Acute and chronic tendon injuries are among the most common musculoskeletal health problems. Typically, an injured tendon experiences fibrotic scarring that leaves the tissue mechanically compromised and prone to debilitating adhesions that impair joint function. In a fibrotic tendon scar, the cell-cell and paracrine signaling between inflammatory cells, such as macrophages, and tendon fibroblasts activate the latter into fibroproliferative myofibroblasts, ultimately differentiating into a senescent phenotype. Our previous studies using next-generation sequencing and gene set enrichment analysis mechanistically linked fibrosis and senescence in injured mouse tendons with TGF-beta activated mTOR signaling. To further elucidate this pathology, the goal of this proposal is to engineer a microfluidic human tendon-on-chip (hToC) system and use it to more accurately model the biological aspects of the inflammation and fibrosis in injured tendons. In the UG3 phase of this proposal, the chip will be fabricated featuring a multicompartmental design and microfluidic channels to incorporate a fibroblast-seeded collagen hydrogel and simulate vascular blood flow, respectively. Ultrathin, highly permeable, and optically transparent porous silicon membranes (SiM) will separate the hydrogel from circulation and provide a substrate for an endothelial barrier in between. The signaling between the fibroblasts, hydrogel-resident- and circulating-macrophages, and endothelial cells will be enabled through nanoporous SiM (~60 nm), while a microporous SiM (~ 8 µm) will allow extravasation of circulating macrophages and infiltration of the hydrogel under TGF-beta stimulation. To allow for a patient-centric chip, tendon fibroblasts will be used to create the tendon hydrogel and to reprogram donor-matching iPSCs to derive the endothelial cells and macrophages, respectively. An additional innovation will be the integration of label- free photonic sensors into the microfluidic device to allow on-chip sensing, which has been long appreciated as a critical, unmet need for organ-on-chip devices. The UG3 studies will use the chip to validate the role of mTOR in the disease model and identify biologically relevant biomarkers. In the UH3 phase, we will utilize the chip as a pre-clinical trial platform for testing efficacy and safety of FDA-approved mTOR inhibitors as potential disease modifying drugs, and as a drug screening platform to identify and prioritize safer and more potent inhibitors of mTOR and senescence in tendon injury for clinical trials. To successfully complete this innovative project, we have assembled a team of accomplished experts in tendon tissue engineering and surgery, immunology, iPSC technology, GMP cell manufacturing, nano- and micro-fabrication, sensor technology, and high throughput screening. The proposed studies will develop a human microphysiological system to catalyze clinical trials and accelerate drug discovery for acute and chronic tendon injuries...