Citation: van den Heever, J.J.; Jordaan, C.J.; Lewies, A.; Goedhals, J.; Bester, D.; Botes, L.; Dohmen, P.M.; Smit, F.E. Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts. Polymers 2022, 14, 3036. https:// doi.org/10.3390/polym14153036 Academic Editors: Antonia Ressler and Inga Urlic Received: 19 May 2022 Accepted: 20 June 2022 Published: 27 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). polymers Article Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts Johannes J van den Heever 1, * , Christiaan J Jordaan 1 , Angélique Lewies 1 , Jacqueline Goedhals 2 , Dreyer Bester 1 , Lezelle Botes 3 , Pascal M Dohmen 1,4 and Francis E Smit 1 1 Department of Cardiothoracic Surgery, Faculty of Health Sciences, University of the Free State (UFS), P.O. Box 339 (Internal Box G32), Bloemfontein 9300, South Africa; jordaancj@ufs.ac.za (C.J.J.); lewiesa@ufs.ac.za (A.L.); besterd@ufs.ac.za (D.B.); pascal.dohmen@med.uni-rostock.de (P.M.D.); smitfe@ufs.ac.za (F.E.S.) 2 Department of Anatomical Pathology, Faculty of Health Sciences, University of the Free State (UFS), P.O. Box 339 (Internal Box G32), Bloemfontein 9300, South Africa; goedhalsj@ufs.ac.za 3 Department of Health Sciences, Central University of Technology, Free State (CUT), Private Bag X20539, P.O. Box 339 (Internal Box G32), Bloemfontein 9300, South Africa; botesl@cut.ac.za 4 Klinikdirektor (k), Klinik und Poliklinik für Herzchirurgie, Universitätsmedizin Rostock, Schillingallee 35, 18057 Rostock, Germany * Correspondence: vdheeverjj@ufs.ac.za; Tel.: +27-834614052 Abstract: 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 uni- form 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. Keywords: homografts; ischaemic harvesting; decellularization; cryopreservation; glutaraldehyde-fixation 1. Introduction End-stage heart valve disease mandates the repair or replacement of a patient’s dis- eased heart valve/s with either mechanical or biological valve prostheses. Mechanical prostheses demonstrate superior durability and longevity in patients; however, recipients require lifelong anticoagulation therapy. In contrast, bioprosthetic valves (including glu- taraldehyde (GA)-fixed porcine valves or bovine pericardium mounted onto a frame, free xenograft valves, or donor homograft valves) do not require continuous anticoagulation therapy but have limited durability and require more frequent reoperation [1]. Currently, cryopreserved pulmonary homografts remain the valve of choice for the replacement of the native pulmonary valve in the Ross procedure [2], as well as for the reconstruction of the right ventricle outflow tract (RVOT) in children with congenital abnormalities [3]. Un- fortunately, the early degeneration of these homografts occurs in younger patients [4], and there is a lack of availability, especially for smaller-sized conduits suitable for neonates [5]. According to the internationally accepted guidelines, homografts from either beating or non-beating heart donors should be harvested and processed within 24 h after death to retain maximum cell viability. This guideline restricts the available post-mortem donor Polymers 2022, 14, 3036. https://doi.org/10.3390/polym14153036 https://www.mdpi.com/journal/polymers