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Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. IMPACT STATEMENT: Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Calcific aortic valve disease (CAVD) is the third leading cause of cardiovascular disease. CAVD exhibits progressive disruption of the normally highly organized and aligned extracellular matrix (ECM) structure within the valve leaflets simultaneously with myofibroblastic and/or osteogenic differentiation of indigenous endogenous valve interstitial cells (VIC). It is unclear how the alignment of VIC within their 3D microenvironment drives VIC phenotype or how alignment affects cellular responses to biochemical cues in physiological or pathological conditions. In this study, we implement a photolithographic technique to control the alignment and elongation of both normal and diseased human aortic VIC (HAVIC) within microengineered 3D hydrogels consisting of methacrylated hyaluronic acid and methacrylated gelatin. Stripe micropatterning created distinct alignment of HAVIC within a 3D culture system, which promoted spreading and enhanced their activation and osteogenic differentiation in pro-osteogenic conditions. HAVIC from a patient with CAVD exhibited greater susceptibility to myofibroblastic and osteogenic differentiation in culture. The roles of conjugated basic fibroblastic growth factor (bFGF) and RhoA/ROCK pathway in regulating HAVIC phenotypes were also investigated in the presence of aligned microtopography. The addition of bFGF was preventative to osteogenic differentiation for healthy HAVIC; however, it promoted osteogenic differentiation in diseased HAVIC. Inhibition of the ROCK pathway only decreased osteogenic differentiation for diseased HAVIC in the aligned formation. Collectively, these results improve our knowledge of the effects that VIC alignment has on VIC phenotypes and valve disease progression. The cell culture platform also enables a better understanding of the interplay between topography, biochemical cues, and VIC differentiation and provides information useful for directing differentiation as well as valve tissue regeneration.

Living tissue-engineered heart valves (TEHV) would be a major benefit for children who require a replacement with the capacity for growth and biological integration. A persistent challenge for TEHV is accessible human cell source(s) that can mimic native valve cell phenotypes and matrix remodeling characteristics that are essential for long-term function. Mesenchymal stem cells derived from bone marrow (BMMSC) or adipose tissue (ADMSC) are intriguing cell sources for TEHV, but they have not been compared with pediatric human aortic valve interstitial cells (pHAVIC) in relevant 3D environments. In this study, we compared the spontaneous and induced multipotency of ADMSC and BMMSC with that of pHAVIC using different induction media within three-dimensional (3D) bioactive hybrid hydrogels with material modulus comparable to that of aortic heart valve leaflets. pHAVIC possessed some multi-lineage differentiation capacity in response to induction media, but limited to the earliest stages and much less potent than either ADMSC or BMMSC. ADMSC expressed cell phenotype markers more similar to pHAVIC when conditioned in basic fibroblast growth factor (bFGF) containing HAVIC growth medium, while BMMSC generally expressed similar extracellular matrix remodeling characteristics to pHAVIC. Finally, we covalently attached bFGF to PEG monoacrylate linkers and further covalently immobilized in the 3D hybrid hydrogels. Immobilized bFGF upregulated vimentin expression and promoted the fibroblastic differentiation of pHAVIC, ADMSC, and BMMSC. These findings suggest that stem cells retain a heightened capacity for osteogenic differentiation in 3D culture, but can be shifted toward fibroblast differentiation through matrix tethering of bFGF. Such a strategy is likely important for utilizing stem cell sources in heart valve tissue engineering applications.