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TAVR for Patients With Rheumatic Heart Disease: Opening the Door for the Many?

P Zilla, DF Williams and D Bezuidenhout
J Am Coll Cardiol (2021) 77(14): 1714-1716


Residual Bioprosthetic Valve Immunogenicity: Forgotten, not Lost

P Human, D Bezuidenhout, E Aikawa and P Zilla
Front Cardiovasc Med (2021)


Long-Term Stability and Biocompatibility of Pericardial Bioprosthetic Heart Valves

DF Williams, D Bezuidenhout, J de Villiers, P Human and P Zilla
Front Cardiovasc Med (2021) 8:728577

The use of bioprostheses for heart valve therapy has gradually evolved over several decades and both surgical and transcatheter devices are now highly successful. The rapid expansion of the transcatheter concept has clearly placed a significant onus on the need for improved production methods, particularly the pre-treatment of bovine pericardium. Two of the difficulties associated with the biocompatibility of bioprosthetic valves are the possibilities of immune responses and calcification, which have led to either catastrophic failure or slow dystrophic changes. These have been addressed by evolutionary trends in cross-linking and decellularization techniques and, over the last two decades, the improvements have resulted in somewhat greater durability. However, as the need to consider the use of bioprosthetic valves in younger patients has become an important clinical and sociological issue, the requirement for even greater longevity and safety is now paramount. This is especially true with respect to potential therapies for young people who are afflicted by rheumatic heart disease, mostly in low- to middle-income countries, for whom no clinically acceptable and cost-effective treatments currently exist. To extend longevity to this new level, it has been necessary to evaluate the mechanisms of pericardium biocompatibility, with special emphasis on the interplay between cross-linking, decellularization and anti-immunogenicity processes. These mechanisms are reviewed in this paper. On the basis of a better understanding of these mechanisms, a few alternative treatment protocols have been developed in the last few years. The most promising protocol here is based on a carefully designed combination of phases of tissue-protective decellularization with a finely-titrated cross-linking sequence. Such refined protocols offer considerable potential in the progress toward superior longevity of pericardial heart valves and introduce a scientific dimension beyond the largely disappointing 'anti-calcification' treatments of past decades.

Neurologic recovery after ten minutes of absent cerebral blood flow at normothermia

T Pennel, F Beyersdorf, E Gates and P Zilla
Perfusion (2021) 36(4): 432-434

Prolonged normothermic cardiac arrest is associated with a high incidence of neurological morbidity and mortality. Whole body temperature-controlled perfusion has been applied to limit reperfusion injury and minimize ischemia. We describe the full recovery of a patient after the application of rapid hypothermia following an intraoperative aortic rupture with ten minutes of absent cerebral blood flow.

A Novel Hybrid Membrane VAD as First Step Toward Hemocompatible Blood Propulsion

A Ferrari, C Giampietro, B Bachmann, L Bernardi, D Bezuidenhhout, P Ermanni, R Hopf, S Kitz, G Kress, C Loosli, V Marina, M Meboldt, G Pellegrini, D Poulikakos, M Rebholz, M Schmid Daners, T Schmidt, C Starck, G Stefopoulos, S Sundermann, B Thamsen, P Zilla, E Potapov, V Falk and E Mazza
Ann Biomed Eng (2021) 49(2): 716-731

Heart failure is a raising cause of mortality. Heart transplantation and ventricular assist device (VAD) support represent the only available lifelines for end stage disease. In the context of donor organ shortage, the future role of VAD as destination therapy is emerging. Yet, major drawbacks are connected to the long-term implantation of current devices. Poor VAD hemocompatibility exposes the patient to life-threatening events, including haemorrhagic syndromes and thrombosis. Here, we introduce a new concept of artificial support, the Hybrid Membrane VAD, as a first-of-its-kind pump prototype enabling physiological blood propulsion through the cyclic actuation of a hyperelastic membrane, enabling the protection from the thrombogenic interaction between blood and the implant materials. The centre of the luminal membrane surface displays a rationally-developed surface topography interfering with flow to support a living endothelium. The precast cell layer survives to a range of dynamically changing pump actuating conditions i.e., actuation frequency from 1 to 4 Hz, stroke volume from 12 to 30 mL, and support duration up to 313 min, which are tested both in vitro and in vivo, ensuring the full retention of tissue integrity and connectivity under challenging conditions. In summary, the presented results constitute a proof of principle for the Hybrid Membrane VAD concept and represent the basis for its future development towards clinical validation.

United in earnest: first pilot sites for increased surgical capacity for rheumatic heart disease announced by cardiac surgery intersociety alliance

ZO Enumah, RM Bolman, P Zilla, P Boateng, B Wilson, AS Kumar, T Chotivatanapong, F Beyersdorf, J Pomar, K Sliwa, JL Eisele, J Dearani and R Higgins
Eur J Cardiothorac Surg (2021) 59(6): 1139-1143

OBJECTIVES: Rheumatic heart disease (RHD) affects >33 000 000 individuals, mostly from low- and middle-income countries. The Cape Town Declaration on Access to Cardiac Surgery in the Developing World was published in August 2018, signalling the commitment of the global cardiac surgery and cardiology communities to improving care for patients with RHD. METHODS: As the Cape Town Declaration formed the basis for which the Cardiac Surgery Intersociety Alliance (CSIA) was formed, the purpose of this article is to describe the history of the CSIA, its formation, ongoing activities and future directions, including the announcement of selected pilot sites. RESULTS: The CSIA is an international alliance consisting of representatives from major cardiothoracic surgical societies and the World Heart Federation. Activities have included meetings at annual conferences, exhibit hall participation for advertisement and recruitment and publication of selection criteria for cardiac surgery centres to apply for CSIA support. Criteria focused on local operating capacity, local championing, governmental and facility support, appropriate identification of a specific gap in care and desire to engage in future research. Eleven applications were received for which 3 finalist sites were selected and site visits conducted. The 2 selected sites were Hospital Central Maputo (Mozambique) and King Faisal Hospital Kigali (Rwanda). CONCLUSIONS: Substantial progress has been made since the passing of the Cape Town Declaration and the formation of the CSIA, but ongoing efforts with collaboration of all committed parties-cardiac surgery, cardiology, industry and government-will be necessary to improve access to life-saving cardiac surgery for patients with RHD.

United in Earnest: First Pilot Sites for Increased Surgical Capacity for Rheumatic Heart Disease Announced by Cardiac Surgery Intersociety Alliance

ZO Enumah, RM Bolman, P Zilla, P Boateng, B Wilson, AS Kumar, T Chotivatanapong, F Beyersdorf, J Pomar, K Sliwa, JL Eisele, J Dearani and R Higgins
Ann Thorac Surg (2021) 111(6): 1931-1936

BACKGROUND: Rheumatic heart disease (RHD) affects more than 33,000,000 individuals, mostly from low- and middle-income countries. The Cape Town Declaration On Access to Cardiac Surgery in the Developing World was published in August 2018, signaling the commitment of the global cardiac surgery and cardiology communities to improving care for RHD patients. METHODS: As the Cape Town Declaration formed the basis for which the Cardiac Surgery Intersociety Alliance (CSIA) was formed, the purpose of this article is to describe the history of the CSIA, its formation, ongoing activities, and future directions, including the announcement of selected pilot sites. RESULTS: The CSIA is an international alliance consisting of representatives from major cardiothoracic surgical societies and the World Heart Federation. Activities have included meetings at annual conferences, exhibit hall participation for advertisement and recruitment, and publication of selection criteria for cardiac surgery centers to apply for CSIA support. Criteria focused on local operating capacity, local championing, governmental and facility support, appropriate identification of a specific gap in care, and desire to engage in future research. Eleven applications were received for which three finalist sites were selected and site visits conducted. The two selected sites were Hospital Central Maputo (Mozambique) and King Faisal Hospital Kigali (Rwanda). CONCLUSIONS: Substantial progress has been made since the passing of the Cape Town Declaration and the formation of the CSIA, but ongoing efforts with collaboration of all committed parties-cardiac surgery, cardiology, industry, and government-will be necessary to improve access to life-saving cardiac surgery for RHD patients.

Blood derived extracellular vesicles as regenerative medicine therapeutics

C de Boer and NH Davies
Biochimie (2021) 10.1016/j.biochi.2021.10.009

The regenerative promise of nanosized extracellular vesicles (EVs) secreted by cells is widely explored. Recently, the capacity of EVs purified from blood to elicit regenerative effect has begun to be evaluated. Blood might be a readily available source of EVs, avoiding need for extensive cell culturing, but there are specific issues that complicate use of the biofluid in this area. We assess the evidence for blood containing regenerative material, progress made towards delivering blood derived EVs as regenerative therapeutics, difficulties that relate to the complexity of blood and the promise of hydrogel-based delivery of EVs.

In silico stress fibre content affects peak strain in cytoplasm and nucleus but not in the membrane for uniaxial substrate stretch

T Abdalrahman, NH Davies and T Franz
Med Biol Eng Comput (2021) 59(9): 1933-1944

Existing in silico models for single cell mechanics feature limited representations of cytoskeletal structures that contribute substantially to the mechanics of a cell. We propose a micromechanical hierarchical approach to capture the mechanical contribution of actin stress fibres. For a cell-specific fibroblast geometry with membrane, cytoplasm and nucleus, the Mori-Tanaka homogenization method was employed to describe cytoplasmic inhomogeneities and constitutive contribution of actin stress fibres. The homogenization was implemented in a finite element model of the fibroblast attached to a substrate through focal adhesions. Strain in cell membrane, cytoplasm and nucleus due to uniaxial substrate stretch was assessed for different stress fibre volume fractions and different elastic modulus of the substrate. A considerable decrease of the peak strain with increasing stress fibre content was observed in cytoplasm and nucleus but not the membrane, whereas the peak strain in cytoplasm, nucleus and membrane increased for increasing elastic modulus of the substrate. Finite element mesh of reconstructed human fibroblast and intracellular strain distribution in cell subjected to substrate stretch.

Progressive Reinvention or Destination Lost? Half a Century of Cardiovascular Tissue Engineering

P Zilla, M Deutsch, D Bezuidenhout, NH Davies and T Pennel
Front Cardiovasc Med (2020) 7(159

The concept of tissue engineering evolved long before the phrase was forged, driven by the thromboembolic complications associated with the early total artificial heart programs of the 1960s. Yet more than half a century of dedicated research has not fulfilled the promise of successful broad clinical implementation. A historical account outlines reasons for this scientific impasse. For one, there was a disconnect between distinct eras each characterized by different clinical needs and different advocates. Initiated by the pioneers of cardiac surgery attempting to create neointimas on total artificial hearts, tissue engineering became fashionable when vascular surgeons pursued the endothelialisation of vascular grafts in the late 1970s. A decade later, it were cardiac surgeons again who strived to improve the longevity of tissue heart valves, and lastly, cardiologists entered the fray pursuing myocardial regeneration. Each of these disciplines and eras started with immense enthusiasm but were only remotely aware of the preceding efforts. Over the decades, the growing complexity of cellular and molecular biology as well as polymer sciences have led to surgeons gradually being replaced by scientists as the champions of tissue engineering. Together with a widening chasm between clinical purpose, human pathobiology and laboratory-based solutions, clinical implementation increasingly faded away as the singular endpoint of all strategies. Moreover, a loss of insight into the healing of cardiovascular prostheses in humans resulted in the acceptance of misleading animal models compromising the translation from laboratory to clinical reality. This was most evident in vascular graft healing, where the two main impediments to the in-situ generation of functional tissue in humans remained unheeded-the trans-anastomotic outgrowth stoppage of endothelium and the build-up of an impenetrable surface thrombus. To overcome this dead-lock, research focus needs to shift from a biologically possible tissue regeneration response to one that is feasible at the intended site and in the intended host environment of patients. Equipped with an impressive toolbox of modern biomaterials and deep insight into cues for facilitated healing, reconnecting to the "user needs" of patients would bring one of the most exciting concepts of cardiovascular medicine closer to clinical reality.

A glimpse of hope: cardiac surgery in low- and middle-income countries (LMICs)

P Zilla, RM Bolman, 3rd, P Boateng and K Sliwa
Cardiovasc Diagn Ther (2020) 10(2): 336-349

Currently, more than five times more people live in low- and middle-income countries (LMICs) than in high-income countries (HICs). As such, the downward trend in cardiac surgical needs in HICs reflects only the situation of one sixth of the world population while the vast majority living in LMICs has still no or limited access to life saving heart operations. In these countries, rheumatic heart disease (RHD) still accounts for a significant proportion of cardiac surgical needs. In low- and lower-middle income countries it remains the single most common cardiovascular disease in young adult and adolescent patients in need of heart surgery outweighing other indications such as congenital cardiac defects almost 4-fold. Compared to HICs with their predominance of calcific aortic stenosis in the elderly mitral valve surgery is required in >90% of the largely young patients with RHD in low-income countries (LICs) and still in 70% of the often middle aged patients in middle-income countries (MICs). Although recent government initiatives in LICs led to the establishment of local, independent cardiac surgical services gradually replacing fly-in missions, these centers still only cover less than 2% of the needs of their populations. In MICs, cardiac surgical needs continually grow with the emergence of degenerative diseases. As such, in spite of the concomitant growth of cardiac surgical capacity, significantly less than half the estimated patients in need have access. Capacities in LICs range from 0.5 to 7 cardiac operations/million population; 100-481/million in MICs and >1,200/million in HICs such as the USA and Germany. While a new level of awareness of the scope and magnitude of the problem has begun to emerge in LICs and the establishment of local cardiac surgical capacity has given rise to a glimpse of hope, the challenges of expanding these fledgling services to a significant proportion of the population still seem insurmountable. Challenges in MICs are on the other hand the widening gap between private cardiac medicine for the affluent few and overwhelmed public services for the many and the rural urban divide with the underappreciation of the ongoing dominance of RHD in the rural and indigent population on the other. Overshadowing all LMICs is the low level of valve-repair skills associated with insufficient cardiac surgical capacity and the unavailability of suitable replacement valves which address the young age of the patients and the difficulties of anticoagulation in a socioeconomic environment distinctly different from the elderly patients of HICs.

A Patient-Specific CFD Pipeline Using Doppler Echocardiography for Application in Coarctation of the Aorta in a Limited Resource Clinical Context

L Swanson, B Owen, A Keshmiri, A Deyranlou, T Aldersley, J Lawrenson, P Human, R De Decker, B Fourie, G Comitis, ME Engel, B Keavney, L Zuhlke, M Ngoepe and A Revell
Front Bioeng Biotechnol (2020) 8(409

Congenital heart disease (CHD) is the most common birth defect globally and coarctation of the aorta (CoA) is one of the commoner CHD conditions, affecting around 1/1800 live births. CoA is considered a CHD of critical severity. Unfortunately, the prognosis for a child born in a low and lower-middle income country (LLMICs) with CoA is far worse than in a high-income country. Reduced diagnostic and interventional capacities of specialists in these regions lead to delayed diagnosis and treatment, which in turn lead to more cases presenting at an advanced stage. Computational fluid dynamics (CFD) is an important tool in this context since it can provide additional diagnostic data in the form of hemodynamic parameters. It also provides an in silico framework, both to test potential procedures and to assess the risk of further complications arising post-repair. Although this concept is already in practice in high income countries, the clinical infrastructure in LLMICs can be sparse, and access to advanced imaging modalities such as phase contrast magnetic resonance imaging (PC-MRI) is limited, if not impossible. In this study, a pipeline was developed in conjunction with clinicians at the Red Cross War Memorial Children's Hospital, Cape Town and was applied to perform a patient-specific CFD study of CoA. The pipeline uses data acquired from CT angiography and Doppler transthoracic echocardiography (both much more clinically available than MRI in LLMICs), while segmentation is conducted via SimVascular and simulation is realized using OpenFOAM. The reduction in cost through use of open-source software and the use of broadly available imaging modalities makes the methodology clinically feasible and repeatable within resource-constrained environments. The project identifies the key role of Doppler echocardiography, despite its disadvantages, as an intrinsic component of the pipeline if it is to be used routinely in LLMICs.

Poorly suited heart valve prostheses heighten the plight of patients with rheumatic heart disease

J Scherman and P Zilla
Int J Cardiol (2020) 318(104-114

Rheumatic heart disease (RHD) still affects more patients globally than degenerative valve disease. The vast majority of these patients live in low- to middle-income countries. Once symptomatic, they will need heart valve surgery. Unfortunately, prosthetic valves perform poorly in these patients given their young age, the high incidence of multi-valve disease, late diagnoses and often challenging socio-economic circumstances. Notwithstanding the fact that better valve designs would ideally be available, ill-informed decision making processes between bioprosthetic and mechanical valves are contributing to the poor results. In the absence of multicentred, randomised clinical trials, comparing the current generations of bioprostheses with mechanical valves across all age groups Western guidelines tend to be uncritically applied. As a consequence, mechanical valves are being implanted into patients who are often not able to deal with anticoagulation while bioprosthetic valves may be overly shunned for fear of reoperations. Almost sixty years after the advent of cardiac surgery heart valve prostheses have eventually undergone improvements and several potentially disruptive developments are on the horizon. Until they materialise, however, choices between contemporary valve prostheses need to be made on the basis of individual risk and life-expectancy rather than an uncritical implementation of guidelines that were derived for very different patients and under distinctly different conditions. Given the fast expansion of cardiac surgery in middle-income countries and a growing number of independently operating centres in low-income countries a critical appraisal of facts underlying the choice of heart valve prostheses for patients with RHD seems opportune.

Intra-myocardial alginate hydrogel injection acts as a left ventricular mid-wall constraint in swine

KL Sack, E Aliotta, JS Choy, DB Ennis, NH Davies, T Franz, GS Kassab and JM Guccione
Acta Biomater (2020) 111(170-180

Despite positive initial outcomes emerging from preclinical and early clinical investigation of alginate hydrogel injection therapy as a treatment for heart failure, the lack of knowledge about the mechanism of action remains a major shortcoming that limits the efficacy of treatment design. To identify the mechanism of action, we examined previously unobtainable measurements of cardiac function from in vivo, ex vivo, and in silico states of clinically relevant heart failure (HF) in large animals. High-resolution ex vivo magnetic resonance imaging and histological data were used along with state-of-the-art subject-specific computational model simulations. Ex vivo data were incorporated in detailed geometric computational models for swine hearts in health (n = 5), ischemic HF (n = 5), and ischemic HF treated with alginate hydrogel injection therapy (n = 5). Hydrogel injection therapy mitigated elongation of sarcomere lengths (1.68 +/- 0.10mum [treated] vs. 1.78 +/- 0.15mum [untreated], p<0.001). Systolic contractility in treated animals improved substantially (ejection fraction = 43.9 +/- 2.8% [treated] vs. 34.7 +/- 2.7% [untreated], p<0.01). The in silico models realistically simulated in vivo function with >99% accuracy and predicted small myofiber strain in the vicinity of the solidified hydrogel that was sustained for up to 13 mm away from the implant. These findings suggest that the solidified alginate hydrogel material acts as an LV mid-wall constraint that significantly reduces adverse LV remodeling compared to untreated HF controls without causing negative secondary outcomes to cardiac function. STATEMENT OF SIGNIFICANCE: Heart failure is considered a growing epidemic and hence an important health problem in the US and worldwide. Its high prevalence (5.8 million and 23 million, respectively) is expected to increase by 25% in the US alone by 2030. Heart failure is associated with high morbidity and mortality, has a 5-year mortality rate of 50%, and contributes considerably to the overall cost of health care ($53.1 billion in the US by 2030). Despite positive initial outcomes emerging from preclinical and early clinical investigation of alginate hydrogel injection therapy as a treatment for heart failure, the lack of knowledge concerning the mechanism of action remains a major shortcoming that limits the efficacy of treatment design. To understand the mechanism of action, we combined high-resolution ex vivo magnetic resonance imaging and histological data in swine with state-of-the-art subject-specific computational model simulations. The in silico models realistically simulated in vivo function with >99% accuracy and predicted small myofiber strain in the vicinity of the solidified hydrogel that was sustained for up to 13 mm away from the implant. These findings suggest that the solidified alginate hydrogel material acts as a left ventricular mid-wall constraint that significantly reduces adverse LV remodeling compared to untreated heart failure controls without causing negative secondary outcomes to cardiac function. Moreover, if the hydrogel can be delivered percutaneously rather than via the currently used open-chest procedure, this therapy may become routine for heart failure treatment. A minimally invasive procedure would be in the best interest of this patient population; i.e., one that cannot tolerate general anesthesia and surgery, and it would be significantly more cost-effective than surgery.

Raising awareness for rheumatic mitral valve disease

LSA Passos, MCP Nunes, P Zilla, MH Yacoub and E Aikawa
Glob Cardiol Sci Pract (2020) 2020(2): e202026

Rheumatic heart disease (RHD) is a major burden in low- to mid-income countries, where each year it accounts for over a million premature deaths associated with severe valve disease. Life-saving valve replacement procedures are not available to the majority of affected RHD patients, contributing to an increased risk of death in young adults and creating a devastating impact. In December 2017, a group of representatives of major cardiothoracic societies and industry, discussed the plight of the millions of patients who suffer from RHD. A comprehensive solution based on this global partnership was outlined in "The Cape Town Declaration on Access to Cardiac Surgery in the Developing World". The key challenge in controlling RHD is related to identification and removal of barriers to the translation of existing knowledge into policy, programs, and practice to provide high-quality care for patients with RHD. This review provides an overview on RHD by emphasizing the disease medical and economic burdens worldwide, risk factors, recent advance for early disease detection, and overall preventive strategies.

Tissue Ingrowth Markedly Reduces Mechanical Anisotropy and Stiffness in Fibre Direction of Highly Aligned Electrospun Polyurethane Scaffolds

H Krynauw, J Buescher, J Koehne, L Verrijt, G Limbert, NH Davies, D Bezuidenhout and T Franz
Cardiovasc Eng Technol (2020) 11(4): 456-468

PURPOSE: The lack of long-term patency of synthetic vascular grafts currently available on the market has directed research towards improving the performance of small diameter grafts. Improved radial compliance matching and tissue ingrowth into the graft scaffold are amongst the main goals for an ideal vascular graft. METHODS: Biostable polyurethane scaffolds were manufactured by electrospinning and implanted in subcutaneous and circulatory positions in the rat for 7, 14 and 28 days. Scaffold morphology, tissue ingrowth, and mechanical properties of the scaffolds were assessed before implantation and after retrieval. RESULTS: Tissue ingrowth after 24 days was 96.5 +/- 2.3% in the subcutaneous implants and 77.8 +/- 5.4% in the circulatory implants. Over the 24 days implantation, the elastic modulus at 12% strain decreased by 59% in direction of the fibre alignment whereas it increased by 1379% transverse to the fibre alignment of the highly aligned scaffold of the subcutaneous implants. The lesser aligned scaffold of the circulatory graft implants exhibited an increase of the elastic modulus at 12% strain by 77% in circumferential direction. CONCLUSION: Based on the observations, it is proposed that the mechanism underlying the softening of the highly aligned scaffold in the predominant fibre direction is associated with scaffold compaction and local displacement of fibres by the newly formed tissue. The stiffening of the scaffold, observed transverse to highly aligned fibres and for more a random fibre distribution, represents the actual mechanical contribution of the tissue that developed in the scaffold.

Decellularization and engineered crosslinking: a promising dual approach towards bioprosthetic heart valve longevity

P Human, C Ofoegbu, H Ilsley, D Bezuidenhout, J de Villiers, DF Williams and P Zilla
Eur J Cardiothorac Surg (2020) 58(6): 1192-1200

OBJECTIVES: While decellularization has previously significantly improved the durability of bioprosthetic tissue, remnant immunogenicity may yet necessitate masking through crosslinking. To alleviate the fears of reintroducing the risk of calcific degeneration, we investigated the application of rationally designed crosslinking chemistry, capable of abrogating mineralization in isolation, in decellularized tissue. METHODS: Bovine and porcine pericardium were decellularized using the standard Triton X/sodium deoxycholate/DNAse/RNAse methodology and thereafter combined incrementally with components of a four-stage high-density dialdehyde-based fixation regimen. Mechanical properties prior to, and calcium levels following, subcutaneous implantation for 6 and 10 weeks in rats were assessed. RESULTS: Enhanced four-stage crosslinking, independent of decellularization, or decellularization followed by any of the crosslinking regimens, achieved sustained, near-elimination of tissue calcification. Decellularization additionally resulted in significantly lower tissue stiffness and higher fatigue resistance in all groups compared to their non-decellularized counterparts. CONCLUSIONS: The dual approach of combining decellularization with enhanced crosslinking chemistry in xenogeneic pericardial tissue offers much promise in extending bioprosthetic heart valve longevity.

Customized stent-grafts for endovascular aneurysm repair with challenging necks: A numerical proof of concept

A Hemmler, A Lin, N Thierfelder, T Franz, MW Gee and D Bezuidenhout
Int J Numer Method Biomed Eng (2020) 36(4): e3316

Endovascular aortic repair (EVAR) is a challenging intervention whose long-term success strongly depends on the appropriate stent-graft (SG) selection and sizing. Most off-the-shelf SGs are straight and cylindrical. Especially in challenging vessel morphologies, the morphology of off-the-shelf SGs is not able to meet the patient-specific demands. Advanced manufacturing technologies facilitate the development of highly customized SGs. Customized SGs that have the same morphology as the luminal vessel surface could considerably improve the quality of the EVAR outcome with reduced likelihoods of EVAR related complications such as endoleaks type I and SG migration. In this contribution, we use an in silico EVAR methodology that approximates the deployed state of the elastically deformable SG in a hyperelastic, anisotropic vessel. The in silico EVAR results of off-the-shelf SGs and customized SGs are compared qualitatively and quantitatively in terms of mechanical and geometrical parameters such as stent stresses, contact tractions, SG fixation forces and the SG-vessel attachment. In a numerical proof of concept, eight different vessel morphologies, such as a conical vessel, a barrel shaped vessel and a curved vessel, are used to demonstrate the added value of customized SGs compared to off-the-shelf SGs. The numerical investigation has shown large benefits of the highly customized SGs compared to off-the-shelf SGs with respect to a better SG-vessel attachment and a considerable increase in SG fixation forces of up to 50% which indicate decreased likelihoods of EVAR related complications. Hence, this numerical proof of concept motivates further research and development of highly customized SGs for the use in challenging vessel morphologies.

Reply to D. Vervoort

P Zilla
Eur J Cardiothorac Surg (2019) 55(5): 1023


Preclinical evaluation of a transcatheter aortic valve replacement system for patients with rheumatic heart disease

J Scherman, C Ofoegbu, A Myburgh, J Swanevelder, B van Breda, H Appa, P Human, D Williams, D Bezuidenhout and P Zilla
EuroIntervention (2019) 15(11): e975-e982

AIMS: Cardiac surgery in middle-income countries differs significantly from that in high-income countries regarding prevailing heart valve pathologies and access to cardiac surgery. Typically, rheumatic aortic regurgitation in the absence of calcification by far outweighs stenosis. As such, entirely different transcatheter aortic valve implantation (TAVI) concepts are required for these regions. The aim of the study was to evaluate the five-month performance of the SAT (Strait Access Technologies, Cape Town, South Africa) pericardial TAVI system in the orthotopic aortic position of juvenile sheep. METHODS AND RESULTS: A self-homing, non-occlusive balloon-expandable TAVI system comprising a hollow balloon, stabilising locator trunks, a scalloped CoCr stent with elevating anchorage arms and decellularised, sandwich-crosslinked pericardium was compared with control surgical valves (Edwards PERIMOUNT) in sheep. The implantation period was five months. The tactile placement of the TAVI valves was accomplished without the need for rapid pacing. At termination, no structural degeneration was observed in either group. The TAVIs were well healed with the stent struts largely embedded in tissue. Correlating with sheep growth (weight gain of 40.4+/-13.0%) during the implantation period, mean transvalvular gradients increased from 3.08+/-1.95 mmHg to 8.50+/-5.38 mmHg (p=0.044) after five months. CONCLUSIONS: A single-stage, balloon-expandable, easy to place TAVI system with antigen-depleted and antigen-masked bioprosthetic leaflets promises to address the distinct needs of low- and middle-income countries with regard to TAVI better than conventional systems.

Isolated mechanical aortic valve replacement in rheumatic patients in a low- to middle-income country

J Scherman, R Manganyi, P Human, T Pennel, A Brooks, J Brink and P Zilla
J Thorac Cardiovasc Surg (2019) 157(3): 886-893

OBJECTIVE: Although the results of aortic valve replacement are well documented for industrialized countries, the outcome in patients with rheumatic aortic valve disease in low- to middle-income countries is less well explored. The aim of this study was to determine the long-term survival and clinical outcomes after isolated aortic valve replacement in patients with rheumatic heart disease in a Sub-Saharan country where follow-up of indigent patients is often challenging. METHODS: A retrospective review of 969 aortic valve replacements performed between 2003 and 2013 was conducted at Cape Town's Groote Schuur Hospital. Patients who underwent concomitant procedures (n = 664) or had nonrheumatic valve pathology (n = 185) were excluded. The mean age of the rheumatic cohort (n = 121) was 43.1 +/- 11.6 years with a mean follow-up period of 6.14 +/- 3.44 years. The primary end points were survival and valve-related complications. RESULTS: A 15% cardiac- or valve-related 10-year mortality after receiving a mechanical prosthesis corresponded with a significantly higher mortality rate than that of a matched population. Overall cumulative survival at 1, 5, and 10 years was 93.5% (87.0-96.9), 86.4% (78.4-91.8), and 78.1% (67.5-86.0), respectively, and the corresponding cumulative freedom from combined thromboembolism and bleeding was 94.4% (88.2-97.5), 87.4% (79.4-92.5), and 86.1% (77.9-91.6), respectively. CONCLUSIONS: In low- to middle-income countries, with their unique mix of indigent and "First World" patients, rheumatic heart disease still accounts for a significant proportion of patients requiring isolated aortic valve replacement. Although mechanical prostheses are often selected in these young adults, survival remains suboptimal. Major bleeding and thromboembolic events account for the majority (77%) of the reported valve-related complications.

After 50 Years of Heart Transplants: What Does the Next 50 Years Hold for Cardiovascular Medicine? A Perspective From the International Society for Applied Cardiovascular Biology

JD Hutcheson, CJ Goergen, FJ Schoen, M Aikawa, P Zilla, E Aikawa and GR Gaudette
Front Cardiovasc Med (2019) 6(8

The first successful heart transplant 50 years ago by Dr.Christiaan Barnard in Cape Town, South Africa revolutionized cardiovascular medicine and research. Following this procedure, numerous other advances have reduced many contributors to cardiovascular morbidity and mortality; yet, cardiovascular disease remains the leading cause of death globally. Various unmet needs in cardiovascular medicine affect developing and underserved communities, where access to state-of-the-art advances remain out of reach. Addressing the remaining challenges in cardiovascular medicine in both developed and developing nations will require collaborative efforts from basic science researchers, engineers, industry, and clinicians. In this perspective, we discuss the advancements made in cardiovascular medicine since Dr. Barnard's groundbreaking procedure and ongoing research efforts to address these medical issues. Particular focus is given to the mission of the International Society for Applied Cardiovascular Biology (ISACB), which was founded in Cape Town during the 20th celebration of the first heart transplant in order to promote collaborative and translational research in the field of cardiovascular medicine.

Cardiac surgery for the forgotten millions: The way forward. Cardiac Surgery Intersociety Alliance (CSIA) Site Selection Criteria

P Boateng, RM Bolman, 3rd, P Zilla and Csia
J Thorac Cardiovasc Surg (2019) 158(3): 818-819


Cardiac Surgery for the Forgotten Millions: The Way Forward. Cardiac Surgery Intersociety Alliance (CSIA) Site Selection Criteria

P Boateng, RM Bolman, 3rd, P Zilla and Csia
Ann Thorac Surg (2019) 108(3): 653


Cardiac surgery for the forgotten millions: the way forward - CSIA site selection criteria

P Boateng, RM Bolman, 3rd and P Zilla
Asian Cardiovasc Thorac Ann (2019) 27(5): 338


Cardiac surgery for the forgotten millions: the way forward. Cardiac Surgery Intersociety Alliance (CSIA) Site Selection Criteria

P Boateng, RM Bolman and P Zilla
Eur J Cardiothorac Surg (2019) 56(2): 217


The African context of the Cape Town Declaration

P Zilla, L Zuhlke, K Sliwa and P Commerford
Cardiovasc J Afr (2018) 29(4): 204

Global Unmet Needs in Cardiac Surgery

P Zilla, M Yacoub, L Zuhlke, F Beyersdorf, K Sliwa, G Khubulava, A Bouzid, AO Mocumbi, D Velayoudam, D Shetty, C Ofoegbu, A Geldenhuys, J Brink, J Scherman, H du Toit, S Hosseini, H Zhang, XJ Luo, W Wang, J Mejia, T Kofidis, RSD Higgins, J Pomar, RM Bolman, BM Mayosi, R Madansein, J Bavaria, AA Yanes-Quintana, AS Kumar, O Adeoye, RF Chauke and DF Williams
Glob Heart (2018) 13(4): 293-303

More than 6 billion people live outside industrialized countries and have insufficient access to cardiac surgery. Given the recently confirmed high prevailing mortality for rheumatic heart disease in many of these countries together with increasing numbers of patients needing interventions for lifestyle diseases due to an accelerating epidemiological transition, a significant need for cardiac surgery could be assumed. Yet, need estimates were largely based on extrapolated screening studies while true service levels remained unknown. A multi-author effort representing 16 high-, middle-, and low-income countries was undertaken to narrow the need assessment for cardiac surgery including rheumatic and lifestyle cardiac diseases as well as congenital heart disease on the basis of existing data deduction. Actual levels of cardiac surgery were determined in each of these countries on the basis of questionnaires, national databases, or annual reports of national societies. Need estimates range from 200 operations per million in low-income countries that are nonendemic for rheumatic heart disease to >1,000 operations per million in high-income countries representing the end of the epidemiological transition. Actually provided levels of cardiac surgery range from 0.5 per million in the assessed low- and lower-middle income countries (average 107 +/- 113 per million; representing a population of 1.6 billion) to 500 in the upper-middle-income countries (average 270 +/- 163 per million representing a population of 1.9 billion). By combining need estimates with the assessment of de facto provided levels of cardiac surgery, it emerged that a significant degree of underdelivery of often lifesaving open heart surgery does not only prevail in low-income countries but is also disturbingly high in middle-income countries.

Celebrating 50 years of heart transplant surgery: A missed opportunity to honour Hamilton Naki

P Zilla, J Brink and T Pennel
S Afr Med J (2018) 108(4): 12270

Reply to Mankahla N, Dlamini S, Taunyane IC, Maqungo S, Cairncross L, Chiliza B. Celebrating 50 years of heart transplant surgery: A missed opportunity to honour Hamilton Naki. S Afr Med J 2018;108(3):151. https://doi.org/10.7196/SAMJ.2018.v108i3.13114.

The Cape Town Declaration on Access to Cardiac Surgery in the Developing World

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RSD Higgins, B Mayosi, A Carpentier and D Williams
Ann Thorac Surg (2018) 106(3): 930-933


The Cape Town Declaration on access to cardiac surgery in the developing world

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RSD Higgins, B Mayosi, A Carpentier and D Williams
J Thorac Cardiovasc Surg (2018) 156(6): 2206-2209


The Cape Town declaration on access to cardiac surgery in the developing world

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RSD Higgins, B Mayosi, A Carpentier and D Williams
Eur J Cardiothorac Surg (2018) 54(3): 407-410


The Cape Town Declaration on Access to Cardiac Surgery in the Developing World

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RSD Higgins, B Mayosi, A Carpentier and D Williams
S Afr Med J (2018) 108(9): 702-704

Twelve years after cardiologists and cardiac surgeons from all over the world issued the 'Drakensberg Declaration on the Control of Rheumatic Fever and Rheumatic Heart Disease in Africa', calling on the world community to address the prevention and treatment of rheumatic heart disease (RHD) through improving living conditions, to develop pilot programmes at selected sites for control of rheumatic fever and RHD, and to periodically review progress made and challenges that remain, RHD still accounts for a major proportion of cardiovascular diseases in children and young adults in low- and middle-income countries, where more than 80% of the world population live. Globally equal in prevalence to human immunodeficiency virus infection, RHD affects 33 million people worldwide. Prevention efforts have been important but have failed to eradicate the disease. At the present time, the only effective treatment for symptomatic RHD is open heart surgery, yet that life-saving cardiac surgery is woefully absent in many endemic regions. In this declaration, we propose a framework structure to create a co-ordinated and transparent international alliance to address this inequality.

The Cape Town Declaration on Access to Cardiac Surgery in the Developing World

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RS Higgins, BM Mayosi, A Carpentier and D Williams
Cardiovasc J Afr (2018) 29(4): 256-259

Mission: to urge all relevant entities within the international cardiac surgery, industry and government sectors to commit to develop and implement an effective strategy to address the scourge of rheumatic heart disease in the developing world through increased access to life-saving cardiac surgery.

The Cape Town Declaration on Access to Cardiac Surgery in the Developing World

P Zilla, RM Bolman, MH Yacoub, F Beyersdorf, K Sliwa, L Zuhlke, RS Higgins, B Mayosi, A Carpentier and D Williams
Asian Cardiovasc Thorac Ann (2018) 26(7): 535-539


Transcatheter valve with a hollow balloon for aortic valve insufficiency

J Scherman, B van Breda, H Appa, C Heerden, C Ofoegbu, D Bezuidenhout and P Zilla
Multimed Man Cardiothorac Surg (2018) 2018(10.1510/mmcts.2018.012

During the past decade transcatheter aortic valve implantation (TAVI) has revolutionized our approach to heart valve disease. Although largely applied to patients with calcific aortic valve stenosis, there is an unmet clinical need to also treat patients with aortic valve insufficiency in patients with non-calcific aortic valve disorders. The following Techno-College tutorial demonstrates our pre-clinical experience with a novel non-occlusive, self-homing TAVI system, developed with Strait Access Technologies, that we hope will improve outcomes for treatment of non-calcific aortic valve insufficiency.

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.

Challenges to paediatric cardiac services in South Africa

J Hewitson and P Zilla
Lancet Child Adolesc Health (2018) 2(8): e15


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


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) 70(71-83

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.

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.

Rapidly Recoverable Thixotropic Hydrogels from the Racemate of Chiral OFm Monosubstituted Cyclo(Glu-Glu) Derivatives

L Wang, X Jin, L Ye, AY Zhang, D Bezuidenhout and ZG Feng
Langmuir (2017) 33(48): 13821-13827

Both chiral OFm monosubstituted cyclo(l-Glu-l-Glu) and cyclo(d-Glu-d-Glu) display a robust gelation ability in a variety of organic solvents and water. In contrast to an individual enantiomer, their racemate can form rapidly recoverable thixotropic hydrogels with a remarkably shorter thixotropic recovery time. This unexpected thixotropic behavior is induced by the random arrangement of d- and l-enantiomers in the cell units, leading to the formation of "pseudoracemate", noncrystalline self-assemblies in the resulting 3D fibrous network.

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


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.

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 Neglected Villain of Bioprosthetic Degeneration: Inflammatory and Immune Processes

P Human and P Zilla
J Long Term Eff Med Implants (2017) 27(2-4): 159-180

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 crosslinking reagent. In this review, we summarize our findings and those of others that 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 vascularized and noncross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we 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 antibodies in bioprosthesis calcification.

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


Cellular mechanosensitivity to substrate stiffness decreases with increasing dissimilarity to cell stiffness

T Abdalrahman, L Dubuis, J Green, N Davies and T Franz
Biomech Model Mechanobiol (2017) 16(6): 2063-2075

Computational modelling has received increasing attention to investigate multi-scale coupled problems in micro-heterogeneous biological structures such as cells. In the current study, we investigated for a single cell the effects of (1) different cell-substrate attachment (2) and different substrate modulus [Formula: see text] on intracellular deformations. A fibroblast was geometrically reconstructed from confocal micrographs. Finite element models of the cell on a planar substrate were developed. Intracellular deformations due to substrate stretch of [Formula: see text], were assessed for: (1) cell-substrate attachment implemented as full basal contact (FC) and 124 focal adhesions (FA), respectively, and [Formula: see text]140 KPa and (2) [Formula: see text], 140, 1000, and 10,000 KPa, respectively, and FA attachment. The largest strains in cytosol, nucleus and cell membrane were higher for FC (1.35[Formula: see text], 0.235[Formula: see text] and 0.6[Formula: see text]) than for FA attachment (0.0952[Formula: see text], 0.0472[Formula: see text] and 0.05[Formula: see text]). For increasing [Formula: see text], the largest maximum principal strain was 4.4[Formula: see text], 5[Formula: see text], 5.3[Formula: see text] and 5.3[Formula: see text] in the membrane, 9.5[Formula: see text], 1.1[Formula: see text], 1.2[Formula: see text] and 1.2[Formula: see text] in the cytosol, and 4.5[Formula: see text], 5.3[Formula: see text], 5.7[Formula: see text] and 5.7[Formula: see text] in the nucleus. The results show (1) the importance of representing FA in cell models and (2) higher cellular mechanical sensitivity for substrate stiffness changes in the range of cell stiffness. The latter indicates that matching substrate stiffness to cell stiffness, and moderate variation of the former is very effective for controlled variation of cell deformation. The developed methodology is useful for parametric studies on cellular mechanics to obtain quantitative data of subcellular strains and stresses that cannot easily be measured experimentally.

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): 10.1002/cnm.2772

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.

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.

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].

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.

The anisotropic mechanical behaviour of electro-spun biodegradable polymer scaffolds: Experimental characterisation and constitutive formulation

G Limbert, R Omar, H Krynauw, D Bezuidenhout and T Franz
J Mech Behav Biomed Mater (2016) 53(21-39

Electro-spun biodegradable polymer fibrous structures exhibit anisotropic mechanical properties dependent on the degree of fibre alignment. Degradation and mechanical anisotropy need to be captured in a constitutive formulation when computational modelling is used in the development and design optimisation of such scaffolds. Biodegradable polyester-urethane scaffolds were electro-spun and underwent uniaxial tensile testing in and transverse to the direction of predominant fibre alignment before and after in vitro degradation of up to 28 days. A microstructurally-based transversely isotropic hyperelastic continuum constitutive formulation was developed and its parameters were identified from the experimental stress-strain data of the scaffolds at various stages of degradation. During scaffold degradation, maximum stress and strain in circumferential direction decreased from 1.02 +/- 0.23 MPa to 0.38 +/- 0.004 MPa and from 46 +/- 11 % to 12 +/- 2 %, respectively. In longitudinal direction, maximum stress and strain decreased from 0.071 +/- 0.016 MPa to 0.010 +/- 0.007 MPa and from 69 +/- 24 % to 8 +/- 2 %, respectively. The constitutive parameters were identified for both directions of the non-degraded and degraded scaffold for strain range varying between 0% and 16% with coefficients of determination r(2)>0.871. The six-parameter constitutive formulation proved versatile enough to capture the varying non-linear transversely isotropic behaviour of the fibrous scaffold throughout various stages of degradation.

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.

Melatonin as a preventive and curative therapy against pulmonary hypertension

G Maarman, D Blackhurst, F Thienemann, L Blauwet, G Butrous, N Davies, K Sliwa and S Lecour
J Pineal Res (2015) 59(3): 343-53

Pulmonary hypertension (PH) is characterized by elevated pulmonary arterial pressure, which leads to right ventricular (RV) hypertrophy and failure. The pathophysiological mechanisms of PH remain unclear but oxidative stress is believed to contribute to RV dysfunction. Melatonin is a powerful antioxidant and is cardioprotective against ischemia-reperfusion injury and hypertension. Therefore, we hypothesized that a chronic treatment with melatonin, given as a curative or preventive therapy, may confer cardiovascular benefits in PH. PH was induced in Long Evans rats (n >/= 6 per group), with a single subcutaneous injection of monocrotaline (MCT, 80 mg/kg). Melatonin was given daily in the drinking water, with the treatment starting either on the day of the injection of MCT (dose testing: melatonin 75 ng/L and 6 mg/kg), 14 days after the injection of MCT (curative treatment: 6 mg/kg), or 5 days before the injection (preventive treatment: 6 mg/kg). The development of PH was assessed by measuring RV hypertrophy, RV function, cardiac interstitial fibrosis, and plasma oxidative stress. Compared with controls, MCT-treated rats displayed RV hypertrophy and dysfunction, increased interstitial fibrosis, and elevated plasma oxidative stress. A chronic melatonin treatment (75 ng/L or 6 mg/kg) reduced RV hypertrophy, improved RV function and reduced plasma oxidative stress. Curative and preventive treatment improved RV functional and plasma oxidative stress parameters and reduced cardiac interstitial fibrosis. Our data demonstrate that melatonin confers cardioprotection in this model of PH. As melatonin is an inexpensive and safe drug, we propose that clinical investigation of the effects of melatonin on RV function in patients with PH should be considered.

Coacervate Delivery of Growth Factors Combined with a Degradable Hydrogel Preserves Heart Function after Myocardial Infarction

NR Johnson, M Kruger, KP Goetsch, P Zilla, D Bezuidenhout, Y Wang and NH Davies
ACS Biomater Sci Eng (2015) 1(9): 753-759

Regenerative therapies to improve prognosis after heart attack and mitigate the onset of heart failure are urgently needed. To this end, we developed a bioactive therapy of sustained release of the morphogen Sonic hedgehog (Shh) and the anti-inflammatory cytokine interleukin-10 (IL-10) from a coacervate delivery vehicle. This is combined with a structural therapy consisting of a biodegradable polyethylene glycol (PEG) hydrogel, harnessing the benefits of both components. Upon injection into the hearts of rats after heart attack, we found that each component synergistically improved the benefit of the other. Furthermore, their combination was critical to preserve heart function. These findings indicate that, when combined, growth factor delivery and an injectable hydrogel represent a promising therapeutic approach for treatment after heart attack.

Assessment of the immunogenicity of mechanically induced interferon aggregates in a transgenic mouse model

P Human, H Ilsley, C Roberson, E Grovender, B Van Antwerp, E Fogt and P Zilla
J Pharm Sci (2015) 104(2): 722-30

Pump delivery of human interferon alpha-2B (IFNalpha2b) has the potential for inducing immunogenic drug aggregates. We therefore evaluated the immunogenicity of mechanically induced IFNalpha2b aggregates to assess this risk. Transgenic human-IFNalpha2b (TG) and wild-type (WT) FVB/N mice (n = 8 and n = 9/group, respectively) were administered mechanically agitated drug [45 Hz for 6 h (LLA) or 24 h (HLA)], chemically modified drug [low pH (pH 4.0) or metal oxidized (OXD)] or unstressed drug (native). Mice received IFNalpha2b (50 mug; 100 mug/mL; s.c.) formulations on days 0, 7, 14, and 21. Drug-binding and neutralizing antibody titers were determined after 28 d. Aggregate concentrations were highest in OXD and HLA formulations but OXD had more dimers/trimers. Geometric mean titers were 1:131, 1:728, 1:1573, 1:871, and 1:10,240 for WT mice (n = 9) and 1:207, 1:587, 1:1810, 1:571, and 1:2,153 for TG mice (n = 8) for native, LLA, HLA, pH4, and OXD groups, respectively. Mechanical agitation of IFNalpha2b induced equivalent titers 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.

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.

Huge left-ventricular pseudoaneurysm compressing coronary artery 10 weeks after stabbing attack

J Scherman, TD Nguyen, P Zilla and MY Emmert
Eur Heart J (2014) 35(6): 385


Massive hemoptysis 18 months after a stabbing attack

J Scherman, TD Linh Nguyen, P Zilla and MY Emmert
Ann Thorac Surg (2014) 98(2): 728


The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models

T Pennel, G Fercana, D Bezuidenhout, A Simionescu, TH Chuang, P Zilla and D Simionescu
Biomaterials (2014) 35(24): 6311-22

There is a significant need for small diameter vascular grafts to be used in peripheral vascular surgery; however autologous grafts are not always available, synthetic grafts perform poorly and allografts and xenografts degenerate, dilate and calcify after implantation. We hypothesized that chemical stabilization of acellular xenogenic arteries would generate off-the-shelf grafts resistant to thrombosis, dilatation and calcification. To test this hypothesis, we decellularized porcine renal arteries, stabilized elastin with penta-galloyl glucose and collagen with carbodiimide/activated heparin and implanted them as transposition grafts in the abdominal aorta of rats as direct implants and separately as indirect, isolation-loop implants. All implants resulted in high patency and animal survival rates, ubiquitous encapsulation within a vascularized collagenous capsule, and exhibited lack of lumen thrombogenicity and no graft wall calcification. Peri-anastomotic neo-intimal tissue overgrowth was a normal occurrence in direct implants; however this reaction was circumvented in indirect implants. Notably, implantation of non-treated control scaffolds exhibited marked graft dilatation and elastin degeneration; however PGG significantly reduced elastin degradation and prevented aneurismal dilatation of vascular grafts. Overall these results point to the outstanding potential of crosslinked arterial scaffolds as small diameter vascular grafts.

Pharmacokinetic evaluation of lisinopril-tryptophan, a novel C-domain ACE inhibitor

P Denti, SK Sharp, WL Kroger, SL Schwager, A Mahajan, M Njoroge, L Gibhard, I Smit, K Chibale, L Wiesner, ED Sturrock and NH Davies
Eur J Pharm Sci (2014) 56(113-9

Angiotensin-converting enzyme (ACE, EC is a metallopeptidase comprised of two homologous catalytic domains (N- and C-domains). The C-domain cleaves the vasoactive angiotensin II precursor, angiotensin I, more efficiently than the N-domain. Thus, C-domain-selective ACE inhibitors have been designed to investigate the pharmacological effects of blocking the C-terminal catalytic site of the enzyme and improve the side effect profile of current ACE inhibitors. Lisinopril-tryptophan (LisW-S), an analogue of the ACE inhibitor lisinopril, is highly selective for the C-domain. In this study, we have analysed the ex vivo domain selectivity and pharmacokinetic profile of LisW-S. The IC50 value of LisW-S was 38.5 nM in rat plasma using the fluorogenic substrate Abz-FRKP(Dnp)P-OH. For the pharmacokinetics analysis of LisW-S, a sensitive and selective LC-MS/MS method was developed and validated to determine the concentration of LisW-S in rat plasma. LisW-S was administered to Wistar rats at a dose of 1 mg/kg bodyweight intravenously, 5 mg/kg bodyweight orally. The Cmax obtained following oral administration of the drug was 0.082 muM and LisW-S had an apparent terminal elimination half-life of around 3.1 h. The pharmacokinetic data indicate that the oral bioavailability of LisW-S was approximately 5.4%. These data provide a basis for better understanding the absorption mechanism of LisW-S and evaluating its clinical application.

Differentiating transmural from transanastomotic prosthetic graft endothelialization through an isolation loop-graft model

T Pennel, P Zilla and D Bezuidenhout
J Vasc Surg (2013) 58(4): 1053-61

BACKGROUND: In humans, transanastomotic endothelial outgrowth onto the surface of prosthetic vascular grafts is limited to the immediate perianastomotic region, even after years of implantation. In contrast, continual transanastomotic outgrowth together with short graft lengths has led to early endothelial confluence in most animal models pre-empting endothelialization through transmural capillary sprouting. We describe an isolation loop-graft model that clearly separates these distinctly different events. METHODS: Baseline transanastomotic endothelialization was assessed by implanting low-porosity expanded polytetrafluoroethylene grafts (ePTFE; internal diameter 1.7 mm; internodal distance 15-25 mum; 14.2 +/- 1.6 mm long) for 2, 4, and 6 weeks (n = 6/time point) in the abdominal aorta of Wistar rats. High-porosity polyurethane (internal diameter 1.7 mm-150 mum pore size) grafts were then interposed ("welded") into the midsection of the ePTFE grafts for 2, 4, 6, and 8 weeks (n = 6/time point). Looping the interposition grafts increased their length to 70.3 +/- 8.3 mm. After implantation periods of 6, 8, 12, and 24 weeks (n = 8/time point) isolation loop grafts were analyzed by light, immune-fluorescence (CD31) and scanning electron microscopy, and endothelialization was expressed as maximal transanastomotic endothelial outgrowth (I(max)), mean transanastomotic outgrowth (I(mean)), and segmental graft coverage (GSE). RESULTS: Transanastomotic outgrowth slowed down between 2 and 6 weeks of implantation (proximal: [I(max) from 0.9 +/- 0.5 to 0.3 +/- 0.3 mm/wk; P < .04; I(mean) from 0.3 +/- 0.1 to 0.2 +/- 0.1 mm/wk; nonsignificant (NS)]; distal: [I(max) from 0.7 +/- 0.3 to 0.3 +/- 0.2 mm/wk; P < .02; I(mean) from 0.3 +/- 0.2 to 0.2 +/- 0.0 mm/wk; NS]) but remained constant thereafter (I(max) = 0.5 +/- 0.2 mm/wk; I(mean) = 0.4 +/- 0.2 mm/wk at 24 weeks NS). In straight composite grafts, the ePTFE separation zones were too short to isolate transmural ingrowth beyond week 4. In contrast, a broad endothelial-free separation zone was preserved in all looped composite grafts even after half a year of implantation (25.9 +/- 5.9 vs 8.7 +/- 4.9 mm proximally and 21.9 +/- 13.4 vs 12.3 +/- 6.2 mm distally at 6 and 24 weeks, respectively). Ninety-three percent of patent loop-grafts showed isolated transmural midgraft endothelium after 4 weeks and 97% after 6 weeks. Midgraft preconfluence was reached by 6 weeks (GSE = 55 +/- 45%) and confluence between week 12 and 24 (GSE = 95.0 +/- 10.0% and 84.0 +/- 30.13%). The subintimal thickness stayed constant with a nonsignificant trend toward regression (91.8 +/- 93.9 mm vs 71.4 +/- 59.4 mm at 6 and 24 weeks, respectively; NS). CONCLUSIONS: Transmural endothelialization can be clearly distinguished from transanastomotic outgrowth in a high throughput rat model. A looped interposition graft model provides sufficient isolation length to separate the two events for up to half a year and does not result in an increase in intimal hyperplasia. CLINICAL RELEVANCE: Although the mode of graft deployment has changed over the years, the problem of an absent surface endothelium remains, whether small- to medium-diameter grafts are surgically implanted or placed endovascularly as "covered stents." In contrast to humans, most animal models experience progressive transanastomotic endothelial outgrowth. Together with graft lengths that were too short, the clinically irrelevant transanastomotic endothelialization inadvertently led to early endothelial confluence in the vast majority of experimental vascular graft studies pre-empting or concealing alternative modes of endothelialization. The isolation loop-graft model we propose allows the long-term differentiation of the different modes of endothelialization in a small animal screening model.

Protective constriction of coronary vein grafts with knitted nitinol

L Moodley, T Franz, P Human, MF Wolf, D Bezuidenhout, J Scherman and P Zilla
Eur J Cardiothorac Surg (2013) 44(1): 64-71

OBJECTIVES: Different flow patterns and shear forces were shown to cause significantly more luminal narrowing and neointimal tissue proliferation in coronary than in infrainguinal vein grafts. As constrictive external mesh support of vein grafts led to the complete suppression of intimal hyperplasia (IH) in infrainguinal grafts, we investigated whether mesh constriction is equally effective in the coronary position. METHODS: Eighteen senescent Chacma baboons (28.8 +/- 3.6 kg) received aorto-coronary bypass grafts to the left anterior descending artery (LAD). Three groups of saphenous vein grafts were compared: untreated controls (CO); fibrin sealant-sprayed controls (CO + FS) and nitinol mesh-constricted grafts (ME + FS). Meshes consisted of pulse-compliant, knitted nitinol (eight needles; 50 mum wire thickness; 3.4 mm resting inner diameter, ID) spray attached to the vein grafts with FS. After 180 days of implantation, luminal dimensions and IH were analysed using post-explant angiography and macroscopic and histological image analysis. RESULTS: At implantation, the calibre mismatch between control grafts and the LAD expressed as cross-sectional quotient (Qc) was pronounced [Qc = 0.21 +/- 0.07 (CO) and 0.18 +/- 0.05 (CO + FS)]. Mesh constriction resulted in a 29 +/- 7% reduction of the outer diameter of the vein grafts from 5.23 +/- 0.51 to 3.68 +/- 0 mm, significantly reducing the calibre discrepancy to a Qc of 0.41 +/- 0.17 (P < 0.02). After 6 months of implantation, explant angiography showed distinct luminal irregularities in control grafts (ID difference between widest and narrowest segment 74 +/- 45%), while diameter variations were mild in mesh-constricted grafts. In all control grafts, thick neointimal tissue was present [600 +/- 63 mum (CO); 627 +/- 204 mum (CO + FS)] as opposed to thin, eccentric layers of 249 +/- 83 mum in mesh-constricted grafts (ME + FS; P < 0.002). The total wall thickness had increased by 363 +/- 39% (P < 0.00001) in CO and 312 +/- 61% (P < 0.00001) in CO + FS vs 82 +/- 61% in ME + FS (P < 0.007). CONCLUSIONS: In a senescent non-human primate model for coronary artery bypass grafts, constrictive, external mesh support of saphenous veins with knitted nitinol prevented focal, irregular graft narrowing and suppressed neointimal tissue proliferation by a factor of 2.5. The lower degree of suppression of IH compared with previous infrainguinal grafts coincided with a lesser reduction of calibre mismatch in the coronary grafts.

Outcomes of myocardial infarction hydrogel injection therapy in the human left ventricle dependent on injectate distribution

R Miller, NH Davies, J Kortsmit, P Zilla and T Franz
Int J Numer Method Biomed Eng (2013) 29(8): 870-84

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.

The effect of hydrogel injection on cardiac function and myocardial mechanics in a computational post-infarction model

J Kortsmit, NH Davies, R Miller, JR Macadangdang, P Zilla and T Franz
Comput Methods Biomech Biomed Engin (2013) 16(11): 1185-95

An emerging therapy to limit adverse heart remodelling following myocardial infarction (MI) is the injection of polymers into the infarcted left ventricle (LV). In the few numerical studies carried out in this field, the definition and distribution of the hydrogel in the infarcted myocardium were simplified. In this computational study, a more realistic biomaterial distribution was simulated after which the effect on cardiac function and mechanics was studied. A validated finite element heart model was used in which an antero-apical infarct was defined. Four infarct models were created representing different temporal phases in the progression of a MI. Hydrogel layers were simulated in the infarcted myocardium in each model. Biomechanical and functional improvement of the LV was found after hydrogel inclusion in the ischaemic models representing the early phases of MI. In contrast, only functional but no mechanical restitution was shown in the scar model due to hydrogel presence.

Cell specific ingrowth hydrogels

M Bracher, D Bezuidenhout, MP Lutolf, T Franz, M Sun, P Zilla and NH Davies
Biomaterials (2013) 34(28): 6797-803

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 diametrically 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.

Covalent incorporation and controlled release of active dexamethasone from injectable polyethylene glycol hydrogels

D Bezuidenhout, A Oosthuysen, N Davies, L Ahrenstedt, S Dobner, P Roberts and P Zilla
J Biomed Mater Res A (2013) 101(5): 1311-8

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.


Remodeling leads to distinctly more intimal hyperplasia in coronary than in infrainguinal vein grafts

P Zilla, L Moodley, J Scherman, H Krynauw, J Kortsmit, P Human, MF Wolf and T Franz
J Vasc Surg (2012) 55(6): 1734-41

BACKGROUND: Flow patterns and shear forces in native coronary arteries are more protective against neointimal hyperplasia than those in femoral arteries. Yet, the caliber mismatch with their target arteries makes coronary artery bypass grafts more likely to encounter intimal hyperplasia than their infrainguinal counterparts due to the resultant slow flow velocity and decreased wall stress. To allow a site-specific, flow-related comparison of remodeling behavior, saphenous vein bypass grafts were simultaneously implanted in femoral and coronary positions. METHODS: Saphenous vein grafts were concomitantly implanted as coronary and femoral bypass grafts using a senescent nonhuman primate model. Duplex ultrasound-based blood flow velocity profiles and vein graft and target artery dimensions were correlated with dimensional and histomorphologic graft remodeling in large, senescent Chacma baboons (n = 8; 28.1 +/- 4.9 kg) during a 24-week period. RESULTS: At implantation, the cross-sectional quotient (Q(c)) between target arteries and vein grafts was 0.62 +/- 0.10 for femoral grafts vs 0.17 +/- 0.06 for coronary grafts, resulting in a dimensional graft-to-artery mismatch 3.6 times higher (P < .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 = .0048), 48.1% +/- 23.6% lower maximal cycling wall shear stress (P = .012), and 62.2% +/- 21.2% lower mean velocity (P = .007) in coronary grafts. After 24 weeks, the luminal diameter of all coronary grafts had contracted by 63%, from an inner diameter of 4.49 +/- 0.60 to 1.68 +/- 0.63 mm (P < .0001; subintimal diameter: -41.5%; P = .002), whereas 57% of the femoral interposition grafts had dilated by 31%, from 4.21 +/- 0.25 to 5.53 +/- 1.30 mm (P = .020). Neointimal tissue was 2.3 times thicker in coronary than in femoral grafts (561 +/- 73 vs 240 +/- 149 mum; P = .001). Overall, the luminal area of coronary grafts was an average of 4.1 times smaller than that of femoral grafts. CONCLUSIONS: Although coronary and infrainguinal bypass surgery uses saphenous veins as conduits, they undergo significantly different remodeling processes in these two anatomic positions.

Blood pressure target attainment in the background of guidelines: the very elderly in Swiss primary care

HH Schafer, I Sudano, GR Theus, P Zilla, G Noll and M Burnier
Fam Pract (2012) 29(5): 511-20

BACKGROUND: There are only a few trials for the very elderly population (>79 years). No consensus, which blood pressure (BP) goals and substances should be applied, has been found yet. This survey was undertaken to investigate how octogenarians are treated and attain BP targets in the Swiss primary care. METHODS: Data from 4594 hypertensive patients were collected within 7 days. Eight hundred and seventy-seven patients met the requirement to be >79 years. We assessed substances/combinations and investigated pulse pressure and target blood pressure attainment (TBPA) using three different recommendations [Canadian Hypertension Education Program (CHEP), Swiss Society of Hypertension (SSH), European Society of Hypertension-European Society of Cardiology (ESH-ESC)]. Secondarily, we compared TBPA attained by angiotensin-converting enzyme inhibitor (ACEI)/diuretic (D), angiotensin receptor blocker (ARB)/D and calcium channel blocker (CCB)/D with any other dual therapy and investigated whether Ds/beta-blockers (BBs) or Ds/renin angiotensin-converting enzyme inhibitors (RAAS-Is) lead to higher TBPA. Finally, we assessed the impact of drug administration, practical work experience, location and specialization of GPs on TBPA. RESULTS: Octogenarians attained target blood pressure (TBP) between 44% (ESH-ESC) and 74% (SSH). Optimal/normal BP was reached in 22.8% of patients. Pulse pressure <65 mmHg was shown in 66.4% of patients. Monotherapy was most commonly applied followed by dual single-pill combination with ARB/D (46.5%) or ACEI/D (36.0%). No benefit in TBPA was found comparing a RAASI/D and CCB/D treatment with any other dual combination. There was also no difference between BB/D and RAAS-I/D combination therapy and between single-pill combination and dual free combinations. CONCLUSIONS: GPs adhere to the use of substances proven in outcome trials and attain high TBP. No difference in meeting BP goals could be found using different drug classes. There is an unmet need to harmonize recommendations and to add additional information for the treatment of octogenarians.

Recommendations for the treatment of hypertension in the elderly and very elderly--a scotoma within international guidelines

HH Schafer, JN De Villiers, I Sudano, S Dischinger, GR Theus, P Zilla and T Dieterle
Swiss Med Wkly (2012) 142(w13574

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

K Kadner, S Dobner, T Franz, D Bezuidenhout, MS Sirry, P Zilla and NH Davies
Biomaterials (2012) 33(7): 2060-6

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 mitral repair versus replacement in a threshold country: the impact of commissural fusion

A Geldenhuys, JJ Koshy, PA Human, JF Mtwale, JG Brink and P Zilla
J Heart Valve Dis (2012) 21(4): 424-32
BACKGROUND AND AIM OF THE STUDY: In developing countries rheumatic heart disease is the predominant indication for cardiac surgery. As the disease tends to progress, reoperation rates for mitral valve repairs are high. Against this background, the predictors of failure were assessed and the overall performance of repairs compared with replacements in a 10-year cohort of rheumatic single mitral valve procedures. METHODS: Between 2000 and 2010, a total of 646 consecutive adult (aged >15 years) patients underwent primary, single mitral valve procedures. All 87 percutaneous balloon valvuloplasties (100%) were rheumatic, compared to 280 of the 345 primary mitral valve replacements (81%) and 69 of the 215 primary mitral valve repairs (32%). As the study aim was to compare the outcome of mitral valve repair versus replacement in rheumatic patients of a threshold country, all 69 repair patients were propensity-matched with 69 of the replacement patients. Based on propensity score analysis, Kaplan-Meier actuarial analysis with log-rank testing was used to evaluate survival and morbidity. RESULTS: The follow up was 100% complete (n = 138), and ranged from 0.6 to 132 months (mean 53.3 +/- 36.5 months). Actuarial freedom from valve-related mortality was 96 +/- 3% and 92 +/- 4% at five years, and 96 +/- 3% and 80 +/- 11% at 10 years for repairs and replacements, respectively (p = NS). Actuarial freedom from all valve-related events (deaths, reoperations and morbidity) was 80 +/- 6% and 86 +/- 5% at five years, and 70 +/- 8% and 69 +/- 11% at 10 years (p = NS). Actuarial freedom from all valve-related events was 57 +/- 11% and 96 +/- 3% at five years (p = 0.0008), and 42 +/- 12% and 96 +/- 3% at 10 years (p < 0.001) for those mitral valve repairs with and without commissural fusion, respectively (p = 0.0002 overall). CONCLUSION: The long-term results for mitral valve replacement in an indigent, rheumatic heart disease population of a developing country were better than generally perceived. Notwithstanding, mitral valve repair has a superior long-term outcome in those patients who do not show commissural fusion at operation.

Pulmonary artery banding: still a valuable option in developing countries?

A Brooks, A Geldenhuys, L Zuhlke, P Human and P Zilla
Eur J Cardiothorac Surg (2012) 41(2): 272-6

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 endpoints 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.

Knitted nitinol represents a new generation of constrictive external vein graft meshes

P Zilla, L Moodley, MF Wolf, D Bezuidenhout, MS Sirry, N Rafiee, W Lichtenberg, M Black and T Franz
J Vasc Surg (2011) 54(5): 1439-50

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.

Degradation-induced changes of mechanical properties of an electro-spun polyester-urethane scaffold for soft tissue regeneration

H Krynauw, L Bruchmuller, D Bezuidenhout, P Zilla and T Franz
J Biomed Mater Res B Appl Biomater (2011) 99(2): 359-68

The aim of this study was the in vitro investigation of the change in mechanical properties of a fast-degrading electro-spun polymeric scaffold for the use in soft tissue regenerative implants. Tubular scaffolds were electro-spun from a DegraPol(R) D30 polyesther-urethane solution (target outer diameter: 5.0 mm; scaffold wall thickness: 0.99 +/- 0.18 mm). Scaffold samples were subjected to hydrolytic in vitro degradation for up to 34 days. The fiber network structure and fiber surfaces were inspected on scanning electron micrographs. Following vacuum drying and determination of mass, flat samples (9.69 +/- 0.21 x 18.47 +/- 2.62 mm, n = 5) underwent uni-axial tensile testing (5 load cycles, strain epsilon = 0 to 20%; final extension to failure) in circumferential scaffold direction after 5, 10, 14, 18, 22, 26, 30, and 34 days of degradation. Scaffold mass did not change with degradation. Maximum elastic modulus, maximum stress and associated strain were E(max) = 1.14 +/- 0.23 MPa, sigma(max) = 0.52 +/- 0.12 MPa and epsilon(max) = 176.8 +/- 21.9% before degradation and E(max) = 0.43 +/- 0.26 MPa, sigma(max) = 0.033 +/- 0.028 MPa and epsilon(max) = 24.6 +/- 3.0% after 34 days of degradation. The deterioration of mechanical properties was not reflected in the ultrastructural surface morphology of the fibers. The current exploratory study provides a basis for the development of constitutive computational models of biodegradable scaffolds with future extension of the investigation most importantly to capture mechanical effects of regenerating tissue. Future studies will include degradation in biological fluids and assessment of molecular weight for an advanced understanding of the material changes during degradation.

Patient-specific prediction of intrinsic mechanical loadings on sub-muscular pectoral pacemaker implants based on an inter-species transfer function

MH de Vaal, J Neville, M Litow, J Scherman, P Zilla and T Franz
J Biomech (2011) 44(14): 2525-31

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.

A mathematical method for constraint-based cluster analysis towards optimized constrictive diameter smoothing of saphenous vein grafts

T Franz, BD Reddy, P Human and P Zilla
Med Biol Eng Comput (2010) 48(6): 519-29

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.

Tailored sizes of constrictive external vein meshes for coronary artery bypass surgery

T Franz, P Human, S Dobner, BD Reddy, M Black, H Ilsley, MF Wolf, D Bezuidenhout, L Moodley and P Zilla
Biomaterials (2010) 31(35): 9301-9

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. 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. 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 (3.0-3.7 mm) 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 (3.0-4.2 mm) 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.

The in vivo assessment of mechanical loadings on pectoral pacemaker implants

MH de Vaal, J Neville, J Scherman, P Zilla, M Litow and T Franz
J Biomech (2010) 43(9): 1717-22

Reduced sizes of implantable cardiac pacemakers and clinical advances have led to a higher feasibility of using such devices in younger patients including children. Increased structural demands deriving from reduced device size and more active recipients require detailed knowledge of in vivo mechanical conditions to ensure device reliability. Objective of this study was the proof of feasibility of a system for the measurement of in vivo mechanical loadings on pacemaker implants. The system comprised the following: implantable instrumented pacemaker (IPM) with six force sensors, accelerometer and radio-frequency (RF) transceiver; RF data logging system and video capture system. Three Chacma baboons (20.6+/-1.15 kg) received one pectoral sub-muscular IPM implant. After wound healing, forces were measured during physical activities. Forces during range of motion of the arm were assessed on the anaesthetized animals prior to device explantation. Mass, volume and dimensions of the excised Pectoralis major muscles were determined after device explantation. Remote IPM activation and data acquisition were reliable in the indoor cage environment with transceiver distances of up to 3m. Sampling rates of up to 1,000 Hz proved sufficient to capture dynamic in vivo loadings. Compressive forces on the IPM in conscious animals reached a maximum of 77.2+/-54.6N during physical activity and were 22.2+/-7.3N at rest, compared with 34.6+/-15.7 N maximum during range of motion and 13.4+/-3.3N at rest in anaesthetized animals. The study demonstrated the feasibility of the developed system for the assessment of in vivo mechanical loading conditions of implantable pacemakers with potential for use for other implantable therapeutic devices.

Mechanical loadings on pectoral pacemaker implants: correlation of in-line and transverse force of the Pectoralis major

MH de Vaal, J Neville, J Scherman, P Zilla, M Litow and T Franz
Ann Biomed Eng (2010) 38(11): 3338-46

Recently we presented a method for the assessment of in vivo forces on pectoral device implants motivated from technological and clinical advancements toward smaller implantable cardiac pacemakers and the altered structural demands arising from the reduced device size. Objective of this study was the investigation of the intra-species proportionality of in-line force and transverse reaction force of the Pectoralis major for the characterization of mechanical in vivo loadings on pectoral implants. Two Chacma baboons (23.9 +/- 1.2 kg) received bilaterally one chronic and one acute pectoral sub-muscular instrumented pacemaker (IPM) implant. The Pectoralis major muscle was electrically stimulated and resulting in-line and transverse muscle force were measured. The correlation of in-line force and transverse force of the Pectoralis major was investigated using linear regression analyses. The proportionality of in-line and transverse force of the Pectoralis major was found to be subject-specific (R(2) = 0.17, p < 0.003). Including morphometric parameters, i.e., length along line of action, width over implant and stress, in the regression analysis provided a strong intra-species correlation between in-line and transverse force (R(2) = 0.71, p < 10(-)(7)). The novel intra-species correlation provides a tool toward the characterization of mechanical in vivo loading conditions of pectoral device implants.

Covalent surface heparinization potentiates porous polyurethane scaffold vascularization

D Bezuidenhout, N Davies, M Black, C Schmidt, A Oosthuysen and P Zilla
J Biomater Appl (2010) 24(5): 401-18

Porous scaffolds play an integral role in many tissue-engineering approaches, and the ability to improve vascularization, without eliciting an excessive inflammatory response, would constitute an important step towards achieving long-term healing and function of devices made from these materials. After having previously optimized the dimensional requirements of the well-defined pores, the present study aimed at a further shift from inflammation to vascularization via surface immobilization with heparin. Porous polyurethane disks were produced to contain well-defined pores (147 +/- 2 microm) with abundant interconnecting windows (67 +/- 2 microm). After heparinization via copolymer grafting and amination to contain 32 microg of heparin, the modification appeared as a uniform layer on all exposed surfaces, with no signs of pore obliteration or significant changes in pore size. After 28 days implantation in a rat subcutaneous model, the scaffolds were assessed for vascularization/arteriolization and inflammation using CD31/actin and ED-1 staining, respectively. Heparinization resulted in a significant increase in vascularization: capillaries increased by 62% in number (66.2 +/- 0.8 to 107.3 +/- 1.4 vessels/mm( 2); p < 0.03) and 56% in total area (0.9 +/- 0.1 to 1.4 +/- 0.02%; p<0.02). Arteriolization also increased in absolute terms (200% in number; p<0.03), but did not change significantly when normalized to capillary number. Heparinization did not significantly affect the inflammatory response at this time-point, as quantified by ED-1 positive macrophage and foreign body giant cell (FBGC) content. Thus, the in vivo vascularization of porous scaffolds could be increased without concomitant increase in the inflammatory response by employing a simple surface modification technique. This could be a valuable tool for in vivo tissue engineering applications where enhanced vascularization is required.

Utilization of shape memory in external vein-graft meshes allows extreme diameter constriction for suppressing intimal hyperplasia: a non-human primate study

P Zilla, M Wolf, N Rafiee, L Moodley, D Bezuidenhout, M Black, P Human and T Franz
J Vasc Surg (2009) 49(6): 1532-42

OBJECTIVE: Constrictive external Nitinol meshes have been shown to suppress neointimal 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. METHODS: 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 <or=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 (Q(C)). RESULTS: At 6 months, all vein grafts without mesh support showed thick eccentric layers of neointimal tissue (group 1: 348 +/- 130 microm [Q(C) median at implant 0.19]; group 2: 318 +/- 142 microm [Q(C) median at implant 0.17]). Fibrin glue-spraying had no effect. In contrast, neointimal tissue was absent in all mesh-supported grafts (P < .007 in all cases) both at 6 weeks/6 months (group 3: 7.5 +/- 8.8 mum and 2.5 +/- 4.4 microm [Q(C) median at implant 1.47]; group 4: 1.3 +/- 0.6 microm and 3.8 +/- 5.6 microm [Q(C) median at implant 3.09]). Except for mild tissue buckling (fold height <356 microm) 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 vs 85 +/- 14%; P < .023 in group 1). Fibrin glue alone (52 +/- 48%) did not preserve endothelialization of control grafts (P < .38). CONCLUSION: 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, extreme constriction may occasionally be required to eliminate luminal irregularities.

Dimensional analysis of human saphenous vein grafts: Implications for external mesh support

P Human, T Franz, J Scherman, L Moodley and P Zilla
J Thorac Cardiovasc Surg (2009) 137(5): 1101-8

OBJECTIVE: Constrictive external mesh support of vein grafts was shown to mitigate intimal hyperplasia in animal experiments. To determine the degree of constriction required for the elimination of dimensional irregularities in clinically used vein grafts, a detailed anatomic study of human saphenous veins was conducted. METHODS: In 200 consecutive patients having coronary artery bypass grafting, harvested saphenous veins (length 34.4 +/- 10.8 cm) were analyzed regarding diameter irregularities, side branch distribution, and microstructure. RESULTS: The mean outer diameter of surgically distended saphenous veins was 4.2 +/- 0.6 mm (men, 4.3 +/- 0.6 mm vs women, 3.9 +/- 0.5 mm; P < .0001). Although the outer diameter significantly decreased over the initial 18 cm (-7.6%; P < .0001), the overall increase between malleolus and thigh was not significant (+11.2%). Smaller-diameter veins (<3.5 mm) had more pronounced diameter fluctuations than larger veins (31.8% +/- 11.0% vs 21.2% +/- 8.8%; P < .0001), with more than 71% of all veins showing caliber changes of more than 20%. There was 1 side branch every 5.4 +/- 4.3 cm, with a significantly higher incidence between 20 and 32 cm from the malleolus (P < .0001 to distal, P < .0004 to proximal). Generally, women had more side branches than men (0.30 +/- 0.15 cm(-1) vs 0.25 +/- 0.12 cm(-1); P = .0190). Thick-walled veins (565.7 +/- 138.4 mum) had a significantly higher number of large side branches (P < .0001), and thin-walled veins (398.7 +/- 123.2 mum) had significantly more small side branches (P < .0001). Pronounced intimal thickening ("cushions") was found in 28% of vessels (119.8 +/- 28.0 mum vs 40.1 +/- 18.2 mum; P < .0001). CONCLUSION: Although the preferential location of side branches may be addressed by the deliberate discarding of infragenicular vein segments, a diameter constriction of 27% on average would eliminate diameter irregularities in 98% of vein grafts.

A synthetic non-degradable polyethylene glycol hydrogel retards adverse post-infarct left ventricular remodeling

S Dobner, D Bezuidenhout, P Govender, P Zilla and N Davies
J Card Fail (2009) 15(7): 629-36

BACKGROUND: 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

L Bezuidenhout, P Zilla and N 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 nonhuman primates

P Zilla, P Human, M Wolf, W Lichtenberg, N Rafiee, D Bezuidenhout, N Samodien, C Schmidt and T Franz
J Thorac Cardiovasc Surg (2008) 136(3): 717-25

OBJECTIVE: External mesh support of vein grafts has been shown to mitigate the formation of intimal hyperplasia. The aim of the present study was to address the issue of optimal mesh size in a nonhuman 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(Sigma) = 32/n = 8 per mesh size). No mesh support (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 seen not only in isolation but also against the background of anastomotic size mismatch at implantation, expressed as quotient of cross-sectional area of host artery to vein graft (Q(C)). RESULTS: Significant amounts of intimal hyperplasia were found in group I (Q(C) median 0.20; intimal hyperplasia 6 weeks = 1.63 +/- 0.34 mm(2); intimal hyperplasia 12 weeks = 1.73 +/- 0.5 mm(2)) and group II (Q(C) median 0.25; intimal hyperplasia 6 weeks = 1.96 +/- 1.64 mm(2); intimal hyperplasia 12 weeks = 2.88 +/- 1.69 mm(2)). In contrast, group III (Q(C) median 0.45; intimal hyperplasia 6 weeks = 0.08 +/- 0.13 mm(2); intimal hyperplasia 12 weeks = 0.18 +/- 0.32 mm(2)) and IV (Q(C) median 1.16; intimal hyperplasia 6 weeks = 0.02 +/- 0.03 mm(2); intimal hyperplasia 12 weeks = 0.11 +/- 0.10 mm(2)) showed dramatically suppressed intimal hyperplasia (P < .01) at both time points. Endothelial integrity was only preserved in group IV (P < .05). There were no significant differences in vascularization and inflammation in either interlayer or intergroup 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.

Prosthetic heart valves: catering for the few

P Zilla, J Brink, P Human and D 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.

The dosage dependence of VEGF stimulation on scaffold neovascularisation

N Davies, S Dobner, D Bezuidenhout, C Schmidt, M Beck, AH Zisch and P 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.

Prosthetic vascular grafts: wrong models, wrong questions and no healing

P Zilla, D Bezuidenhout and P Human
Biomaterials (2007) 28(34): 5009-27

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.

Bioprosthetic tissue preservation by filling with a poly(acrylamide) hydrogel

A Oosthuysen, PP Zilla, PA Human, CA Schmidt and D Bezuidenhout
Biomaterials (2006) 27(9): 2123-30

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 BPT and the mitigation/prevention of calcification by filling aortic wall samples with a synthetic poly(acrylamide) (pAAm) hydrogel, 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 pre-fixed tissue. The observed decrease in amino-group content (0.157+/-0.012-0.123+/-0.021 micromol/mg, p<0.03) and corresponding increase in shrinkage temperature (67.2+/-0.8-78.1+/-1.8 degrees 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 88% (p<0.0001) 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.

Diamine-extended glutaraldehyde- and carbodiimide crosslinks act synergistically in mitigating bioprosthetic aortic wall calcification

P Zilla, D Bezuidenhout, M Torrianni, M Hendriks and P Human
J Heart Valve Dis (2005) 14(4): 538-45
BACKGROUND AND AIM OF THE STUDY: The extension of glutaraldehyde (GA) crosslinks with diamine bridges was shown previously to reduce bioprosthetic heart valve calcification to a significant degree. The aim of the present study was to investigate whether the additional crosslinking 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 h of cold storage. Crosslinking of amino-functional groups was achieved either by GA fixation alone (0.2% or 0.7%) or with an interim treatment with the diamine L-lysine (25, 50 or 100 mM; 37 degrees C; 2 days). Carboxyl groups were activated with carbodiimide (N'-{3-dimethylaminopropyl}-N-ethyl carbodiimide hydrochloride (EDC), 240 mM) and crosslinked with an oligomeric diamine (polypropylene glycol-bis-aminopropyl ether (Jeffamine), 60 mM, 230D). By permutation of treatments and combinations thereof, a total of 17 groups was compared. Aortic wall discs (12 mm 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 GA or carbodiimide fixation was used alone. Equally, the combined crosslinking 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 (25 mM), additional EDC/Jeffamine crosslinking 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 100 mM 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 in bioprosthetic aortic wall calcification can be achieved by combining diamine-extended conventional GA fixation with a diamine-extended carbodiimide based crosslinking step.

Carbodiimide treatment dramatically potentiates the anticalcific effect of alpha-amino oleic acid on glutaraldehyde-fixed aortic wall tissue

P Zilla, D Bezuidenhout and P Human
Ann Thorac Surg (2005) 79(3): 905-10

BACKGROUND: Bifunctional amines were previously found to act as bridging molecules between the terminal ends of incomplete glutaraldehyde (GA) 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 alpha-amino oleic acid (AOA) to act as a bridging molecule between -NH2- and COOH-dependent cross-links was hypothesized to similarly augment the anticalcification effect of the AOA molecule. METHODS: Porcine aortic wall tissue from Medtronic Freestyle valve bioprostheses incorporating the AOA anticalcification process additionally underwent carboxyl-group cross-linking with Jeffamine (poly[propylene glyco]-bis-[aminopropyl ether]) using 1-ethyl-3-(3-dimethylaminopropyl) 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 (25 mmol/L carbodiimide) before implantation. Tissue calcification was assessed by atomic absorption spectroscopy and histochemical staining. RESULTS: Aldehyde end-capping with AOA 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 AOA was essential for the beneficial effect of the additional EDC/Jeffamine cross-linking step. CONCLUSIONS: Potentially utilizing both the amino- and the carboxyl moieties of AOA for tissue binding dramatically reduces aortic wall calcification of GA-fixed tissue.

The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides

MH Fittkau, P Zilla, D Bezuidenhout, MP Lutolf, P Human, JA Hubbell and N 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.

Bioprosthetic heart valves: the need for a quantum leap

P Zilla, P Human and D Bezuidenhout
Biotechnol Appl Biochem (2004) 40(Pt 1): 57-66

More than 250,000 bioprosthetic heart valves are being implanted annually. Although the majority of recipients are elderly developed-world patients, the most urgent need for tissue valves is in younger patients, where rapid degeneration of contemporary prostheses remains a serious obstacle. After decades of empirical and mostly futile attempts to extend the longevity of tissue valve prostheses, new insights and solutions are on the horizon. Aetiologically, a shift of focus from mineralization to immune responses and inflammation emerges. On the development side, new engineering approaches to both selective extraction of tissue components and cross-links are increasingly defining the new direction. In order to dramatically improve the performance of bioprosthetic heart valves, these new developments need to lead to a broad consensus for a paradigm shift in a hitherto rather stagnant field of medical research.

Detoxification on top of enhanced, diamine-extended glutaraldehyde fixation significantly reduces bioprosthetic root calcification in the sheep model

AE Trantina-Yates, P Human and P Zilla
J Heart Valve Dis (2003) 12(1): 93-100; discussion 100-1
BACKGROUND AND AIM OF THE STUDY: Increased concentrations of glutaraldehyde (GA), diamine-extension (DA) of crosslinks and subsequent extraction of excess GA all reduce bioprosthetic calcification in the subdermal rat model. The study aim was to demonstrate the combined effect of all three treatments in a circulatory sheep model. METHODS: Two fixation treatments were used for GA detoxification (urazole in acetate buffer, 0.1 M; pH 4.5; 37 degrees C; 7 days): (i) conventional 0.2% GA fixation (4 degrees C; 7 days); and (ii) enhanced 3.0% GA fixation (4 degrees C; 2 days, followed by a DA interim step; 100 mM L-lysine; 37 degrees C; 2 days, followed by GA; 3.0%; 37 degrees C; 5 days). Entire porcine root prostheses were implanted in the distal aortic arch of young sheep for 12 weeks (n = 5 per group). Non-detoxified 0.2% GA-treated roots served as controls (n = 5). Calcium analysis was based on atomic absorption spectrophotometry; morphology was assessed using light and transmission electron microscopy. RESULTS: Detoxification alone resulted in an 83% reduction of leaflet calcification (p = 0.086), but achieved only 23% (p = 0.145) and 12% (p = 0.362) mitigation of calcification in aortic wall and sinus tissue, respectively. When combined with DA-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 subadventitial layer of aortic wall tissue, whereas enhanced fixation seemed to affect all structures. CONCLUSION: It was shown in a circulatory sheep model that a combination of DA-enhanced fixation with an extraction process of excess GA leads to a distinct mitigation of leaflet and aortic wall calcification.

Optimization of diamine bridges in glutaraldehyde treated bioprosthetic aortic wall tissue

P Huma, D Bezuidenhout, M Torrianni, M Hendriks and P Zilla
Biomaterials (2002) 23(10): 2099-103

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; 0, 25, 50 or 100 mM; 37 degrees C; 2 days) was followed by completion of the GA fixation (37 degrees C; 5 days). Aortic wall coupons (12 mm) 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 25 mM 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 (0 mM 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 100 mM. 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 100 mM DA has the potential to practically halve aortic wall calcification.

The challenge of pediatric cardiac services in the developing world

J Hewitson, J Brink and P Zilla
Semin Thorac Cardiovasc Surg (2002) 14(4): 340-5

Pediatric cardiac services are too expensive for most developing nations. Problems other than cardiac disease take priority when it comes to budget allocations. Poor health infrastructure and referral systems, malnutrition, and the HIV/AIDS pandemic aggravate the situation, and the increasing economic divide is threatening what services do exist. We highlight how the practice of pediatric cardiac surgery in South Africa compares with first-world standards and outline some of the problems faced by pediatric cardiac services in developing nations.

The anticalcific effect of glutaraldehyde detoxification on bioprosthetic aortic wall tissue in the sheep model

P Zilla, C Weissenstein, M Bracher and P Human
J Card Surg (2001) 16(6): 467-72

BACKGROUND: Increasing concentrations of glutaraldehyde (GA) lead to a decreased rather than increased calcification of bioprosthetic aortic wall tissue. This study determined 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% for 1 week at 4 degrees C). Subsequently, roots underwent a GA extraction process using high volumes of Urazole solution (acetic acid buffer, pH 4.5, 37 degrees C, 1 week) followed by NaBH4 reduction (2 days, 37 degrees 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 anticalcification effect of GA detoxification after 6 weeks (56.8% to 97.9%; 95% confidence interval [CI]), which stabilized 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 cross-link 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 +/- 11.9 microg/mg without detoxification to 119.3 +/- 19.3 microg/mg with detoxification) as 3.0% fixation (114.8 +/- 10.0 microg/mg without detoxification to 91.3 +/- 11.5 microg/mg with detoxification). CONCLUSION: We were able to determine in the circulatory sheep model to what degree the intrinsic procalcific effect of GA counteracts the protective effect of higher cross-link density. Our study also established that the effect of detoxification is particularly pronounced in commercial low-grade fixation.

Diamine extension of glutaraldehyde crosslinks mitigates bioprosthetic aortic wall calcification in the sheep model

P Zilla, D Bezuidenhout, C Weissenstein, A van der Walt and P Human
J Biomed Mater Res (2001) 56(1): 56-64

We previously have been able to 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 that introduced diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT degrees ), resistance to protease digestion (RPD), residual amine analysis (RA), and tensile modulus (E(10)). 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 degrees from 85.7 degrees +/- 0.3 degrees to 87.5 degrees +/- 0.3 degrees C, p < 0.002; RPD from 24.2 +/- 1.2 to 29.1 +/- 0.7%, p < 0.003; RA from 0.069 +/- 0.004 to 0.058 +/- 0.003 micromol/mg, p < 0.03, and E(10) from 1.9 +/- 0.11 to 2.94 +/- 0.34 MPa, p < 0.01), but it was distinctly enhanced when diamine bridges were introduced (SrT degrees from 87.5 degrees +/- 0.3 degrees to 93.4 degrees +/- 0.3 degrees C, p << 0.0001; RPD from 29.1 +/- 0.7 to 68.4 +/- 1.8%, p << 0.0001; and E(10) from 2.94 +/- 0.34 to 6.80 +/- 0.61 MPa, p < 0.0003). Aortic wall calcification was reduced significantly 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 6 weeks and 24 weeks, respectively. The correlation coefficients between calcification and SrT degrees, RDP, and E(10) was -0.9767, -0.9460, and -0.9740, respectively (6 weeks). The inflammatory host reaction regularly found in 0.2% fixed tissue was practically abolished through the introduction of diamine bridges. Our study demonstrated a distinct correlation between the mitigation of aortic wall calcification and three parameters used to assess crosslink density.

Prosthetic heart valves: why biological?

UO von Oppell and P Zilla
J Long Term Eff Med Implants (2001) 11(3-4): 105-13
The replacement of heart valves only became feasible after the development of the heart-lung machine in 1953. Two groups of prosthetic heart valves were subsequently developed: biological valves that do not require anticoagulation and mechanical valves that require life-long anticoagulation with Coumadin. The incidence of heart surgery and the demographics of patients who require heart valve surgery vary worldwide; these factors influence the choice of prosthetic valve for the individual patient and are briefly reviewed. Improved biological tissue-fixation methods are also increasing the durability of biological prosthetic valves and will further favor the implantation of biological valves in the future.

Mitigation of bioprosthetic heart valve degeneration through biocompatibility: in vitro versus spontaneous endothelialization

AE Trantina-Yates, P Human, M Bracher and P Zilla
Biomaterials (2001) 22(13): 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 (0.1 M) or detoxified and in vitro lined with cultured, autologousjugular 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 significantly 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.

Stentless bioprosthetic heart valve research: sheep versus primate model

A Trantina-Yates, C Weissenstein, P Human and P Zilla
Ann Thorac Surg (2001) 71(5 Suppl): 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+/-3 kg) and Chacma baboons (n = 5; 17+/-3 kg). After 6 weeks of tissue calcification, pannus outgrowth and inflammation were assessed by atomic absorption spectrophotometry, histologic 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 microg/mg in the sheep model versus 4.1+/-5.9 microg/mg in the primate model; p > 0.005). In both models, leaflet calcification was negligible (2.6+/-2.4 microg/mg in the sheep versus 2.5+/-1.9 microg/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 whereas the inflammatory demarcation often resembled a granulocyte-dominated xenograft response in the primate. Pannus outgrowth was comparable in length (8.4+/-2.3 mm versus 9.1+/-4.3 mm proximally and 7.1+/-3.4 mm versus 7.4+/-5.1 mm distally, in the sheep and baboon, respectively; p > 0.05). CONCLUSIONS: 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 xenograft-type reaction in the primate model.

Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides?

C Merzkirch, N Davies and P Zilla
Anat Rec (2001) 263(4): 379-87

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 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.

Characterization of the immune response to valve bioprostheses and its role in primary tissue failure

P Human and P Zilla
Ann Thorac Surg (2001) 71(5 Suppl): 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 glutaraldehyde-treated tissue and the possible role of graft-specific antibody in graft mineralization, we performed immune-calcification studies in the rabbit and correlated those results with the analysis of specific antibodies. METHODS: Aortic wall buttons (6 mm) were punched from porcine aortic wall tissue fixed with 0.2% glutaraldehyde and detoxified with urazole and then subsequently perforated under sterile conditions. The perforated buttons were then incubated with either immune serum prepared by immunization 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 preimmune sera obtained before immunization 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 buttons 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 with control samples treated with preimmune sera. Overall, the mean calcium level was significantly increased (p < 0.0001) when tissue was treated with immune sera (66.0+/-10.0 microg/mg versus 22.6+/-4.8 microg/mg in control tissue). Graft specificity of immune sera was confirmed by Western blot analysis. CONCLUSIONS: These results strongly suggest a role of circulating graft-specific antibody in the disease of bioprosthetic graft calcification.

The possible role of immune responses in bioprosthetic heart valve failure

P Human and P Zilla
J Heart Valve Dis (2001) 10(4): 460-6

Inflammatory and immune processes: the neglected villain of bioprosthetic degeneration?

P Human and P Zilla
J Long Term Eff Med 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 crosslinking reagent. In this review, we summarize our findings and those of others that 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 vascularized and non-cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we 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 antibodies in bioprosthesis calcification.

Fixation-related autolysis and bioprosthetic aortic wall calcification

P Human, C Weissenstein, A Trantina and P Zilla
J Heart Valve Dis (2001) 10(5): 656-65
BACKGROUND AND AIM OF THE STUDY: It has been established previously that immediate fixation and increased glutaraldehyde (GA) concentrations are required to prevent severe autolytic tissue damage during bioprosthetic aortic root production. The study aim was to verify that structure-preserving fixation also reduces aortic wall calcification. METHODS: Porcine aortic roots were fixed either instantly or after being kept on ice for 48 h (phosphate-buffered saline, PBS). Two concentrations of GA (0.2% and 3.0%) were chosen (4 degrees C, seven days, PBS). Discs of aortic wall tissue (1.2 cm diameter) were implanted subcutaneously in rats for 60 days (n = 10 per group), while aortic roots were implanted in the distal aortic arch of sheep for six weeks (n = 3 per group) and six months (n = 4 per 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 with 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, vacuolization and vesicle shedding on the one hand, and calcification on the other. CONCLUSION: Coincidental fixation-related ultrastructural damage and increased calcification was demonstrated in bioprosthetic aortic wall tissue.

Matrix metalloproteinases and tissue valve degeneration

M Bracher, D Simionescu, A Simionescu, N Davies, P Human and P Zilla
J Long Term Eff Med Implants (2001) 11(3-4): 221-30
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 because of structural deterioration. The role of proteolytic degradation, with particular reference to the matrix metalloproteinases (MMPs) in the degeneration of aortic bioprostheses, is appraised in this minireview. It is clear that both the intrinsic and host-derived proteolytic activities present in heart-valve bioprostheses may combine with mechanical stress to bring about valve failure.

High glutaraldehyde concentrations mitigate bioprosthetic root calcification in the sheep model

P Zilla, C Weissenstein, P Human, T Dower and UO von Oppell
Ann Thorac Surg (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. 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 qualitatively assessed. RESULTS: By increasing the GA concentration from 0.2% to 3.0%, aortic wall calcification could be reduced by 38% after 6 weeks and 34% after 6 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 6 weeks but in a few leaflets presented as heterogeneous, nodular spongiosa deposits after 6 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. CONCLUSIONS: We have shown in the circulatory sheep model that the anticalcific effect of better cross-linking seems to outweigh the intrinsic pro-calcific effect of GA accumulation in bioprosthetic aortic wall tissue.

Glutaraldehyde detoxification in addition to enhanced amine cross-linking dramatically reduces bioprosthetic tissue calcification in the rat model

C Weissenstein, P Human, D Bezuidenhout and P Zilla
J Heart Valve Dis (2000) 9(2): 230-40
BACKGROUND AND AIM OF THE STUDY: Enhanced fixation of bioprosthetic tissue by both increased concentrations of glutaraldehyde (GA) and the introduction of additional cross-links with L-lysine significantly reduces calcification. We have previously reported that prolonged exposure to high-volume amino-compounds under warm, acidic conditions leads to thorough, non-rebounding GA detoxification. The aim of the present study was to prove that removal of excess GA can amplify the benefits of enhanced GA cross-linking with regard to bioprosthetic tissue calcification. METHODS: Porcine ascending aortas and leaflet tissue, and bovine pericardium were immediately fixed using three GA concentrations (0.2%, 1.0%, 3.0% (v/v)) for seven days at 4 degrees C. Samples were allocated to nine groups. Groups I to III received no further treatment (one at each GA concentration); groups IV to IX underwent an additional L-lysine interim step (48 h/37 degrees C/0.1 M) two days before completion of standard seven-day GA fixation; and groups VII to IX were additionally treated with a GA extraction process using high-volume urazole solution (acetic acid buffer, pH 4.5, 37 degrees C, one week) followed by NaBH4 reduction (2 days, 37 degrees C). Samples were implanted subcutaneously in rats (six per group) for six weeks. Tissue calcium was measured by atomic absorption spectrophotometry and examined histologically after von Kossa staining. RESULTS: Calcification was reduced in all three tissue types by enhanced cross-linking and by extraction of excess GA. Increasing the GA concentration from 0.2% to 3.0% led 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 tissues (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 with 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: Removal of excess GA from fixed bioprosthetic tissue was capable of markedly improving the anti-calcific effect of enhanced GA cross-linking.

Endothelial cell transplantation

P Zilla, M Deutsch and J Meinhart
Semin Vasc Surg (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.

Tissue adhesives in cardiovascular surgery

UO von Oppell and P Zilla
J Long Term Eff Med Implants (1998) 8(2): 87-101
Tissue adhesives are increasingly being used in cardiovascular surgery as adjuncts to obtain more rapid hemostasis, as tissue reinforcing agents, as carriers for prolonged local release of antibiotics, to spatially fix long saphenous vein grafts that could otherwise possibly kink, and to promote endothelialization of prosthetic graft surfaces. The available tissue adhesives, their current indications for use, and possible future trends are discussed.

Improved ultrastructural preservation of bioprosthetic tissue

P Zilla, Y Zhang, P Human, W Koen and U von Oppell
J Heart Valve Dis (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

P Zilla, C Weissenstein, M Bracher, Y Zhang, W Koen, P Human and U von Oppell
J Heart Valve Dis (1997) 6(5): 502-9
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.

Glutaraldehyde detoxification of aortic wall tissue: a promising perspective for emerging bioprosthetic valve concepts

P Zilla, L Fullard, P Trescony, J Meinhart, D Bezuidenhout, M Gorlitzer, P Human and U von Oppell
J Heart Valve Dis (1997) 6(5): 510-20
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.

Acute traumatic rupture of the thoracic aorta. A comparison of techniques

UO Von Oppell, J Brink, J Hewitson, P Pinho and P Zilla
S Afr J Surg (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

JO Fulton, P Zilla, KM De Groot and UO Von Oppell
Eur J Cardiothorac Surg (1996) 10(10): 922-4

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.

Aortic dissection repair with GRF glue complicated by heart block

UO Von Oppell, D Chimuka, JG Brink and P Zilla
Ann Thorac Surg (1995) 59(3): 761-3

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.

Traumatic aortic rupture: twenty-year metaanalysis of mortality and risk of paraplegia

UO von Oppell, TT Dunne, MK De Groot and P Zilla
Ann Thorac Surg (1994) 58(2): 585-93

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

UO von Oppell, TT Dunne, KM De Groot and P Zilla
J Card Surg (1994) 9(6): 685-91

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.

Successful management of aortoesophageal fistula due to thoracic aortic aneurysm

UO von Oppell, M de Groot, C Thierfelder, P Zilla and JA Odell
Ann Thorac Surg (1991) 52(5): 1168-70

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.

Adenosine cardioplegia: reducing reperfusion injury of the ischaemic myocardium?

DH Boehm, PA Human, U von Oppell, P Owen, H Reichenspurner, LH Opie, AG Rose and B Reichart
Eur J Cardiothorac Surg (1991) 5(10): 542-5

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.

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining

P Zilla, R Fasol, U Dudeck, S Siedler, P Preiss, T Fischlein, W Muller-Glauser, G Baitella, D Sanan, J Odell and et al.
J Vasc Surg (1990) 12(2): 180-9
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).

Endothelial cell toxicity of solid-organ preservation solutions

UO von Oppell, S Pfeiffer, P Preiss, T Dunne, P Zilla and B Reichart
Ann Thorac Surg (1990) 50(6): 902-10

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.

Effect of pharmacologic immunosuppression on donor heart survival in a closely related nonhuman primate xenograft model

H Reichenspurner, PA Human, AG Rose, B Reichart and DK Cooper
Transplant Proc (1990) 22(3): 1086-7

Reply to: Blood Platelets and Bypass

P Zilla
J Thorac Cardiovasc Surg (1989) 98(5 Pt 1): 797-800

Optimalization of immunosuppression after xenogeneic heart transplantation in primates

H Reichenspurner, PA Human, DH Boehm, AG Rose, R May, DK Cooper, P Zilla and B Reichart
J Heart Transplant (1989) 8(3): 200-7; discussion 207-8
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)

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

DK Cooper, PA Human, AG Rose, J Rees, M Keraan, B Reichart, E Du Toit and R Oriol
J Thorac Cardiovasc Surg (1989) 97(3): 447-55
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?

DK Cooper, PA Human, AG Rose, J Rees, M Keraan, E DuToit and R Oriol
Transplant Proc (1989) 21(1 Pt 1): 549-50

Anomalous left superior vena cava in combined heart-lung transplantation

UO von Oppell, JA Odell, H Reichenspurner, B Reichart, P Zilla and R Fasol
J Heart Transplant (1988) 7(6): 445-7
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.

Inotropic effect of triiodothyronine following myocardial ischemia and cardiopulmonary bypass: an experimental study in pigs

D Novitzky, PA Human and DK Cooper
Ann Thorac Surg (1988) 45(1): 50-5

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 pigs (Subgroup A) had died of low cardiac output, but all 5 treated pigs (Subgroup B) survived. After 3 hours of ischemia, both groups showed some reduced function at 10 minutes, though the reduction was more marked in untreated animals. By 70 minutes, 4 of 6 untreated pigs had died of myocardial failure and all treated pigs remained alive (p less than 0.03). Surviving pigs in both groups still demonstrated some reduced function compared with values obtained before CPB. When all pigs are considered 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)

Effect of triiodothyronine (T3) on myocardial high energy phosphates and lactate after ischemia and cardiopulmonary bypass. An experimental study in baboons

D Novitzky, PA Human and DK Cooper
J Thorac Cardiovasc Surg (1988) 96(4): 600-7
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.

Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon

DK Cooper, PA Human, G Lexer, AG Rose, J Rees, M Keraan and E Du Toit
J Heart Transplant (1988) 7(3): 238-46
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.

Twenty years of heart transplantation at Groote Schuur Hospital

H Reichenspurner, JA Odell, DK Cooper, D Novitzky, PA Human, U Von Oppell, E Becerra, DH Boehm, A Rose, R Fasol and et al.
J Heart Transplant (1987) 6(6): 317-23
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

BA Reichart, HC Reichenspurner, JA Odell, DK Cooper, D Novitzky, PA Human, UO Von Oppell, EA Becerra, DH Boehm and AG Rose
S Afr Med J (1987) 72(11): 737-9
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

BA Reichart, PA Human, AG Rose, D Novitzky and DK Cooper
J Heart Transplant (1987) 6(4): 238-43
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.

Prolongation of cardiac xenograft (vervet monkey to baboon) function by a combination of total lymphoid irradiation and immunosuppressive drug therapy

DK Cooper, PA Human and B Reichart
Transplant Proc (1987) 19(6): 4441-2