ABSTRACT
Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries. Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls. Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold. Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology.
ABSTRACT
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.
