Matrix Stability and Composition of Cryopreserved and Decellularized Heart Valves
K. Theodoridis, J. Müller, R. Ramm, T. Goecke, S. Korossis, I. Tudorache, S. Cebotari, A. Haverich, A. Hilfiker
HannoverMedical School, Division of Thoracic and Cardiovascular Surgery, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
For heart valve replacement therapy, non-fixed decellularized allogeneic heart valves seem to be grafts of choice due to their minimal immunological burden and their ability for growth adaption. The current lack of donor material demands a method for long-term storage, making an optimal patient-matched use possible. Thus, we analysed the impact of cryopreservation, either before or after decellularization, on the structure of porcine pulmonary valves.
Porcine pulmonary valves were either cryopreserved (-150°C) and then decellularized (0.5% TritonX-100 for 24 h, followed by 0.5% SDS for 24 h) (CD) or first decellularized and afterwards cryopreserved (DC). CD and DC valves were histologically, biochemically and biomechanically compared to only decelluarized (D), to only cryopreserved (C) and to fresh non-treated valves (F).
Biomechanical stability was analyzed applying uniaxial tensile-testing to samples of truncus pulmonalis in the circumferential direction. While differences were seen in tissue thickness of all treated valves compared to F valves, only D valves showed significant differences to F valves regarding UTS and E modulus. CD and DC valves show no significant difference when compared to each other or to F valves.
HE- staining revealed no intact nuclei in DC and D valves, while small residues of nuclei were found in CD valves. DNA measurement in lyophilized valvular wall also revealed, that decellularization resulted in a strong reduction of DNA content, DC valves showed significantly less DNA than CD valves.
Typical three-layered structure was maintained in all grafts as seen by Movat´s pentachrome staining. Blue staining evident for Glucosaminoglycans (GAGs) seemed reduced in D, DC and CD. Those grafts also revealed significantly reduced chondroitin sulphate by biochemical accession of lyophilized valvular wall, indicating a reduction of GAGs by decellularization. The increase of hydroxyproline in C, DC and CD can be explained not by loss of collagens, but by the loss of cells and the shift of the matrix composition towards extracellular matrix proteins, as no reduction was observed in histology. (yellow: collagen, blue: GAGs)
SDS, a detergent used in decellularization, was detected in all decellularized grafts. After in vitro culture of porcine aortal endothelial cells (RFP-expression) on DC and CD cusps for 3 days in endothelial growth medium (20% FCS) a monolayer formed on the surface was found in both groups equally confluent and comparable to the monolayer created on the control culture plate. Thus, remaining SDS did not prevent cell growth and the endothelial cells maintained their characteristics as demonstrated by the expression of VE-cadherin in cell-cell-contacts.
DC and CD valves resemble histologically native grafts in ECM-composition and bear similar biomechanical properties. In spite of remaining SDS in grafts´ matrix, no harmful effect was evident when seeded with allogeneic cells. While cryopreservation did not alter the ECM composition of the heart valve, decellularization itself removed GAGs. Although decellularization after cryopreservation was effective, decellularization prior to cryopreservation led to a higher reduction of nuclei. Data show that DC should be preferred to CD, which is also more practical in clinical use. In vivo testing for long term stability needs to be conducted, especially before considering decellularizing valves already cryopreserved in cryobanks.