Development and testing of a transcatheter heart valve with reduced calcification potential.

Introduction: Patients from developing countries who require heart valve surgery are younger and have less access to open heart surgery than those from developed countries. Transcatheter heart valves (THVs) may be an alternative but are currently unsuitable for young patients because of their inadequate durability. We developed and tested a THV utilizing two new types of decellularized bovine pericardial leaflets in an ovine model. Methods: The two decellularized tissues [one with a very low dose (0.05%) of monomeric glutaraldehyde (GA) fixation and detoxification (DF) and the other without glutaraldehyde (DE)] were compared to an industry standard [Glycar-fixed with the standard dose (0.625%) of glutaraldehyde]. THVs were manufactured with the three tissue types and implanted in the pulmonary position of nine juvenile sheep for 180 days. Baseline and post-explantation evaluations were performed to determine the hemodynamic performance of the valves and their dynamic strength, structure, biological interaction, and calcification. Results: Heart failure occurred in one animal due to incompetence of its Glycar valve, and the animal was euthanized at 158 days. The gradients over the Glycar valves were higher at the explant than at the implant, but the DE and DF valves maintained normal hemodynamic performance throughout the study. The DF and DE tissues performed well during the mechanical testing of explanted leaflets. Glycar tissue developed thick pannus and calcification. Compared to Glycar, the DF tissue exhibited reduced pannus overgrowth and calcification and the DE tissue exhibited no pannus formation and calcification. All tissues were endothelialized adequately. There was a striking absence of host ingrowth in the DE tissue leaflets, yet these leaflets maintained integrity and mechanical function. Conclusion: In the juvenile sheep THV model, Glycar tissue developed significant pannus, calcification, and hemodynamic deterioration. Using a very low dose of monomeric GA to fix the decellularized bovine pericardium yielded less pannus formation, less calcification, and better hemodynamic function. We postulate that the limited pannus formation in the DF group results from GA. Bovine pericardium decellularized with our proprietary method resulted in inert tissue, which is a unique finding. These results justify further development and evaluation of the two decellularized tissue types in THVs for use in younger patients.

Monomeric glutaraldehyde fixation and amino acid detoxification of decellularized bovine pericardium for production of biocompatible tissue with tissue-guided regenerative potential.

The effect of monomeric glutaraldehyde fixation and amino acid detoxification on biocompatibility and tissue-guided regenerative potential of decellularized bovine pericardium was evaluated. The degree of cross-linking, porosity, enzymatic degradation, alpha-galactosyl content, the efficacy of detoxification, and cytotoxicity towards human epithelial cells were assessed. Tissue was subcutaneously implanted for eight weeks in male juvenile Sprague-Dawley rats, and mechanical properties, host cell infiltration, and calcification were evaluated. Three groups were compared i) decellularized tissue, ii) decellularized, monomeric glutaraldehyde fixed and amino acid detoxified tissue, and iii) commercial glutaraldehyde fixed non-decellularized tissue (Glycar®) (n = 6 rats per group). The fixation process gave a high degree of cross-linking (>85%), and was resistant to enzymatic degradation, with no significant effect on porosity. The detoxification process was effective, and the tissue was not toxic to mammalian cells in vitro. Tissue from both decellularized groups had significantly higher (p < 0.05) porosity and host cell infiltration in vivo. The process mitigated calcification. A non-significant decrease in the alpha-galactosyl content was observed, which increased when including the alpha-galactosidase enzyme. Mechanical properties were maintained. The fixation and detoxification process adequately removes free aldehyde groups and reduces toxicity, preventing enzymatic degradation and allowing for host cell infiltration while mitigating calcification and retaining the mechanical properties of the tissue. This process can be considered for processing decellularized bovine pericardium with tissue-guided regeneration potential for use in cardiovascular bioprostheses; however, methods of further reducing antigenicity, such as the use of enzymes, should be investigated.

Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts.

Homografts are routinely stored by cryopreservation; however, donor cells and remnants contribute to immunogenicity. Although decellularization strategies can address immunogenicity, additional fixation might be required to maintain strength. This study investigated the effect of cryopreservation, decellularization, and decellularization with additional glutaraldhyde fixation on the strength and structure of ovine pulmonary homografts harvested 48 h post-mortem. Cells and cellular remnants were present for the cryopreserved group, while the decellularized groups were acellular. The decellularized group had large interfibrillar spaces in the extracellular matrix with uniform collagen distribution, while the additional fixation led to the collagen network becoming dense and compacted. The collagen of the cryopreserved group was collapsed and appeared disrupted and fractured. There were no significant differences in strength and elasticity between the groups. Compared to cryopreservation, decellularization without fixation can be considered an alternative processing technique to maintain a well-organized collagen matrix and tissue strength of homografts.

Comparison of the function and structural integrity of cryopreserved pulmonary homografts versus decellularized pulmonary homografts after 180 days implantation in the juvenile ovine model.

Homograft availability and durability remain big challenges. Increasing the post-mortem ischaemic harvesting time beyond 24 h increases the potential donor pool. Cryopreservation, routinely used to preserve homografts, damages the extracellular matrix (ECM), contributing to valve degeneration. Decellularization might preserve the ECM, promoting host-cell infiltration and contributing towards better clinical outcomes. This study compared the performance of cryopreserved versus decellularized pulmonary homografts in the right ventricle outflow tract (RVOT) of a juvenile ovine model. Homografts (n = 10) were harvested from juvenile sheep, subjected to 48 h post-mortem cold ischaemia, cryopreserved or decellularized and implanted in the RVOT of juvenile sheep for 180 days. Valve performance was monitored echocardiographically. Explanted leaflet and wall tissue evaluated histologically, on electron microscopical appearance, mechanical properties and calcium content. In both groups the annulus diameter increased. Cryopreserved homografts developed significant (¾) pulmonary regurgitation, with trivial regurgitation (») in the decellularized group. Macroscopically, explanted cryopreserved valve leaflets retracted and thickened while decellularized leaflets remained thin and pliable with good coaptation. Cryopreserved leaflets and walls demonstrated loss of interstitial cells with collapsed collagen, and decellularized scaffolds extensive, uniform ingrowth of host-cells with an intact collagen network. Calcific deposits were shown only in leaflets and walls of cryopreserved explants. Young fibroblasts, with vacuoles and rough endoplasmic reticulum in the cytoplasm, repopulated the leaflets and walls of decellularized scaffolds. Young's modulus of wall tissue in both groups increased significantly. Cryopreserved valves deteriorate over time due to loss of cellularity and calcification, while decellularized scaffolds demonstrated host-cell repopulation, structural maintenance, tissue remodelling and growth potential.

Cadaver donation: structural integrity of pulmonary homografts harvested 48 h post mortem in the juvenile ovine model.

Cryopreserved pulmonary homograft (CPH) implantation remains the gold standard for reconstruction of the right ventricular outflow tract (RVOT). Harvesting homografts < 24-h post mortem is the international norm, thereby largely excluding cadaveric donors. This study examines the structural integrity and stability of ovine pulmonary homografts harvested after a 48-h post mortem period, cryopreserved and then implanted for up to 180 days. Fifteen ovine pulmonary homografts were harvested 48-h post mortem and cryopreserved. Five CPH served as a control group (group 1; n = 5). CPH were implanted in the RVOT of juvenile sheep and explanted after 14 days (group 2; n = 5) and 180 days (group 3; n = 5). Leaflet integrity was evaluated by strength analysis, using tensile strength (TS), Young's modulus (YM) and thermal denaturation temperature (Td), and morphology, including haematoxylin and eosin (H&E), Picrosirius red staining, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and von Kossa stains. Echocardiography confirmed normal function in all implants. In explants, no reduction in TS, YM or Td could be demonstrated and H&E showed mostly acellular leaflet tissue with no difference on Picrosirius red. TEM demonstrated consistent collagen disruption after cryopreservation in all three groups, with no morphological deterioration during the study period. von Kossa stains showed mild calcification in group 3. No deterioration of structural integrity could be demonstrated using strength or morphological evaluations between the controls and implant groups over the study period. Extending the post mortem harvesting time of homografts beyond 24 h did not appear to negatively affect the long-term performance of such transplanted valves in this study.

Does prolonged post-mortem cold ischemic harvesting time influence cryopreserved pulmonary homograft tissue integrity?

This study investigated cryopreserved pulmonary homograft (CPA) structural integrity after prolonged cold ischemic harvesting times in a juvenile sheep model. Three groups with different post-mortem cold ischemic harvesting times were studied, i.e. Group 1 (24 h, n = 10); group 2 (48 h, n = 10); group 3 (72 h, n = 10). In each group, 5 CPAs were studied in vitro after cryopreservation and thawing. The other 5 CPAs were implanted in juvenile sheep for a minimum of 180 days. Serology samples were obtained and echocardiography was performed before euthanasia. Hematoxylin and eosin (H&E), scanning electron microscopy (SEM), von Kossa, Picrosirius red, α-actin, immunohistochemistry [von Willebrand factor (vWF), CD4, CD31 and CD34] and calcium content analyses were performed on explanted CPAs. The in vitro and in vivo studies failed to demonstrate any change in tensile strength, Young's Modulus and thermal denaturation (Td) results between the groups. SEM demonstrated a reduction in endothelial cells (50 % at 24 h, 60.9 % at 48 h and 40.9 % at 72 h), but H&E could not demonstrate autolysis in any CPA in vitro. All cultures were negative. In the explanted groups, IgE, IgM and IgG results were inconclusive. Echocardiography demonstrated normal valve function in all groups. H&E and Picrosirius red staining confirmed tissue integrity. vWF, CD31 and CD34 staining confirmed a monolayer of endothelial cells in all explanted valves. Calcium content of explanted CPA leaflets was similar. This experimental study supports the concept of prolonging the cold ischemic harvesting time of cryopreserved homografts to reduce homograft shortage.

Cardiac allografts: a 24-year South African experience.

The history of using homologous cardiac valves dates back more than 30 years. Through the years emphasis was placed on the optimization of graft retrieval, preservation techniques and clinical application. A cardiac homograft valve bank was established at the Department of Cardiothoracic Surgery, University of the Free State, Bloemfontein in 1982. A retrospective analysis was performed on all allograft data since 1984. Since the first valve was successfully procured and transplanted in 1984, 2,540 aortic and pulmonary homografts were harvested from 1,792 donors, of which 1,545 [989 (64%) aortic and 556 (36%) pulmonary] were released for clinical use. Valves were discarded for various reasons, the main reasons being Human Immunodeficiency Virus (32.4%), Hepatitis B (9.6%) and venereal diseases (8.9%). The mean donor age was 26.98 years with a male predominance of 1,368 males versus 424 females. The average ischemic time was 33 h mainly due to medico-legal autopsies exceeding the desired 24 h time limit. The valves were disinfected in an antibiotic cocktail of Mefoxin, Piperacillin, Amikacin and Amphotericin B prior to cryopreservation. The surgical procedures utilizing the majority of homografts were aortic valve replacements (42.9%), aortic root replacements (19.3%) and right ventricular-pulmonary artery conduits (33.3%). The bank also supplied 23 other centers with homografts (402 aortic and 301 pulmonary). The Bloemfontein bank has established itself over the years as a viable and functional cardiac homograft bank. However, with increasing activity in the procurement arena and widened applications in the operating room the role of the homograft seems assured but availability still remains a major concern.