The impact of extended harvesting times on tissue integrity of cryopreserved ovine pulmonary homograts

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Bester, Dreyer
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University of the Free State
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.
Afrikaans: Die gebruik van aorta-homotransplantate in kardiale chirurgie het deur die pogings van die baanbrekers Donald Ross en Barratt-Boyes (Ross, 1962; Barratt-Boyes, 1964) gewildheid bereik en tot vandag toe nog is pulmonale homotransplantate die eerste keuse wanneer dit kom by die gebruik van kleppe vir die rekonstruksie van die regterventrikel-uitvloeikanaal (RVUK) wat noodsaaklik is in die behandeling van algemene kongenitale hart-toestande. Aanvanklik is homotransplantate oor die algemeen binne twee-en-sewentig uur na afsterwe in ‘n nie-steriele omgewing van kadawers geoes en daarna vars gepreserveer in ‘n steriele antibiotiese middel teen 4°C. Hierdie homotransplantate is dan binne weke vanaf verkryging gebruik. Kriobewaring is deur Marc O’Brien (O’Brien et al.,1987) gewild gemaak en het aanleiding gegee tot die totstandkoming van homotransplantaatbanke. Daar is aangevoer dat hierdie kleppe ‘n mate van lewensvatbaarheid behou, wat hul langtermyn bestendigheid na inplanting verhoog. Vars, onverwerkte kleppe wat onder steriele toestande van kloppende-hart skenkers of kort na afsterwe geoes is, is kort daarna ingeplant (onverwerk) (Yacoub et al., 1995). Hierdie studies het tot die heengaan van kadawerprogramme gelei en programme wat die kriobewaring van homotransplantate wat geoes is van kloppende-hart skenkers, of minder as ses ure tot ‘n maksimum van vier-en-twintig uur na afsterwe voorstaan, het die norm geword. Dit het egter ook terselfdertyd duidelik geword dat die immuunreaksie op weefsel-lewensvatbaarheid, veral lewensvatbare endoteel, tot vroeër verwerping van homotransplantate gelei het, veral wat kinders aanbetref (Yankah et al., 1995). Langtermyn resultate van vars, antibioties gesteriliseerde kleppe wat teen 4°C bewaar is en met vroeë kriobewaring van lewensvatbare kleppe vergelyk is, het boonop nie daarin geslaag om aanvanklike verwagtings te bevestig of te ondersteun nie en was soortgelyk in verskeie studies, noemenswaardig dié in 2001 deur O’Brien et al. Uit ‘n hele aantal uitplanting-studies is dit afgelei dat homotransplantate binne maande na inplanting nie-lewensvatbaar en hoofsaaklik asellulêr word, wat dit wesentlik nie-lewensvatbare raamwerke maak (Mitchell et al., 1998, Koolbergen et al., 2002). Die primêre rol van immunologiese prosesse in die oorlewing van homotransplantate is dus bevraagteken. Die skade-effek van kriobewaring op homotransplantaatweefsel gedurende die kriobewaringsproses is ook beskryf (Schenke-Layland et al., 2006). Gedurende die laaste vyftig jaar van die bestaan van homotransplantaatbanke, het kriobewaring sodoende die gekose tegniek gebly met verskeie studies wat te kenne gegee het dat vroeë post mortem–oes ‘n voordelige effek op die oorlewing van homotransplantate na inplanting het. Dit kon egter nie deur verskeie langtermyn studies bewys word nie. Die verwoestende uitwerking van ware lewensvatbare kleppe en geassosieerde immuun-prosesse op die oorlewing van homotransplantate is ook beskryf. Verskeie studies het ook bewys dat uitgeplante kleppe essensieel asellulêr en dus onlewensvatbaar was. In realiteit is dit heel algemeen vir die tydperk, vanaf ‘n post mortem-kardiektomie of ontvangs deur die homotransplantaatbank voor die verwerking en kriobewaring, om na agt-en-veertig uur verleng te word soos gerapporteer is in the Directory of European Cardiovascular Tissue Banks and Tissue Bank Addresses World Wide (2013). Dit suggereer dat die onafwendbare koue iskemiese tyd wat kriobewaring voorafgaan buitendien met drie tot vier dae verleng word – in ‘n aansienlike aantal gevalle. Dít, tesame met die kompleksiteit van kwessies rondom die lewensvatbaarheid van homotransplantate asook gebrekkige bewyse in publikasiereekse ten opsigte van die langtermyn voordele van die lewensvaatbaarheid van homotransplantate, dwing die vraag na die herevaluering of nie van kadawerprogramme af. Die homotransplantaatbank in Bloemfontein is ‘n bykans algehele kadawer-gebaseerde program, met gemiddelde post mortem-oestye wat vier-en-twintig uur oorskry (gemiddeld dertig uur). Ongepubliseerde kliniese resultate wat die uitkoms van pulmonale homotransplantate in die regterventrikel-uitvloeikanaal van kinders jonger as veertien jaar evalueer, kon nie ‘n verskil in die oorbodigheid van heroperasie toon tussen homotransplantate wat meer as vier-en-twintig uur na afsterwe geoes is en homotransplantate wat minder as vier-en-twintig uur na afsterwe geoes is nie. Aangesien die Bloemfontein homotransplantaatbank huidiglik die enigste homotransplantaatbank in Suid-Afrika is, is ‘n aantal studies in ‘n skaapmodel aangepak om die bank se praktyk, en per implikasie, die kadawergebaseerde program van die bank te staaf. Vier studies wat die impak van verlengde post mortem-oestye en kriobewaring op homotransplantaatweefsel se integriteit en in vivo-prestasie evalueer, word hier voorgelê. Tydens die eerste studie, word die impak van verlengde post mortem-oestye beskryf in skape waarvan die kriobewaarde, pulmonale homotransplantate vier-en-twintig uur, agt-en-veertig uur en twee-en-sewentig uur post mortem geoes is. In die in vitro-studies wat die morfologie en weefselsterkte voor inplanting evalueer, kon geen verskil tussen die groepe waargeneem word nie, tot en met twee-en-sewentig uur post mortem-oes. In die in vivo-studies kon geen verskil in kliniese werking, immunologiese prosessse, morfologie, weefselsterkte en verkalking na inplanting op 180 dae opgemerk word nie. Daaruit is afgelei dat post mortem-oestye van homotransplantate met veiligheid tot en met twee-en-sewentig dae verleng kan word. In die tweede studie word die morfologie van onbewerkte en kriobewaarde pulmonale homotransplantaatklepsuile met post mortem-oestye van tot en met twee-en-sewentig uur beskryf. Die impak van kriobewaring op spesifiek klepsuile is in ‘n kontrolegroep beskryf, sowel as in weefsel wat vier-en-twintig uur, agt-en-veertig uur en twee-en-sewentig uur na afsterwe geoes is. Daar kon weereens geen impak deur die verlenging van post mortem-oestye waargeneem word nie, behalwe vir ‘n toename in edeem op die transmissie elektron mikroskopie (TEM) in die twee-en-sewentig uur-groep. Deur middel van Picrosirius-rooi is daar gedemonstreer dat kriobewaring ‘n samepersende en afplattende effek op kollageen in alle groepe het. Kollageenskeuring is op die transmissie elektron mikroskopie van alle kriobewaarde groepe waargeneem. Dit het gedemonstreer dat kriobewaring ‘n onmiddellike impak op weefselmorfologie het en dat dit meer ultra-strukturele weefselskeuring as die verlenging van post mortem-oestye veroorsaak. Tydens die derde studie is die impak van verlengde post mortem-oestye in vitro bestudeer deur onverwerkte en kriobewaarde klepsuile ten opsigte van weefselsterkte te vergelyk. Geen verskil tussen die onbewerkte klepsuile van die kontrolegroep, die vier-en-twintig uur-, agt-en-veertig uur- en twee-en-sewentig uur-groepe ten opsigte van sterkte kon deur middel van rekbaarheidsterktetoeste, Young se moduletoets en termiese denaturering temperatuur waargeneem word nie. Benewens dit, kon geen verskil tussen klepsuile wat na vier-en-twintig uur, agt-en-veertig uur en twee-en-sewentig uur post mortem geoes, verwerk en kriobewaar is, onderskei word nie. Wanneer dit met onverwerkte klepsuile vergelyk is, is rekbaarheidsterkte moontlik deur kriobewaring verminder, maar dit was nie in enige van die gevalle statisties beduidend nie. In die finale studie is ‘n homotransplantaatgroep wat agt-en-veertig uur na afsterwe geoes is bewerk en kriobewaar vir inplantingsdoeleindes. Dit boots spesifiek die kliniese omstandighede van kadawerprogramme na. Die doelwit van hierdie studie was om die stabiliteit van homotransplantaatklepsuilweefsel na twee tydperke van inplanting te evalueer. Kontroleweefsel (verwerk, kriobewaar en ontdooi) is met weefsel wat na twee weke en 180 dae in die skaapmodel uitgeplant is vergelyk. Ten spyte van die skeuringseffek van kriobewaring wat deur transmissie–elektronmikroskopie in alle groepe gedemonstreer is, het die weefsel teen 180 dae regdeur die tydperk bestendig en stabiel gebly met normale kliniese funksie en minimale verkalking. Danksy die voorgenoemde skaapmodelstudies wat uitgevoer is ten einde eksperimentele bewyse te lewer dat dit veilig is om koue post mortem-oestye te verleng, is daar afgelei dat in vitro- en in vivo-studies geen nadelige effek getoon het op die weefselintegriteit van homotransplantate wat ten minste agt-en-veertig uur en moontlik selfs tot twee-en-sewentig uur post mortem geoes is. Die veiligheid van homotransplantate wat agt-en-veertig uur na afsterwe geoes is en daarna kriobewaarde pulmonale homotransplantate is spesifiek bestudeer deur die menslike kliniese omstandighede na te boots waartydens die stabiliteit van die homotransplantate tydens twee studietydperke wel bevestig is. Daar word tot die slotsom gekom dat hierdie studies eksperimentele, wetenskaplike bewyse lewer om post mortem homotransplantaat-oestye tot na ten minste agt-en-veertig uur te verleng. Hierdie studies voorsien verder kollektief eksperimentele steun om menslike kadawer homotransplantaatskenkerbanke te herevalueer in ‘n poging om internasionale homotransplantaat-tekorte te verminder.
Heart -- Surgery, Aorta -- Surgery, Congenital heart disease, Donation of organs, tissues, etc., Thesis (Ph.D. (Cardiothoracic Surgery))--University of the Free State, 2017