The Stiffness-Sensitive Transcriptome of Human Tendon Stromal Cells.

Extracellular matrix stiffness is a major regulator of cellular states. Stiffness-sensing investigations are typically performed using cells that have acquired "mechanical memory" through prolonged conditioning in rigid environments, e.g., tissue culture plastic (TCP). This potentially masks the full extent of the matrix stiffness-driven mechanosensing programs. Here, a biomaterial composed of 2D mechanovariant 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 elasticity (E.) approximating tendon microscale stiffness range (E. ≈ 35 kPa) distinctly favors transcriptional programs related to chromatin remodeling and Hippo signaling; whereas compliant stiffnesses (E. ≈ 2 kPa) are enriched in processes related to cell stemness, synapse assembly, and angiogenesis. While tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, mechanovariant substrates abrogate this activation with tenogenic stiffnesses inducing a transcriptional program that strongly correlates with established tendon tissue-specific expression signature. Computational inference predicts that AKT1 and ERK1/2 are major stiffness-sensing signaling hubs. Together, these findings highlight how matrix biophysical cues may dictate the transcriptional identity of tendon cells, and how matrix mechano-reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon fibroblasts.

Extrinsic Macrophages Protect While Tendon Progenitors Degrade: Insights from a Tissue Engineered Model of Tendon Compartmental Crosstalk.

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.

An adhesion G protein-coupled receptor is required in cartilaginous and dense connective tissues to maintain spine alignment.

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs, and dense connective tissues (i.e., ligaments and tendons) is essential for the maintenance of spinal alignment. We show that the adhesion G protein-coupled receptor ADGRG6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine alignment in mice. Furthermore, we show that ADGRG6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with a cAMP agonist could mirror aspects of receptor function in culture, thus defining core pathways for regulating these axial cartilaginous and connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis.

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown.

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.