heart surgery program at Groote Schuur Hospital and the University of Cape Town in 1958. In 1967, he led the team that performed the world's first human-to-human heart transplant. Although the first heart transplant patient survived only 18 days, four of Groote Schuur hospital's first 10 patients survived for more than one year, two living for 13 and 23 years, respectively. This relative success amid many failures worldwide did much to generate guarded optimism that heart transplantation would eventually become a viable therapeutic option, Barnard then developed the operation of heterotopic heart transplantation (the socalled "piggy-back" transplant), which had some advantages in the pre-cyclosporine era when immunosuppressive therapy was limited. His group was the first to successfully transport donor hearts using a hypothermic perfusion storage device in 1981. Several studies on the haemodynamic and metabolic sequelae of brain death were carried out in his Department's cardiovascular research laboratories at the University of Cape Town, and the concept of hormonal replacement therapy in organ donors was developed. An active heart transplant program still continues in the Chris Barnard Division of Cardiothoracic Surgery at Groote Schuur Hospital and the University of Cape Town, but the thrust of clinical activity within the Division and the research within its state-of-the-art cardiovascular research laboratories is now directed towards valvular and ischaemic heart diseases, which are common in the African population.

The Limitations of Bioprosthetic Heart Valves.
Peter Zilla, Paul Human, Deon Bezuidenhout.
Encyclopaedia of Biomaterials and Biomedical Engineering (2004)

This is a review paper and does not include an abstract.

Why Bioprosthetic Heart Valves Fail.
Peter Zilla, Paul Human, Deon Bezuidenhout.
Encyclopaedia of Biomaterials and Biomedical Engineering (2004)

This is a review paper and does not include an abstract.

Vascular Grafts.
Deon Bezuidenhout, Peter Zilla.
Encyclopaedia of Biomaterials and Biomedical Engineering (2004)

This is a review paper and does not include an abstract.

Gene Activated Matrix.
Neil Davies.
Encyclopaedia of Biomaterials and Biomedical Engineering (2004)

This is a review paper and does not include an abstract.

Bioprosthetic heart valves: The need for a quantum leap.
Peter Zilla, Paul Human, Deon Bezuidenhout.
Biotechnol Appl Biochem (2004) 40(1): 57-66

Factors affecting outcome in penetrating oesophageal trauma.
N Smakman, AJ Nicol, Gabi Walther, Andre Brooks, PH Navsaria, R Zellweger.
British Journal of Surgery (2004) 91(11): 1513-1519

BACKGROUND: Penetrating oesophageal trauma is rare and the risk factors affecting outcome have not been clearly identified. Delayed management has been cited as a factor contributing to the high rates of morbidity and mortality, but evidence for this is lacking. METHODS: A retrospective study was undertaken of patients with penetrating oesophageal trauma presenting to a level I trauma centre over 8 years. Outcome was assessed in terms of mortality, morbidity (oesophageal and non-oesophageal), and length of hospital and intensive care unit (ICU) stays. RESULTS: Fifty-two patients with oesophageal injury who reached the operating theatre were included. The overall mortality rate was 6 per cent. Fifteen patients (29 per cent) developed oesophageal injury-related complications. Time from injury to management was the only important risk factor for the development of oesophageal complications (P = 0.001), but did not affect the length of ICU (P = 0.560) or hospital (P = 0.329) stay, incidence of non-oesophageal injury-related complications (P = 0.963) or death (P = 0.937). Patients with gunshot injuries spent longer in the ICU (P = 0.007) and the duration of hospital stay was longer for those with higher-grade oesophageal injuries (P = 0.025). CONCLUSION: The risk of oesophageal injury-related complications was directly related to the interval between the trauma and definitive management of the oesophageal injury.

Detoxification on top of enhanced, diamine-extended glutaraldehyde treatment significantly reduces bioprosthetic root calcification in the sheep model.
Ameli Trantina-Yates, Paul Human, Peter Zilla.
Journal of Heart Valve Disease (2003) 12: 93-101

Objective: Increased concentrations of glutaraldehyde (GA), diamine-extension (DA) of crosslinks and subsequent extraction of excess GA were all shown to significantly reduce bioprosthetic calcification in the subdermal rat model. The present study aimed at demonstrating the combined effect of all three treatments in the circulatory sheep model.
Methods: Two different fixation treatments were chosen for GA detoxification (Urazole in acetic buffer, 0.1M; pH 4.5; 37C; 7d): conventional 0.2 % GA fixation (4°C; 7d) and enhanced 3.0% GA fixation (4°C; 2d – followed by a DA interim step; 100mM L-Lysine; 37°C; 2 d – followed by GA; 3,0%; 37°C; 5 d). Entire porcine root prostheses were implanted in the distal aortic arch of young sheep for 12 weeks (n=5/group). Non-detoxified 0.2% GA treated roots served as controls (n=5). Calcium analysis was based on atomic absorption spectrophotometry, light microscopy and transmission electron microscopy.
Results: Detoxification alone resulted in an 83% reduction of leaflet calcification (p=0.086) but only achieved a 23% (p=0.145) and 12% (p=0.362) mitigation of calcification in aortic wall and sinus tissue, respectively. Combined with diamine-enhanced 3% GA fixation, detoxification led to a 95% reduction in leaflet calcification (p=0.057), followed by 79% in sinus (p=0.003) and 79% in aortic wall tissue (p=0.0003). Morphologically, detoxification primarily affected leaflets and the sub-adventitial layer of aortic wall tissue, whereas enhanced fixation seemed to equally affect all structures.
Conclusion: Although group sizes were limited by logistical constraints in this large animal model and thus failed to confirm that detoxification alone, or on top of diamine extension of GA crosslinks, was able to achieve a statistically significant reduction in leaflet calcification despite the magnitude of that reduction, the statistically significant effect of these combined treatments on aortic wall and sinus tissue clearly demonstrated the relevance of this procedure in the circulatory model.

Left ventricular sub-valvar mitral aneurysms.
Henning Du Toit, Ulrich von Oppell, John Hewitson, John Lawrenson, John Davies.
Interactive Cardiovascular and Thoracic Surgery (2003) 2(4): 547-551

We retrospectively reviewed the surgical treatment of 12 patients (nine female, mean age 16.1±8.7 years) with sub-mitral aneurysms managed in our institution between 1991 and 2002. We identified three groups of patients in accordance with the degree of posterior mitral annular involvement by the aneurysm. A single aneurysm neck was found in seven patients, multiple necks in two and involvement of the entire posterior mitral annulus in three patients. Involvement of the entire posterior annulus by the aneurysmal process has not been previously described. The mean age of this latter group 29±5.1 years was significantly older than the former (P=0.001), suggesting a possible progressive nature of sub-mitral aneurysms. An intracardiac surgical approach was used in six patients and a combined intra and extracardiac approach in the remainder. There was no operative mortality. The mitral valve was initially repaired in eight patients. Failure of closure of the aneurysm necessitating reoperation occurred in four patients (33.3%). An understanding of the inter-relationship between the aneurysm and mitral valve is essential for successful surgical repair. Histology of the aneurysm tissue showed rheumatic heart disease in two patients and tuberculosis in two patients. Hence, although sub-valvar aneurysms are thought to be congenital, a third of our patients had evidence of co-existent rheumatic heart disease or tuberculosis.

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth.
AH Zisch, MP Lutolf, M Ehrbar, GP Raeber, SC Rizzi, Neil Davies, H Schmokel, Deon Bezuidenhout, V Djonov, Peter Zilla.
FASEB J (2003) 17(15): 2260-2262

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Optimization of diamine-bridges in glutaraldehyde treated bioprosthetic aortic wall tissue.
Paul Human, Deon Bezuidenhout, Mark Torrianni, Marc Hendriks, Peter Zilla.
Biomaterials  (2002) 23: 2099-2103

Objective: Bioprosthetic calcification can be significantly mitigated by both increased concentrations of glutaraldehyde (GA) and the introduction of diamine (DA) bridges. The purpose of the present study was to evaluate whether an optimal effect of DA-enhanced fixation can be achieved by titration of dialdehyde and diamine concentrations.
Methods: Porcine aortic roots were fixed at 0.05%GA (under-fixation) or 0.2%GA and 0.7%GA (commercial fixation). An interim step of DA treatment (L-Lysine; 0mM, 25mM, 50mM or 100mM; 37°C; 2 days) was followed by completion of the GA fixation (37°C; 5 days). Aortic wall coupons (12mm) were punched out and implanted subcutaneously into seven-week old Long-Evans rats for 60 days. Calcium content was assessed by atomic absorption spectroscopy and histology
Results: Increasing the L-Lysine concentrations beyond 25mM was essential to achieve the anti-calcific effect of DA-enhanced fixation. This effect was proportional to the GA concentrations applied. Compared to non-enhanced GA fixation (0mM DA), calcification increased by 17.4% (p=0.2114) in 0.05% fixed tissue but decreased by 32.0% (p<0.0001) and 45.1% (p<0.0002) in 0.2% and 0.7% GA respectively when the DA concentration was 100mM. Histologically the extent, but not the pattern of calcification, was affected.
Conclusion: The calcium mitigating effect of diamine-treatment as an interim step of glutaraldehyde fixation is proportional to the GA concentration applied. Within commercial 0.7%GA fixation 100mM DA have the potential to practically halve aortic wall calcification.

The damaging effrect of serum lipids on EC growth is particularly caused by  triglycerides.
Johann Meinhart, Walter-Michael Halbmayer, Manfred Deutsch, Peter Zilla.
Endothelium (2002)

Background:  Patient-related risk factors for the growth of autologous endothelial cells were assessed in a clinical series of 100 consecutive recipients of in-vitro endothelialized prosthetic vascular grafts. For all patients the indication for bypass operation was arteriosclerotic occlusive disease of the distal arteries.
Methods and Results: Endothelial cells were harvested from a small piece of subdermal vein and cultured in medium containing 20% of autologous serum. Growth was continually monitored. In cultures that failed to grow, the autologous serum supplement to the culture medium was replaced by pooled homologous serum from young healthy donors.
The comparison of a multitude of serum parameters between patients whose endothelial cells failed to grow and those showing normal growth revealed a significant difference in serum lipid content: Triglycerides: 4.76 ± 3.36 mmol/L vs to 2.83 ± 2.28 mmol/L (P=0.001); cholesterol: 6.78 ± 1.69 mmol/L vs 5.69 ± 1.32 mmol/L (P=0.003) and lipoprotein (a): 35.9 ± 28.3 mg/dL vs 22.2 ± 26.6 mg/dL (P=0.04). Following serum exchange with low-lipid pool serum which contained 1.74 mmol/L triglycerides; 4.86 mmol/L cholesterol and 5 mg/dl lipoprotein (a) and 5.79 mmol glucose, a remarkable recovery occurred in 85% of these cultures resulting in fully restored proliferative capacity. As a consequence population doubling time did not differ between the two groups at any point in time and mass cultures sufficient for confluent graft endothelialization were obtained with hardly any delay.
Conclusion:  We conclude that hyperlipidaemia may lead to growth impairment of cultured human endothelial cells. This growth inhibition is reversible if the supplemented autologous serum is replaced by pooled serum with low lipid content.

Effect of well-defined dodecahedral porosity on inflammation and angiogenesis.
Deon Bezuidenhout, Neil Davies, Peter Zilla.
ASAIO (2002) 48: 465-471

Porosity is an important factor in the healing of prosthetic devices.  To better understand this phenomenon, porous polyurethane scaffolds were produced by a variation of the phase inversion / porogen extraction technique in which a pre-packed column of spherical porogen particles was infiltrated with a polymer solution prior to polymer precipitation and porogen extraction.  Scaffolds contained pores of well-defined shape (approaching pentagonal dodecahedrons), narrow size distributions (66.1±1.3, 84.2±1.7 and 129.0±1.8µm) and high interconnectivity (interconnecting windows of 30.1±0.8, 41.9±1.5 and 76.4±2.0µm respectively).  A high degree of accessible macroporosity (greater than 80%) could be achieved while limiting the inaccessible microporosity to below 2%. 
The neo-vascularization and inflammatory responses to the scaffolds were evaluated in the subcutaneous rat model for 4 weeks.  The inflammatory response index (IRI) and foreign body giant cell index (FBGCI) could be reduced by and 56% (p<0.05) and 21% (p<0.02) respectively when the pore size was increased from 66 to 157µm, while the vascularization index (VI) and arteriolar index (AI) remained unchanged.
Thus a significant decrease in inflammatory response could be achieved without adversely affecting the degree of neo-vascularization by increasing the size of the pores. 

Carboxyl group cross-linking further mitigates bioprosthetic aortic wall calcification of diamine-enhanced glutaraldehyde treated tissue.
Peter Zilla, Deon Bezuidenhout, Paul Human, Mark Torrianni, Marc Hendriks.
Cardiovascular Pathology (2002) 11: 47

Objective: The extension of glutaraldehyde (GA) cross-links between functional amine groups with diamine (DA) bridges was shown to significantly reduce bioprosthetic heart valve calcification. The present study investigated whether the additional cross-linking of functional carboxyl groups with a diamine spacer could augment the anticalcific effect.
Methods: Entire aortic roots of medium sized pigs were fixed in GA concentrations typically used in commercial fixation regimens (0.2% or 0.7%; PBS; 7d). Cross-linking of amino functional groups was achieved by GA fixation for 48h (4˚C) followed by an interim step of DA treatment (L-Lysine; 0mM, 25mM, 50mM or 100mM; 37˚C, 2 days) and a final phase of GA fixation (37˚C; 5d). A second identical set of samples were prepared but subsequently underwent additional carboxyl group cross-linking (carbodiimide/60mM jeffamine) treatment. Aortic wall discs (12mm diameter) were implanted subcutaneously into five week old Long Evans rats for 60 days. Tissue calcification was determined by atomic absorption spectrophotometry.
Results: When L-Lysine enhancement of GA was omitted, the application of EDC/Jeffamine cross-linking exerted a calcium mitigating effect on 0.2% GA-fixed tissue (21% reduction; p<0.005) but was ineffective in the case of 0.7% GA. The introduction of 25mM L-Lysine enhancement of GA together with EDC/Jeffamine cross-linking of carboxyl groups resulted in a distinct additive effect in both 0.2% (-31%; p=0.0002) and 0.7% (-36%; p=0.0073) GA-fixed tissue. Relative to conventional GA fixation, this combination mitigated aortic wall calcification by 43% (p<0.0001) and 34% (p<0.0001) in 0.2% and 0.7% GA-fixed tissue respectively. An increase in L-Lysine concentration to 100mM further reduced calcification of 0.7% GA-fixed tissue (18.5%; p=0.016), but had no additional effect on 0.2% GA-fixed tissue (0.6%; p=0.463).
Conclusion: A distinct reduction of bioprosthetic aortic wall calcification can be achieved by combining mildly DA-enhanced GA fixation with a carbodiimide based cross-linking step.

Investigations into vascular cell mobility under normoxic and hypoxic conditions.
Matthias Fittkau, Peter Zilla, Neil Davies.
Cardiovascular Pathology (2002) 11: 41

lNTRODUCTlON: The development of surfaces that allow the manipulation of cell movement and thus the possible control of tissue ingrowth would be desirable in tissue engineering. In many situations where scaffolds are implanted, the environment will initially be hypoxic. We have therefore designed an experimental set-up that allows the quantification of cell migration on defined surfaces under hypoxic conditions. As an initial trial we have examined the mobility of human microvascular endothelial cells (MVEC), endothelial cells on tissue culture treated (tct) polystyrene and fibronectin in normoxia and hypoxia.
METHODS: Human MVEC were isolated from foreskins by immunodepletion of fibroblasts, smooth muscle cells and macrophages. Cells were seeded on tct polystyrene and fibronectin coated polystyrene (20 µg/mI with subsequent bovine serum albumin blocking). A PeCon climate controlled microscope stage incubator was modified through the addition of a N2 port to allow precise control of the O2 content. Migration was monitored under conditions of 1% 02,5% CO2 or 20%, 0,5% CO2. Computer-aided time-lapse video-microscopy was used to quantify single cell migration on tct polystyrene and fibronectin coated polystyrene. Migration speed was determined as the sum of the quarter hourly distances divided by the total time of 8h.
RESULTS: The mobility of MVEC was reduced by 25% on both tct polystyrene and fibronectin by decreasing the 02 content. The observed changes were significant with p<0.05.
CONCLUSION: The mobility of MVEC is altered by the oxygen content of the environment. Therefore, investigations into the migration of other vascular cell types such as fibroblasts, smooth muscle cells and macrovascular endothelial cells in hypoxia and normoxia on a range of adhesive substrates are presently being carried Out. This should allow optimization of surfaces under conditions more accurately simulating those that tissue engineered implants would be exposed to in vivo.

Variations in amine blockers successfully mitigates calcification of carbodiimide cross-linked tissue.
Frank Everaerts, Mark Torrianni, Paul Human, Peter Zilla, Pauline van Wachem, Linda Brouwer, Marc Hendriks.
Cardiovascular Pathology (2002) 11: 41

Introduction: A two-step fixation process for porcine aortic root tissue has been developed and found to reduce calcification > 85% in walls and > 95% in leaflets. Initial screening studies included the evaluation of a number of mono-aldehydes in an effort to find blockers with appropriate solubilities for aqueous based reactions.
Methods: Porcine aortic root tissue (ART) was obtained from slaughter houses, rinsed free of blood with a buffered, physiological saline, trimmed to remove excess myocardium and adventitial tissue and divided into groups for treatment. These groups were processed with selected mono-aldehydes, varying in their solubilities. Excess aldehyde was removed with extensive rinsing followed by a second step cross-linking process with a water-soluble carbodiimide (EDC) that utilized Jeffamine as a spacer molecule. The cross-linked tissue was rinsed to remove residual reagents followed by sterilization. The efficacy of the blocking reaction was monitored by TNBS analysis, while stabilization of the tissue valve matrix was studied by DSC and enzymatic hydrolysis. ART (leaflet and wall) were evaluated for the potential to calcify in a subdermal implant model in two separate laboratories. 3-week-old Sprague-Dawley rats were used in Groningen, whereas 5-week old Long Evans rats were used in Cape Town. Inflammatory response to the various tissue treatments was monitored by an in vitro test utilizing human macrophages. Production of pro-inflammatory cytokines IL-I and TNF-  were followed by ELISA assay.
Results: TNBS data showed slightly higher blocking potential for mono-aldehydes having greater hydrophobic character. Amine blockers used in this study did not have much of an impact on pro-inflammatory cytokine production, but were all lower than the controls. Calcification data did show a trend in calcification mitigation, with the hydrophobic blockers being more successful at inhibiting calcification than their hydrophilic counterparts. This trend was especially notable in the aortic wall, with up to a 15-fold reduction in calcium content noted when hydrophobic blockers were used (unblocked controls = 87.6 µg Ca+2 /mg wall tissue, glyceraldehyde blocked = 47.7 µg Ca+2 /mg wall tissue, propionaldehyde blocked = 5.6 µg Ca+2 /mg wall tissue).
Conclusion: In the developed two-step fixation process for porcine aortic root tissue, data from this study suggests that there may be a correlation between mono-aldehyde solubility and the potential to block calcification.

Carbodiimide treatment dramatically potentiates the anti-calcific effect of AOA on glutaraldehyde fixed aortic wall tissue.
Peter Zilla, Paul Human, Deon Bezuidenhout, Mark Torrianni, Marc Hendriks.
Cardiovascular Pathology (2002) 11: 32

Objective: Bridging with bifunctional amines enables additional amino-amino cross-links in glutaraldehyde (GA) fixed tissue and significantly mitigates bioprosthetic calcification. In analogy, the present study investigated the possibility of utilizing the GA-end capping molecule alpha-amino oleic acid (AOA) for the subsequent introduction of amino-carboxyl-bridging cross-links.
Methods: Aortic roots of medium-sized pigs were fixed in 0.2% GA prior to additional treatment with either AOA (GA+AOA), EDC/Jeffamine (GA+EDC/Jeff) or AOA plus EDC/Jeffamine (GA+AOA+EDC/Jeff). Tissue treated with 0.2%GA alone served as controls. Subsequently, 12rnm circular discs were punched from the supracommissural aortic wall and stored in either GA or EDC-based sterilant (25mM carbodiimide) before subcutaneous implantation in five-week old Long Evans male rats for 60 days. Tissue calcification was assessed by atomic absorption spectroscopy.
Results: Both aldehyde end capping with AOA (GA+AOA) and the inclusion of tissue-carboxyl-carboxyl cross-links (GA+EDC/Jeff) achieved a modest reduction of calcification in GA-treated aortic wall tissue (16.0%; AOA; p=0.024 / 19.5%; EDC/Jeff; p=0.015). Using an EDC based sterilant instead of GA had no additional effect on GA+EDC/Jeff treated tissue, but led to a further 11.5% mitigation of calcification in GA+AOA treated tissue (p=0.011). Adding an EDC/Jeff cross-linking step to such GA+AOA treatment led to a distinct further reduction of calcification by 46.2% (p =0.011). Overall, aortic wall calcification was 67% lower if GA+AOA treated tissue underwent an additional EDC step before storage in an EDC sterilant (GA+AOA+EDC/Jeff+EDC).
Conclusion: Utilizing both the amino- and the carboxyl functionalities of AOA for tissue binding dramatically reduces aortic wall calcification of AOA treated, glutaraldehyde fixed tissue.

Antihypertensive effects of angiotensin converting enzyme inhibition by lisinopril in post-transplant patients.
Lionel Opie, Matthias Haus, Patrick Commerford, Basil Levetan, Karen Moore, Johan Brink.
American Journal of Hypertension (2002) 15: 911-916

It is not known whether strict control of blood pressure (BP) in mild post-transplant hypertension gives any benefit. Our primary objective was to test the antihypertensive effects of lisinopril added to standard therapy on ambulatory BP (ABP) of post-transplant patients. The secondary objective was to monitor echocardiographic and hemodynamic end points.Post-transplant patients with an abnormality of the 24-h ABP recording were recruited to this double-blind randomized prospective study that started 2 to 3 months after transplantation. Patients were then evaluated at 6, 12, 18, and 24 months after transplantation.Lisinopril decreased the clinic BP and ABP, the latter from 134/85 to 126/82 mm Hg at 6 months (P =.01 v placebo) and 121/79 mm Hg after 2 years (P =.03 v placebo). Fewer patients in the lisinopril group required added amlodipine to control the BP (P =.01). Data on left ventricular (LV) mass are difficult to interpret because by coincidence in this small study, the lisinopril group had lower initial values than placebo. However, in the lisinopril group mean LV mass decreased by 10% (P =.02) and mass index by 13% (P =.01), whereas placebo LV mass and index did not change. The LV end-diastolic diameter increased only in the placebo group (P =.008). There were no significant changes in any of the other secondary outcomes, including the cardiac index and systemic vascular resistance.Thus, in these post-transplant patients, stricter BP control to normal levels by the addition of lisinopril to existing therapy, reduced BP and modestly decreased LV mass without altering cardiac hemodynamic function.

Tissue engineering of vascular prostheses: Beyond the hype.
Peter Zilla.
International Journal of Artificial Organs (2002) 25(7): 629-632

Evaluation of peripheral blood CD4 and CD8 lymphocyte subsets, CD69 expression and histologic rejection grade as diagnostic markers for the presence of cardiac allograft rejection.
Pauline Creemers, Johan Brink, Helen Wainwright, Karen Moore, Enid Shephard, Del Kahn.
Transplant Immunology (2002) 10(4): 285-292

We investigated the dynamics of the CD4+ and CD8+ lymphocyte subsets, and the expression of activation markers in cardiac transplant recipients. We tested 132 peripheral blood samples from 62 cardiac transplant recipients using fluorescent staining and flow cytometry analysis. The results were correlated with histological rejection grade of concurrently taken biopsies, and 5-year survival of the recipients. A decrease in the total T lymphocyte subset, and in CD4+ lymphocytes was associated with higher rejection grade and lesser survival. An increase (5–11%) of double positive CD4+CD8+ lymphocytes was observed; these were mostly CD4brightCD8dim. The CD4/CD8 ratio was significantly (p<0.001) lower in the transplant recipients than in normal individuals. CD69 expression was higher than CD54 and CD154 expression on CD4 and CD8 lymphocytes of cardiac transplant recipients; correlation between these activation markers was excellent (p<0.001). Fluorescent staining for CD69 was often of low intensity. Multiple regression for % CD8+CD69+ cells and survival, and for % CD69+ T cells and rejection grade yielded a significant correlation (p<0.050). Both % CD8+CD69+ and % CD69+ T cells were significantly higher in samples with severe and moderate rejection grade (grades 3A, 3B and 4) than in samples which showed no, minimal or mild rejection (grades<=2); p-values were 0.052 and 0.003, respectively. Preliminary results indicated that false negative results could be contributed to increased immunosuppression. We conclude that CD69 expression on circulating CD4 and CD8 lymphocytes is a useful parameter for the diagnosis of moderate and severe rejection.

Dissected aortic sinuses repaired with gelatin-resorcin-formaldehyde (GRF) glue are not stable on follow-up.
Ulrich von Oppell, Zead Karani, Andre Brooks, Johan Brink.
Journal of Heart Valve Disease (2002) 11(2): 249-57

BACKGROUND AND AIM OF THE STUDY: The chemical glue, gelatin, resorcin and formaldehyde (GRF) is widely used to obliterate the false lumen of acute dissected aortic wall tissue.
METHODS: A retrospective review of 41 consecutive patients operated upon for ascending aortic dissection between 1993 and 2000 was conducted. This study focused on 19 patients with acute aortic dissection in whom the aortic valve was resuspended and GRF glue used in the proximal aortic sinuses. These patients were compared with ascending aortic dissection patients in whom the aortic valve was not resuspended. In total, nine acute and 13 chronic dissections were performed in which aortic valve replacement, valve-sparing root reconstruction (without GRF glue), or no aortic valve surgery was carried out.
RESULTS: The operative mortality for ascending aortic dissections was 24.4%; identified risk factors included the specific surgeon involved. Third-degree heart block occurred only in patients in whom GRF glue was used in the proximal aortic sinus (15% incidence). Operative survivors in whom the aortic valve was resuspended and GRF glue used in the proximal aortic sinus, had a 64% incidence of late recurrent aortic regurgitation requiring reoperation due to recurrent aortic sinus aneurysm formation with or without recurrent proximal aortic dissection. No recurrence of aortic regurgitation or proximal disease occurred in the other two groups (p <0.01). Actuarial survival of patients in whom the aortic valve was resuspended with GRF glue was 52.1+/-11.6% at five years and 27.8+/-14.3% at eight years, compared with 55.6+/-16.6% at five years if the aortic valve was not resuspended using GRF glue.
CONCLUSION: The use of GRF glue to repair acute dissected aortic sinuses combined with the resuspension of  the aortic valve is associated with an unacceptable incidence of failure of aortic valve repair and recurrence of aortic regurgitation. It may be more appropriate to resect all acute dissected aortic sinus tissue.

Tissue engineering of vascular prostheses: Beyond the hype.
Peter Zilla.
European Journal of Artificial Organs (2002)

The challenge of paediatric cardiac services in the developing world.
John Hewitson, Johan Brink, Peter Zilla.
Semin Thorac Cardiovasc Surg (2002) 14(4): 340-5

Hyperlipidemia coincides with reversible growth impairment of cultured human autologous endothelial cells.
Johann Meinhart, Walter-Michael Halbmayer, Manfred Deutsch, Peter Zilla.
Endothelium (2002) 9(4): 239-246

Patient-related risk factors for the growth of autologous endothelial cells were assessed in a clinical series of 100 consecutive recipients of in vitro endothelialized prosthetic vascular grafts. For all patients, the indication for bypass operation was arteriosclerotic occlusive disease of the distal arteries. Endothelial cells were harvested from a small piece of subdermal vein and cultured in medium containing 20% of autologous serum. Growth was continually monitored. In cultures that failed to grow, the autologous serum supplement to the culture medium was replaced by pooled homologous serum from young healthy donors. The comparison of a multitude of serum parameters between patients whose endothelial cells failed to grow and those showing normal growth revealed a significant difference in serum lipid content: triglycerides: 4.76 +/- 3.36 versus 2.83 +/- 2.28 mmol/L (p = .001); cholesterol: 6.78 +/- 1.69 versus 5.69 +/- 1.32 mmol/L (p = .003); and lipoprotein (a): 35.9 +/- 28.3 versus 22.2 +/- 26.6 mg/dl (p = .04). Following serum exchange with low-lipid pool serum that contained 1.74 mmol/L triglycerides, 4.86 mmol/L cholesterol, 5 mg/dl lipoprotein (a), and 5.79 mmol/L glucose, a remarkable recovery occurred in 85% of these cultures, resulting in fully restored proliferative capacity. As a consequence, population doubling time did not differ between the two groups at any point in time and mass cultures sufficient for confluent graft endothelialization were obtained with hardly any delay. The authors conclude that hyperlipidemia may lead to growth impairment of cultured human endothelial cells. This growth inhibition is reversible if the supplemented autologous serum is replaced by pooled serum with low lipid content.

SMC secretion of VEGF is upregulated by stretching.
James Smith, Neil Davies, Bauer Sumpio, AI Willis, Peter Zilla.
Endothelium (2001) 8(1): 41-48

Objective: Accumulating evidence links the release of vascular endothelial growth factor (VEGF) by vascular smooth muscle cells (VSMC) to normal endothelial cell (EC) function, repair and maintenance. Using an in vitro model we investigate the role of cyclic stretch on both the release of VEGF by VSMC and the phosphorylation of a VEGF receptor on EC.
Methods: Bovine VSMC and EC were exposed to 10% cyclic strain for 4 hours. VEGF mRNA steady-state levels of VSMC were analysed by northern blot hybridisation. The presence of secreted VEGF from VSMC was determined by assaying the migration of EC. VEGF receptor phosphorylation on stretched EC was assayed by immunoblotting.

Results: The steady-state level of VEGF mRNA in stretched VSMC increased 3.3 (± 0.6) fold above that of unstretched VSMC (p< 0.005). Migration of EC was stimulated 8.3 (± 1.1) and 14.6 (± 1.3) fold by media from unstretched and stretched VSMC respectively, demonstrating a 1.8 fold increase due to stretch alone (p< 0.05). Cyclic stretch resulted in phosphorylation of the VEGF receptor KDR.
Conclusion: Exposure of VSMC to physiological levels of stretch induces a biologically significant increase in VEGF secretion and may provide an arterial stimulus for maintenance of steady state levels of VEGF essential for EC survival.

Diamine extension of glutaraldehyde crosslinks mitigates bioprosthetic wall calcification in the sheep model.
Peter Zilla, Deon Bezuidenhout, Christoph Weissenstein, Anel van der Walt, Paul Human.
Journal of Biomedical Materials Research (2001) 56: 56-64

We could previously show that fixation at increasing concentrations of glutaraldehyde (GA) leads to mitigated rather than facilitated tissue calcification. The purpose of the present study was to introduce additional crosslinks and provide evidence that crosslink density may be an underlying inhibitory principle. Entire aortic roots were chosen to verify the concept on the challenging aortic wall tissue.
Porcine aortic roots were crosslinked with 0.2% GA, 3%GA, and 3% GA containing an interim step introducing diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT°), resistance to protease digestion (RPD), residual amine analysis (RA) and tensile modulus (E10). Calcium levels, calcification patterns and inflammation were assessed after 6 and 24 weeks of implantation in a sheep circulatory model.
Crosslink efficiency in aortic wall tissue was moderately affected by increasing the fixative  concentration from 0.2% GA to 3% GA (SrT° from 85.7±0.3 to 87.5±0.3°C, p<0.002; RPD from 24.2±1.2 to 29.1±0.7%, p<0.003; RA from 0.072±0.004 to 0.061±0.002µmol/mg, p<0.03 and E10 from 1.76±0.26 to 3.11±0.55MPa, p<0.05), but distinctly enhanced when diamine bridges were introduced (SrT° from 87.5±0.3 to 93.4±0.3°C, p<<0.0001; RPD from 29.1±0.7 to 68.4±1.8%, p<<0.0001; RA from 0.061±0.002 to 0.065±0.002µmol/mg, p=0.087 and E10 from 3.11±0.55 to 7.19±0.85MPa, p<0.008).  Aortic wall calcification was significantly reduced by increasing the GA concentration from 0.2% to 3% (37.8%; p=0.076 [6 weeks] and 34.0%; p=0.008 [24 weeks] ) and further reduced by the introduction of additional diamine (84.0%; p=0.006 [6 weeks] and 29.8%; p=0.037 [24 weeks] ). The combined effect of increased  GA concentration plus an interim diamine step on aortic wall tissue resulted in a 90% and 53.7% reduction of calcification after six weeks and 24 weeks, respectively.  The correlation coefficients between calcification and SrT°, RDP and E10 was  -0.9767, -0.9460 and -0.9820 respectively. The inflammatory host reaction regularly found in 0.2% fixed tissue could practically be abolished through the introduction of diamine bridges.
In conclusion, our study demonstrated a distinct correlation between  the mitigation of aortic wall calcification and three parameters used to assess crosslink density.

Mitigation of bioprosthetic heart valve degeneration through biocompatibility: in-vitro versus spontaneous endothelialization.
Ameli Trantina-Yates, Mona Bracher, Paul Human, Peter Zilla.
Biomaterials  (2001) 22: 1837-46

Background: Glutaraldehyde-related cytotoxicity and transanastomotic ingrowth inhibition prevent the spontaneous endothelialization of bioprosthetic heart valves. In order to evaluate the ability of improved biocompatibility to reduce tissue degeneration, conventionally fixed aortic root prostheses were both glutaraldehyde-detoxified and in vitro endothelialized.
Methods: Entire aortic roots were fixed in 0.2% glutaraldehyde (GA)(control group) and either detoxified in acetic acid-buffered Urazole (0.1M) or detoxified and in vitro lined with cultured, autologous jugular vein endothelial cells. The valved roots were inserted in the distal aortic arch of 15 juvenile Merino sheep for a period of 12 weeks. Upon explant, leaflets, sinuses and aortic wall of the prostheses were analysed by SEM to assess the surface endothelium, histologically regarding tissue inflammation, and by atomic absorption spectrophotometry to determine the content of tissue calcium.
Results: There was no endothelium on control grafts, except for a short anastomotic pannus. The detoxified group showed an incomplete patchy endothelium on the aortic wall but hardly any on the leaflets, whereas the in vitro lined group had aortic wall, sinuses and most of the leaflets confluently endothelialized. Tissue inflammation was prominent in the control group and least expressed in the endothelialized group. (p<0.05). Detoxification reduced leaflet calcification. In the aortic wall, both detoxification and endothelial lining were required to significantly mitigate calcification.
Conclusion: In the 12 week circulatory sheep model, the calcium mitigating effect of detoxification was more pronounced than that of in vitro endothelialization. Nevertheless, there was a distinct overall benefit if detoxification was combined with endothelialization.

Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides?
Christoph Merzkirch, Neil Davies, Peter Zilla.
Anatomical Records (2001) 263: 379-387

Anastomotic intimal hyperplasia and surface thrombogenicity are the main reasons for the high failure rate of prosthetic small diameter vascular grafts. While anastomotic intimal hyperplasia is a multifactorial event, ongoing surface thrombogenicity is primarily caused by the lack of an endothelium, even after years of clinical implantation.
After decades of poorly performing synthetic artery-grafts, tissue engineering has emerged as a promising approach to generate biologically functional bio-synthetic hybrid grafts mimicking native arteries regarding the presence of an endothelial lining on the blood surface. “In vitro endothelialization” represented the first generation of such tissue engineered vascular grafts, utilising cell culture techniques for the creation of a confluent autologous endothelium on ePTFE grafts. The clinical long-term results with this method in almost 200 patients are highly encouraging, showing patencies equal to vein grafts. Since “in vitro endothelialization” requires cell culture facilities, it will always be confined to large centres. Therefore, research of the 1990s turned to the development of spontaneously endothelializing implants, to make tissue engineered grafts amenable to the entire vascular-surgical community.
Apart from scaffold designs allowing transmural ingrowth, biological signalling through a facilitating ingrowth matrix holds a key to spontaneous endothelialization. In biological signalling, the increasingly deeper understanding of bio-active molecules and the discovery of domains and peptide sequences during the 1980s created the expectation in the 1990s that peptide signalling may be all that is needed. This present review article highlights the possible problems associated with such a reductionist approach. Using the fibronectin molecule we demonstrated that domains may be more suitable modules in tissue engineering than peptide sequences.

Inflammatory and immune processes: The neglected villain of bioprosthetic heart valve failure?
Paul Human, Peter Zilla.
Journal of Long Term Effects of Medical Implants (2001) 11(3&4): 199-220

In an attempt to avoid the destructive process of bioprosthetic heart valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the cross-linking reagent. In this review, we summarise our findings and those of others which confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularised and non-crosslinked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response and furthermore examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibody in the role of bioprosthesis calcification.

The possible role of immune responses in bioprosthetic heart valve failure.
Paul Human, Peter Zilla.
Journal of Heart Valve Disease (2001) 10(4): 460-46

Better cross-linking resulted in significantly reduced inflammation and tissue erosion.
Conventional cross-linking leads to both a strong macrophage/foreign body giant cell response underneath pannus tissue and distinct pannus outgrowth. Higher cross-link density significantly mitigates both the mononuclear reaction and the anastomotic tissue overgrowth.
Cross-link density correlates inversely with calcification - the higher the cross-link density, the lower the mineralisation.
Conventionally fixed tissue elicits a strong, specific antibody response which can be suppressed by better cross-linking. Tissue exposure to specific antibodies facilitates calcification.

In summary, we are still in a phase of observation rather than elucidation. However, although the mechanisms of immune-degeneration of heart valve prostheses are far from being established, sufficient proof exists for the involvement of immune mechanisms in bioprosthetic heart valve degeneration to date, to break the existing taboo.

Prosthetic heart valves: Why biological?
Ulrich von Oppell, Peter Zilla.
Journal of Long Term Effects of Medical Implants (2001) 11(3&4): 105-113

Matrix metalloproteinases and tissue valve degeneration.
Mona Bracher, Dan Simionescu, Agnetha Simionescu, Neil Davies, Paul Human, Peter Zilla.
Journal of Long Term Effects of Medical Implants (2001) 11(3&4): 221-230

Bioprosthetic heart valves have been used as replacements for diseased heart valves for over 30 years. More than 50% of bioprosthetic valves fail within 15 years due to structural deterioration. The role of proteolytic degradation, with particular reference to the matrix metalloproteinases (MMP’s) in the degeneration of aortic bioprostheses is appraised in this mini-review. It is clear that both the intrinsic and host-derived proteolytic activities present in heart valve bioprostheses may be able to combine with mechanical stress to bring about valve failure.

The bridge graft: a new concept for infrapopliteal surgery.
Manfred Deutsch, Johann Meinhart, Norbert Howanietz, A Froschl, B Heine, R Moidl, H Mendel, A Sisel, A Stumpfen, Peter Zilla.
European Journal of Vascular and Endovascular Surgery (2001) 21(6): 508-512

BACKGROUND: The long-term results of ePTFE grafts are particularly poor in crural reconstructions. We report on a novel surgical technique, whereby both run-off and anastomotic mismatches are concomitantly addressed. PATIENTS AND METHODS: Short segments of vein grafts (5-15 cm in length) were used to bridge two crural artery segments. Subsequently, a femoro-distal ePTFE graft was anastomosed to the bridge graft. Venous valves were made incompetent to allow bi-directional flow. In a retrospective series of 45 patients with crural bridge grafts, 12 patients were in stage III and 33 in stage IV. In 18 patients the reconstruction was the first procedure and in the remaining 28 patients it was the first or second re-operation.
RESULTS: The primary patency rate at 1, 2, 3 and 4 years was 53, 44, 35 and 26% respectively. The secondary patency rate was 67, 53, 49 and 39% respectively. The corresponding limb salvage rate was 70, 61, 56 and 45%. In a small subgroup of patients, in which the crural bridge was the first reconstructive procedure, the primary patency was 76 at 1 year and 64 at 4 years.
CONCLUSION: convincing long-term crural bridge grafts should be considered in those patients who have more than one crural or pedal artery available for grafting and an insufficient length of saphenous vein.

The anticalcific effect of glutaraldehyde detoxification on bioprosthetic aortic wall tissue in the sheep model.
Peter Zilla, Christoph Weissenstein, Mona Bracher, Paul Human.
Journal of Cardiac Surgery (2001) 16(6): 467-472

Background: Increasing concentrations of glutaraldehyde (GA) were shown to lead to a decreased rather than increased calcification of bioprosthetic aortic wall tissue.  The purpose of the present study was to determine to what extent the benefit of better cross-linking is masked by the intrinsic propensity of GA towards calcification.
Materials and Methods: Porcine aortic roots were immediately fixed at the abattoir at three different concentrations of GA (0.2%, 1.0% and 3.0% / 1 week / 4°C). Subsequently roots underwent a GA extraction process using high volumes of Urazole solution (acetic acid buffer, pH 4.5, 37°C, 1 week) followed by NaBH4 reduction (2 days, 37°C). Roots were implanted in the distal aortic arch of young sheep for 6 weeks and 6 months. Calcium analysis was quantitatively done by atomic absorption spectrophotometry and qualitatively assessed by light microscopy on Von Kossa stains.
Results: There was a distinct anti-calcification effect of glutaraldehyde-detoxification after 6 weeks (56.8 to 97.9%; 95%CI) which stabilised on a more moderate level after 6 months of implantation (19.1 to 31.6%; 95%CI). The most pronounced effect of GA extraction was seen in 0.2% fixed tissue where aortic wall calcification was mitigated by 97% and 32% after 6 weeks and 6 months, respectively. Mitigation of aortic wall calcification was 71% (6 weeks) and 21% (6 months) in the 3.0% GA group. The combined effect of higher crosslink density and detoxification achieved an 82% (6 weeks) and 48% (6 months) reduction of calcium levels in the 3.0% GA group. In long-term implants (6 months), detoxification alone on top of standard 0.2% GA fixation was as effective (from 174.1±11.9µg/mg without detoxification to 119.3±19.3µg/mg with detoxification) as 3.0% fixation (114.8±10.0µg/mg without detoxification to 91.3±11.5µg/mg with detoxification).
Conclusion: We were able to determine in the circulatory sheep model to what degree the intrinsic pro-calcific effect of glutaraldehyde counteracts the protective effect of higher crosslink density. Our study also established that the effect of detoxification is particularly pronounced in commercial low-grade fixation.

Fixation related autolysis augments bioprosthetic aortic wall calcification.
Paul Human, Christoph Weissenstein, Ameli Trantina-Yates, Peter Zilla.
Journal of Heart Valve Disease (2001) 10: 656-665

Background: We had previously established that immediate fixation and increased glutaraldehyde concentrations are required to prevent severe autolytic tissue damage during the process of bioprosthetic aortic root production. In the present study we wanted to verify that this structure-preserving fixation procedure also reduces aortic wall calcification.
Materials and Methods: Porcine aortic roots were either instantly fixed at the abattoir or after having been kept on ice for 48 hours (Phosphate buffered saline, PBS). Two concentrations of glutaraldehyde (GA) (0.2% and 3.0%) were chosen (4°C, 7 days, PBS). Discs of aortic wall tissue (1.2cm diameter) were subcutaneously implanted in rats for 60 days (n=10/group) whereas aortic roots were implanted in the distal aortic arch of sheep for 6 weeks (n=3/group) and 6 months (n=4/group). Calcification was assessed by atomic absorption spectrophotometry and light microscopy. Fixation related tissue damage was determined by transmission electron microscopy and correlated with calcification.
Results: No significant difference in calcification was found between immediate and delayed fixation if tissue was fixed at 0.2% GA. In the 3.0%GA group, both animal models showed a significantly lower level of calcification if tissue was immediately fixed. In the subcutaneous rat model, immediate fixation reduced calcification by 26% (p<0.0001). In the circulatory sheep model immediate fixation did not affect calcification in the short-term six week implants but markedly lowered it by 37% (p=0.035) after six months. Ultrastructurally, there was a significant correlation between membrane damage, vacuolisation and vesicle shedding on the one hand and calcification on the other.
Conclusion: We could demonstrate a coincidence of fixation-related ultrastructural damage and increased calcification in bioprosthetic aortic wall tissue.

Characterization of the immune response to valve bioprostheses and its role in primary tissue failure.
Paul Human, Peter Zilla.
Annals of Thoracic Surgery (2001) 71: S385-8

Background:  The role of an immune response in the failure of bioprosthetic heart valves is poorly understood and disregarded by many. To elucidate the nature of the immune response to GA-treated tissue and the possible role of graft-specific antibody in graft mineralisation we performed immune-calcification studies in the rabbit and correlated those results with the analysis of specific antibodies.
Methods: Aortic wall coupons (6mm) were punched from porcine aortic wall tissue fixed with 0.2% glutaraldehyde and detoxified with urazol and then subsequently perforated under sterile conditions. The perforated coupons were then incubated with either immune serum prepared by immunisation of New Zealand White rabbits (n=5) with Freund’s incomplete adjuvant emulsions of tissue homogenates of similarly treated aortic wall tissue, or incubated with the corresponding control pre-immune sera obtained prior to immunisation of the same animals. The tissue was then implanted subdermally on the back of unrelated New Zealand White rabbits (n=8) for a period of 3 weeks. After the coupons were explanted, tissue calcium levels were determined by atomic absorption spectroscopy.
Results: Tissue calcium was increased in all five immune serum-treated replicates (range 61.8% to 431.2%, mean 225.9% ± 73.2) when compared to control samples treated with pre-immune sera. Overall, the mean calcium level was significantly increased (p<<0.0001) when tissue was treated with immune sera (66.0µg/mg ± 10.0 versus 22.6µg/mg ± 4.8 in controls). Graft specificity of immune sera was confirmed by Western blot analysis.
Conclusion:  These results strongly suggest a role of circulating graft-specific antibody in the pathology of bioprosthetic graft calcification.

Stentless bioprosthetic heart valve research: sheep vs primate model.
Ameli Trantina-Yates, Christoph Weissenstein, Paul Human, Peter Zilla.
Annals of Thoracic Surgery (2001) 71: S422-7

Background: The mild inflammatory response against stented bioprosthetic heart valves in the sheep model is often opposed by a more distinct response in failing human implants. With the emergence of stentless root prostheses with their significantly larger proportion of tissue interacting with the immune system of the host, a more relevant animal model than the sheep may be needed.
Methods: Valved, porcine aortic roots of 5 cm length were fixed in 0.2% glutaraldehyde and implanted in the upper descending aorta of Merino sheep (n=5; 43 ± 3kg) and Chacma baboons (n=5; 17 ± 3kg). After 6 weeks tissue calcification, pannus outgrowth and inflammation were assessed by atomic absorption spectrophotometry, histological damage-scoring (0 to 3), image analysis and transmission electron microscopy.

Results: The main difference between the two animal models was in aortic wall calcification (64.8 ± 39.8 mg/mg in the sheep model versus 4.1 ± 5.9 mg/mg in the primate model; p>0.005). In both models, leaflet calcification was negligible (2.6 ± 2.4 mg/mg in the sheep versus 2.5 ± 1.9 mg/mg in the primate) and the overall extent of inflammation was comparable (1.2+ 0,8 versus 0.98+ 0,7; p=0,18 in the sheep and the primate, respectively). Qualitatively, the sheep demonstrated a macrophage-dominated reaction while the inflammatory demarcation often resembled a granulocyte-dominated xeno-response in the primate. Pannus outgrowth was comparable in length (8.4 ± 2.3mm versus 9.1 ± 4.3mm proximally and 7.1 ± 3.4mm versus 7.4 ± 5.1mm distally, in the sheep and baboon, respectively; p>0.05).
Conclusion: Our results confirm the sheep as a significantly stronger calcification model for stentless aortic heart valves than the primate. Remaining antigenicity of porcine tissue as a result of incomplete cross-linking, however, elicits a distinctly stronger xeno-reaction in the primate model.

Clinical autologous in vitro endothelialization of  153 infrainguinal ePTFE grafts.
Johann Meinhart, Manfred Deutsch, Teddy Fischlein, Norbert Howanietz, Peter Zilla.
Annals of Thoracic Surgery (2001) 71

Background Over the past 17 years, our group has developed and clinically applied an in-vitro endothelialization procedure whereby infrainguinal ePTFE prostheses are confluently lined with cultured autologous endothelial cells prior to implantation. After a successful randomised pilot study from 1989 to 1993, the procedure was adopted for routine surgery.
Methods: Since June 1993 153 endothelialized ePTFE grafts were implanted in infrainguinal position in 136 patients (102 AK and 51 BK / 89 male and 47 female / mean age 64.7+9.4 years). Seventeen patients received an endothelialized prosthesis bilaterally. Autologous endothelial cells were harvested from 4-5cm segments of a subcutaneous vein (in 86% the cephalic vein), grown to first passage mass cultures and confluently lined onto 6mm (n=113) or 7mm (n=40) ID ePTFE grafts, precoated with fibrin glue. The observation period for 6mm grafts was 7 years, that for 7mm grafts 4 years. Patency assessment for Kaplan-Meier survivorship analyses was based on duplex sonography and angiography.
Results: Phase 1:  Kaplan-Meier-Survivorship function revealed a primary patency rate of 62.8% after 7 years (S.E.=0.05) for all infrainguinal reconstructions (60% AK / 70.8% BK). The primary patency for stage II and III patients was 64.4% after 7 years. The more recent group of  7mm ID grafts showed a primary patency of 83.7% after 4 years.
Conclusion: Our data provide strong evidence that autologous endothelial cell lining distinctly improves the patency of small diameter vascular grafts.

Postpneumonectomy aortic arch mycotic aneurysm.
Francois Stemmet, J. Davies, Ulrich von Oppell.
Annals of Thoracic Surgery (2001) 71(3): 1030-1032

A 31-year-old woman who had undergone left pneumonectomy for a tuberculosis-destroyed left lung 3 years previously presented in respiratory distress after a pregnancy complicated by preeclampsia and aspiration pneumonia. Investigation revealed a large aortic arch aneurysm as well as a filling defect in the descending thoracic aortic lumen. Emergency aortic arch reconstruction was performed for a massive pseudoaneurysm or contained rupture filling the entire postpneumonectomy space. Pathologic and microbiological examination demonstrated Aspergillus fumigatus and active inflammation.

Biocor No-React stentless aortic valve--short-term results.
Ulrich von Oppell, F. Stemmet, Basil Levetan, S. Heijke, Johan Brink.
Cardiovascular Journal of South Africa (2001) 12(3): 152-8

OBJECTIVES: Short-term results of the bioprosthetic Biocor No-React composite porcine stentless aortic valve (Biocor Industria e Pesquisas LTDA, Belo Horizonte, Brazil) implanted in patients in whom anticoagulation was thought to be contraindicated or expected to be non-compliant.
METHODS: Retrospective review of 52 consecutive prospective patients in whom this valve was implanted, between September 1994 and May 1998.
RESULTS: Average age was 44 +/- 17 years; 75% of patients were operated on for rheumatic heart disease and combined procedures were done in 40% of cases. Early mortality was 5.8%, and related to pre-operative ejection fraction (P < 0.03), New York Heart Association (NYHA) class (P < 0.01), and bacterial endocarditis (P < 0.04). On discharge, 84% of survivors were in NYHA class I and 16% in class II. The average postoperative prosthetic valve peak gradient on echocardiography was 19.9 +/- 11 mmHg and was related to pre-operative ejection fraction and smaller valve sizes. Postoperative residual trivial or mild aortic regurgitation was seen in 19 patients (36.6%), resolved on follow-up in 10 cases, and did not correlate with structural deterioration, re-operation, mortality, or widening of the non-coronary sinus. The non-coronary aortic sinus was widened on closure, because of perceived crowding of the adjacent stentless valve commisures, in 52% of cases. This was thought to be related to the use of an oblique as opposed to transverse aortotomy. Patient survival, inclusive of operative deaths, was 88.5%, and event-free survival was 80.0% at 4 years.
CONCLUSION: The short-term results of this stentless aortic valve in a young predominantly third-world population group are acceptable, and appear to be superior to the results for mechanical valves in a similar patient group. We would recommend a       transverse aortotomy above the sinotubular ridge to be the more appropriate aortotomy incision when using stentless aortic       valves.

Christian (Chris) Neethling Barnard, 1922-2001.
David Dent, Johan Brink, John Terblanche.
South African Medical Journal (2001) 91(10): 840-1

Cyclic stretch induces the expression of vascular endothelial growth factor in vascular smooth muscle cells.
James Smith, Neil Davies, AI Willis, Bauer Sumpio, Peter Zilla.
Endothelium (2001) 8(1): 41-48

OBJECTIVE: Accumulating evidence links the release of vascular endothelial growth factor (VEGF) by vascular smooth muscle cells (VSMC) to normal endothelial cell (EC) function, repair and maintenance. Using an in vitro model we investigate the role of cyclic stretch on both the release of VEGF by VSMC and the phosphorylation of a VEGF receptor on EC.
METHODS: Bovine VSMC and EC were exposed to 10% cyclic strain for 4 hours. VEGF mRNA steady-state levels of VSMC were analysed by northern blot hybridisation. The presence of secreted VEGF from VSMC was determined by assaying the migration of EC. VEGF receptor phosphorylation on stretched EC was assayed by immunoblotting.
RESULTS: The steady-state level of VEGF mRNA in stretched VSMC increased 3.3 (+/- 0.6) fold above that of unstretched VSMC (p < 0.005). Migration of EC was stimulated 8.3 (+/- 1.1) and 14.6 (+/- 1.3) fold by media from unstretched and stretched VSMC respectively, demonstrating a 1.8 fold increase due to stretch alone (p < 0.05). Cyclic stretch resulted in phosphorylation of the VEGF receptor KDR.
CONCLUSION: Exposure of VSMC to physiological levels of stretch induces a biologically significant increase in VEGF secretion and may provide an arterial stimulus for maintenance of steady state levels of VEGF essential for EC survival.

Excision of a giant hydatid cyst of the lung under thoracic epidural anaesthesia.
RA Dyer, PC Gordon, Mark De Groot, Gabi Walther, Mike James.
Anaesth Intensive Care (2001) 29(2): 181-184

We present a patient with a large pulmonary hydatid cyst compressing underlying lung, with previous pulmonary tuberculosis, who presented in respiratory failure. After institution of thoracic epidural anaesthesia employing 0.25% bupivacaine, 1% lignocaine and fentanyl, the patient was placed in the sitting position and the hydatid cyst excised and drained after a limited rib resection. An air leak persisted until the 16th postoperative day. A marked improvement in symptoms as well as in spirometly and arterial blood gases occurred, and the patient was discharged on the 20th day. Thoracic epidural anaesthesia may be a safer method than general anaesthesia for removal of a hydatid cyst in a patient with severe respiratory compromise.

Glutaraldehyde detoxification in addition to enhanced amine cross-linking dramatically reduces bioprosthetic tissue calcification in the rat model.
Christoph Weissenstein, Paul Human, Deon Bezuidenhout, Peter Zilla.
Journal of Heart Valve Disease (2000) 9(2): 230-40

Background: Enhanced fixation of bioprosthetic tissue through both increased concentrations of glutaraldehyde (GA) and the introduction of additional cross-links through L-Lysine significantly reduces calcification. We have previously described that prolonged exposure to high-volume amino-compounds under warm, acidic conditions is capable of thorough and non-rebounding GA detoxification. The purpose of the present study is to prove that the removal of excess GA can amplify the beneficial effect of enhanced GA cross-linking with regard to bioprosthetic tissue calcification.
Materials and Methods: Porcine ascending aortas and leaflet tissue as well as bovine pericardium were immediately fixed using three different GA concentrations (0.2%, 1.0%, 3.0%; v:v) for seven days at 4°C. The samples were distributed into nine groups. The first three groups received no further treatment (one at each GA concentration). Groups four to nine underwent an additional L–Lysine interim step (48 hours/37°C/0.1M) two days prior to the completion of the standard seven day GA fixation process. Finally groups seven to nine were additionally treated with a GA extraction process using high volume Urazole solution (acetic acid buffer, pH4.5, 37°C, one week) followed by NaBH4 reduction (2 days, 37°C). Samples were subcutaneously implanted in rats (6 per group) for 6 weeks. Calcium was determined by atomic absorption spectrophotometry and examined histologically by Von Kossa staining.
Results: Calcification was reduced in all three tissue types by enhanced cross-linking and by the additional extraction of excess GA.  Increasing the GA concentration from 0.2% to 3.0% lead to a reduction in calcification of 11.5% (p=0.074, Student’s t-test) in leaflets, 63.6% (p<0.0001) in pericardium and 17.5% (p=0.034) in aortic wall tissue.  The introduction of additional cross-links with L-Lysine resulted in a significant reduction of calcium in all tissue types (maximally 42.5%; p=0.0003 in leaflets, 79.3%; p=0.005 in pericardium and 49.6%; p<<0.0001 in aortic wall, Student’s t-test). Optimal reduction in calcification could be achieved with the combined effect of 3.0% GA fixation, L-Lysine enhancement and urazole detoxification.  When compared to 0.2% GA fixed tissue, calcification could be reduced by 99.1% in leaflets, 95.9% in pericardium and 90.8% in aortic wall tissue (p<<0.0001 for all tissue types, Student’s t-test).
Conclusion: We were able to demonstrate that the removal of excess GA from fixed bioprosthetic tissue is capable of markedly improving the anti-calcific effect of enhanced GA cross-linking.

High GA concentrations mitigate bioprosthetic root calcification in the sheep model.
Peter Zilla, Christoph Weissenstein, Paul Human, Terri Dower, Ulrich von Oppell.
Annals of Thoracic Surgery (2000) 70(6): 2091-5

Background: Fixation at high glutaraldehyde (GA) concentrations mitigated bioprosthetic calcification in the rat model. The present study intended to verify this observation in the circulatory sheep model.
Materials and Methods: Porcine aortic roots were either fixed in 0.2%, 1.0% or 3.0% GA. Eight roots per group were implanted in the distal aortic arch of sheep. After six weeks and six months calcification and inflammation were quantitatively and qulitatively assessed..
Results: By increasing the GA concentration from 0.2% to 3.0%, aortic wall calcification could be reduced by 38% after six weeks and 34% after six months of implantation (p<0.01). 
Mineralization coincided with the presence of elastin although calcium was predominantly found in cell nuclei and membranes. Leaflet calcification was absent in all groups after six weeks but in a few leaflets presented as heterogeneous, nodular spongiosa deposits after six months. Overall, differences between 0.2%, 1.0% and 3.0%-fixed tissue were quantitative but not qualitative regarding distribution patterns. There was no significant difference in inflammatory host reaction between all groups.
Conclusion: We have shown in the circulatory sheep model that the anti-calcific effect of better cross-linking seems to outweigh the intrinsic pro-calcific effect of glutaraldehyde  accumulation  in bioprosthetic aortic wall tissue.

Penetrating thoracic injuries: what we have learnt.
U. von Oppell, P. Bautz, M. de Groot.
Journal of Thoracic and Cardiovascular Surgery (2000) 48(1): 55-61

BACKGROUND: Thoracic injuries, especially cardiac, vascular, and transmediastinal injuries, are amongst the most lethal of penetrating injuries. METHOD: Our experience at Groote Schuur Hospital is reviewed, where up to 1,000 patients were admitted annually with penetrating chest wounds between 1982 and 1997. Results: The approximate pre-hospital mortality was 86% with penetrating cardiac injuries, 92 % with extrapericardial vascular injuries, and 11 % with pulmonary injuries. Less than 2% of pneumothorax cases and less than 10% of haemothorax cases required surgical intervention. Thoracoscopic evacuation of retained clots was successful in the majority of the latter. Most penetrating injuries of the thoracic duct required surgical exploration. The mortality of penetrating cardiac injuries varied according to clinical presentation (moribund 52%, hypovolaemia 20% and tamponade 2-5%) and the chamber involved. Higher mortalities were associated with atrial injuries. CONCLUSIONS: The appropriate use of intercostal drains and therapeutic thoracoscopy are important considerations in penetrating non-cardiac thoracic trauma. Rapid transportation, immediate triage, open-minded use of emergency room thoracotomy, and aggressive surgical management with liberal use of sub-xiphisternal pericardial windows are important factors in improving the survival of penetrating cardiac trauma.

Ischemic mitral valve repair surgery.
Ulrich von Oppell, F. Stemmet, Johan Brink, Patrick Commerford, S. Heijke.
Journal of Heart Valve Disease (2000) 9(1): 64-73; discussion 73-64

BACKGROUND AND AIM OF THE STUDY: The management of concomitant moderate or severe ischemic mitral regurgitation in the presence of ischemic heart disease is important for long-term prognosis. Mitral repair by either a suture or ring annuloplasty method has been advocated, although clear superiority of either method has not been established. METHODS: Combined coronary artery bypass and mitral valve surgery for ischemic mitral incompetence was performed on 68 consecutive patients between January 1996 and December 1998. The outcome in 63 of these patients (35 females, 28 males) who underwent mitral valve repair was reviewed. RESULTS: Average patient age was 61+/-9.4 years (range: 39-81 years). Average left ventricular ejection fraction (LVEF) was 42.1%; a suture annuloplasty was used in 84% and a ring in 16%. The average number of distal anastomoses was 3.9+/-1.1 (range: 1-6) and aortic cross-clamp time was 131+/-35 min (range: 58-238 min). Operative mortality rate (<30 days or in-hospital) was 12.7% and only requirement for intra-aortic balloon pumping either before or during surgery (21%) was predictive (p<0.05). On discharge, 98.2% of patients were in NYHA class I or II. Follow up (range: 1-35 months) was complete in 95% of cases. Moderate mitral regurgitation on discharge occurred in nine patients and was not related to the type of annuloplasty. Predictive risk factors were preoperative severe mitral regurgitation (p<0.04), poor LVEF (p = 0.05), and was predictive of deterioration of NYHA class (p<0.02), progression of regurgitation (p<0.05), and poor outcome (p<0.01). Poor outcome was also related to surgeon's experience. Structural valvular deterioration occurred in 21.8% of operative survivors, and there was one reoperation and four late deaths. The survival rate (including operative deaths) at 35 months was 68.3 +/- 13.1%, and event-free survival rate (no mortality, reoperation or angina) 65.2+/-6.2%. CONCLUSIONS: The type of annuloplasty used did not influence outcome. The risk of structural mitral valve dysfunction on follow up was related to severe preoperative mitral regurgitation, poor LVEF, surgeon's experience, and was predictive of poor outcome.

The surgical management of cricopharyngeal achalasia in children.
Andre Brooks, AJ Millar, Hans Rode.
International Journal of Pediatric Otorhinolaryngology (2000) 56(1): 1-7

The objective of this study was to evaluate the clinical presentation of children with cricopharyngeal achalasia and to document the diagnostic process followed and evaluate the immediate and long-term results of those children treated with a cricopharyngeal myotomy. METHODS: Five children who underwent cricopharyngeal myotomies since 1976 were identified and the hospital records were reviewed in detail. Four patients were followed up to establish the long-term results of surgery. RESULTS: The age of initial presentation ranged from birth to 6 months with a universal delay in establishing the diagnosis ranging from 11 to 138 months. Two children had pre- and post-operative manometry of the upper esophageal sphincter. Post-operative upper esophageal sphincter pressures were reduced to 29 and 47% in relation to pre-operative values. Nissen fundoplications were performed in two patients to control documented gastro esophageal reflux. No post-operative complications were noted and complete symptomatic relief was obtained in all children. The long-term follow up was 2, 10, 12 and 14 years with all children remaining free of symptoms. CONCLUSION: Cricopharyngeal achalasia is an important but relatively seldom diagnosed cause of dysphagia in children. The diagnosis is almost always delayed because the condition is not widely recognised amongst physicians. If the diagnosis and effective treatment is delayed significant morbidity or even mortality, mainly due to pulmonary aspiration, may result. Cricopharyngeal myotomy is a safe and effective operation with excellent results. Symptomatic relief is immediate and complete with no long-term recurrence documented in this series.

Endothelial cell transplantation.
Peter Zilla, Manfred Deutsch, Johann Meinhart.
Seminars in Vascular Surgery (1999) 12(1): 52-63

After more than 20 years of autologous endothelial cell transplantation, controversy is slowly giving way to consensus. The ongoing discussion regarding the optimal methods of creating an endothelial layer on synthetic vascular prostheses has been replaced by the realization that both in vitro endothelialization with cultured venous endothelial cells and mixed microvascular sodding result in equilibrated luminal tissue layers covered by a persistant endothelium. Clinical trials with almost 200 in vitro endothelialized prostheses are in their 10th year, and patency results are distinctly better than in nonendothelialized prostheses, particularly in below-the-knee grafts.

Mycotic aneurysm of the left pulmonary artery in a child with tetralogy of Fallot and Streptococcus viridans infective endocarditis.
J. Lawrenson, J. Stirling, John Hewitson.
Heart (1999) 82: 88

Blunt cardiac rupture caused by zip gun backfire.
James Fulton, Mark de Groot, Ulrich von Oppell, Tom Ruttman.
Annals of Thoracic Surgery (1998) 65(3): 837-839

A 16-year-old boy who sustained right ventricular rupture after backfiring of a homemade zip gun is reported. The unusual nature of this case together with the mechanisms and management of blunt cardiac rupture are briefly discussed.

Mistaken angiographic diagnosis of traumatic aorto-atrial fistula.
S. Williams-Jones, S. Beningfield, Johan Brink.
Australas Radiol (1998) 42(3): 264-6
A case of angiographically occult traumatic aorto-caval fistula masquerading as aorto-right atrial fistula is presented. The importance of angiographic vigilance in the imaging of arterio-venous fistula is emphasized.

Thoracoscopy in undiagnosed pleural effusions.
Mark de Groot, Gabi Walther.
South African Medical Journal (1998) 88(6): 706-711

OBJECTIVE: To review the indications and accuracy of diagnostic thoracoscopy for pleural effusions of unknown origin. DESIGN: Retrospective review of consecutive patients referred for diagnostic thoracoscopy over a 5-year period from 1 January 1989 to 31 December 1993. SETTING: Tertiary referral cardiothoracic unit. PATIENTS: Thirty-four patients referred from either medical or oncology services within a university-affiliated academic complex. INTERVENTIONS: All patients had diagnostic thoracoscopy performed under general anaesthesia. Retrospective data were collected in respect of presenting symptoms, gross findings, final pathological findings, amount of drainage, length of hospital stay and complications of the procedure. In 7 patients (21%), iodised talc was insufflated at the same time to create pleurodesis. MAIN RESULTS: Final diagnoses were: 17 (50%) malignant disease, 6 (18%) tuberculosis and 9 (26%) 'negative' pathology. In 2 (6%), further intervention was required to make a conclusive diagnosis. The diagnostic sensitivity for malignant disease was 89% and the specificity 100%. For pleural tuberculosis both the sensitivity and specificity were 100%. For 'negative' diagnoses the negative predictive value was 82%. A history of fever and sweats had a marked association (P = 0.002) with the final diagnosis of tuberculosis. No association could be identified between the gross observations at the time of thoracoscopy and the final diagnosis. The average length of hospital stay was 6.7 (range 1-25) days. There was 1 in-hospital death (3%), and 9 patients (26%) had major complications related to the procedure. CONCLUSIONS: Diagnostic thoracoscopy is a useful modality for obtaining a diagnosis in effusions of unknown origin where other methods have failed. The presence of symptoms such as fever and sweats is highly associated with a final diagnosis of tuberculosis.

Blunt cardiac rupture.
James Fulton, L. Nel, Mark de Groot, Ulrich von Oppell.
South African Medical Journal (1998) 36(4): 132-135

Cardiac rupture as a result of blunt trauma is not commonly encountered. Seven patients with this injury have been treated at Groote Schuur Hospital over the past 14 years. All presented with cardiovascular collapse and 4 developed signs of cardiac tamponade. A clinical diagnosis was made in 4 patients and echocardiography was done in 3. Pericardiocentesis was used in 1 patient to confirm the diagnosis. Significant diagnostic delay occurred in 1 patient with associated liver rupture. Two patients required emergency room thoracotomy. All other patients were approached using a median sternotomy. Five patients survived, giving an overall survival rate of 71%. Five patients had right atrial ruptures and 2 right ventricular ruptures. One patient with right ventricular rupture died in the operating room, while another patient with multiple right atrial ruptures died from multiple organ failure after 11 days. We also briefly review the history, mechanisms and pathology.

Glutaraldehyde detoxification of aortic wall tissue: a promising perspective for emerging bioprosthetic valve concepts.
Peter Zilla, Lynn Fullard, Paul Trescony, Johann Meinhart, Deon Bezuidenhout, Michael Gorlitzer, Paul Human, Ulrich von Oppell.
Journal of Heart Valve Disease (1997) 6(5): 510-520

BACKGROUND AND AIMS OF THE STUDY: Due to its superb crosslinking activity, glutaraldehyde (GA) is still the most widely used fixative for bioprosthetic heart valves. At the same time, however, GA is also believed to be partly responsible for tissue calcification and the lack of surface re-endothelialization, both of which may contribute to valve degeneration. Although excess GA has previously been extracted from thin leaflet tissue, this treatment proved insufficient for the detoxification of thick aortic wall tissue of stentless valves or root prostheses. METHODS: In order to establish a detoxification procedure which thoroughly extracts biologically active GA from aortic wall tissue, we used a highly sensitive bioassay where endothelial cells were seeded onto glutaraldehyde-fixed aortic wall discs following various detoxification procedures. Absolute cell numbers and morphologic shape were correlated with shrinkage temperature and shrinkage extent of the tissue to determine the potential of the treatments to reverse crosslinks. To optimize treatment conditions, pH (3.2 versus 4.5), temperature (22 degrees C versus 37 degrees C) and incubation time (48 h versus one week) were varied. In order to identify an optimal detoxification agent, 12 different amino-reagents from four chemical groups were compared: low pKa aromatic amines, amino acids, low pKa N-heterocyclic compounds and amino sugars. RESULTS: Amino-reagent treatment required warm temperature (37 degrees C), prolonged reaction time (one week) and a pH of 4.5 to achieve long-term cell growth on glutaraldehyde-fixed aortic wall. All 12 amino-reagents were able to detoxify aortic tissue satisfactorily; and all mildly reversed crosslinks, although there were differences between candidates. When summarized data were ranked correlating cell growth and quality with shrinkage temperature and shrinkage extent, seven reagents had a rank sum above the overall mean value, and five below with statistically significant differences between candidates. The additional stabilization of the detoxification reaction through borohydride-reduction had no further effect on tissue biocompatibility and crosslinks. CONCLUSIONS: Efficient detoxification of thick aortic wall tissue is possible if a one-week incubation in an acetic acid buffer-based amino-reagent is carried out at 37 degrees C.

Improved ultrastructural preservation of bioprosthetic tissue.
Peter Zilla, Yinxing Zhang, Paul Human, Willie Koen, Ulrich von Oppell.
Journal of Heart Valve Disease (1997) 6(5): 492-501

BACKGROUND AND AIMS OF THE STUDY: Poor ultrastructural tissue preservation of bioprosthetic heart valves is associated with a higher propensity for calcification. In spite of this realization, commercial valve fixation remains suboptimal. METHODS: In an attempt to maintain tissue integrity through improved cross-linking procedures, transmission electron microscopy and a 21-point damage score were applied to assess the ultrastructural preservation of aortic wall tissue-the main component of contemporary aortic valve bioprostheses. An ideal glutaraldehyde (GA) concentration was assessed by immediate tissue fixation at 4 degrees C comparing 0.2%, 0.5%, 0.65%, 1.0%, 2.0%, 3.0% and 4.0% GA in phosphate-buffered saline (PBS). Subsequently, an optimal concentration of 3.0% GA was used to determine the effect of fixation temperature (4 degrees, 22 degrees and 37 degrees C). Finally, the superior glutaraldehyde concentration (3.0%) and cross-linking temperature (4 degrees C) were used to assess tolerance towards delayed fixation. RESULTS: When different GA concentrations were used almost identical damage scores of 6.3 and 5.8 were found for 0.2% and 0.65% fixation. The first significant improvement was found at a concentration of 1.0% (score 3.3; p < 0.01) followed by a further improvement at 3.0% (score 2.6; p = 0.05). The optimal fixation temperature was 4 degrees C (3.7) with the worst results obtained at room temperature (score 9.2; p < 0.03). When fixation was delayed, the most significant damage occurred during the initial 30 min after slaughter (from 2.3 to 7.4; p < 0.02) followed by another significant deterioration between 4 and 16 h (from 5.6 to 9.7; p < 0.02). CONCLUSIONS: In summary, the prerequisites for an ideal ultrastructural preservation of bioprosthetic aortic wall tissue are immediate fixation (within 30 min), high GA concentrations (> 1.0%) and cold-temperature fixation (4 degrees C).

High glutaraldehyde concentrations reduce rather than increase the calcification of aortic wall tissue.
Peter Zilla, Christoph Weissenstein, Mona Bracher, Yinxing Zhang, Willie Koen, Paul Human, Ulrich von Oppell.
Journal of Heart Valve Disease (1997) 6(5): 502-509

BACKGROUND AND AIMS OF THE STUDY: This study was performed in order to: (i) determine whether a similar reduction of tissue calcification as seen after prolonged storage can be achieved through higher concentrations of glutaraldehyde (GA); and (ii) verify that well-preserved tissue integrity can suppress calcification. METHODS: Before fixation in 0.2% GA (PBS, 4 degrees C, seven days) porcine aortas were kept on ice for 48 h. Alternatively, tissue was immediately fixed at the abattoir in 0.2%, 1.0% or 3% glutaraldehyde (PBS, 4 degrees C, seven days). A second group of immediately fixed tissue (0.2%, 1.0%, 3.0% GA) (PBS, 4 degrees C, two days) had an interim step of L-lysine treatment (0.1M, 37 degrees C, acetic acid buffer, two days) in order to enhance cross-linking followed by warm-temperature fixation (PBS, 37 degrees C, five days). Two animal models were compared: subcutaneous implantation in rats (12 weeks) and vascular implantation in non-human primates, Chacma baboons (six weeks). RESULTS: In both animal models the highest level of calcification was found in the group with delayed fixation in 0.2% GA. In the rat model there was an inverse correlation between tissue calcification and the GA concentration used, with 3% GA-fixed tissue showing the lowest level of tissue calcium. Overall, increasing GA concentration had a significant benefit on calcification (p < 0.0001; two-factor analysis of variance). Enhancement of cross-linking with L-lysine further abrogated tissue calcium levels at all GA concentrations (p < 0.0001; two- factor analysis of variance). Although the short-term baboon model showed lower tissue calcium levels, the trend seen in the rat model was confirmed. CONCLUSIONS: Our results demonstrate the detrimental effect of delayed fixation and further suggest that, against previous beliefs, fixation at higher glutaraldehyde concentrations reduces the calcification tendency of cross-linked aortic tissue.

Pulmonary resection as an adjunct in the treatment of multiple drug-resistant tuberculosis.
M. van Leuven, Mark de Groot, K. Shean, Ulrich von Oppell, P. Willcox.
Annals of Thoracic Surgery (1997) 63(5): 1368-1372; discussion 1372-1363

BACKGROUND: Over the past decade the incidence of pulmonary disease due to drug-resistant strains of Mycobacterium tuberculosis has increased worldwide. We reviewed our local experience to clarify the benefits and risks of pulmonary resection in the management of drug-resistant strains of Mycobacterium tuberculosis. METHODS: A retrospective review was performed of 62 patients undergoing pulmonary resection for drug-resistant strains of Mycobacterium tuberculosis between January 1990 and November 1995. RESULTS: Fifty-three percent were men and 47% women with an average age of 34 years (range, 16 to 72 years). There was one postoperative death, for a perioperative (30-day) mortality of 1.6%. Sixteen complications occurred in 14 patients for an overall morbidity of 23%. Eighteen of 24 patients (75%) who were persistently sputum positive at the time of operation immediately converted to a negative sputum smear and culture. For all patients who were sputum negative after operation 80% remain relapse-free by actuarial analysis. CONCLUSIONS: We believe that operation plays an important ancillary role in the treatment of drug-resistant strains of Mycobacterium tuberculosis. The operation can be performed with acceptable morbidity and mortality and must be combined with appropriate and well-monitored pre- and postoperative antituberculous drug therapy.

Pulmonary artery banding: adequacy and long-term outcome.
Paulo Pinho, Ulrich von Oppell, Johan Brink, John Hewitson.
European Journal of Cardio-Thoracic Surgery (1997) 11(1): 105-111

OBJECTIVE: Pulmonary artery banding remains a palliative option for patients with congenital heart disease and excessive pulmonary blood flow, if there is unfavourable anatomy or frail condition. In contrast to more developed countries, our patients at Red Cross Children's Hospital, Cape Town, often present to medical services late and in poor nutritional condition. We retrospectively reviewed patients undergoing pulmonary artery banding to determine major variables that influenced long-term outcome. METHODS: In a 10-year period ending June 1992, 135 consecutive patients underwent pulmonary artery banding; 89 with ventricular septal defect type non-mixing disorders, and 46 with mixing or complex disorders. The median age was 3.0 months and weight 3.5 kg with 74.8% of patients weighing less than the third percentile (NCHS adapted), and 39.3% had an additional serious medical illness. RESULTS: Pulmonary banding mortality was 8.1%, and was higher in neonates (22.2%), P = 0.04) but was not related to congenital disorder, associated medical illness, or associated coarctation or interrupted aortic arch. The pulmonary band was inadequate at follow-up in 28.9%, which occurred more commonly if banding was necessary before 3 months of age (41.5%, P = 0.003) but was not related to weight, congenital disorder or associated respiratory infection. Sixty patients (44.4%) have now proceeded to definitive repair with a mortality of 23.3%, which was increased if the pulmonary band was inadequate at the time of definitive repair (44.4%: P = 0.02), but was not related to the congenital disorder. CONCLUSIONS: An inadequate pulmonary artery band adversely affects outcome and demands further aggressive management prior to definitive repair.

Eight years of clinical endothelial cell transplantation. Closing the gap between prosthetic grafts and vein grafts.
Johann Meinhart, Manfred Deutsch, Peter Zilla.
ASAIO (1997) 43(5): M515-521

After years of in vitro studies and non human primate implantations, the authors commenced their clinical program of autologous in vitro endothelialization of polytetrafluoroethylene (ePTFE) grafts in 1989. Based on the successful 3 year results of a pilot study (Phase I) with 49 patients (1:2 randomization, assigning 33 patients to the endothelialized group and 16 to the control group), the authors offered in vitro endothelialized grafts to all patients who did not have a suitable saphenous vein available from June 1993 onward (Phase II). Another 72 patients received 81 successfully endothelialized ePTFE grafts in this second phase of the transplantation program. In the Phase I randomized trial, the Kaplan-Meier survivorship analysis showed a primary 3 year patency rate of 84.7% for endothelialized grafts and 55.4% for control grafts. After 5 years, it was 73.8% for the endothelialized group and 20.8% for the controls. At the end of the 7 year follow-up period, the primary patency rate for endothelialized grafts remained high at 73.8%, whereas that for the control grafts dropped to zero (log-rank test; p = 0.001 and Wilcoxon test; p = 0.003). The subsequent Phase II routine clinical implantation of endothelialized ePTFE grafts showed a 3 1/2 year primary patency rate of 72.9% for all femoropopliteal reconstructions. The authors' overall 7 year follow-up with endothelialized femoropopliteal ePTFE grafts (n = 108) shows a patency of 66.0%. When pretreated with fibronectin (n = 43), the 7 year patency was 72.1%. In the above-knee group, the 7 year patency for fibronectin treated grafts was as high as 75.8% (n = 36). After 8 years of clinical endothelial cell transplantation, the authors conclude that in vitro endothelialization of ePTFE grafts results in a patency rate of arterial prostheses comparable with that of vein grafts.

Role of thoracic surgery for childhood tuberculosis.
John Hewitson, Ulrich von Oppell.
World Journal of Surgery (1997) 21(5): 468-474

Lymphadenopathy is the hallmark of intrathoracic tuberculosis in children. The role of the thoracic surgeon in treating childhood tuberculosis is to relieve the more severe symptoms of lymphadenopathy, prevent the more long-term secondary damage that lymphadenopathy may cause to the lung, and treat the sequelae of thoracic tuberculosis. We reviewed the role of surgery in childhood tuberculosis at Red Cross Children Hospital from January 1981 to January 1996 in 161 children under 13 who were admitted for 168 therapeutic surgical interventions for proved intrathoracic tuberculosis and its related complications. We classified patients according to the pathophysiology of their disease to clarify the role of surgery in their management. Successful decompression of lymph nodes that were acutely compromising major airways was done in 25 children, and decompression for chronic airway compression was successful in 8 of 11 children. Therapeutic bronchoscopy successfully opened an airway obstructed by intraluminal tissue in 68% of 28 patients, with long-term pulmonary reexpansion in 50%. Pulmonary resections for postprimary tuberculous damage were done in 72 patients with a mortality of 2.7% and morbidity of 16.7%. Another 17 patients were operated on for pleural disease and 15 for other tuberculosis-related problems. The mortality for all patients undergoing surgery for complications of tuberculosis during childhood was 1.9% (3/161), suggesting that when indicated, an aggressive surgical approach is relatively safe.

Complex thoracic vascular injury repair using deep hypothermia and circulatory arrest.
James Fulton, Johan Brink.
Annals of Thoracic Surgery (1997) 63(2): 557-559

A 61-year-old man with a penetrating injury to the innominate artery, left common carotid artery, and left subclavian artery at their origins from the aortic arch with associated injuries to both innominate veins and an innominate artery to vein fistula after a single stab wound is described. The patient was managed successfully using cardiopulmonary bypass together with deep hypothermia and circulatory arrest. Presentation and management are discussed.

In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation.
Manfred Deutsch, Johann Meinhart, M. Vesely, Teddy Fischlein, Peter Groscurth, Ulrich von Oppell, Peter Zilla.
Journal of Vascular Surgery (1997) 25(4): 757-763

PURPOSE: Forty-one months after we performed bilateral implantation of in vitro endothelialized femoropopliteal bypass grafts in a 69-year-old patient, we obtained a central graft segment for histologic and ultrastructural investigation. METHODS: Before implantation the grafts were confluently lined with autologous first passage mass cultures of pure cephalic vein endothelial cells. The precoating of the expanded polytetrafluoroethylene prosthesis was done with fibrinolytically inhibited fibrin glue. Reoperation became necessary because of symptomatic unilateral atherosclerotic lesions located in the center of one of the two in vitro lined grafts. A 21 cm long graft segment was removed and replaced by a new in vitro endothelialized expanded polytetrafluoroethylene graft. RESULTS: On scanning electron microscopy a confluently covering mature endothelium was found throughout the whole length of the removed prosthesis. The endothelial identity was confirmed by a positive immunohistochemical CD 34, von Willebrand factor-staining, and the ultrastructural demonstration of Weibel Pallade bodies. The endothelium rested on a collagen IV positive basement membrane. Histologic cross sections revealed uniformly developed subintimal tissue of 1.21 +/- 0.19 mm thickness, which was separated from the intima by a distinct internal elastic membrane. The cells of this cell-rich matrix stained strongly positive for actin. Ultrastructurally, this matrix was dominated by highly contractile myofibroblasts loaded with peripherally located well-developed actin fillaments. A number of these cells also showed signs of secretory cells with a distinct endoplasmic reticulum and a Golgi complex. In areas of atherosclerotic lesions the subendothelial matrix was partially exposed, and the internal elastic membrane had to a certain extent disintegrated. Only in these areas KP-1 and MG-M1 positive foamy macrophages and CD 34 positive capillaries were found. The myofibroblasts of this diseased part of the subintimal tissue contained large lipid vacuoles. CONCLUSIONS: We conclude that the confluent in vitro lining of synthetic vascular grafts with pure autologous endothelial cells facilitates graft healing, which may result in a hybrid structure with features of a native vessel.

Postpneumonectomy stump fistula in a ventilated patient.
Mark de Groot, W. Douie.
Annals of Thoracic Surgery (1997) 63(2): 552-554

The development of a postpneumonectomy stump fistula in a ventilated patient is a feared and frequently fatal event. Furthermore, the necessity of a pneumonectomy from sequelae of blunt trauma is rare. We describe the salvage of a young patient with a combination of the above events. The method involves the use of a simple intravenous bag "plombage" in combination with a regional thoracoplasty to buttress a resutured bronchial stump.

Interaction between panel reactive antibodies, auto- and cold reactive antibodies, and a positive B cell cross-match in renal and cardiac allograft survival.
Pauline Creemers, Johan Brink, Del Kahn.
Clin Transplant (1997) 11(2): 134-8

Penetrating injuries involving the intrathoracic great vessels.
James Fulton, Mark de Groot, Jullian Buckels, Ulrich von Oppell.
South African Medical Journal (1997) 35(2): 82-86

Forty-four consecutive patients with injuries to the intrathoracic great vessels admitted to our department from January 1982 to June 1994 were reviewed retrospectively. Forty-two patients (95%) sustained stabwounds and 2 (5%) patients had gunshot wounds. The most frequent radiological abnormality was mediastinal widening in 26 patients (59%). Eighteen patients (41%) were haemodynamically stable on admission with the remainder being unstable (46%), agonal (11%) or lifeless (2%). Twenty-two patients (50%) underwent angiography with 1 false-negative study. A total of 48 arterial and 16 venous injuries were identified with the innominate artery (N = 17, 39% of patients) and left innominate vein (N = 8, 18% of patients) the most frequently injured structures. Associated injuries to thoracic viscera occurred in 13 patients (30%). Two patients required cardiopulmonary bypass to repair their injuries. Arterial shunts were not used in any case. Overall mortality was 5% (2/44) and complications occurred in 7 patients (16%).

Profound hypothermnia and total circulatory arrest in the management of a massive false aneurysm of the Subclavian artery:  A case report.
James Fulton, HD Louwrens, J Marr, John Hewitson.
Journal of Endovascular Surgery (1997) 31(2): 199-203

The surgical management of a massive traumatic false aneurysm of the junction of the right subclavian artery with the innominate artery following a stab wound four months earlier is described. Surgical alternatives are discussed.

Acute traumatic rupture of the thoracic aorta. A comparison of techniques.
Ulrich von Oppell, Johan Brink, John Hewitson, Paulo Pinho, Peter Zilla.
South African Journal of Surgery (1996) 34(1): 19-24

Twenty-eight patients were treated for acute blunt thoracic aortic rupture at Groote Schuur Hospital between January 1984 and March 1994. Aortic arch ruptures occurred in 2 patients and were successfully repaired by means of hypothermic circulatory arrest. Descending aortic ruptures were repaired more safely by insertion of an interposition graft as opposed to direct suture reapproximation, and with the aid of partial heparinless bypass as opposed to simple aortic cross-clamping.

Syphilitic aortic aneurysm eroding through the sternum.
James Fulton, Peter Zilla, Mark de Groot, Ulrich von Oppell.
European Journal of Cardio-Thoracic Surgery (1996) 10(10): 922-924

Syphilitic aortic aneurysms are uncommon today. A syphilitic aneurysm eroding through the anterior chest wall with successful surgical treatment is reported. The large size these aneurysms reach, in conjunction with the overlying pressure-induced skin necrosis necrosis can represent a technical challenge to the surgeon, both in the method of repairing the aneurysm as well as reconstructing the chest wall. Syphilitic aortic disease is also briefly reviewed.

Stab wounds of the innominate artery.
James Fulton, Mark de Groot, Ulrich von Oppell.
Annals of Thoracic Surgery (1996) 61(3): 851-853

BACKGROUND: Innominate artery stab wounds are rarely encountered, and the optimal management of this injury is different from that of blunt innominate injury in that permanent bypass shunting should not be necessary. METHODS: The records of 19 patients with stab wounds of the innominate artery who were treated by our department from January 1982 to June 1995 were reviewed. RESULTS: Eighteen patients (95%) sustained zone 1 neck stabs, with a similar proportion having only a single stab wound. Seventeen (89%) of the 18 patients having chest roentgenograms had mediastinal widening. Thirteen patients (68%) were hemodynamically stable at admission; the remainder were unstable (26%) or moribund (5%). Fourteen patients (74%) underwent angiography, with no false-negative studies for arterial injury. Associated injuries to thoracic viscera occurred in 4 patients (21%). All injuries were repaired with either direct suture (18 of 19) or prosthetic interposition grafting (1 of 19). One patient required cardiopulmonary bypass to repair complex injuries. The overall mortality rate was 5% (1 of 19), and complications occurred in 2 patients (11%). CONCLUSIONS: Innominate artery stab wounds can be managed successfully without permanent bypass shunting and with a low mortality rate.

The surgical cure of atrial fibrillation/flutter.
Ulrich von Oppell, Rob Scott-Miller, James Fulton, Peter Zilla.
South African Medical Journal (1996) 86(Supplement 1): 22-24

Isolated thoracic duct injury after penetrating chest trauma.
Mike Worthington, Mark de Groot, Alf Gunning, Ulrich von Oppell.
Annals of Thoracic Surgery (1995) 60(2): 272-274

BACKGROUND. Isolated thoracic duct injuries as a result of penetrating chest trauma without any major vascular or tracheoesophageal injury seldom are seen. METHODS. A retrospective 13-year review identified 8 patients with this injury. RESULTS. Seven had supraclavicular or suprascapular knife stabs, and the eighth had a low-velocity gunshot injury entering the mid-lateral right chest wall. All 7 stab victims presented with left-sided chylothoraces, and the site of injury of the thoracic duct was within Poirier's triangle, the borders of which are the arch of aorta, the left subclavian artery, and the vertebral column as seen from a lateral approach. Five patients initially were treated conservatively for 13.4 +/- 4.4 days without success. Surgical intervention thus was necessary and was successful in all 8 patients. The thoracic duct injury was controlled successfully through a left posterolateral thoracotomy in 6 patients. A supraclavicular repair was attempted in 1 patient but failed to control the leak and required reexploration via the supraclavicular approach. The right chylothorax from the gunshot injury was explored via a right posterolateral thoracotomy; the leak into the pleura was identified and obliterated. CONCLUSIONS. As conservative management was uniformly unsuccessful, we advocate early operative management through a thoracotomy on the side of the chylothorax for this relatively rare injury.

Limited private practice--the other issue [letter; comment].
Ulrich von Oppell, Johan Brink, Mark de Groot, John Hewitson, Mike Worthington, Peter Zilla.
South African Medical Journal (1995) 85(9): 929

Aortic dissection repair with GRF glue complicated by heart block.
Ulrich von Oppell, D. Chimuka, Johan Brink, Peter Zilla.
Annals of Thoracic Surgery (1995) 59(3): 761-763

Gelatin-resorcin-formaldehyde-glutaraldehyde (GRF) biologic glue is an available adjunct to repair acute ascending aortic dissections. Permanent complete heart block complicated the operative repair of 2 of 6 patients. The pathophysiology of heart block resulting from either the acute dissecting process or the technique of applying GRF glue is discussed.

Clinical in vitro endothelialization of femoropopliteal bypass grafts: an actuarial follow-up over three years.
Peter Zilla, Manfred Deutsch, Johann Meinhart, R. Puschmann, T. Eberl, E. Minar, R. Dudczak, H. Lugmaier, P. Schmidt, I. Noszian.
Journal of Vascular Surgery (1994) 19(3): 540-548

PURPOSE: The creation of an endothelial coverage on prosthetic vascular surfaces may improve the performance of synthetic small diameter vascular grafts. In vitro lining with cultured autologous endothelial cells offers a confluent endothelium at the time of implantation. METHODS: Between June 1989 and December 1991, 49 patients who had no saphenous vein available entered the study. Indication for operation was disabling claudication in 37 patients and critical ischemia in 12 patients. With a random 1:2 assignment, 33 patients were admitted to the endothelialized group and 16 control patients received an untreated polytetrafluoroethylene prosthesis. Cultured autologous endothelial cells from the external jugular vein were confluently lined onto polytetrafluoroethylene grafts precoated with fibrinolytically inhibited fibrin glue. The follow-up was based on angiography, platelet labeling studies with indium 111-labeled oxine, assessment of the ankle-brachial index, and duplex sonography. RESULTS: First-passage mass cultures of 16 million endothelial cells-required for the confluent lining of a 70 cm long 6 mm graft-were reached 25.1 +/- 11.2 days after vein excision. Growth failure occurred in 27.3%. After 32 months, the actuarial patency was 84.7% for endothelialized grafts and 55.4% for control grafts (p < 0.041 by Breslow test; p < 0.068 by Mantel-Cox test). The ankle-brachial index was continually diverging, reaching significantly lower values in the control group at 24 months (0.98 +/- 0.14 in the endothelialized group versus 0.70 +/- 0.12 in the control; p < 0.0023). The uptake of indium 111-labeled platelets--measured at 9 days, 3 months, 6 months, and 12 months--was significantly lower in the endothelialized group during the entire observation period.

In vitro-lined endothelium: initial integrity and ultrastructural events.
Peter Zilla, Petra Preiss, Peter Groscurth, Frank Rosemeier, Manfred Deutsch, John Odell, C. Heidinger, Roland Fasol, Ulrich von Oppell.
Surgery (1994) 116(3): 524-534

BACKGROUND. The early fate of in vitro-endothelialized prosthetic vascular grafts was assessed in the nonhuman primate. METHODS. Each of 17 male chacma baboons received a control and a confluently endothelialized 4 mm polytetrafluoroethylene graft in femoro-femoral positions (8.2 +/- 0.8 cm). All experimental grafts were precoated with fibrinolytically inhibited fibrin glue and lined with cultured autologous endothelial cells (EC) from the external jugular vein. The average time period needed to obtain first-passage mass-cultures sufficient for preconfluent graft endothelialization was 19.8 +/- 5.2 days. Before implantation in vitro-lined grafts were kept in culture for another 16.1 +/- 4.3 days to achieve complete confluence and maturation of the EC cytoskeleton. RESULTS. After 9 days of implantation, endothelial-lined grafts still showed a confluent endothelium that was free of any fibrin deposits. However, the EC density was significantly lower than at implantation (39.7 +/- 7.6 x 10(3) versus 59.9 +/- 8.5 x 10(3) EC/cm2; p < 0.05), and occasional 10-microns-wide intercellular gaps with adherent platelets and leukocytes were visible. Transmission electron microscopy showed leukocytes and cell debris in the underlying fibrin glue. After 4 weeks of implantation, the endothelium of experimental prostheses had regained a high cell density (72.7 +/- 10.5 x 10(3) EC/cm2) with a mature and well-differentiated morphologic appearance. At both observation periods, the surface of control grafts showed a wide range from fibrin deposits to an amorphous protein coverage containing spread platelets. CONCLUSIONS. The endothelium of in vitro-endothelialized vascular prostheses remains confluent after implantation and is nonthrombogenic in spite of a moderate initial cell loss.

Traumatic aortic rupture: twenty-year metaanalysis of mortality and risk of paraplegia.
Ulrich von Oppell, Tim Dunne, Mark de Groot, Peter Zilla.
Annals of Thoracic Surgery (1994) 58(2): 585-593

A metaanalysis of articles concerning the surgical management of acute traumatic rupture of the descending thoracic aorta published in the English-language literature between 1972 and July 1992 was performed. The overall mortality of 1,742 patients who arrived at the hospital alive was 32.0%, one-third died before surgical repair was started. Paraplegia was noted preoperatively in 2.6% of these hospitalized patients, and paraplegia complicated the surgical repair in 9.9% of 1,492 patients who reached the operating room in a relatively stable condition. Patients then were analyzed according to the surgical intervention used. Simple aortic cross-clamping (n = 443) was associated with a hospital mortality of 16.0% and incidence of paraplegia of 19.2%, despite lower average mean cross-clamp times (32 minutes; p < 0.01 versus passive or active methods of providing distal perfusion). In a subset of 290 patients in whom individual data were available, the cumulative risk of paraplegia was shown to increase substantially if the duration of aortic cross-clamping exceeded 30 minutes, but only when distal perfusion was not augmented (p < 0.00001). "Passive" perfusion shunts (n = 424) were associated with a mortality of 12.3%, and the incidence of paraplegia decreased to 11.1% (p < 0.001). However, shunts inserted from the apex of the left ventricle had a contradictory high 26.1% incidence of paraplegia compared with shunts from the ascending aorta (8.2%; p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Spinal cord protection in the absence of collateral circulation: meta-analysis of mortality and paraplegia.
Ulrich von Oppell, Tim Dunne, Mark de Groot, Peter Zilla.
Joutnal of Cardiac Surgery (1994) 9(6): 685-691

A meta-analysis of paraplegia complicating aortic surgery on patients having neither intercostal nor spinal collaterals, epitomized by patients with acute traumatic aortic rupture, was done. Index Medicus and Medline were searched for all suitable English publications between 1972 and 1992. New paraplegia occurred in 9.9% of 1492 patients who underwent surgery. However, 19.2% of patients undergoing surgery with only simple aortic cross-clamping developed paraplegia, in contrast to 6.1% if distal aortic perfusion was augmented by either "passive" or "active" methods (p < 0.00001). The risk of paraplegia increased progressively as cross-clamp times lengthened if simple aortic cross-clamping was used (p < 0.00001), but only once did the cross-clamp time exceed 30 minutes (p < 0.05). Paraplegia occurred in 8.2% of patients with "passive" shunts from the ascending aorta (p < 0.001 vs simple cross-clamping). Shunts from the left ventricular apex, however, had an incidence of paraplegia of 26.1% and, therefore, did not decrease the risk of paraplegia. "Active" augmentation of distal perfusion had the lowest risk of paraplegia: 2.3% (p < 0.00001 vs simple cross-clamping or "passive" shunts). Mortality, however, was higher in these potentially polytraumatized patients when they were perfused distally using methods requiring full systemic heparinization (18.2%), compared to mortality with methods not requiring heparin (11.9%; p < 0.01). In conclusion, simple aortic cross-clamping has a high risk of paraplegia if the cross-clamp time extends beyond 30 minutes. "Active" modalities of augmenting distal perfusion provide optimal spinal protection.

High-dose aprotinin in cardiac surgery--a prospective, randomized study.
M. Swart, P. Gordon, P.B.  Hayse-Gregson, R. Dyer, A. Swanepoel, N. Buckels, R. Schall, John Odell.
Anaesth Intensive Care (1994) 22(5): 529-533

Fifty patients undergoing primary coronary artery bypass surgery and 50 patients undergoing valve surgery received either high-dose aprotinin (2 million units loading dose, 2 million units added to the CPB prime, and 500,000 units/hr maintenance infusion) or placebo. Mean postoperative blood loss in the first six hours was reduced from 321 ml in the placebo group to 172 ml in the aprotinin group (95% confidence interval (CI) for difference = 95 to 189 ml). Seven patients in the placebo group and 16 patients in the aprotinin group did not require transfusion with homologous blood. This study adds to the growing body of evidence that the administration of high-dose aprotinin reduces blood loss and blood transfusion requirements associated with primary cardiac surgery.

In vitro endothelialization of a mesosystemic shunt: a clinical case report.
Teddy Fischlein, Peter Zilla, Johann Meinhart, R. Puschmann, M. Vesely, T. Eberl, R. Balon, Manfred Deutsch.
Journal of Vascular Surgery (1994) 19(3): 549-554

The existence of a confluently covering endothelium that is free of any thrombotic appositions can be proved 30 days after clinical implantation of an in vitro endothelialized expanded polytetrafluoroethylene graft. The recipient of the mesosystemic H-graft was a 69-year-old man who had a thrombosed portal vein following pancreatitis. Autologous endothelial cells were obtained from the external jugular vein under local anesthesia, applying the in situ cannulation technique. After low-density plating, first-passage mass cultures of 1.22 x 10(6) endothelial cells were obtained 14 days after vein excision. After precoating was accomplished with fibrinolytically inhibited fibrin glue, a 10 mm expanded polytetrafluoroethylene graft was confluently lined with the autologous endothelial cells at a seeding density of 1.2 x 10(5) cells/cm2. After a maturation period of an additional 9 days and the microbiologic exclusion of a possible infection, an 11 cm graft segment was implanted between the superior mesenteric vein and the inferior vena cava. In spite of a patent shunt the patient had a repeat bleeding episode, needed parenteral nutrition, and died of sepsis on day 30. Immediately after the graft had been taken out, specimens were processed by scanning electron microscopy and light microscopy for the immunohistochemical proof of the endothelial nature of the surface-covering cell layer. The entire graft surface displayed a confluent cell lining that was free of any thrombotic appositions. A strongly positive stain result for both factor VIII-related antigen and the fixation-resistant CD34 molecule identified these cells as endothelial. No alpha-actin-positive cells could be detected. The underlying protein matrix was well preserved and unaltered in thickness and appearance, compared with preimplantation samples. None of the specimens showed any evidence of infection. This human demonstration of an intact endothelium on a patent venous prosthesis further establishes in vitro lining as a method that actually creates a persistent and functioning endothelium on a synthetic graft surface.

The endothelium: a key to the future.
Peter Zilla, Ulrich von Oppell, Manfred Deutsch.
Joutnal of Cardiac Surgery (1993) 8(1): 32-60

The vascular endothelium is a complex modulator of a variety of biological systems and may well be the key to definitive success in the treatment of cardiovascular disorders. Surgically-induced endothelial injury may occur preoperatively during cardiac catheterization and intraoperatively from mechanical manipulation, ischemia, hypothermia, and exposure to cardioplegic solutions. The normal endothelium is antithrombogenic and yet promotes platelet aggregation and coagulation if injured. Vasospasm, occlusive intimal hyperplasia, and accelerated arteriosclerosis can also all occur as a result of endothelial injury. Furthermore, endothelial injury is harmful even in the absence of disruption of its monolayer integrity. Thus, preservation of the endothelium should be an additional objective for all cardiovascular surgeons. Synthetic vascular grafts, cardiac valves, and artificial ventricles do not spontaneously endothelialize and thus usually require some form of anticoagulation to maintain patency. Hence, endothelialization of prosthetic implants became an attractive concept. A number of different methods of obtaining an endothelial lining of prosthetic material has since been developed; these include facilitated endothelial cell migration, and endothelial cell seeding by using either venous or microvascular endothelial cells. Manipulating the endothelium might well provide the next major advancement for therapeutic and preventive measures for cardiovascular disease.

Surgical relief of acute airway obstruction due to primary tuberculosis.
Mike Worthington, Johan Brink, John Odell, Jullian Buckels, Mark de Groot, M. Klein, Alf Gunning.
Annals of Thoracic Surgery (1993) 56(5): 1054-1062

Primary pulmonary tuberculosis in children remains a leading cause of mortality and morbidity in developing countries. Thirteen children requiring urgent thoracotomy for relief of acute respiratory distress resulting from critical major airway narrowing caused by enlarged tuberculous mediastinal lymph nodes were admitted to two hospitals over a 4-year period. Ages ranged from 2 months to 10 years. The condition of each patient had deteriorated despite appropriate antituberculosis therapy and an oral corticosteroid. At operation, the enlarged tuberculous subcarinal or paratracheal lymph nodes or both were decompressed. Surgical complications included a bronchial tear and a pulmonary artery laceration. Additional procedures included a right upper lobectomy, two pneumonectomies, plication of a hemidiaphragm, and mobilization of two muscle flaps. Postoperatively all children showed dramatic improvement. The trachea to main bronchi diameter ratio improved by 49.1% on the left and 44.9% on the right in the immediate postoperative period. In children with respiratory distress produced by compression of the main bronchi between enlarged subcarinal and paratracheal lymph nodes, surgical decompression of the lymph nodes is indicated if there is no marked initial response to appropriate medical therapy. At operation, lymph nodes should be decompressed only by incision and curettage. Attempts at lymph node excision are associated with increased complications.

Immediate shear stress resistance of endothelial cell monolayers seeded in vitro on fibrin glue-coated ePTFE prostheses.
W. Muller-Glauser, Peter Zilla, M. Lachat, B. Bisang, F. Rieser, L. von Segesser, M. Turina.
European Journal of Vascular Surgery (1993) 7(3): 324-328

The shear stress resistance of endothelial cells (EC) previously seeded onto ePTFE grafts was assessed by morphometric determination of the number of cells per cm2 of graft surface before and after exposure of 6 h of arterial blood flow interposed in the canine femoral artery. Autologous venous endothelial cells (AVEC) were harvested from the extrajugular veins of five dogs. The AVEC were cultured in vitro and seeded at a density of 150 x 10(3) cells per cm2 onto 4 mm ID ePTFE grafts precoated with fibrin glue and human fibronectin. Subsequently, the AVEC monolayers on the grafts were cultured for 8 days using a perfusion system and then implanted end-to-end in the femoral artery. All grafts remained patent (5/5). Scanning electron microscopy demonstrated complete, thrombus-free monolayers of AVEC after 6 h of arterial blood flow. The cell densities were 124 +/- 14 and 129 +/- 7 x 10(3) cells per cm2 respectively before and after implantation. It is concluded that in vitro lining of 4 mm ePTFE vascular prostheses is feasible and results in EC monolayers on the graft surface which are shear stress resistant and athrombogenic.

Massive thymic hyperplasia.
A. Linegar, John Odell, W. Fennell, P. Close, Mark de Groot, D. Casserly, J. Perold.
Annals of Thoracic Surgery (1993) 55(5): 1197-1201

Massive thymic hyperplasia is an extremely rare form of true thymic hyperplasia most often described in infants and children. Hyperplasia of this order is not known to occur in any other organ, and its etiology and prognostic significance remain unknown. As there is no accurate way of preoperatively differentiating massive thymic hyperplasia from other tumors of the thymus and anterior mediastinum, we advise excision in all cases for histological analysis and relief of mediastinal compression. This description of 4 cases updates the 30 previously reported cases, and includes a literature review.

Extended cardiopulmonary preservation: University of Wisconsin solution versus Bretschneider's cardioplegic solution.
Paul Human, Jurgen Holl, Susan Vosloo, John Hewitson, Johan Brink, Herman Reichenspurner, Dieter Boehm, Alan Rose, John Odell, Bruno Reichart.
Annals of Thoracic Surgery (1993) 55(5): 1123-1130

Application of the University of Wisconsin cold storage solution has rapidly expanded to include medium-term to long-term preservation of virtually all intraabdominal organs. Its use in intrathoracic organ transplantation has also been suggested. We therefore examined the efficacy of the University of Wisconsin solution in a primate allotransplantation model for preservation of hearts, and as a simple single-solution system for static preservation of heart-lung blocks, for periods of ischemia ranging from 6 to 24 hours. For comparison, we employed the histidine-tryptophane-ketoglutarate cardioplegic solution of Bretschneider. University of Wisconsin solution provided superior results with regard to clinical outcome and hemodynamic recovery of hearts after ischemic periods of up to 16 hours. This was in contrast to Bretschneider's solution, which allowed storage of hearts for periods of only up to 10 hours. Heart-lung blocks were equally well preserved with either University of Wisconsin or Bretschneider's solution after 6 to 12 hours, although the University of Wisconsin solution group exhibited a more notable increase in pulmonary water content. This was in accordance with histological data, which suggested that, although hemodynamic recovery of hearts stored for periods longer than 10 hours was poor, preservation of pulmonary ultrastructure was far superior using Bretschneider's solution as compared with University of Wisconsin solution after an ischemic period of up to 16 hours.

Long-term results of the Ivalon baffle mitral valve repair.
John Odell, J. Schurek, C. Barnard, Patrick Commerford.
Annals of Thoracic Surgery (1992) 54(2): 283-285

In the evolution of mitral valve surgery, Ivalon sponge was sutured to the posterior leaflet of the mitral valve to obtain competency. Between August 1959 and October 1962, 18 patients had this procedure. All patients were discharged home. Three patients were lost to follow-up 5 to 10 years after operation. Valve replacement was necessary in 7 patients 10.4 +/- 8.5 years after repair. Bacterial endocarditis causing late death occurred in 5 patients within 4 years. Five embolic episodes occurred. The estimated probability of survival and need for valve replacement at 28 years were 29.2% +/- 12.3% and 12.4% +/- 6.7%, respectively.

Clinical lining of prosthetic femoropopliteal bypass grafts with cultured autologous endothelial cells.
Teddy Fischlein, Peter Zilla, Johann Meinhart, Manfred Deutsch.
Transplantation Proceedings (1992) 24(6): 3043

Experimental in vitro endothelialization of cardiac valve leaflets [see comments].
T. Eberl, S. Siedler, B. Schumacher, Peter Zilla, K. Schlaudraff, Roland Fasol.
Annals of Thoracic Surgery (1992) 53(3): 487-492

This study reports our results with vitro endothelialization of fresh nonpreserved homograft valve leaflets compared with mild alternatively preserved valves and valves treated by preservation procedures commonly used for commercially available tissue valves. In vitro lining of biological heart valves with cultured autologous endothelial cells might help prevent the detrimental effects of degeneration on valve durability. To investigate the growth characteristics of endothelial cells on valve bioprostheses, three different methods of storage and preservation were compared. After precoating with fibronectin and seeding of 4.4 x 10(4) endothelial cells/cm2 onto the different leaflet surfaces, primary adherence, growth kinetics, morphology, and maintenance of monolayer integrity were studied over a period of 10 days. On valve leaflet surfaces of group 1 (fresh nonpreserved homograft valve leaflets) and group 2 (mild alternatively preserved valves), endothelial cells grew to persistent monolayers between days 6 and 10. In contrast, endothelial cell proliferation with monolayer growth could not be achieved on the group 3 leaflets (preserved like commercially available biological valve prostheses). In that group, no viable endothelial cells could be found on the valve surfaces 2 days after seeding. These results demonstrate the theoretical feasibility of endothelializing biological heart valve leaflets in vitro if they are not preserved and stored according to commonly used procedures. Provided such an endothelium can withstand the mechanical forces after implantation in vivo, in vitro endothelialization might contribute either to the development of new biological heart valves for modern cardiac surgery or to the improvement of clinical results with homograft valve transplants.

Donor heart coronary sinus ostium atresia in a successful cardiac transplant.
N. Buckels, Susan Vosloo, Alan Rose, John Odell.
Annals of Thoracic Surgery (1992) 53(6): 1096-1097

Heart transplantation in South Africa - a critical appraisal.
John Odell, Johan Brink.
South African Medical Journal (1992) 82(6): 394-6

Growth properties of cultured human endothelial cells on differently coated artificial heart materials.
Peter Zilla, Roland Fasol, M. Grimm, Teddy Fischlein, T. Eberl, Petra Preiss, O. Krupicka, Ulrich von Oppell, Manfred Deutsch.
Journal of Thoracic and Cardiovascular Surgery (1991) 101(4): 671-680

The cultivation of autologous endothelial cells on the blood surface of artificial hearts might prevent their detrimental thromboembolic complications. To investigate the growth characteristics of endothelial cells on theoretically suitable biomaterials, we compared three polyurethanes (Pellethane, Biomer, Enka) and three silicone rubbers (Elastosil, 3145 RTV, Medical Adhesive). All synthetic surfaces were precoated with an extracellular matrix (group 1), fibronectin (group 2), or a glutaraldehyde-preserved cellular matrix (group 3). After the seeding of 2.5 x 10(4)/cm2 human endothelial cells into the various surfaces, primary adherence, growth kinetics, and maintenance of monolayer integrity were studied for 13 days. On the three polyurethanes all precoating procedures resulted in endothelial cell proliferation and the formation of persistent monolayers. In contrast, on silicone rubbers a persistent coverage with a confluent endothelium could be achieved only on the glutaraldehyde-preserved cellular matrix. When endothelial cell growth was quantitatively assessed on all precoating substrates, the glutaraldehyde-preserved cellular matrix proved to be far superior on each of the synthetics (p less than 0.001). These results demonstrate the theoretical feasibility of endothelialization of artificial hearts in vitro. Provided such an endothelium can withstand the mechanical forces within an artificial heart, in vitro endothelialization might contribute to a regained attractiveness of the elective long-term implantation of artificial hearts.

Successful management of aortoesophageal fistula due to thoracic aortic aneurysm.
Ulrich von Oppell, Mark de Groot, C. Thierfelder, Peter Zilla, John Odell.
Annals of Thoracic Surgery (1991) 52(5): 1168-1170

Aortoesophageal fistulas due to atherosclerotic thoracic aneurysms are usually fatal, with few reported survivors. We report an aortoesophageal fistula managed successfully in one stage by resection and replacement of the aortic aneurysm with a prosthetic graft and total esophageal resection. Immediate esophageal reconstruction was attained using orthotopic gastric interposition with omentopexy around the prosthetic aortic graft.

St. Thomas' Hospital cardioplegic solution. Beneficial effect of glucose and multidose reinfusions of cardioplegic solution.
Ulrich von Oppell, Eugene Du Toit, Linda King, Patricia Owen, Tim Dunne, Bruno Reichart, Lionel Opie.
Journal of Thoracic and Cardiovascular Surgery (1991) 102(3): 405-412

The intention of this study was to determine whether glucose is beneficial in a cardioplegic solution when the end products of metabolism produced during the ischemic period are intermittently removed. The experimental model used was the isolated working rat heart, with a 3-hour hypothermic 10 degrees C cardioplegic arrest period. Cardioplegic solutions tested were the St. Thomas' Hospital No. 2 and a modified Krebs-Henseleit cardioplegic solution. Glucose (11 mmol/L) was beneficial when multidose cardioplegia was administered every 30 minutes. Including glucose in Krebs-Henseleit cardioplegic solution improved postischemic recovery of aortic output from 57.0% +/- 1.8% to 65.8% +/- 2.2%; p less than 0.025. The addition of glucose to St. Thomas' Hospital No. 2 cardioplegic solution improved aortic output from 74.6% +/- 1.9% to 87.4% +/- 1.9%; p less than 0.005. Furthermore, a dose-response curve showed that a glucose concentration of 20 mmol/L gave no better recovery than 0 mmol/L, and glucose in St. Thomas Hospital No. 2 cardioplegic solution was beneficial only in the range of 7 to 11 mmol/L. In addition, we showed that multidose cardioplegia was beneficial independent of glucose. Multidose St. Thomas' Hospital No. 2 cardioplegia, as opposed to single-dose cardioplegia, improved aortic output recovery from 57.4% +/- 5.2% to 74.6% +/- 1.9%; p less than 0.025, and with St. Thomas' Hospital No. 2 cardioplegic solution plus glucose (11 mmol/L) aortic output recovery improved from 65.9% +/- 2.9% to 87.4% +/- 1.9%; p less than 0.005. Hence, at least in this screening model, the St. Thomas' Hospital cardioplegic solution should contain glucose in the range of 7 mmol/L to 11 mmol/L, provided multidose cardioplegia is given. We cautiously suggest extrapolation to the human heart, on the basis of supporting clinical arguments that appear general enough to apply to both rat and human metabolisms.

Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts.
Alan Rose, David Cooper, Paul Human, Herman Reichenspurner, Bruno Reichart.
Journal of Heart and Lung Transplantation (1991) 10(2): 223-234

The histologic findings in a total of 112 experimental heart transplants comprising allografts (baboon to baboon: n = 37), concordant xenografts (vervet monkey to baboon: n = 52), and discordant xenografts (pig to baboon: n = 23), in which the roles of ABO blood group incompatibility, corcordance, and immunosuppression were evaluated, are described. Hyperacute (vascular, humoral) rejection was characterized by disruption of the microcirculation, with interstitial hemorrhage and edema, rather than by intravascular thrombosis; the features were basically similar whether hyperacute rejection occurred in an ABO-incompatible allograft, concordant xenograft, or discordant xenograft. Hyperacute rejection was noted in all 23 discordant xenografts, in 12 to 52 concordant xenografts, and in four of 17 ABO-incompatible allografts. A unique mixture of acute and hyperacute rejection was observed in three ABO-incompatible allografts and in 10 concordant xenografts. Intensive antirejection therapy was associated with a reduced incidence of hyperacute rejection in corcordant xenografts but also with a significant number of fatal treatment-related complications.

FK 506: short- and long-term treatment after cardiac transplantation in nonhuman primates.
Andreas Hildebrandt, Bruno Meiser, Paul Human, Herman Reichenspurner, Alan Rose, John Odell, Bruno Reichart.
Transplantation Proceedings (1991) 23(1 Pt 1): 509-510

Adenosine cardioplegia: reducing reperfusion injury of the ischaemic myocardium?
Dieter Boehm, Paul Human, Ulrich von Oppell, Patricia Owen, Herman Reichenspurner, Lionel Opie, Alan Rose, Bruno Reichart.
European Journal of Cardio-Thoracic Surgery (1991) 5(10): 542-545

Hyperkalaemia-induced hypopolarization of the sarcolemnal membrane during standard crystalloid cardioplegic arrest potentiates calcium influx during reperfusion and is associated with depletion of high-energy phosphate reserves. Adenosine has been shown to induce fast cardiac arrest whilst preserving membrane hyperpolarization in an isolated rat heart model. In this study we compared the efficacy of adenosine, both as an arresting agent and as an ultrastructural, haemodynamic and high-energy phosphate preserving agent, in an in situ global ischemia model in the baboon with St. Thomas' Hospital solution No. 2 (ST2; n = 8) and with Krebs-Henseleit buffer (KHB; n = 7). The addition of 10 mM adenosine to the non-cardioplegic KHB (ADO; n = 8) improved haemodynamic recovery significantly in terms of cardiac index (91.6% +/- 7.2 vs 59.9% +/- 9.9) and stroke volume index (101.6% +/- 8.9 vs 55.6 +/- 10.0) and was not statistically distinguishable from the ST2 with regard to cardiac index (91.6% +/- 7.2 vs 94.8% +/- 5.8), stroke volume index (101.6% +/- 8.9 vs 114.0% +/- 8.3) or left ventricular dP/dt (73.1% +/- 9.9 vs 87.0% +/- 12.4). Adenosine triphosphate was best preserved with ADO (103.5% +/- 21.1 vs 67.9% +/- 9.3 and 48.5% +/- 8.7) although this was not statistically significant. This suggests therefore that the mechanism of cardioprotection by adenosine occurs by means other than its role as high-energy phosphate precursor.

Traumatic rupture of the descending thoracic aorta.
Ulrich von Oppell, C. Thierfelder, S. Beningfield, Johan Brink, John Odell.
South African Medical Journal (1991) 79(10): 595-8

The management of acute traumatic rupture of the descending thoracic aorta at Groote Schuur Hospital between January 1984 and December 1989 is reviewed. Aortic rupture was diagnosed angiographically in 18 of 150 patients (12%), who underwent aortography because this injury was suspected. However, 3 of these patients had false-positive angiograms. The diagnosis was initially missed in 31% of patients, and this contributed to morbidity and mortality. Simple aortic cross-clamping (N = 8) was used before September 1988 and 3 patients died--1 intra-operatively from cardiac arrhythmia and 2 postoperatively, where major peri-operative haemorrhage had occurred. In contrast, partial heparin-less bypass (N = 5) using a centrifugal vortex pump was used after September 1988, and there were no haemorrhagic or paraplegic complications or mortality in this group. This technique is safe and appears to be superior to simple aortic cross-clamping in managing this condition.

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.
Peter Zilla, Roland Fasol, Ute Dudeck, S. Siedler, Petra Preiss, Teddy Fischlein, W. Muller-Glauser, G. Baitella, D. Sanan, John Odell.
Journal of Vascular Surgery (1990) 12(2): 180-189

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Inflow occlusion in the surgical management of a penetrating aortic arch injury: case report.
Susan Vosloo, Bruno Reichart.
Journal of Trauma (1990) 30(4): 514-515

The acute surgical management of a 22-year-old male patient with a stab wound below the left sternoclavicular junction causing partial transsection of the origin of the innominate artery, laceration of the posterior wall of the ascending aorta, as well as an innominate artery-vein fistula, is described and the value of inflow occlusion in the surgical repair discussed.

Endothelial cell toxicity of solid-organ preservation solutions.
Ulrich von Oppell, S. Pfeiffer, Petra Preiss, Tim Dunne, Peter Zilla, Bruno Reichart.
Annals of Thoracic Surgery (1990) 50(6): 902-910

Endothelial cell damage caused by myocardial cardioplegic solutions (Bretschneider HTK and St. Thomas' Hospital No. 2) or renal and hepatic cold storage solutions (modified Collins and University of Wisconsin solution) was assessed in monolayer cultures of adult human venous endothelial cells at 4 degrees to 10 degrees C with phase-contrast microscopy. St. Thomas' Hospital solution caused the cells to contract, resulting in disruption of monolayer integrity and opening of intercellular gaps, and resulted in a 24-hour postexposure survival of 51.0% +/- 2.4%. Bretschneider HTK solution altered cellular morphology less and produced the best postexposure survival (80.2% +/- 2.6%; p less than 0.001). Although morphology was altered the least with University of Wisconsin solution, postexposure survival with this solution, which was similar to that with modified Collins solution, was superior to that with St. Thomas' (p less than 0.01) but inferior to that with Bretschneider HTK (p less than 0.05). The superior protection provided by Bretschneider HTK was due to its additives histidine, tryptophan, and KH-2-oxygluterate (p less than 0.005), and to its low chloride content (p less than 0.005). Furthermore, modifying St. Thomas' solution by decreasing its chloride content improved cell survival to 71.2% +/- 2.3% (p less than 0.001). Normothermic (37 degrees C) exposure to Bretschneider HTK, modified Collins, and University of Wisconsin solution was cytotoxic, whereas normothermic exposure to St. Thomas' cardioplegia was not. In conclusion, the preservation solution that is the least harmful to endothelial cells at hypothermia is Bretschneider HTK cardioplegic solution.

Mechanical simulation of shear stress on the walls of peripheral arteries.
H. Schima, S. Tsangaris, Peter Zilla, M. Kadletz, E. Wolner.
Journal of Biomechanics (1990) 23(8): 845-851

In the last few years many attempts were made to line artificial vascular grafts with in vitro grown endothelial cell layers and thereby to minimize the risk of thromboembolism. However, adherence and resistance against shear stress forces were not tested under physiological pulsatile shear stress forces. In this paper, a mock-circulation apparatus is described, which simulates various forms of pulsatile shear stress, and which at the same time meets the requirements of cell cultivation. It can be sterilized and needs less than 700 ml of culture medium for priming. The generated flow profile can be adapted to a wide range of shear stress and also to different viscosities of used media. To take account of the different viscosities of culture medium and blood, a computerized calculation of the shear stress pattern was performed. Using the results of this computer model, the flow pattern was modified to obtain normal physiological shear stress when using culture medium. Results of pulse generation and simulation for the superficial femoral artery are presented.

15-Deoxyspergualin for induction of graft nonreactivity after cardiac and renal allotransplantation in primates.
Herman Reichenspurner, Andreas Hildebrandt, Paul Human, Dieter Boehm, Alan Rose, John Odell, Bruno Reichart, H. Schorlemmer.
Transplantation (1990) 50(2): 181-185

In order to assess the immunosuppressive potentials of 15-deoxyspergualin (15-DS) in a preclinical experiment, heterotopic cardiac (n = 27, group I) and classic renal (n = 25, group II) allotransplantations were performed in Chacma baboons. The following immunosuppressive regimens were applied: Groups IB and IIB were treated with 15-DS alone (4 mg/kg/day) for p.o. days 0-9. Groups IC and IIC were treated with cyclosporine A (10-40 mg/kg/day) for p.o. days 0-30. Groups ID and IID received a combination of 15-DS (for p.o. days 0-9) and CsA (for p.o. days 0-30). Groups IA and IIA served as control and received no medication. The mean graft survival was 11.0 days for group IA, 28.2 days for group IB (P less than 0.05; IB vs. IA), 32.4 days for group IC, and 43.1 days for group ID (P less than 0.025; ID vs. IA). After renal transplantation, the corresponding figures were 12.3 days for group IIA, 8.5 days for group IIB, 30.4 days for group IIC and 148.9 days for group IID (P less than 0.025; IID vs. IIA). After cardiac and renal transplantation, acute rejection was the main cause of graft failure. Treatment-related side effects, mainly gastrointestinal complications, were observed only in primates, who were treated with 15-DS alone. After cardiac transplantation, permanent graft non-reactivity was not achieved, but a delayed rejection occurred within a mean of 21.8 days after immunosuppression had been stopped. Following renal transplantation, graft nonreactivity was also not achieved in groups IIB and IIC. In group IID, however, 4 of 8 animals (50%) were graft-tolerant 340, 256, 244, and 164 days after treatment discontinuation. Thus, the combination of 15-DS and CsA led to a significant prolongation of graft survival in both groups. Long-term nonreactivity was achieved only after renal transplantation, when initially treated with 15-DS and CsA.

15-Deoxyspergualin after cardiac and renal allotransplantation in primates.
Herman Reichenspurner, Andreas Hildebrandt, Paul Human, Dieter Boehm, Alan Rose, H. Schorlemmer, Bruno Reichart.
Transplantation Proceedings (1990) 22(4): 1618-1619

Effect of pharmacologic immunosuppression on donor heart survival in a closely related nonhuman primate xenograft model.
Herman Reichenspurner, Paul Human, Alan Rose, Bruno Reichart, David Cooper.
Transplantation Proceedings (1990) 22(3): 1086-1087

[In vitro endothelialization of ePTFE vascular prostheses in clinical use: preliminary results].
Manfred Deutsch, T. Eberl, Teddy Fischlein, Johann Meinhart, E. Minar, R. Puschmann, P. Schmid, Peter Zilla.
Vasa Suppl (1990) 30: 219-220

Accelerated cardiac allograft rejection associated with administration of liver cell extract in the baboon.
David Cooper, N. Harris, Alan Rose, Dimitri Novitzky.
Transplantation Proceedings (1990) 22(4): 1966-1969

Adenosine and its role in cardioplegia: effects on postischemic recovery in the baboon.
Dieter Boehm, Paul Human, Herman Reichenspurner, Ulrich von Oppell, Patricia Owen, Lionel Opie, Bruno Reichart.
Transplantation Proceedings (1990) 22(2): 545-546

Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts.
Peter Zilla, Roland Fasol, Petra Preiss, M. Kadletz, Manfred Deutsch, H. Schima, S. Tsangaris, Peter Groscurth.
Surgery (1989) 105(4): 515-522
The shear stress resistance of cultured human endothelium was investigated on 6 mm polytetrafluoroethylene vascular grafts. Endothelial cell attachment was promoted by precoating the grafts with fibrin glue, which contained human fibronectin and inhibitors of fibrinolysis (aprotinin and tranexam acid). To evaluate the possible effect of fibrinolysis on cell detachment, seven grafts were lined with adult human saphenous vein endothelial cells (AHSVEC) and 11 with fibrinolytically almost inactive human umbilical vein endothelial cells (HUVEC). Endothelial cell seeding was performed in a microprocessor-controlled rotation device, allowing a low inoculum of 12 X 10(4) endothelial cells/cm2. Grafts were then cultivated for 9 days to enable the maturation of the cytoskeleton, before they were exposed to pulsatile shear stress for 48 hours. A mock circulation simulated the flow patterns and the wall shear forces of the femoral artery. After a 3-hour seeding process, 45% of AHSVEC and 43% of HUVEC were attached to the fibrin matrix, forming a confluent monolayer. After 24 hours of perfusion, a cell loss of 23% in AHSVEC- and of 42% in HUVEC-lined grafts was encountered. In spite of a further cell loss during the following 24 hours of perfusion, the majority of the graft surface was still covered by endothelial cells. Therefore we conclude that fibrin glue is a suitable substrate for the formation of a shear stress-resistant endothelial cell monolayer on polytetrafluoroethylene vascular grafts.

Blood platelets in cardiopulmonary bypass operations. Recovery occurs after initial stimulation, rather than continual activation [see comments].
Peter Zilla, Roland Fasol, Peter Groscurth, W. Klepetko, Herman Reichenspurner, E. Wolner.
Journal of Thoracic and Cardiovascular Surgery (1989) 97(3): 379-388

The ultrastructure of blood platelets was related to platelet function and secretion products before, during, and after cardiopulmonary bypass. Circulating platelets from 15 patients undergoing aorta-coronary bypass operations were investigated at ten predetermined points of time by scanning and transmission electron microscopy. Simultaneously, platelet adenosine triphosphate, diphosphate, and serotonin, as well as plasma levels of platelet factor 4, beta-thromboglobulin, serotonin, thromboxane B2, lactic dehydrogenase, and free hemoglobin were measured. Moreover, platelet responsiveness toward adenosine diphosphate and collagen was determined by optical aggregometry. By scanning electron microscopy, the number of unactivated platelets dropped from 96% +/- 4% to 54% +/- 19% (p less than 0.05) 8 minutes after the onset of bypass. Simultaneously, the percentage of "shape changed" platelets significantly increased. No major release reaction was detected at this time. After the initial activation, platelet morphology began to recover although the bypass continued. During the late period of bypass, a highly significant correlation between increasing plasma levels of alpha-granule compounds (platelet factor 4 and beta-thromboglobulin) and lysis parameters (lactic dehydrogenase and free hemoglobin) was found. However, transmission electron microscopic analysis of the arterial filter and scanning electron microscopic findings of circulating platelets indicated that the release products in plasma were due not only to platelet lysis but also to a limited extent to secondary aggregation. In an inverse and probably causative manner, platelet morphology recovered, whereas the sensitivity of platelets to adenosine diphosphate and collagen decreased toward the end of bypass.

Reduced reproductive capacity of freshly harvested endothelial cells in smokers: a possible shortcoming in the success of seeding?
Peter Zilla, S. Siedler, Roland Fasol, J. Sharefkin.
Journal of Vascular Surgery (1989) 10(2): 143-148

In an attempt to explain the failure of first clinical trials of autologous endothelial seeding in smokers, the initial reproductive capacity of saphenous vein endothelial cells from smokers and nonsmokers was studied by a replicate microwell technique. Endothelial cells were enzymatically harvested from saphenous vein segments of patients with coronary bypasses (21 smokers and 18 nonsmokers). After 15 minutes (group A) and 7 minutes (group B) of collagenase exposure, the endothelial cell harvest from donors who smoked was 41% (p less than 0.02) lower for group A and 30% (p less than 0.2) lower for group B than that from nonsmokers. In analogy, the viable cell yield was 32% (p less than 0.04) and 29% (p less than 0.05) lower for groups A and B, respectively, in cultures from donors who smoked. Daily cell counts over an ensuing 10-day period also revealed a significant difference in the proliferative behavior of endothelial cells from smokers and nonsmokers. Whereas endothelial cells from nonsmokers regularly entered the exponential phase of proliferation on day 4.4 +/- 1.8 (group A) and day 4.6 +/- 1.3 (group B), endothelial cells from smokers reached the logarithmic growth phase either with delay (day 6.8 +/- 2.1, group A) or remained completely quiescent (group B). Lower harvest efficiency and suppressed reproductive capacity of endothelial cells in smokers--on top of an already critically low inoculum in single-staged endothelial cell seeding--might explain the failure of initial clinical trials.

Reply to: Blood Platelets and Bypass.
Peter Zilla.
Journal of Thoracic and Cardiovascular Surgery (1989) 98(5 Pt 1): 797-800

Heterotopic heart transplantation in 1988--recent selective indications and outcome.
Herman Reichenspurner, Andreas Hildebrandt, Dieter Boehm, H. Kaulbach, S. Willems, John Odell, A. Horak, Bruno Reichart.
Journal of Heart Transplantation (1989) 8(5): 381-386

Considering a worldwide average 1-year survival rate of nearly 90% after orthotopic heart transplantation, the question arises as to whether there is still an indication for heterotopic heart transplantation. Since 1967, 132 heart transplantations have been performed at our institution. From 1974 to 1983 only heterotopic transplantations were performed. Since 1985, quadruple-drug therapy has been used for immunosuppression. This consists of low dose cyclosporine in combination with azathioprine, methylprednisolone (in lower dosages), and rabbit antithymocyte globulin (for the first 4 to 6 days after operation and as rescue therapy for severe rejections). Fifty-five transplantations have been performed with this therapy (44 orthotopic and 11 heterotopic). The indications for heterotopic transplantations were either elevated pulmonary vascular resistance (4 to 6 Wood units, n = 6), or a gross donor and recipient weight mismatch (more than 20%) in candidates who showed signs of severe cardiac decompensation (n = 6). One patient had both indications. The 1-year survival rate for those patients was 83%. Currently seven of the 11 patients are alive with life spans ranging from 6 months to 2.5 years after operation. Causes of deaths were infections (n = 3) and chronic graft rejection (n = 1). The recipients were restudied with right-sided heart catheterizations performed from 2 months to 2 years after transplantation. In all patients the cardiac output increased significantly from a mean of 4.0 to 5.8 L/min (p less than 0.0005). In patients with elevated pulmonary vascular resistance, this value decreased after heterotopic transplantation from a mean of 4.9 to 2.4 Wood units.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimalization of immunosuppression after xenogeneic heart transplantation in primates.
Herman Reichenspurner, Paul Human, Dieter Boehm, Alan Rose, Robin May, David Cooper, Peter Zilla, Bruno Reichart.
Journal of Heart Transplantation (1989) 8(3): 200-207; discussion 207-208

Xenogeneic heart transplantation is becoming increasingly attractive because of the shortage of suitable donor organs. In small infants and neonates, for whom suitable human grafts are difficult to obtain, this may play a particularly important role. To evaluate the optimal immunosuppressive regimen after xenogeneic transplantation, cervical heterotopic heart transplantation was performed with vervet monkeys as donors and chacma baboons as recipients. The following groups were investigated: group 1 (n = 9): control, no immunosuppressive medication; group 2 (n = 5): cyclosporine in combination with azathioprine and methylprednisolone; group 3 (n = 6): cyclosporine, azathioprine, and methylprednisolone in combination with antithymocyte globulin for postoperative days 0 to 9; group 4 (n = 7): cyclosporine, azathioprine, and methylprednisolone in combination with 15-deoxyspergualin for postoperative days 0 to 9. Because of severe treatment-related side effects that were observed in group 4, further immunosuppression was modified as follows: group 5 (n = 5): 15-deoxyspergualin was combined with cyclosporine and methylprednisolone only. Acute rejection episodes were diagnosed by cytoimmunologic monitoring on alternate days and weekly myocardial biopsies and were treated with 500 mg methylprednisolone intravenously for 3 to 5 consecutive days. The graft survival after xenogeneic heart transplantation was best in group 3 with 43.3 days compared with 10.3 days in the control group. Still 2.3 acute rejections occurred, which in most cases led to graft failure in these animals. In group 4 the graft survival was prolonged to 20.1 days on average. Only 0.5 acute rejections per animal occurred, but severe gastrointestinal complications and infections were observed that made further experiments necessary to minimize these treatment-related complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Inotropic effect of triiodothyronine (T3) in low cardiac output following cardioplegic arrest and cardiopulmonary bypass: an initial experience in patients undergoing open heart surgery.
Dimitri Novitzky, David Cooper, A. Swanepoel.
European Journal of Cardio-Thoracic Surgery (1989) 3(2): 140-145

A significant reduction in plasma free triiodothyronine (T3) (P less than 0.0001) has been observed in patients undergoing open heart surgery. The beneficial effect of T3 would appear to be associated with increased synthesis and utilization of myocardial high energy stores. We have therefore administered T3 (4-10 micrograms iv) to 10 patients either when difficulty was being experienced in weaning from cardiopulmonary bypass (CPB) support (n = 5), or when myocardial function remained extremely poor (n = 5), despite inotropic and intraaortic balloon pump support. Mean preoperative NYHA functional class of the 10 patients was 3.2, left ventricular enddiastolic pressure (LVEDP) 20 mm Hg and ejection fraction (EF) 40%. The mean myocardial ischaemia time was 72 min (range 40-120 min). Within 1 h of T3 administration the mean plasma free T3 level had risen from 1.03 to 3.56 micrograms/ml and CPB was discontinued in all 5 cases. Balloon pump support (n = 2) was no longer essential within 3 h. At 1 h, the mean arterial pressure (MAP) had risen from 42 to 78 mm Hg, and heart rate (HR) from 90 to 104 beats/min; the left atrial pressure (LAP) had fallen from 30 to 14 mm Hg, and the central venous pressure (CVP) from 20 to 11 cm H2O. (All changes significant at P less than 0.0001.) Inotropic support had been significantly reduced or discontinued. To our knowledge, T3 has not been administered previously as an inotropic agent to patients who have undergone cardiac surgery.

Heart transplantation - the treatment of choice for patients with end-stage ischaemic heart disease.
Andreas Hildebrandt, Herman Reichenspurner, Bruno Reichart.
Journal of Thoracic and Cardiovascular Surgery (1989) 37(1): 37-41

Between December, 1967 and August, 1988, 147 heart transplants (64 orthotopic, 68 heterotopic procedures; 15 heart-and-lung replacements) were performed on 128 patients. In the majority of the recipients, dilated cardiomyopathy or end-stage ischaemic heart disease was diagnosed. From 1985 to the present, 70 transplants (45 orthotopic, 11 heterotopic and 14 heart-lung) took place. Seventeen of these patients (mean age 46.6 years) suffered from end-stage disabling (NYHA IV) coronary artery disease; in each case the angiogram verified severe stenosis or occlusion of the three main coronary artery systems. Their histories revealed a total of 22 previous myocardial infarctions; 8 patients needed a total of 9 surgical revascularization procedures. The left ventricular ejection fraction (LVEF) ranged from 9% to 24% (mean 15.3%). Before transplantation three patients required intraaortic balloon pump (IABP) support. Fourteen of the 17 patients are at present still alive with post operative periods ranging from 8 weeks to 3.5 years (the actuarial 90-days and 1-year-survival rates being respectively 91.7% and 81.5%). Twelve of the patients are in NYHA class I; 2 are in class II. Three late deaths occurred: one from pneumocystic carinii/cytomegalovirus pneumonia, a second from atypical pneumonia and a third from chronic graft rejection. Radionuclide ventricular studies demonstrated postoperative left ventricular ejection fractions ranging from 54%-81% (mean 71%).(ABSTRACT TRUNCATED AT 250 WORDS)

Human endothelial cell seeding: evaluation of its effectiveness by platelet parameters after one year.
Roland Fasol, Peter Zilla, Manfred Deutsch, M. Grimm, Teddy Fischlein, G. Laufer.
Journal of Vascular Surgery (1989) 9(3): 432-436

Platelet parameters were assessed in patients after implantation of polytetrafluoroethylene (PTFE) grafts, either nonseeded or seeded with autologous endothelial cells or reversed saphenous vein grafts, during the follow-up period of 1 year. The objective of this investigation was to compare the sensitivity of different noninvasive platelet parameters with the assessment of the result of endothelial cell seeding. Scanning electron microscopic investigation of circulating blood platelets showed a significantly higher incidence of activated platelets in patients with seeded or nonseeded PTFE grafts, compared with patients after reversed vein graft implantation. "Shape-changed" platelets, as well as membrane-perforated thrombocytes, were found exclusively in the circulating blood of patients after seeded or nonseeded PTFE graft implantation, indicative of platelet trauma by the artificial graft surface. Plasma levels of platelet factor IV and beta-thromboglobulin, as well as the uptake of indium-111-labeled platelets, failed to show statistically significant differences after 1 year of follow-up. The similar results for seeded and nonseeded grafts indicate the failure of endothelial cell seeding to induce the development of a nontraumatizing surface, comparable to that of saphenous vein grafts.

Surface morphology of circulating platelets: a suggested parameter for the monitoring of endothelial seeded grafts.
Roland Fasol, Peter Zilla, Teddy Fischlein, G. Laufer, Manfred Deutsch.
Journal of Cardiovascular Surgery (Torino) (1989) 30(3): 398-401

For the clinical follow-up of endothelial cell seeded grafts, non-invasive but sensitive parameters are required in order to determine the extent and effectiveness of the endothelialization process. Such a very sensitive parameter was found by investigating the surface morphology of circulating platelets. This technique is based on the previous observation that characteristic alterations of platelet morphology are closely associated with the course of the aggregometer tracings. Applying these ultrastructural criteria we investigated 16 patients after distal femoro-popliteal and femoro-crural bypass surgery. Reinforced 6 mm non-seeded PTFE vascular grafts were compared with reversed autologous saphenous vein grafts. As early as 4 hours after graft implantation, a significant higher percentage of discoid platelets with marginal pseudopods--representing the earliest stage of platelet activation--as well as of spheroid "shape changed" platelets was found in patients with PTFE grafts. Moreover, perforated platelet membranes were exclusively found in the PTFE group. These findings were most distinguished 10 days postoperatively but persisted over the follow-up period of 1 year. Considering the sensitivity of the described technique it might provide a simple, reliable, and non-invasive means for the clinical assessment of successful graft endothelialization.

Vascular injuries caused by anti-personnel mines.
Roland Fasol, S. Irvine, Peter Zilla.
Journal of Cardiovascular Surgery (Torino) (1989) 30(3): 467-472

During a 3 month period 94 patients, injured by anti-personnel mines on the Thailand-Cambodian border, underwent emergency surgical treatment in the ICRC (International Committee of the Red Cross) hospital in Khao-I-Dang, Thailand. As a result of difficult evacuation facilities at the border, the mean time between injury caused by an anti-personnel mine and admission to the hospital was 8.3 hours (range: 2.5 to 14 hours). However, in 14 patients a penetrating vessel injury was sustained, and these underwent surgical treatment. Peripheral vascular repair was performed in 10 patients. The vessels involved in these reconstructions were: popliteal artery: 4, femoral artery: 2, anterior tibial artery: 3, femoral vein: 1. Vascular repair of the iliac artery and vein was performed in 1 patient. Limb amputation had to be performed in 3 patients, due to the long duration of tourniquet application in these cases. In the field conditions at Khao-I-Dang hospital many surgical facilities normally present in Western hospitals were unavailable. However, our series shows that satisfactory results could be obtained, despite adverse working conditions.

[Infrarenal aortic aneurysm: results of surgical treatment].
Manfred Deutsch, C. Holzinger, B. Krisch, H. Magometschnigg, Roland Fasol, Peter Zilla, C. Schwarz, M. Staudacher.
Wien Klin Wochenschr (1989) 101(2): 66-69

Between 1976 and 1987 93 patients with an infrarenal aortic aneurysm underwent surgical correction. In 62 patients the procedure was performed electively, whilst 13 displayed an unstable aneurysm and in 18 cases a ruptured aneurysm was present at operation. During the past 5 years the mortality was lowered to 2% in elective cases, whereas in cases of ongoing rupture only moderate improvement took place. The most frequent cause of a lethal outcome was pump failure of the heart (6 times), followed by renal insufficiency and haemorrhagic shock and bleeding complications. Among the non-lethal complications, relaparotomy on the basis of postoperative bleeding ranks first, followed by pulmonary insufficiency, peripheral emboli and partial ischemia of the spine. Resection of infrarenal aneurysms should be performed in the stable state of disease, since insufficiency of multiple vital organ systems increases the mortality by up to 20 fold.

The role of ABO blood group compatibility in heart transplantation between closely related animal species. An experimental study using the vervet monkey to baboon cardiac xenograft model.
David Cooper, Paul Human, Alan Rose, Jimmy Rees, M. Keraan, Bruno Reichart, Ernette DuToit, R. Oriol.
Journal of Thoracic and Cardiovascular Surgery (1989) 97(3): 447-455

The role of ABO blood group compatibility on graft survival when transplantation is performed between closely related animal species is uncertain. Heart transplants (in the neck) were performed between donor vervet monkeys and recipient baboons; no immunosuppressive therapy was given. Survival in ABO-compatible pairs (group 1, n = 9) was for a mean of 10.3 (+/- 5.2) days, which was not significantly different from that in ABO-incompatible pairs (group 2, n = 9: mean survival 7.3 +/- 5.6 days). In group 2, however, three hearts were rejected hyperacutely within 60 minutes, whereas in group 1 only one heart was rejected within 24 hours (not significant). Preformed anti-vervet monkey antibody was present in only one of 18 baboons, but developed in eight others. ABO-specific antibodies were present in all nine group 2 baboons and increased in titer in six cases. Histopathologic features of vascular (humoral) rejection, sometimes associated with cellular infiltration, were seen in a majority of hearts in both groups. Though the number of animals in this study was small, ABO-incompatibility would not appear to be a major factor in cardiac xenograft survival when transplantation is performed between closely related primate species, though early hyperacute rejection would seem more likely to occur when blood group incompatibility is present.

Can cardiac allografts and xenografts be transplanted across the ABO blood group barrier?
David Cooper, Paul Human, Alan Rose, Jimmy Rees, M. Keraan, Ernette DuToit, R. Oriol.
Transplantation Proceedings (1989) 21(1 Pt 1): 549-550

The pathophysiological effects of brain death on potential donor organs, with particular reference to the heart.
David Cooper, Dimitri Novitzky, Winston Wicomb.
Annals of the Royal College of Surgeons of England (1989) 71(4): 261-266

Major electrocardiographic, haemodynamic, and histopathological changes take place during the development of brain death; myocardial and pulmonary injury may result. Significant depletion of certain circulating hormones occurs, resulting in an inhibition of mitochondrial function, leading to reduced aerobic metabolic oxidative processes, affecting the body as a whole. Major organ energy stores are therefore diminished, leading to deterioration of function. Replacement of the depleted hormones, in particular triiodothyronine (T3), cortisol, and insulin, leads to rapid replacement of organ energy stores, associated with a return to normal function. T3 alone leads to reactivation of the mitochondria, stimulating aerobic metabolism. Hormonal therapy to brain-dead potential organ donors has been shown to lead to metabolic and haemodynamic stability, resulting in no wastage of organs, and in improved function after transplantation.

Coronary artery plaque rapidly induced by local electromagnetic stimulation in the baboon.
Hans Breuer, John Fincham, Paul Hinrichsen, C. Uys, H. Weich, Bruno Reichart.
European Surgical Research (1989) 21(2): 123-128

A reliable method has been developed to produce stenosis of the right coronary artery of baboons as a consequence of electrostimulation of the vessel at a chosen position. At that site a pair of electrodes were implanted and activated with a train of 9-volt pulses (length: 10 ms, separation: 100 ms) for 30 min, 5 days/week, up to 6 weeks. 13 animals were included in the experiment, 2 of those served as controls. Pathohistologically the structure of the artificially produced constrictions is similar to atherosclerotic lesions. On average the stenoses occupied 55% of the available lumen; total occlusion and no stenosis were observed in 1 case each.

Whole blood aggregometry and platelet adenine nucleotides during cardiac surgery.
Peter Zilla, Roland Fasol, Manfred Deutsch, G. Laufer, G. Wollenek, M. Muller, P. Knobl, T. Vukovich, A. Hammerle, Ulrich von Oppell.
Scandinavian Journal of Thoracic and Cardiovascular Surgery (1988) 22(2): 165-169

The influence of extracorporeal circulation (ECC) on human platelet adenine nucleotides has been studied in 28 coronary bypass patients before, during and after operation. An oscillating pattern of transient increases and decreases in total platelet ATP was observed following the sternotomy until the end of the operation. A highly significant increase in platelet ATP (20% +/- 14 of the pre-anaesthesia values) occurred during the first 24 h after surgery. Total platelet ADP however, did not show this oscillation nor was there any significant release of ADP from the platelets during ECC. Following collagen activation, increased amounts of 'releaseable ATP' were found after protaminization (124% +/- 38 of pre-anaesthesia values) (p less than 0.05), although whole blood aggregability was slightly reduced (89% +/- 18 of pre-anaesthesia values). This study indicates that 1) the metabolic ATP pool of circulating platelets underwent rapid changes during open heart surgery; 2) the majority of platelets did not release inert ADP during ECC; 3) there may be a compensatory enhancement of platelet function by other blood cells, which could explain the discrepancy between our aggregatory results in whole blood and those reported in platelet rich plasma (PRP) aggregometry.

Effects of hormonal therapy on subsequent organ (kidney) storage in the experimental animal.
Winston Wicomb, Dimitri Novitzky, David Cooper.
Transplantation Proceedings (1988) 20(5 Suppl 7): 55-58

Anomalous left superior vena cava in combined heart-lung transplantation.
Ulrich von Oppell, John Odell, Herman Reichenspurner, Bruno Reichart, Peter Zilla, Roland Fasol.
Journal of Heart Transplantation (1988) 7(6): 445-447

An anomalous left superior vena cava (SVC) was identified in two recipients during combined heart-lung transplantation. In the first patient an interposition Gore-Tex graft was used to reconstitute the venous drainage from the aberrant left SVC to the right atrium. In the second patient a new method of reconstituting the drainage from the left SVC with the donor innominate vein is described. It is recommended that excision of the donor heart and lung should include the innominate vein, as it may be used to create a venous channel for an aberrant left SVC if present in the recipient.

Triiodothyronine therapy for heart donor and recipient.
Dimitri Novitzky, David Cooper, Paul Human, Bruno Reichart, N. Zuhdi.
Journal of Heart Transplantation (1988) 7(5): 370-376

Both (1) brain-dead donors and (2) transplant recipients on cardiopulmonary bypass suffer a depletion in plasma-free triiodothyronine (T3), which leads to metabolic changes (from inhibition of mitochondrial function), resulting in myocardial energy store depletion. Replacement therapy with T3 reverses these changes in both donor and recipient. Donor heart energy stores and function will be maintained at optimum levels if T3 therapy is administered to both donor and recipient at the time of transplantation.

The effects of denervation and acute rejection on left ventricular volumes measured by radionuclide ventriculography following cardiac transplantation in the chacma baboon.
Dimitri Novitzky, David Cooper, Alan Rose, Sedic Isaacs, J. Boniaszczuk, J. Smith, Bruno Reichart, M. Iturralde.
Seminars in Nuclear Medicine (1988) 18(3): 213-220

Seven baboons underwent autotransplantation of the heart or heart and both lungs (group A). Eleven allografts were performed (group B) (nine orthotopic heart transplants and two en bloc transplants of the heart and both lungs). Radionuclide ventriculography was performed both pretransplant and at intervals posttransplant in all animals, and provided measurements of ejection fraction (EF) and left ventricular volumes (LVv) (end-diastolic volume [EDV], end-systolic volume [ESV], and stroke volume [SV]). In seven animals, a total of 20 endomyocardial biopsies were taken. Correlation was made between histopathological features of acute rejection seen on endomyocardial biopsy and changes in EF and LVv measured by radionuclide imaging. A significant increase of 12% in the EF (P less than 0.01) and significant falls in the LVv were observed in all animals (groups A and B) on the first posttransplant day, presumably a result of total cardiac denervation. EDV was reduced by 50% (P less than 0.005), ESV by 62% (P less than 0.0001), and SV by 43% (P less than 0.0001). In autografted baboons (group A) EF and LVv showed no further changes until reinnervation of the heart had occurred, when they reverted to pretransplant levels. In the allografted baboons (group B) further significant reductions in the LVv occurred as acute cardiac rejection progressed. From the first post-transplant day to the time of the final study before the animals' death, the EF decreased by 10% (P less than 0.01), the EDV by 38% (P less than 0.005), and SV by 73% (P less than 0.003): the decrease in ESV did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of triiodothyronine (T3) on myocardial high energy phosphates and lactate after ischemia and cardiopulmonary bypass. An experimental study in baboons [see comments].
Dimitri Novitzky, Paul Human, David Cooper.
Journal of Thoracic and Cardiovascular Surgery (1988) 96(4): 600-607

Cardiopulmonary bypass is associated with a reduction in plasma free triiodothyronine in patients undergoing cardiac operations. A previous experimental study in pigs demonstrated a marked inotropic effect when triiodothyronine was administered after a period of myocardial ischemia and cardiopulmonary bypass; this was associated with a significant reduction in mortality compared with the mortality in control pigs. To clarify the effect of triiodothyronine on myocardial high energy phosphate stores and lactate, a series of experiments was done in baboons undergoing 3 hours of myocardial ischemia while supported by cardiopulmonary bypass. Seven baboons received no triiodothyronine and six received 6 micrograms of triiodothyronine at the end of the ischemic period. Seventy minutes after cardiopulmonary bypass, the myocardial adenosine triphosphate level was significantly higher (p less than 0.01) in the treated animals. In untreated animals, a steady increase in myocardial lactate occurred after cardiopulmonary bypass; by 120 minutes after ischemia (70 minutes after cardiopulmonary bypass) there was a significant difference in lactate levels between the two groups (p less than 0.01). We postulate that a combination of global ischemia and depletion of triiodothyronine results in reduced mitochondrial function, inhibition of the tricarboxylic acid cycle, and increased anaerobic metabolism and depletion of myocardial phosphates. Triiodothyronine replacement therapy leads to improved mitochondrial function and increased aerobic metabolism, which results in increased synthesis of myocardial phosphates. We suggest that there may be a place for the administration of triiodothyronine in patients undergoing cardiac operations with a prolonged myocardial ischemic period or in whom there is any evidence of low cardiac output after discontinuation of cardiopulmonary bypass.

Endocrine changes and metabolic responses.
Dimitri Novitzky, David Cooper, Winston Wicomb.
Transplantation Proceedings (1988) 20(5 Suppl 7): 33-38

Inotropic effect of triiodothyronine following myocardial ischemia and cardiopulmonary bypass: an experimental study in pigs.
Dimitri Novitzky, Paul Human, David Cooper.
Annals of Thoracic Surgery (1988) 45(1): 50-55

A significant reduction (p less than 0.0001) in plasma-free triiodothyronine (T3), which is known to have an inotropic effect, has been documented in patients undergoing open-heart procedures. To investigate the effect of this observation, 22 pigs underwent 2 hours (Group 1, r = 10) or 3 hours (Group 2, r = 12) of myocardial ischemia during cardiopulmonary bypass (CPB) at 26 degrees C; the myocardium was protected by cardioplegic solution and cold saline solution at 30-minute intervals. After the pig was rewarmed to 37 degrees C, CPB was discontinued, and measurements of hemodynamic function were made 10 and 70 minutes later. Half of the pigs (Subgroup B) received 6 micrograms of T3 intravenously immediately after removal of the aortic cross-clamp; the remainder (Subgroup A) received no T3. After 2 hours of ischemia, untreated pigs showed significantly reduced myocardial function 10 minutes after discontinuation of CPB. By 70 minutes after the end of CPB, 2 of 5 untreated pnsidered together, overall survival of those that did not receive T3 was significantly less than those that did (p less than 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

Prediction of acute cardiac rejection by changes in left ventricular volumes.
D. Novitzky, D. Cooper, J. Boniaszczuk.
Journal of Heart Transplantation (1988) 7(6): 453-455

Sixteen patients underwent heart transplantation (11 orthotopic, five heterotopic). Monitoring for acute rejection was by both endomyocardial biopsy (EMB) and multigated equilibrium blood pool scanning with technetium 99m-labelled red blood cells. From the scans information was obtained on left ventricular volumes (stroke, end-diastolic, and end-systolic), ejection fraction, and heart rate. Studies (208) were made in the 16 patients. There was a highly significant correlation between the reduction in stroke volume and end-diastolic volume (and a less significant correlation in end-systolic volume) and increasing acute rejection seen on EMB. Heart rate and ejection fraction did not correlate with the development of acute rejection. Correlation of a combination of changes in stroke volume and end-diastolic volume with EMB showed a sensitivity of 85% and a specificity of 96%. Radionuclide scanning is therefore a useful noninvasive tool for monitoring acute rejection.

Results of hormonal therapy in human brain-dead potential organ donors.
Dimitri Novitzky, David Cooper.
Transplantation Proceedings (1988) 20(5 Suppl 7): 59-62

Closure of subarachnoid-pleural fistulae with fibrin sealant.
J. Morgan.
European Journal of Cardio-Thoracic Surgery (1988) 2(1): 56-57

Subarachnoid-pleural fistulae are rare and require closure if conservative therapy has failed. A simple and effective method is described using a pleural graft sealed with fibrin glue. The closure is immediate and long lasting.

Experimental evaluation of the mitroflow pericardial heart valve prosthesis. Part II. Pathologic examination.
J. Hassoulas, A. Rose.
Angiology (1988) 39(8): 733-741

The results of the morphologic and histopathologic examination of 37  bovine pericardial heart valve prostheses that were retrieved after  experimental implantation in Chacma baboons for periods of one to twelve  months are presented in this study. The implanted prostheses consisted of  33 Mitroflow valves, size 21 mm, that belonged to four different groups  according to the method of preparation of the pericardium (process I to  IV) and 4 commercially available Ionescu-Shiley valves, size 19 mm. All  implantations were in the mitral position, except 1 Mitroflow and 1  Ionescu-Shiley prosthesis, which were implanted in the tricuspid position.  Moderate or abundant noninfected thrombotic deposits were found on the  cusps of 13 of the 33 Mitroflow valves (39%). The Ionescu-Shiley valves  shared this tendency, perhaps to a lesser extent, for the formation of  thrombus on the valve cusps. Results of transmission electron microscopy,  scanning electron microscopy, and calcification analysis are also  presented. In this experimental study, the authors did not identify any  significant characteristics in the Mitroflow bovine pericardial heart  valve prosthesis that would lead to improved or extended durability of  this device over the commercially available Ionescu-Shiley bovine  pericardial heart valve prosthesis and probably over any other  glutaraldehyde-treated bioprosthesis. They would suggest, therefore, that  any clinical investigations of the prosthesis be undertaken very cautiously.

Experimental evaluation of the mitroflow pericardial heart valve prosthesis. Part I. In vivo hemodynamic evaluation.
J. Hassoulas, O. Ayzenberg.
Angiology (1988) 39(8): 725-732

Evaluation of possible enhanced durability of the Mitroflow pericardial  heart valve prosthesis was undertaken in an experimental animal model  using the Chacma baboon. For comparison purposes a small series of 4  Ionescu-Shiley pericardial valves were also implanted. The 33 Mitroflow  prostheses implanted were all manufacturer's size 21mm and belonged to 4  different groups according to the process used for the preparation of the  pericardium (Process I to IV). The Ionescu-Shiley prostheses were all  manufacturer's size 19mm and were commercially available valves. The  valves were implanted in the mitral position, except one each Mitroflow  and Ionescu-Shiley prosthesis that was implanted in the tricuspid  position. Four animals died in the early postoperative period, between 2  and 14 days. Two died from a probable viral infection, one from a low  cardiac output state and the fourth one from bacterial endocarditis. The  clinical evaluation of the animals for an implantation time of up to  twelve months was unremarkable. Hemodynamic studies were performed  immediately after implantation (n = 29), at an intermediate cardiac  catheterization at 9 months after implantation (n = 4) and at the time of  terminal elective sacrifice of the animals (n = 33). From the data  obtained the resultant prosthetic valve area at the time of elective  sacrifice of the animals was also calculated. The data obtained for each  of the 5 groups of valves tested are presented in the text. Transvalvular  gradients measured at the time of sacrifice of the animals were  elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac allotransplantation across major blood group barriers in the baboon.
D. Cooper, G. Lexer, A. Rose, M. Keraan, J. Rees, T. E. Du Toit, R. Oriol.
Journal of Medical Primatology (1988) 17(6): 333-346

In heterotopic heart transplantation experiments in Chacma baboons, some of the animals were significantly immunosuppressed with cyclosporine, resulting in prolonged cardiac allograft survival. ABO blood group incompatibility between recipient and donor did not significantly influence mean allograft survival, but early hyperacute (vascular) or acute (cellular) rejection occurred only when ABO incompatibility was present.

Hemodynamic and electrocardiographic responses.
D. Cooper, D. Novitzky, W. Wicomb.
Transplantation Proceedings (1988) 20(5 Suppl 7): 25-28

Initial experimental experience with a "replaceable" cardiac valve prosthesis.
D. Cooper, W. Wicomb, G. Gould, D. Boonzaier.
Annals of Thoracic Surgery (1988) 45(5): 554-558

An easily "replaceable" cardiac valve prosthesis has been designed. It consists of two parts: (1) a sewing ring incorporating a circlip and (2) a functioning valve (either mechanical or tissue). The circlip is encased in a sewing ring, which is sutured into the natural valve annulus, and grips the functional part of the prosthesis, thereby preventing dislodgment. A simple instrument has been designed to open the circlip a few millimeters to allow easy removal or insertion of the functional element. This sewing ring/circlip with the functional element of a Bjork-Shiley prosthesis was used in 10 baboons undergoing mitral valve replacement. Removal and replacement of the functional element was carried out at a second operation between 1 and 12 weeks later. There were no operative deaths. Baboons were electively killed one day to twelve months after the second operation. There were no complications related to the prosthesis; cardiac catheterization showed normal hemodynamics before and after the second operative procedure.

Hormonal therapy in the brain-dead experimental animal.
D. Cooper, D. Novitzky, W. Wicomb.
Transplantation Proceedings (1988) 20(5 Suppl 7): 51-54

Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon.
D. Cooper, Paul Human, G. Lexer, A. Rose, J. Rees, M. Keraan, T. E. Du Toit.
Journal of Heart Transplantation (1988) 7(3): 238-246

The problem of donor heart supply would be solved if hearts could be transplanted from readily available animals such as the pig or sheep. We have investigated heterotopic heart transplantation (in the neck) with the pig as donor and baboon as recipient. Five experimental groups were studied. Control hearts (group 1, n = 4) were rejected within 4 minutes to 8 hours. Splenectomy done before transplantation (group 2, n = 3) did not extend survival significantly (30 minutes to 8 hours). Donor heart survival in baboons receiving immunosuppressive therapy of cyclosporine and methylprednisolone (group 3, n = 5) was from 15 to 75 minutes only in four animals and for 5 days in one animal. Anti-pig antibody adsorption from baboon blood by pretransplant donor-specific kidney hemoperfusion (group 4, n = 7) resulted in cardiac function for 6 to 12 hours in three cases and from 4 to 5 days in four cases (p less than 0.02). A combination of pretransplant antibody adsorption and immunosuppression (group 5, n = 4) resulted in graft survival of 8 to 20 hours in three cases and of 4 days in one case (p less than 0.03). Histopathologic features of vascular (hyperacute) rejection were seen in all hearts except one (the 5-day survivor in group 3). Pretransplant adsorption of antibody clearly prolonged survival of discordant cardiac xenografts in some cases. Further exploration of this technique appears justified.

Experimental evaluation of the mitroflow pericardial heart valve  prosthesis. Part II. Pathologic examination.
J. Hassoulas, A.G. Rose.
Angiology (1988) 39(8): 733-741

The results of the morphologic and histopathologic examination of 37  bovine pericardial heart valve prostheses that were retrieved after  experimental implantation in Chacma baboons for periods of one to twelve  months are presented in this study. The implanted prostheses consisted of  33 Mitroflow valves, size 21 mm, that belonged to four different groups  according to the method of preparation of the pericardium (process I to  IV) and 4 commercially available Ionescu-Shiley valves, size 19 mm. All  implantations were in the mitral position, except 1 Mitroflow and 1  Ionescu-Shiley prosthesis, which were implanted in the tricuspid position.  Moderate or abundant noninfected thrombotic deposits were found on the  cusps of 13 of the 33 Mitroflow valves (39%). The Ionescu-Shiley valves  shared this tendency, perhaps to a lesser extent, for the formation of  thrombus on the valve cusps. Results of transmission electron microscopy,  scanning electron microscopy, and calcification analysis are also  presented. In this experimental study, the authors did not identify any  significant characteristics in the Mitroflow bovine pericardial heart  valve prosthesis that would lead to improved or extended durability of  this device over the commercially available Ionescu-Shiley bovine  pericardial heart valve prosthesis and probably over any other  glutaraldehyde-treated bioprosthesis. They would suggest, therefore, that  any clinical investigations of the prosthesis be undertaken very cautiously.

Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: a preliminary report.
P. Zilla, R. Fasol, M. Deutsch, T. Fischlein, E. Minar, A. Hammerle, O. Krupicka, M. Kadletz.
Journal of Vascular Surgery (1987) 6(6): 535-541

The importance of initial human trials with autologous endothelial seeding lies not only in the implementation of a promising idea but also in the fact that canine data are only partially applicable to humans. The surface area of jugular veins in humans is much smaller than in dogs and considerably longer grafts are needed. Moreover, the reproductive capacity of adult human endothelial cells under in vivo conditions, which probably determines the success of seeding more than the seeding density, is also uncertain. Therefore the efficiency of autologous endothelial seeding in humans was investigated in 18 patients undergoing distal femoropopliteal bypass surgery. The average surface area of the jugular veins was 4.9 +/- 1.7 cm2 with an average cell yield of 32.6 +/- 18.0 x 10(4). The mean number of seeded cells per square centimeter of graft surface was 3.1 x 10(3). In a follow-up extending for 14 weeks, plasma levels of platelet factor 4 and beta-thromboglobulin as well as the platelet function in the whole blood aggregometer showed significantly better results in the seeded group. Plasma thromboxane B2, uptake and survival of indium 111-labeled platelets, and Doppler ultrasound investigations also favored the seeded group, but the results were statistically insignificant. No difference at all was found for the platelet dense granule compounds, releasable adenosine triphosphate and platelet serotonin. Thus our findings did not indicate the development of a closed endothelialized surface after 14 weeks, which is a period three times as long as the one required for confluent endothelial cell coverage in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

PGI2 and PGE1 induce morphological alterations in human platelets similar to those of the initial phase of activation.
P. Zilla, P. Groscurth, G. Varga, T. Fischlein, R. Fasol.
Experimental Hematology (1987) 15(7): 741-749

The effects of PGI2 and PGE1 on the ultrastructure of human platelets were studied by scanning (SEM) and transmission (TEM) electron microscopy in relation to the record of an optical aggregometer. Addition of PGI2 or PGE1 to citrated platelet-rich plasma (C-PRP) resulted in a permanent slight decrease in percent light transmission (%T) recorded by the aggregometer. SEM investigation of the platelets showed marginal pseudopods and occasional large stomata after application of prostaglandins. These alterations occurred within the initial 30 s and remained constant during the subsequent 20 min of incubation. TEM studies revealed morphological changes of alpha granules and moderately electron-dense material in the dilated profiles of the surface-connected canalicular system (SCCS). Addition of 10 microM ADP to C-PRP preincubated for 30 s with either 2 ng/ml (5 nmol/liter) PGI2 or 30 ng/ml (85 nmol/liter) PGE1 resulted in a further decrease of %T followed by a slight increase. The alterations of the aggregometer tracing were characterized in SEM by platelet shape change and the generation of primary aggregates. C-PRP samples preincubated with 3 and 9 ng/ml (8 nmol/liter and 24 nmol/liter) PGI2 or 40 and 120 ng/ml (113 nmol/liter and 338 nmol/liter) PGE1 did not produce additional changes in the aggregometer curves or in the ultrastructure of platelets in response to ADP. Our morphological study indicates that antiaggregatory prostaglandins induce an early phase of platelet activation but inhibit "shape change" and the formation of aggregates.

Virological implications of the use of primates in xenotransplantation.
F. Van der Riet, Paul Human, D. Cooper, B. Reichart, J. Fincham, S. Kalter, P. Kanki, M. Essex, D. Madden, M. Lai-Tung.
Transplantation Proceedings (1987) 19(5): 4068-4069

Twenty years of heart transplantation at Groote Schuur Hospital.
H. Reichenspurner, J. Odell, D. Cooper, D. Novitzky, Paul Human, U. von Oppell, E. Becerra, D. Boehm, A. Rose, R. Fasol.
Journal of Heart Transplantation (1987) 6(6): 317-323

Between December 1967 and July 1987, 110 heart transplantations (61 heterotopic and 49 orthotopic) and 12 heart-lung transplantations were done at Groote Schuur Hospital in Cape Town, South Africa. Twelve procedures were retransplantations, including two third interventions. The patients were divided into three groups: Group A (n = 55) from 1967 to 1982 received so-called conventional treatment of azathioprine, methylprednisolone, and antithymocyte globulin. Group B (n = 15) from 1983 to 1984 had cyclosporine in high dosages together with methylprednisolone. Group C (n = 30) received quadruple drug therapy of low-dosage cyclosporine, together with azathioprine, methylprednisolone in lower dosages, and antithymocyte globulin (for the first 4 to 6 days and rescue antithymocyte globulin for severe rejection). From Group A, nine of 55 patients are alive up to 17 years after transplantation. The main causes of death were acute rejections and infections (in 60% altogether). From group B, six of 15 patients are alive. Acute rejections and infections were the causes of death in 12% of the patients, but multiple organ failure was a major cause in 24% most probably because of the high dosages of cyclosporine. From group C, 23 of 30 patients have survived. In this group the results after heterotopic heart transplantation do not differ significantly from orthotopic transplantation, which justifies this procedure in particular situations. If all heterotopic and orthotopic transplantations are compared, orthotopic procedures have a substantially better outcome. With the modified immunosuppressive regimen (group C) combined with precise donor and recipient selection and more sophisticated rejection monitoring, the actuarial survival rate within the last 12 months is 94%.

Heart transplantation at Groote Schuur Hospital, Cape Town. Twenty years' experience.
B. Reichart, H. Reichenspurner, J. Odell, D. Cooper, D. Novitzky, Paul Human, U. von Oppell, E. Becerra, D. Boehm, A. Rose.
South African Medical Journal (1987) 72(11): 737-739

Human allogeneic heart transplantation was started at Groote Schuur Hospital in Cape Town in 1967. Since then 110 hearts (61 heterotopic and 49 orthotopic) and 12 heart-lung transplantations have been performed in the unit. Ten procedures were retransplantations including 2 third interventions. The patients fall into three groups according to their immunosuppressive therapy: group A (N = 55) from 1967 to 1982 received the so-called 'conventional treatment' (azathioprine, methylprednisolone and antithymocyte globulin (ATG)); group B (N = 15) from 1983 to 1984 received cyclosporin A in high dosage, together with methylprednisolone; and group C (N = 30) received quadruple drug therapy of low-dose cyclosporin A, together with azathioprine, methylprednisolone in lower dosages and antithymocyte globulin (for the first 4-6 days and rescue-ATG for severe rejection). The results have improved significantly over the years. The actuarial survival rate after heart transplantation within the last 12 months is 94%. Several important steps have been inaugurated: in 1973 heterotopic heart transplantation was initiated and in 1984 hormonal therapy of brain-dead organ donors was started. Radionuclide scanning, in combination with endomyocardial biopsies, has proved to be a very sensitive means of monitoring rejection.

Is pulmonary ischemia a factor in the reperfusion response? An experimental study in the chacma baboon.
B. Reichart, Paul Human, A. Rose, D. Novitzky, D. Cooper.
Journal of Heart Transplantation (1987) 6(4): 238-243

A reimplantation or reperfusion response has been described in both the experimental animal and the human patient after various procedures involving pulmonary ischemia. We have investigated this phenomenon in a primate model. Ten chacma baboons were placed on cardiopulmonary bypass and cooled to 20 degrees C. Circumferential segments of the right main bronchus and pulmonary artery were denuded of all surrounding tissue. Each structure was then cross-clamped, which rendered the lung ischemic, during which time the organ was immersed in cold saline solution. Ischemia was maintained for 1.5 to 5 hours; after reperfusion and discontinuation of bypass, the right lung was biopsied and the chest closed. Chest radiographs, lung biopsies, and arterial blood gases were taken at intervals for up to 16 to 28 days. Right lung shadowing on chest radiography with concomitant histopathologic changes, indicative of a reperfusion reaction, were seen in only one animal, which had undergone lung ischemia for 1.5 hours. In one other animal that was ischemic for 5 hours, patchy opacification of the lung was seen on two occasions (days 8 and 15) with concomitant mild histopathologic changes. In conclusion, therefore, a major reperfusion response after pulmonary ischemia in the chacma baboon is possible but unusual. This would suggest that the appearance of pulmonary opacification on chest radiography within the first 4 weeks after heart-lung transplantation in humans is most likely attributable to some other condition, such as isolated lung rejection or infection.

The value of hormonal therapy in improving organ viability in the transplant donor.
D. Novitzky, D. Cooper, B. Reichart.
Transplantation Proceedings (1987) 19(1 Pt 3): 2037-2038

Cardiac transplantation using discordant xenografts in a nonhuman primate model.
G. Lexer, D. Cooper, W. Wicomb, A. Rose, J. Rees, M. Keraan, B. Reichart, E. Du Toit.
Transplantation Proceedings (1987) 19(1 Pt 2): 1153-1154

The protein C system in patients undergoing cardiopulmonary bypass.
P. Knobl, P. Zilla, R. Fasol, M. Muller, T. Vukovich.
Journal of Thoracic and Cardiovascular Surgery (1987) 94(4): 600-605

Fifteen men undergoing extracorporeal circulation for aorta-coronary bypass grafting were investigated for alterations of the plasma levels of cross-linked fibrin degradation products, protein C, free protein S, coagulation factor II, immunoglobulin G, and albumin. Although all patients were given heparin, a progressive increase of cross-linked fibrin degradation products was recorded during extracorporeal circulation, which indicates an activation of the plasmatic coagulation system. This increase was most pronounced in the late phase of extracorporeal circulation after reperfusion of the lung and in the early postoperative period. The levels of all other investigated plasma proteins decreased drastically after the patient was connected to the bypass circuit, which was primed with saline solution. These levels increased after termination of extracorporeal circulation and administration of fresh-frozen plasma. To study the consumption of protein C, protein S, and factor II during extracorporeal circulation, we formed ratios of the values of these parameters to the value of immunoglobulin G. After this volume correction, protein C was found to decrease significantly in the late phase of extracorporeal circulation, remaining low in the early postoperative period; protein S increased significantly soon after the onset of extracorporeal circulation and decreased after termination of extracorporeal circulation; factor II was unaffected by extracorporeal circulation, showing only a slight, insignificant increase in the postoperative phase. These results suggest a disturbance of the protein C system by extracorporeal circulation, which is possibly linked to the reported high bleeding tendency in patients undergoing operations with extracorporeal circulation.

In vitro lining of fibronectin coated PTFE grafts with cryopreserved saphenous vein endothelial cells.
M. Kadletz, R. Moser, P. Preiss, M. Deutsch, P. Zilla, R. Fasol.
Journal of Thoracic and Cardiovascular Surgery (1987) 35 Spec No 2: 143-147

In an attempt to produce instant endothelial cell (EC) monolayers on graft surfaces, cryopreserved cultivated human saphenous vein endothelial cells (HSVEC) were cultivated on reinforced PTFE prostheses. The graft surface was precoated with 40 micrograms/ml human fibronectin (HFN) prior to seeding with 200 x 10(3) EC/cm2. The seeding procedure was performed in a specially designed rotation device. After a cultivation period of 9 days, the seeded endothelial cells on the PTFE prostheses were exposed to shear stresses in a perfusion circuit, containing a bubble oxygenator, a roller pump and a tygon perfusion loop. The applied shear forces were 3 and 6 dyn/cm2, respectively. In control grafts, spontaneous cell detachment occurred from day 11 onwards and only 50% of the graft surface remained endothelialized on day 16. When the grafts were exposed to 3 dyn/cm2 only small cell-free areas less than 1000 micron in diameter were found after 4 hours of perfusion. In contrast, exposure to 6 dyn/cm2 produced discouraging results: as early as 4 hours after the onset of perfusion 50% of the graft surface was cell free. After 16 hours only 20% endothelial cell coverage was seen under the stereo microscope. However, assuming that an ideal precoating substratum can be found, the two stage technique of in vitro endothelialization of vascular grafts with autologous endothelial cells may offer a promising clinical method. Moreover, the fact that our grafts were lined with cryopreserved EC implies the possible prospect of cell banks supplying unlimited numbers of EC for subsequent bypass operations.

Is ABO compatibility essential in xenografting between closely related species?
D. Cooper, Paul Human, A. Rose.
Transplantation Proceedings (1987) 19(6): 4437-4440

Cardiac allograft survival in ABO blood group incompatible baboons.
D. Cooper, G. Lexer, A. Rose, J. Rees, M. Keraan, T. E. Du Toit, R. Oriol.
Transplantation Proceedings (1987) 19(1 Pt 2): 1036-1038

Prolongation of cardiac xenograft (vervet monkey to baboon) function by a combination of total lymphoid irradiation and immunosuppressive drug therapy.
D. Cooper, Paul Human, B. Reichart.
Transplantation Proceedings (1987) 19(6): 4441-4442

Are there indications for heterotopic heart transplantation today?
E. Becerra, D. Cooper, D. Novitzky, B. Reichart.
Transplantation Proceedings (1987) 19(1 Pt 3): 2512-2513

Experimental in vitro cultivation of human endothelial cells on artificial surfaces.
R. Fasol, P. Zilla, P. Groscurth, E. Wolner, R. Moser.
Trans Am Soc Artif Intern Organs (1985) 31: 276-283

Surface morphology of human platelets during in vitro aggregation.
P. Zilla, P. Groscurth, K. Rhyner, A. von Felten.
Scandinavian Journal of Haematology (1984) 33(5): 440-447

The alterations of surface morphology of human platelets during the course of the aggregometer tracing were studied by scanning electron microscopy (SEM). Prior to activation the platelet rich plasma was pre-incubated at 37 degrees C for at least 30 min in order to obtain a sufficient number of discoid platelets. Immediately following the addition of collagen or ADP platelets showed slender pseudopods which thereafter were replaced by bulbous protrusions. The decrease in percent light transmission (%T) in the aggregometer tracing was characterized in SEM by a significant enlargement of bulbous protrusions and by the transformation of platelet shape from disc to sphere. During the increase in %T platelets forming primary aggregates displayed spiny pseudopods at their surface indicating that, during in vitro aggregation induced by collagen or ADP, two generations of pseudopods are formed. Using a low dose of ADP, the return of aggregometer tracing to the base line was regularly accompanied by dissociation of primary aggregates, but platelets remained spheroid and displayed pseudopods for a long time. Our study indicates that the course of aggregometer tracing is closely associated to the surface morphology of platelets. Single morphological changes, however, are not reflected by the aggregometer method.