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Development and Revitalization of a Mitral Valve Scaffold for Tissue-Engineered Regeneration

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Development and Revitalization of a Mitral Valve Scaffold for Tissue-Engineered Regeneration

Christopher deBordea, Dan Simionescua Lee Sieradc Jun Liaob, Christopher Wrightd, Agneta Simionescua

Cardiovascular Tissue Engineering and Regenerative Medicine, Department of Bioengineering, Clemson University, Clemson, SC, USA

a Clemson University, South Carolina; b Mississippi State University, Mississippi; c Aptus Bioreactors d Greenville Health System

•Mitral valve prolapse is the most common cause of regurgitation referred for surgical repair or replacement of the mitral valve in the western world, but currently available substitutes do not adequately comply with the performance and flow pattern requirements of the left ventricle.
•Mitral valve tissue engineering and regenerative medicine approaches using scaffolds, cells, and bioreactors might provide permanent implant solutions for MV surgery; they could also serve as reliable in vitro models for studying MV biology and pathology.
•An extracellular matrix-based scaffold was generated, based on the native porcine mitral valve as starting material and a technique for porcine cell removal without damaging the matrix components.
•We hypothesize that PGG, by virtue of its collagen and elastin-stabilizing abilities will preserve the composition and structure of the tissue engineered valve ECM, and protect the scaffold from matrix peptide liberation and, consequently, seeded cell activation.

Evaluation of Decellularization: Histological analysis using H&E (row 1), DAPI (row 2), and immunohistochemistry for the cellular protein actin (row 3) show complete removal of all xenogeneic nuclei and protein from both chordae tendinae and leaflets.

Evaluation of ECM Retention:

(from top): Movat’s Pentachrome and VVG stains were used to evaluate ECM quality for fresh and decellularized chordae and leaflets. Collagen and elastin were well preserved after decellularization.

(from left): IHC for basal lamina proteins collagen type IV and laminin show positive results indicating retention of basal lamina after decellularization.

Evaluation of Mechanical Properties and Protease-Resistance:

(top): Biaxial mechanical test of leaflets indicates recovery of physiological strength post-decellulariation. (left): DSC shows loss of collagen cross-links after decellularization. This is regained after PGG treatment. (below): Scaffold resistance to collagenase and elastase was compared among fresh, decellularized, and PGG-treated scaffolds. PGG greatly increased resistance to the proteases.

Scaffold Cytocompatibility and Recellularization: Externally seeded hADSC’s survived and proliferated on scaffolds. Scaffolds also allow interstitial seeding through direct injection.

Pre-differentiation of hADSC’s in vitro: hADSC’s were pre-differentiated into SMC’s and fibroblasts using growth factors. Markers for SMC’s (α-SMA, SM22, MHC) and fibroblasts (HSP-47, Prolyl 4-hydroxylase, vimentin) were expressed.

TGF-β Expression and MMP Activity in Seeded Constructs: (top, left): TGF-β expression significantly lower in PGG-treated, cell-seeded constructs. (top, right): MMP expression is significantly higher in untreated, cell-seeded constructs. (bottom, right): Silver stain shows much lower protein expression, and thus a decreased release of peptides from the seeded constructs.

Heart Valve Bioreactor Development: Heart valve bioreactor fitted for mitral valve pulsatile pressures and flow. Panel A shows complete, multi-chamber bioreactor assembly. Panels (C, E) illustrates placement of valve in system. Panels (D, F) show valve opening and closing in bioreactor.

Mitral Valve Bioreactor Development: Modifications have and are being created to utilize the mitral annular anatomy for proper force distribution. Proper tensional forces will be re-created in bioreactor to provide anatomically-similar forces for tissues.

Bioreactor Study 1: hADSC’s were seeded interstitially and constructs pre-conditioned for 4 weeks. Markers commonly found in VICs were used to evaluate differentiation. Positive expression for most markers were found. These pre-conditioned constructs were compared to statically seeded constructs.

Bioreactor Study 2: Pre-differentiated hADSC’s were seeded interstitially and externally. Constructs were pre-conditioned in bioreactor for 4 weeks. Expression of markers for each type of cell was found.

PGG, by virtue of its collagen and elastin-stabilizing abilities preserved the composition and structure of the ECM of the acellular leaflets and chordae, required by the valve’s complex mechanical effort. Biaxial testing showed similar mechanical characteristics of the PGG-treated scaffold to the fresh tissue. Cell-seeded scaffolds conditioned in the bioreactor showed good viability and cell differentiation. PGG-treated scaffolds released a reduced amount of matrix peptides that might mitigate the activation of fibroblasts seeded on the scaffold, and reduced the level of activated MMP, TGF- β1, and α-smooth muscle actin, compared to untreated scaffolds.

Levine, R. A., Hagége, A. A., Daniel, P., Padala, M., Jacob, P., Aikawa, E., … Song, J. K. (2015). Mitral Valve Disease - Morphology and Mechanisms. Nature Publishing Group, 12(12), 689–710. doi:10.1038/nrcardio.2015.161

Sierad, L. N., Simionescu, A., Albers, C., Chen, J., Maivelett, J., Tedder, M. E., … Simionescu, D. T. (2010). Design and Testing of a Pulsatile Conditioning System for Dynamic Endothelialization of Polyphenol-Stabilized Tissue Engineered Heart Valves. Cardiovascular Engineering and Technology, 1(2), 138–153. doi:10.1007/s13239-010-0014-6

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