Development of a Decellularized Tissue Model of Early Heart Valve Lesions
Maleki, Hoda 1, 2, 3; Simmons, Craig A. 1, 2, 3, 4
1. Translational Biology and Engineering Program, The Ted Rogers Centre for Heart Research; 2. Institute of Biomaterials & Biomedical Engineering, University of Toronto; 3. Department of Mechanical & Industrial Engineering, University of Toronto; 4. Faculty of Dentistry, University of Toronto
Heart valve dysfunction in disease results from maladaptive remodeling of the extracellular matrix (ECM) that initially results in focal proteoglycan-rich lesions, but ultimately progresses to fibrotic, calcific, and stiffened valve leaflets. We showed previously that cells within early lesions express genes and proteins associated with valve calcification, suggesting that the lesion ECM may influence valve cell phenotypes in early lesions to drive disease development. To test this hypothesis and others related to valve pathobiology, we are developing a decellularized scaffold model that will enable, for the first time, in vitro mechanistic studies under conditions that accurately represent lesion and non-lesion microenvironments.
Our top-down approach involves decellularizing porcine aortic valve leaflets to yield ECM that can be re-seeded with naïve valve interstitial cells (VICs) to test the effect of lesion vs. non-lesion ECM on cell phenotypic expression. Decellularization of valve leaflets using different sodium dodecyl sulfate (SDS) concentrations (0.075% or 0.1%) and incubation times (4-9 hours) yielded >97% effective decellularization in all cases. The two decellularization treatments with the shortest incubation times (0.075% SDS for 7 hours (Treatment #757) and 0.1% SDS for 4 hours (#014)) were tested further, as the most likely to preserve the ECM. Preservation of ECM mechanical properties and composition relative to untreated control leaflet ECM was assessed by atomic force microscopy (AFM) and histomorphometric analysis of Movat’s pentachrome-stained sections, respectively. Preliminary studies qualitatively assessed VIC adhesion, viability, and migration on decellularized models.
AFM demonstrated that the elastic moduli of lesions were significantly lower than those of non-lesion tissue (p<0.001) regardless of treatment. Decellurization had no effect on the elastic moduli of lesions (p>0.5), but the elastic moduli of the 757-treated non-lesion ECM were significantly higher those of the control and 014-treated non-lesion regions (p<0.01). Histomorphometric analyses showed that the area fractions of collagen (p=0.91) and glycosaminoglycans (p=0.61) were not altered by either decellularization treatment relative to the untreated control samples in lesion and non-lesion areas. VICs cultured on decelullarized lesion and non-lesion ECM attached, migrated throughout the tissue, and remained viable for at least 14 days. On the basis of these data, the 014 treatment offers the best combination of cell removal efficiency and preserved ECM mechanics and composition. On-going studies are using scanning electron microscopy and biochemical assays to further confirm that the 014 decelullarization treatment preserves ECM microstructure and composition.
This novel platform will provide insights into the role the ECM plays in regulating native valve (dys)function, with potential implications towards better understanding valve disease development.