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Extracellular matrix stiffness is a major regulator of cellular states in health and disease. Stiffness-sensing investigations are typically performed using cells that have acquired "mechanical memory" through prolonged conditioning in rigid mechanical environments, e.g. tissue culture plastic (TCP). This potentially masks the full extent of the matrix stiffness-driven mechanosensing programs. Here, a biomaterial system composed of two-dimensional mechano-variant silicone substrates with simplified and scalable surface biofunctionalization chemistry is developed to facilitate large-scale cell culture expansion processes. Using RNA sequencing, stiffness-mediated mechano-responses of human tendon-derived stromal cells are broadly mapped. Matrix rigidities approximating tendon microscale stiffness range (E. ∼35 kPa) distinctly favor transcriptional programs related to chromatin remodeling and Hippo signaling; whereas more compliant stiffnesses (E. ∼2 kPa) are enriched in responses related to cell stemness, synapse assembly and angiogenesis. While tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, mechano-variant substrates abrogate this activation with tenogenic stiffnesses inducing a transcriptional program that strongly correlates with established tendon tissue-specific expression signature. Computational inference predicted that AKT1 and ERK1/2 are major signaling hubs mediating stiffness-sensing in tendon cells. Together, these findings highlight how extracellular matrix biophysical cues may dictate the transcriptional identity of tendon resident cells, and how matrix mechano-reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon fibroblasts. This article is protected by copyright. All rights reserved.

Tendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146+ tendon stem/progenitor cell and CD45+ F4/80+ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80+ "tenophages" from the intrinsic tissue core.