Concurrent with a progressive loss of regenerative capacity, connective tissue aging is characterized by a progressive accumulation of Advanced Glycation End-products (AGEs). Besides being part of the typical aging process, type II diabetics are particularly affected by AGE accumulation due to abnormally high levels of systemic glucose that increases the glycation rate of long-lived proteins such as collagen. Although AGEs are associated with a wide range of clinical disorders, the mechanisms by which AGEs contribute to connective tissue disease in aging and diabetes are still poorly understood. The present study harnesses advanced multiscale imaging techniques to characterize a widely employed in vitro model of ribose induced collagen aging and further benchmarks these data against experiments on native human tissues from donors of different age. These efforts yield unprecedented insight into the mechanical changes in collagen tissues across hierarchical scales from molecular, to fiber, to tissue-levels. We observed a linear increase in molecular spacing (from 1.45 nm to 1.5 nm) and a decrease in the D-period length (from 67.5 nm to 67.1 nm) in aged tissues, both using the ribose model of in vitro glycation and in native human probes. Multiscale mechanical analysis of in vitro glycated tendons strongly suggests that AGEs reduce tissue viscoelasticity by severely limiting fiber-fiber and fibril-fibril sliding. This study lays an important foundation for interpreting the functional and biological effects of AGEs in collagen connective tissues, by exploiting experimental models of AGEs crosslinking and benchmarking them for the first time against endogenous AGEs in native tissue.

Athletic performance relies on tendons, which enable movement by transferring forces from muscles to the skeleton. Yet how load-bearing structures in tendon sense and adapt to physical demands is not understood. Here, by performing calcium (Ca 2+ ) imaging in mechanically loaded tendon explants from rats and in primary tendon cells from rats and humans, we show that tenocytes detect mechanical forces via the mechanosensitive ion channel PIEZO1, which senses shear stresses induced by collagen-fibre sliding. Via tenocyte-targeted loss-of-function and gain-of-function experiments in rodents, we show that reduced PIEZO1 activity decreased tendon stiffness and that elevated PIEZO1 mechanosignalling increased tendon stiffness and strength, seemingly through upregulated collagen crosslinking. We also show that humans carrying the PIEZO1 E756del gain-of-function mutation display a 13.2% average increase in normalized jumping height, presumably owing to a higher rate of force generation or to the release of a larger amount of stored elastic energy. Further understanding of the PIEZO1-mediated mechanoregulation of tendon stiffness should aid research on musculoskeletal medicine and on sports performance.

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Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.

1Aberrant matrix turnover with elevated matrix proteolysis is a hallmark of tendon pathology. While 2 tendon disease mechanisms remain obscure, mechanical cues are central regulators. Unloading of 3 tendon explants in standard culture conditions provokes rapid cell-mediated tissue breakdown. Here we 4 show that biological response to tissue unloading depends on the mimicked physiological context. Our 5 experiments reveal that explanted tendon tissues remain functionally stable in a simulated avascular 6 niche of low temperature and oxygen, regardless of the presence of serum. This hyperthermic and 7 hyperoxic niche-dependent catabolic switch was shown by whole transcriptome analysis (RNA-seq) to 8 be a strong pathological driver of an immune-modulatory phenotype, with a stress response to reactive 9 oxygen species (ROS) and associated activation of catabolic extracellular matrix proteolysis that 10 involved lysosomal activation and transcription of a range of proteolytic enzymes. Secretomic and 11 degradomic analysis through terminal amine isotopic labeling of substrates (TAILS) confirmed that 12 proteolytic activity in unloaded tissues was strongly niche dependent. Through targeted 13 pharmacological inhibition we isolated ROS mediated oxidative stress as a major checkpoint for matrix 14 proteolysis. We conclude from these data that the tendon stromal compartment responds to traumatic 15 mechanical unloading in a manner that is highly dependent on the extrinsic niche, with oxidative stress 16 response gating the proteolytic breakdown of the functional collagen backbone. tendon fibroblasts in a collagen-rich extracellular matrix (ECM). The fact that tendons bear 20 physiologically extreme mechanical stresses is reflected by its tightly packed and highly structured 21 ECM. The load bearing tendon core is comprised by hierarchically organized type-I collagen fibrils, 22 fibres and fascicles (or fibre bundles) [1], with fascicles representing the basic functional load-bearing 23 unit of stromal tissue [2,3]. Physiological mechanical signals are understood to regulate the lifelong 24 adaption of the tissue to individual functional demands [4][5][6][7][8], with a narrow gap between beneficial and 25 detrimental mechanical loads [9][10][11][12]. This threshold is often exceeded, with tendon disorders 26 accounting for 30 -50% of all musculoskeletal clinical complaints associated with pain [13]. 27Our basic knowledge of the molecular and cellular mechanisms behind tendon physiology and 28 pathology is limited [14][15][16], yet damage and inadequate repair are considered to be central to tendon 29 disease onset and progression [17][18][19]. The stimuli that trigger tissue healing responses remain unclear, 30 potentially being a direct effect of cellular activation by overloading or by unloading after isolated fibre 31 rupture with subsequently altered cell-matrix interaction [20]. However, the mechanisms of 32 overloading and underloading may not be mutually exclusive, with tissue damage resulting in localized 33 regions...

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc−/− mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc−/− mice. Tendon hypoplasia was attenuated in Sparc−/− mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc−/− three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc−/− tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

Tendons enable movement by transferring muscle forces to the skeleton, and athletic performances critically rely on mechanically-optimized tendons. How load-bearing structures of tendon sense and adapt to physical demands is an open question of central importance to musculoskeletal medicine and human sports performance. Here, with calcium imaging in tendon explants and primary tendon cells we characterized how tenocytes detect mechanical forces and determined collagen fiber-sliding-induced shear stress as a key stimulus. CRISPR/Cas9 screening in human and rat tenocytes identified PIEZO1 as the crucial shear sensor. In rodents, elevated mechano-signaling increased tendon stiffness and strength both in vitro by pharmacological channel activation and in vivo by a Piezo1 gain-of-function mutation. Strikingly, humans carrying the PIEZO1 gain-of-function E756del mutation revealed a 16% average increase in normalized jumping height, with more effective storage of potential energy released during dynamic jumping maneuvers. We propose that PIEZO1-mediated mechano-signaling regulates tendon stiffness and impacts human athletic performance.

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