There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for wholeroot decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.

The goal of this study was to test the hypothesis that stem cells, as a response to valve-specific extracellular matrix “niches” and mechanical stimuli, would differentiate into valvular interstitial cells (VICs). Porcine aortic root scaffolds were prepared by decellularization. After verifying that roots exhibited adequate hemodynamics in vitro, we seeded human adipose-derived stem cells (hADSCs) within the interstitium of the cusps and subjected the valves to in vitro pulsatile bioreactor testing in pulmonary pressures and flow conditions. As controls we incubated cell-seeded valves in a rotator device which allowed fluid to flow through the valves ensuring gas and nutrient exchange without subjecting the cusps to significant stress. After 24 days of conditioning, valves were analyzed for cell phenotype using immunohistochemistry for vimentin, alpha-smooth muscle cell actin (SMA) and prolyl-hydroxylase (PHA). Fresh native valves were used as immunohistochemistry controls. Analysis of bioreactor-conditioned valves showed that almost all seeded cells had died and large islands of cell debris were found within each cusp. Remnants of cells were positive for vimentin. Cell seeded controls, which were only rotated slowly to ensure gas and nutrient exchange, maintained about 50% of cells alive; these cells were positive for vimentin and negative for alpha-SMA and PHA, similar to native VICs. These results highlight for the first time the extreme vulnerability of hADSCs to valve-specific mechanical forces and also suggest that careful, progressive mechanical adaptation to valve-specific forces might encourage stem cell differentiation towards the VIC phenotype.

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Introduction. Xenogeneic tissues decellularization represents the obtaining process of extracellular matrix derived scaffolds. Most antigens being cell based, non-immunogenicity is obtained by cells removal. Scaffolds are temporary structures with biologic and mechanical role. Scaffolds, stem cells and bioreactors represent premise of regenerative medicine, aiming towards the ideal valvular substitute. In previous studies, we decellularized pulmonary valves root by immersion histology revealing cellular residue, requiring a more efficient approach. We hypothesized that immersion is insufficient and thus a pressure gradient was added. Material and Method. This is part of a grant approved by the UMFTS. Eleven porcine pulmonary valves were included in the study: n=6 underwent immersion decellularization and n=5 were cyclically perfused with a 20-25mmHg pressure gradient during a 10-day protocol. The acellular valves obtained underwent a quality control using DAPI (4′,6-diamidino-2-phenylindol) nuclear staining, histological Haematoxylin-Eosin, DNA extraction and quantification, harvested from different structural levels: arterial wall, sinus, cusp. Results. Histological assessments highlighted integrity of extracellular matrix in both groups and overall cells absence at the different levels of valvular structures analyzed. Immersion decellularized valves exhibited DAPI positive structures identified as potential residual nucleic material. Comparatively, the perfusion decellularized valves, lacked in those structures, result confirmed by DNA extraction and quantitation procedure. Conclusions. Perfusion decellularization represents a feasible approach to obtain acellular cardiac valvular scaffolds derived from the extracellular matrix, being superior to immersion decellularization method. Their nonimmunogenic potential is underlined by total absence of nuclei. The process is fast, allowing production of an abundant number of valvular biomaterials in a short time.