Smit, F. E.Dohmen, P. M.Van den Heever, Johannes Jacobus2021-03-242021-03-242020-02http://hdl.handle.net/11660/10979The availability of pulmonary homografts with improved biomechanical properties, tissue stability, reduced calcification and improved durability for right ventricular outflow tract (RVOT) reconstruction is desired. In paediatric patients, a valve with growth potential will be advantageous. Extending the post-mortem ischaemic time will enlarge the donor pool. Cryopreservation of homografts remains the gold standard, but it damages the extracellular matrix (ECM) and reduces the cellularity, contributing to early valve degeneration. Decellularization of homografts might reduce immunogenicity, promote recellularization and tissue remodeling, maintain mechanical stability and improve clinical outcomes. The decellularization process should not compromise the durability and strength of the homograft, and alternative stabilization of the scaffold might be required. The current study evaluated the effect of the further processing of pulmonary homografts, following a 48 h cold ischaemic postmortem harvesting time, on the structural integrity and function when implanted in the RVOT position in the juvenile ovine model. Sheep pulmonary homografts (n = 30) were subjected to 48 h cold ischaemia to simulate the clinical homograft donor circumstances, and equally divided into three groups. Homografts in group 1 were cryopreserved, decellularized in group 2 and decellularized, GA-fixed and detoxified in group 3. Decellularization consists of a multi-detergent and enzymatic protocol with numerous washout steps, and additional fixation and detoxification were done with EnCap technology. The study was divided into three parts. In study 1, the histological (DAPI, H&E, von Kossa, Modified von Gieson, SEM, TEM) and mechanical (TS and YM) properties of the processed homografts (n = 15, 5 per group) were compared. Study 2 involved implantation of cryopreserved and decellularized pulmonary homografts (n = 5 per group) in the RVOT of juvenile sheep for 180 days, monitored with echocardiography and compared on histology, mechanical properties and calcification after explantation. Study 3 involved the same parameters, however, decellularized and decellularized plus EnCap treated homografts (n = 5 per group) were implanted and compared. Cryopreserved homografts demonstrated collapsed and disrupted/fractured collagen with cells and cellular remnants. Homografts in the decellularized group were acellular with large interfibrillar spaces and a loosely arranged collagen network, while decellularized plus EnCap treated homograft were acellular with a compacted collagen network. Decellularization did not reduce tensile strength and tissue stiffness, but EnCap treatment did increase tissue stiffness. Implanted cryopreserved homografts demonstrated significant regurgitation due to leaflet thickening and retraction, loss of interstitial cells, calcification and increased tissue stiffness. Decellularized homografts showed increased annulus diameter with trivial regurgitation, excellent haemodynamics, remained soft and pliable, recellularized extensively with young fibroblasts exhibiting rough endoplasmic reticulum, and mitigated calcification. Decellularized and EnCap treated homografts became rigid and stenotic, showed poor haemodynamic characteristics, development of bacterial endocarditis and premature death, no leaflet recellularization, and fibrous encapsulation. Cryopreserved homografts remain the valve of choice for RVOT reconstruction surgery, however, cryopreservation causes cell death and collagen disruption, and loss of cellularity and calcification during implantation, which will result in early valve degeneration. Our proprietary decellularization protocol proved to be effective for complete decellularization of pulmonary homografts with a post-mortem ischaemic time of 48 h, while maintaining a well-organized collagen matrix and tissue strength and stiffness. Implanted decellularized homografts repopulated extensively without signs of inflammation, maintained structural integrity and strength, calcification was mitigated, and the potential for remodeling and growth in size with somatic growth was observed. Additional fixation of the decellularized homograft scaffold will be counterproductive in growing individuals, and should only be performed on adult size homografts where valve growth is not required. GA-fixation restricts valve repopulation with host cells and tissue remodeling, and defies the purposes and advantages of decellularization. Additional fixation may not be necessary when using decellularization methods that achieve complete acellularity without altering the ECM structure and mechanical properties of homografts. Successful decellularization of donor homograft heart valves and other collagenous tissues holds exciting new prospects and possibilities for tissue processing, and can open a new era in supply of substitution valves and tissues with improved properties and advantages to medical patients in South Africa.enThesis (Ph.D. (Cardiothoracic Surgery))--University of the Free State, 2020CryopreservationPulmonary homograftsIschaemic timeValve degenerationDecellularizationRecellularizationTissue remodelingCalcificationRight ventricular outflow tractTissue stabilizationThesisUniversity of the Free State