Triglycidyl amine crosslinking combined with ethanol inhibits bioprosthetic heart valve calcification.

BACKGROUND: One of the most important factors responsible for the calcific failure of bioprosthetic heart valves is glutaraldehyde crosslinking. Ethanol (EtOH) incubation after glutaraldehyde crosslinking has previously been reported to confer anticalcification efficacy for bioprostheses. The present studies investigated the anticalcification efficacy in vivo of the novel crosslinking agent, triglycidyl amine (TGA), with or without EtOH incubation, in comparison with glutaraldehyde. METHODS: The TGA crosslinking (±EtOH) was used to prepare porcine aortic valves for both rat subdermal implants and sheep mitral valve replacements, for comparisons with glutaraldehyde-fixed controls. Thermal denaturation temperature, an index of crosslinking, cholesterol extraction, and hydrodynamic properties were quantified. Explant endpoints included quantitative and morphologic assessment of calcification. RESULTS: Thermal denaturation temperatures after TGA were intermediate between unfixed and glutaraldehyde-fixed. EtOH incubation resulted in almost complete extraction of cholesterol from TGA or glutaraldehyde-fixed cusps. Rat subdermal explants (90 days) demonstrated that TGA-EtOH resulted in a significantly greater level of inhibition of calcification than other conditions. Thus, TGA-ethanol stent mounted porcine aortic valve bioprostheses were fabricated for comparisons with glutaraldehyde-pretreated controls. In hydrodynamic studies, TGA-EtOH bioprostheses had lower pressure gradients than glutaraldehyde-fixed. The TGA-ethanol bioprostheses used as mitral valve replacements in juvenile sheep (150 days) demonstrated significantly lower calcium levels in both explanted porcine aortic cusp and aortic wall samples compared with glutaraldehyde-fixed controls. However, TGA-EtOH sheep explants also demonstrated isolated calcific nodules and intracuspal hematomas. CONCLUSIONS: The TGA-EtOH pretreatment of porcine aortic valves confers significant calcification resistance in both rat subdermal and sheep circulatory implants, but with associated structural instability.

Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts.

The composition and organization of the vocal fold extracellular matrix (ECM) provide the viscoelastic mechanical properties that are required to sustain high-frequency vibration during voice production. Although vocal injury and pathology are known to produce alterations in matrix physiology, the mechanisms responsible for the development and maintenance of vocal fold ECM are poorly understood. The objective of this study was to investigate the effect of physiologically relevant vibratory stimulation on ECM gene expression and synthesis by fibroblasts encapsulated within hyaluronic acid hydrogels that approximate the viscoelastic properties of vocal mucosa. Relative to static controls, samples exposed to vibration exhibited significant increases in mRNA expression levels of HA synthase 2, decorin, fibromodulin and MMP-1, while collagen and elastin expression were relatively unchanged. Expression levels exhibited a temporal response, with maximum increases observed after 3 and 5 days of vibratory stimulation and significant downregulation observed at 10 days. Quantitative assays of matrix accumulation confirmed significant increases in sulphated glycosaminoglycans and significant decreases in collagen after 5 and 10 days of vibratory culture, relative to static controls. Cellular remodelling and hydrogel viscosity were affected by vibratory stimulation and were influenced by varying the encapsulated cell density. These results indicate that vibration is a critical epigenetic factor regulating vocal fold ECM and suggest that rapid restoration of the phonatory microenvironment may provide a basis for reducing vocal scarring, restoring native matrix composition and improving vocal quality.

Tissue engineering therapies for the vocal fold lamina propria.

The vocal folds are laryngeal connective tissues with complex matrix composition/organization that provide the viscoelastic mechanical properties required for voice production. Vocal fold injury results in alterations in tissue structure and corresponding changes in tissue biomechanics that reduce vocal quality. Recent work has begun to elucidate the biochemical changes underlying injury-induced pathology and to apply tissue engineering principles to the prevention and reversal of vocal fold scarring. Based on the extensive history of injectable biomaterials in laryngeal surgery, a major focus of regenerative therapies has been the development of novel scaffolds with controlled in vivo residence time and viscoelastic properties approximating the native tissue. Additional strategies have included cell transplantation and delivery of the antifibrotic cytokine hepatocyte growth factor, as well as investigation of the effects of the unique vocal fold vibratory microenvironment using in vitro dynamic culture systems. Recent achievements of significant reductions in fibrosis and improved recovery of native tissue viscoelasticity and vibratory/functional performance in animal models are rapidly moving vocal fold tissue engineering toward clinical application.

Mechanomimetic hydrogels for vocal fold lamina propria regeneration.

Vocal fold injury commonly leads to reduced vocal quality due to scarring-induced alterations in matrix composition and tissue biomechanics. The long-term hypothesis motivating our work is that rapid restoration of phonation and the associated dynamic mechanical environment will reduce scarring and promote regenerative healing. Toward this end, the objective of this study was to develop mechanomimetic, degradable hydrogels approximating the viscoelastic properties of the vocal ligament and mucosa that may be photopolymerized in situ to restore structural integrity to vocal fold tissues. The tensile and rheological properties of hydrogels (targeting the vocal ligament and mucosa, respectively) were varied as a function of macromer concentration. PEG diacrylate-based hydrogels exhibited linear stress-strain response and elastic modulus consistent with the properties of the vocal ligament at low strains (0-15%), but did not replicate the non-linear behavior observed in native tissue at higher strains. Methacrylated hyaluronic acid hydrogels displayed dynamic viscosity consistent with native vocal mucosa, while elastic shear moduli values were several-fold higher. Cell culture studies indicated that both hydrogels supported spreading, proliferation and collagen/proteoglycan matrix deposition by encapsulated fibroblasts throughout the 3D network.

A novel synthetic route for the preparation of hydrolytically degradable synthetic hydrogels.

A variety of approaches have been described for the modification of synthetic, water soluble polymers with hydrolytically degradable bonds and terminal vinyl groups that can be crosslinked in situ by photo- or redox-initiated free radical polymerization. However, changes in macromer concentration, functionality, and molecular weight commonly used to achieve variable degradation rates simultaneously alter hydrogel mechanical properties. Herein, we describe a novel, two-step synthetic route for the preparation of hydrolytically degradable, crosslinkable PEG-based macromers based on chemical intermediaries that form ester linkages with variable alkyl chain length. Changes in the concentration of a single macromer were shown to provide effective variation of degradation, but with corresponding significant changes in tensile properties. Through variation in the alkyl chain length of the chemical intermediary, variable degradation times ranging from weeks to months are achieved, without significantly affecting initial gelation efficiency, swelling, or tensile properties. When modified with adhesive ligands, hydrogels supported viability of encapsulated and adherent cells. Controlled release of a model protein (Immunoglobulin G) was attained as a function of hydrogel degradation rate. Independent control of hydrogel degradation and mechanical properties will offer improved flexibility for studying the effect of these material characteristics on cellular function and may be useful in the design of matrices for tissue engineering and controlled release of bioactive molecules.

The effect of hyaluronic acid incorporation on fibroblast spreading and proliferation within PEG-diacrylate based semi-interpenetrating networks.

The nanometer-scale mesh size of many synthetic crosslinked hydrogel networks restricts encapsulated cells to a rounded morphology that can inhibit cellular processes such as proliferation and migration that are essential for the early stages of remodeling and tissue formation. The objective of these studies was to investigate an approach for accelerating cellular remodeling based on the creation of semi-interpenetrating networks (IPNs) composed of hydrolytically degradable poly(ethylene glycol) (PEG) diacrylate macromers and native, enzymatically degradable extracellular matrix (ECM) components (collagen, gelatin and hyaluronic acid (HA)). Among the three ECM components investigated, addition of HA at concentrations of 0.12% w/v and greater supported fibroblast spreading throughout the three-dimensional network and significantly increased proliferation relative to control hydrogels without HA. Incorporation of HA resulted in relatively small changes in hydrogel physical/chemical properties such as swelling, degradation rate, and elastic modulus. Fibroblast spreading was eliminated by the addition of hyaluronidase inhibitors, demonstrating that cell-mediated enzymatic degradation of HA is a necessary mechanism responsible for the observed increases in fibroblast activity. By accelerating early cellular remodeling and growth, these semi-IPNs may be useful vehicles for cell transplantation in a variety of tissue engineering applications.

Photopatterned polymer brushes promoting cell adhesion gradients.

The ability to spatially control cellular adhesion in a continuous manner on a biocompatible substrate is an important factor in designing new biomaterials for use in wound healing and tissue engineering applications. In this work, a novel method of engineering cell-adhesive RGD-ligand density gradients to control specific cell adhesion across a substrate is presented. Polymer brushes exhibiting spatially defined gradients in chain density are created and subsequently functionalized with RGD to create ligand density gradients capable of inducing cell adhesion on an otherwise weakly adhesive substrate. Cell studies indicate that these ligand-functionalized surfaces are noncytotoxic, with cellular adhesion increasing with RGD-ligand density across the gradient brush surface.