To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices (ECM), we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibers, and bulk contraction of the material. While increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation, increasing fiber stiffness instead suppressed spreading and proliferation depending on network architecture. Lower fiber stiffness permitted active cellular forces to recruit nearby fibers, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signaling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fiber recruitment as a novel mechanism by which cells probe and respond to mechanics in fibrillar matrices.

The patterning of chemical and mechanical signals within hydrogels permits added complexity towards their use as cell microenvironments for biomedical applications. Specifically, photopatterning is emerging to introduce heterogeneity in hydrogel properties; however, currently employed systems are limited in the range of properties that can be obtained, as well as in decoupling mechanical properties from changes in chemical signals. Here, we present an orthogonal photopatterning system that utilizes thiol-norbornene chemistry and permits extensive hydrogel modification, including with multiple signals, due to the number of reactive handles accessible for secondary reaction. Hyaluronic acid was functionalized with norbornene groups (NorHA) and reacted with di-thiols to create non-toxic hydrogels with a wide range of mechanical properties. For example, for 4 wt% NorHA at 20% modification, hydrogel mechanics from ~1 kPa up to ~70 kPa could be obtained by simply changing the amount of crosslinker. By limiting the initial extent of crosslinking, NorHA gels were synthesized with remaining pendent norbornene groups that could be reacted with thiol containing molecules in the presence of light and an initiator, including with spatial control. Secondary reactions with a di-thiol crosslinker changed mechanical properties, whereas reaction with mono-thiol peptides had no influence on the gel elastic modulus. This orthogonal chemistry was used sequentially to pattern multiple peptides into a single hydrogel, demonstrating the robustness of this system for the formation of complex hydrogels.

Hyaline cartilage serves as a low-friction and wear-resistant articulating surface in load-bearing, diarthrodial joints. Unfortunately, as the avascular, alymphatic nature of cartilage significantly impedes the body’s natural ability to regenerate, damage resulting from trauma and osteoarthritis necessitates repair attempts. Current clinical methods are generally limited in their ability to regenerate functional cartilage, and so research in recent years has focused on tissue engineering solutions in which the regeneration of cartilage is pursued through combinations of cells (e.g., chondrocytes or stem cells) paired with scaffolds (e.g., hydrogels, sponges, and meshes) in conjunction with stimulatory growth factors and bioreactors. A variety of synthetic and natural materials have been employed, most commonly in the form of hydrogels, and these systems have been tuned for optimal nutrient diffusion, connectivity of deposited matrix, degradation, soluble factor delivery, and mechanical loading for enhanced matrix production and organization. Even with these promising advances, the complex mechanical properties and biochemical composition of native cartilage have not been achieved, and engineering cartilage tissue still remains a significant challenge. Using hyaluronic acid hydrogels as an example, this review will follow the progress of material design specific to cartilage tissue engineering and propose possible future directions for the field.

Electrospinning has recently gained much interest due to its ability to form scaffolds that mimic the nanofibrous nature of the extracellular matrix, such as the size and depth-dependent alignment of collagen fibers within hyaline cartilage. While much progress has been made in developing bulk, isotropic hydrogels for tissue engineering and understanding how the microenvironment of such scaffolds affects cell response, these effects have not been extensively studied in a nanofibrous system. Here, we show that the mechanics (through intrafiber crosslink density) and adhesivity (through RGD density) of electrospun hyaluronic acid (HA) fibers significantly affect human mesenchymal stem cell (hMSC) interactions and gene expression. Specifically, hMSC spreading, proliferation, and focal adhesion formation were dependent on RGD density, but not on the range of fiber mechanics investigated. Moreover, traction-mediated fiber displacements generally increased with more adhesive fibers. The expression of chondrogenic markers, unlike trends in cell spreading and cytoskeletal organization, was influenced by both fiber mechanics and adhesivity, in which softer fibers and lower RGD densities generally enhanced chondrogenesis. This work not only reveals concurrent effects of mechanics and adhesivity in a fibrous context, but also highlights fibrous HA hydrogels as a promising scaffold for future cartilage repair strategies.

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