Bioreactor conditioning of valve scaffolds seeded internally with adult stem cells

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.

TGF-β Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish

“…Multiple cell types have been proposed as VIC precursors including ECs, myofibroblasts, and several stem cell types (Schenke-Layland et al, 2003;Sutherland et al, 2005;Dohmen et al, 2006;Schmidt et al, 2010;Harrington et al, 2011;Kennamer et al, 2016). Here, we show that in uninjured valves a small number of cells derived from ECs, the main source of VICs in amniotes (Snarr et al, 2008;Jain et al, 2011;Wessels et al, 2012;MacGrogan et al, 2014;Liu et al, 2018), express nfatc1, suggesting that some ECs undergo EndoMT and contribute to the replacement and maintenance of VICs in homeostatic conditions.…”

Culture Into Perfusion-Assisted Bioreactor Promotes Valve-Like Tissue Maturation of Recellularized Pericardial Membrane

“…Despite this work is not the first to employ a bioreactor system to assess cell viability inside a decellularized scaffold for cardiac valve engineering (33,34), it is the first to address the problem of valve maturation of a fully recellularized tissue using a systematic proteomic approach. In fact, our mass spectrometry data clearly showed that compared to the native pericardial membrane, a number of proteins were upregulated in the material as a result of de-novo protein synthesis by the introduced valve cells.…”

“…We are now planning new studies with human-derived bone marrow/adipose-derived mesenchymal stem cells and scaled up bioreactors able to efficiently repopulate decellularized pericardial patches with surgical size. It will be also interesting to assess whether mounting the recellularized pericardial material onto stent posts and valve pulse duplicators will promote further biological maturation of the living pericardium material due to mechanical conditioning (41), and will maintain the viability of the cells under tissue mechanical stress (33), as a step forward in translation of the living pericardium material for clinical employment.…”

Oscillatory fluid-induced mechanobiology in heart valves with parallels to the vasculature

“…Mechanical heart valves require patients to be on life-long anticoagulant medication; bioprosthetic valves are less durable and more prone to re-calcification. Scientists and engineers have been exploring alternative treatments such as tissue engineered heart valves using bioreactor systems to deliver the proper flow, flex, and stretch conditioning (116,117).…”