Recapitulating the Native Tendon Environment in a Synthetic 3D Anisotropic Hydrogel as an Engineered Extracellular Matrix

The ability of tendons to transmit forces from muscle to bone is fundamentally attributed to the hierarchical anisotropy of the tissue. After injury, disorganized fibrotic scar tissue forms during the natural healing process, resulting in inferior mechanical properties that often lead to reinjury and limited restoration of function. Therefore, intervention is necessary to facilitate regenerative healing of the tendon. Polymeric biomaterials have historically been used to guide cell behavior, showing promise for the use of topological guidance and cell-mediated matrix remodeling as mechanisms for promoting regeneration. Here, we fabricated 3D scaffolds for tenocytes using anisotropic poly(ethylene glycol)-based hydrogels that recapitulate both the biophysical and biochemical properties of the native tendon. These materials were synthesized using a two-stage polymerization strategy that includes an initial cross-linking step facilitated by thiol-Michael addition, an intermediate mechanical stretching step to align the polymer network, and a second-stage crosslinking step facilitated by a thiol-ene reaction. The application of 300% strain during the mechanical alignment of the network resulted in highly oriented materials (<i>S</i> = 0.38). Furthermore, a matrix metalloproteinase (MMP)-degradable peptide was incorporated into the network to facilitate cell-mediated remodeling of the scaffold. After 14 days of exposure to exogenous MMP2, a sufficient number of cross-links were degraded for alignment to be lost (<i>S</i> = 0.03). When tenocytes were encapsulated in the 3D anisotropic hydrogels, they adopted the anisotropic morphology of the polymer network and deposited an extracellular matrix mainly comprised of type I collagen, indicating a pro-regenerative environment. Comparatively, isotropic materials of the same composition induced a random orientation of encapsulated tenocytes, and a matrix primarily comprised of collagen III was deposited, indicating a fibrotic environment. Collectively, these results demonstrate the successful use of a synthetic scaffold with tunable biophysical and biochemical properties for recapitulating the native tendon environment and promoting regenerative cell behavior.