Doctoral Degrees (Cardiothoracic Surgery)
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Browsing Doctoral Degrees (Cardiothoracic Surgery) by Author "Bester, Dreyer"
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Item Open Access The impact of extended harvesting times on tissue integrity of cryopreserved ovine pulmonary homograts(University of the Free State, 2017-02) Bester, Dreyer; Smit, F. E.; Botes, L.; Dohmen, P. M.English: The use of aorta valve homografts in cardiac surgery was pioneered by Donald Ross and Barratt-Boyes (Ross, 1962; Barratt-Boyes, 1964) and today, pulmonary valve homografts remain the valved conduit of choice for reconstruction of the right ventricle outflow tract (RVOT) required in the treatment of common congenital cardiac conditions. Initially, homografts were harvested from cadavers generally within seventy-two hours after death, in a non-sterile environment, and then freshly preserved in a sterile antibiotic medium at 4°C. These homografts were then used within six to eight weeks after procurement (Botes et al., 2012). Cryopreservation was popularised by Marc O’Brien (O’Brien et al., 1987), which saw the introduction of the development of homograft banks. It was claimed that these valves retained a degree of viability, which enhances long term durability after implantation. Freshly unprocessed valves that were harvested under sterile conditions from beating heart donors or within hours after death, were implanted (unprocessed) shortly afterwards (Yacoub et al., 1995). These studies resulted in the demise of cadaver programmes and programmes cryopreserving homografts harvested from beating heart donors, or less than six hours to a maximum of twenty-four hours post mortem became the norm. On the other hand, it became clear that immune response to viable tissue, especially viable endothelium, resulted in earlier rejection of homografts, especially in children (Yankah et al., 1995). Furthermore, long term results of fresh antibiotic sterilised valves stored at 4°C compared to early cryopreservation of viable valves failed to confirm or support earlier expectations and were similar in several studies, notably in that of O’Brien et al, in 2001. In, a number of explant studies it was also concluded that homografts become nonviable and essentially acellular within months of implantation and are essentially nonviable scaffolds (Mitchell et al, 1998, Koolbergen et al, 2002). The primary role of immunological processes on homograft survival was therefore questioned. The damaging effect on homograft tissue during the cryopreservation process was also described (Schenke-Layland et al., 2006). Thus, during the last fifty years of homograft banking, cryopreservation remained the technique of choice with various studies suggesting that early post mortem harvesting has a beneficial effect on homograft survival after implantation. This could however not be demonstrated in several long term studies. The deleterious effect of truly viable valves and associated immune processes on homograft survival were also described. In addition, several studies showed that explanted valves were essentially acellular and thus nonviable. In reality, the time from post mortem cardiectomy or homograft bank receipt before processing and cryopreservation commonly extend to fourty-eight hours as reported in the Directory of European Cardiovascular Tissue Banks and Tissue Bank Addresses World Wide (2013). This implies that the inevitable cold ischaemic time before cryopreservation is extended to three to four days in a significant percentage of cases anyway. This, and the complexity of issues of homograft viability as well as inconclusive long term advantages of homograft viability in published series, beg the question whether cadaver programmes should not be re-evaluated. The Bloemfontein homograft bank is an almost exclusively cadaver donor based programme, with average post mortem harvest times exceeding twenty-four hours (mean thirty hours). Unpublished clinical results evaluating outcomes of pulmonary homografts implanted in the RVOT of children less than fourteen years, could not show a difference in freedom from reoperation between homografts harvested more than twenty-four hours post mortem and those harvested less than twenty-four hours post mortem. As the Bloemfontein homograft bank is presently the only homograft bank in South Africa, it embarked on a number of experimental studies in the ovine model in order to validate its practise and by implication, also that of cadaver based programmes. Four studies are presented evaluating the impact of increased post mortem harvest times and cryopreservation on homograft tissue integrity and in vivo performance. In the first study, the impact of increased post mortem homograft harvest times is described in cryopreserved ovine pulmonary homografts harvested twenty-four hours, fourty-eight hours and seventy-two hours post mortem. In the in vitro studies evaluating the morphology and tissue strength before implantation, no differences could be observed between the groups up to seventy-two hours post mortem harvest times. In the in vivo study no differences could be discerned in clinical performance, immunological processes, morphology, tissue strength and calcification after 180 days implantation. It was concluded that post mortem harvest times of pulmonary homografts can safely be extended up to seventy-two hours. In the second study, the morphology of unprocessed and cryopreserved pulmonary homograft leaflets with post mortem harvest times up to seventy-two hours was described. The impact of cryopreservation on leaflets per se was described in a control group as well as in tissue harvested at twenty-four hours, fourty-eight hours and seventy-two hours post mortem. Once again, no impact of extended post mortem harvest times could be perceived, except for increased oedema on TEM in the seventy-two hour group. Picrosirius red staining demonstrated that cryopreservation had a compressing and flattening impact on collagen in all groups. Disruption of collagen was observed on TEM in all cryopreserved groups. It demonstrated that cryopreservation had an immediate impact on tissue morphology and produced more ultrastructural tissue disruption than extending post mortem harvest times. In the third study, the impact of increased post mortem harvest times was studied in vitro comparing unprocessed and cryopreserved leaflets in relation to tissue strength. No difference in strength using tensile strength, Young’s modulus and thermal denaturation temperture, could be observed between the control group and the twenty-four hour, fourty-eight hour and seventy-two hour groups in the unprocessed leaflets. In addition, no difference could be discerned between leaflets processed and cryopreserved after twenty-four hours, fourty-eight hours and seventy-two hours post mortem harvesting. Tensile strength was potentially reduced by cryopreservation when compared to unprocessed leaflets, but did not reach statistical significance in all instances. In the final study, a fourty-eight hour post mortem homograft harvested group was processed and cryopreserved for implantation. This mimicks the clinical circumstances of cadaver programmes. The objective of this study was to evaluate the stability of homografts’ leaflet tissue after two periods of implantation. Control tissue (processed, cryopreserved and thawed) was compared to tissue explanted after two weeks and after 180 days in the ovine model. Despite the disruptive effect of cryopreservation demonstrated by TEM in all groups, the tissue remained stable throughout the period with normal clinical function and minimal calcification at 180 days. Through these studies conducted in the ovine model in order to provide experimental evidence for the safe extension of cold post mortem harvest times, it was concluded that in vitro and in vivo studies could not reveal detrimental effects on tissue integrity up to at least fourty-eight hours and possibly to seventy-two hours post mortem harvesting. The safety of fourty-eight hour post mortem harvested and thereafter cryopreserved pulmonary homografts was specifically studied in order to mimic the human clinical scenario wherein the stability of the homografts was confirmed in two study periods. It is concluded that these studies provide experimental scientific evidence to increase post mortem homograft harvest times to at least fourty-eight hours. Furthermore, these studies collectively provide experimental support for the re-evaluation of human cadaver homograft donor banks in order to attenuate international homograft shortages.