Hydrogel-Supported, Engineered Model of Vocal Fold Epithelium.

There is a critical need for the establishment of an engineered model of the vocal fold epithelium that can be used to gain understanding of its role in vocal fold health, disease, and facilitate the development of new treatment options. Toward this goal, we isolated primary vocal fold epithelial cells (VFECs) from healthy porcine larynxes and used them within passage 3. Culture-expanded VFECs expressed the suprabasal epithelial marker cytokeratin 13 and intercellular junctional proteins occludin, E-cadherin, and zonula occludens-1. To establish the engineered model, we cultured VFECs on a hyaluronic acid-derived synthetic basement membrane displaying fibronectin-derived integrin-binding peptide (RGDSP) and/or laminin 111-derived syndecan-binding peptide AG73 (RKRLQVQLSIRT). Our results show that matrix stiffness and composition cooperatively regulate the adhesion, proliferation, and stratification of VFECs. Cells cultured on hydrogels with physiological stiffness (elastic shear modulus, G' = 1828 Pa) adopted a cobblestone morphology with close cell-cell contacts, whereas those on softer matrices (G' = 41 Pa) were spindle shaped with extensive intracellular stress fibers. The development of stratified epithelium with proliferating basal cells and additional (1-2) suprabasal layers requires the presence of both RGDSP and AG73 peptide signals. Supplementation of cytokines produced by vimentin positive primary porcine vocal fold fibroblasts in the VFEC culture led to the establishment of 4-5 distinct cell layers. The engineered vocal fold epithelium resembled native tissue morphologically; expressed cytokeratin 13, mucin 1, and tight/adherens junction markers; and secreted basement membrane proteins collagen IV and laminin 5. Collectively, our results demonstrate that stiffness matching, cell-matrix engagement, and paracrine signaling cooperatively contribute to the stratification of VFECs. The engineered epithelium can be used as a versatile tool for investigations of genetic and molecular mechanisms in vocal fold health and disease.

Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-ß/ALK5 signaling.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-ß)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-ß/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

Biocompatibility and Viscoelastic Properties of Injectable Resilin-Like Polypeptide and Hyaluronan Hybrid Hydrogels in Rabbit Vocal Folds.

Vocal fold scar, characterized by alterations in the lamina propria extracellular matrix, disrupts normal voice quality and function. Due to a lack of satisfactory clinical treatments, there is a need for tissue engineering strategies to restore voice. Candidate biomaterials for vocal fold tissue engineering must match the unique biomechanical and viscoelastic properties of native tissue without provoking inflammation. We sought to introduce elastomeric properties to hyaluronic acid (HA)-based biomaterials by incorporating resilin-like polypeptide (RLP) into hybrid hydrogels. Physically crosslinked RLP/HA and chemically crosslinked RLP-acrylamide/thiolated HA (RLP-AM/HA-SH) hydrogels were fabricated using cytocompatible chemistries. Mechanical properties of hydrogels were assessed in vitro using oscillatory rheology. Hybrid hydrogels were injected into rabbit vocal folds and tissues were assessed using rheology and histology. A small number of animals underwent acute vocal fold injury followed by injection of RLP-AM/HA-SH hydrogel alone or as a carrier for human bone marrow mesenchymal stem cells (BM-MSCs). Rheological testing confirmed that mechanical properties of materials in vitro resembled native vocal fold tissue and that viscoelasticity of vocal fold mucosa was preserved days 5 and 21 after injection. Histological analysis revealed that hybrid hydrogels provoked only mild inflammation in vocal fold lamina propria with demonstrated safety in the airway for up to 3 weeks, confirming acute biocompatibility of crosslinking chemistries. After acute injury, RLP-AM/HA-SH gel with and without BM-MSCs did not result in adverse effects or increased inflammation. Collectively, results indicate that RLP and HA-based hybrid hydrogels are highly promising for engineering the vocal fold lamina propria.

Rapid Bioorthogonal Chemistry Enables in Situ Modulation of the Stem Cell Behavior in 3D without External Triggers.

Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.

Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D.

Current treatments for chronic xerostomia, or "dry mouth", do not offer long-term therapeutic benefits for head and neck cancer survivors previously treated with curative radiation. Towards the goal of creating tissue-engineered constructs for the restoration of salivary gland functions, we developed new hyaluronic acid (HA)-based hydrogels using thiolated HA (HA-SH) and acrylated HA (HA-AES) with a significant molecular weight mismatch. Four hydrogel formulations with varying HA concentration, (1-2.4 wt%) and thiol/acrylate ratios (2/1 to 36/1) and elastic moduli (G': 35 to 1897 Pa, 2 h post-mixing) were investigated. In our system, thiol/acrylate reaction was initiated rapidly upon mixing of HA-SH/HA-AES to establish thioether crosslinks with neighboring ester groups, and spontaneous sulfhydryl oxidation occurred slowly over several days to install a secondary network. The concurrent reactions cooperatively create a cell-permissive network to allow for cell expansion and aggregation. Multicellular spheroids formed readily from a robust ductal epithelial cell line (Madin-Darby Canine Kidney, MDCK cells) in all hydrogel formulations investigated. Primary salivary human stem/progenitor cells (hS/PCs), on the other hand, are sensitive to the synthetic extracellular environment, and organized acini-like structures with an average diameter of 50 µm were obtained only in gels with G' ≤ 216 Pa and a thiol/acrylate ratio ≥18. The spheroid size and size distribution were dependent on the HA content in the hydrogel. Cells in hS/PC spheroids formed tight junctions (occludin), remained viable and proliferative, secreted structural proteins (collagen IV and laminin) found in the basement membrane and maintained key stem/progenitor markers. We conclude that incorporation of time-dependent, dynamic features into a covalently crosslinked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of salivary morphogenesis, key to reconstitution of a fully functional implantable salivary gland.