Project Summary Following injury and repair, tendons rarely exhibit full restoration of function and reinjuries are prominent. Poor clinical outcomes are due to deposition of disorganized scar tissue rather than tissue with the anisotropic, hierarchical structure as in uninjured tendon. Prior work has demonstrated that anisotropic materials as scaffolds to guide cells promote expression of tenogenic markers and provide a template for cell orientation, showing promise for promoting regenerative healing. However, biomaterials used in these studies lack tunability in biophysical and biochemical cues critical for fully recapitulating the native tendon environment in an engineered extracellular matrix (eECM) and translation of the eECM to in vivo applications. To address these limitations, we have developed a novel approach to fabricate anisotropic poly(ethylene glycol) (PEG) hydrogel-based eECM to promote the alignment of human tenocytes. These anisotropic hydrogels are formed using a two-stage polymerization that eliminates complications of using additives or complex processing techniques to introduce anisotropy and enables scaling of the biomaterial without compromising material properties. In the first stage, a network is formed via a Michael-addition reaction of 4-arm PEG-maleimide and dicysteine peptides. The network is strained to introduce anisotropy, followed by a secondary thiol-ene photocrosslinking of remaining peptide thiols and 8-arm PEG-norbornene to retain strain-induced alignment. Notably, the eECM includes MMP-degradable crosslinks to balance structural cues and matrix remodeling. We propose herein that hydrogel-mediated anisotropic guidance and biochemical cues to tenocytes in balance with hydrogel remodeling will orchestrate native-like tendon deposition. In Aim 1, material properties of anisotropic eECM will be characterized. Mechanical properties will be tested via tensile testing parallel and perpendicular to alignment. Temporal retention of alignment as a function of MMP-mediated degradation will be analyzed via wide angle x-ray diffraction. Human tenocytes will be seeded in the eECM and cell and matrix anisotropy will be analyzed using microscopy. Matrix deposition will be analyzed by comparing ratios and organization of type I to type III collagen deposition and gene expression of scleraxis, tenomodulin, mohawk, and ⍺SMA as measures of regenerative versus fibrotic characteristics. Isotropic hydrogels will be used as controls with analysis over 14 days in vitro. In Aim 2, tenocyte gene expression profiles induced by anisotropic, degradable PEG hydrogel-based eECM will be comprehensively analyzed via RNAseq. Differential gene expression will be assessed on day 14 samples to compare gene profiles between eECM groups and freshly isolated cells from healthy and fibrotic tendons. Completing these aims will provide insight into materials design parameters for eECM to develop a pro-regenerative environment for tenocytes. The hydrogel...