The effect of bioactive hydrogels on the secretion of extracellular matrix molecules by valvular interstitial cells.

Valvular interstitial cells (VICs) were encapsulated in enzymatically degradable, crosslinked hydrogels formed from hyaluronic acid (HA) and poly(ethylene glycol) (PEG) macromolecular monomers. Titration of PEG with HA allowed for the synthesis of gels with a broad compositional spectrum, leading to a range of degradation behavior upon exposure to bovine testes hyaluronidase. The rate of mass loss and release of HA fragments from the copolymer gels depended on the PEG content of the network. These hydrogels were shown to have the dual function of permitting the diffusion of ECM elaborated by 3D cultured VICs and promoting the development of a specific matrix composition. Initial cleavage of hydrogel crosslinks, prior to network mass loss, permit the diffusion of collagen, while later stages of degradation promote elastin elaboration and suppress collagen production due to HA fragment release. Exogenous HA delivery through the cell culture media further demonstrated the utility of delivered HA on manipulating the secretory properties of encapsulated VICs.

Macromolecular Monomers for the Synthesis of Hydrogel Niches and Their Application in Cell Encapsulation and Tissue Engineering.

Hydrogels formed from the photoinitiated, solution polymerization of macromolecular monomers present distinct advantages as cell delivery materials and are enabling researchers to three-dimensionally encapsulate cells within diverse materials that mimic the extracellular matrix and support cellular viability. Approaches to synthesize gels with biophysically and biochemically controlled microenvironments are becoming increasingly important, and require strategies to control gel properties (e.g., degradation rate and mechanism) on multiple time and size scales. Furthermore, biological responses of gel-encapsulated cells can be promoted by hydrogel degradation products, as well as by the release of tethered biologically relevant molecules.

Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells.

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.

Designing scaffolds for valvular interstitial cells: cell adhesion and function on naturally derived materials.

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.

Valvular myofibroblast activation by transforming growth factor-beta: implications for pathological extracellular matrix remodeling in heart valve disease.

The pathogenesis of cardiac valve disease correlates with the emergence of muscle-like fibroblasts (myofibroblasts). These cells display prominent stress fibers containing alpha-smooth muscle actin (alpha-SMA) and are believed to differentiate from valvular interstitial cells (VICs). However, the biological factors that initiate myofibroblast differentiation and activation in valves remain unidentified. We show that transforming growth factor-beta1 (TGF-beta1) mediates differentiation of VICs into active myofibroblasts in vitro in a dose-dependent manner, as determined by a significant increase in alpha-SMA and the dramatic augmentation of stress fiber formation and alignment. Additionally, TGF-beta1 and increased mechanical stress function synergistically to enhance contractility. In turn, contractile valve myofibroblasts exert tension on the extracellular matrix, resulting in a dramatic realignment of extracellular fibronectin fibrils. TGF-beta1 also inhibits valve myofibroblast proliferation without enhancing apoptosis. Our results are consistent with activation of a highly contractile myofibroblast phenotype by TGF-beta1 and are the first to connect valve myofibroblast contractility with pathological valve matrix remodeling. We suggest that the activation of contractile myofibroblasts by TGF-beta1 may be a significant first step in promoting alterations to the valve matrix architecture that are evident in valvular heart disease.