Exercise entrainment of musculoskeletal connective tissue clocks.

The musculoskeletal system, crucial for movement and support, relies on the delicate balance of connective tissue homeostasis. Maintaining this equilibrium is essential for tissue health and function. There has been increasing evidence in the last decade that shows the circadian clock as a master regulator of extracellular matrix (ECM) homeostasis in several connective tissue clocks. Very recently, exercise has emerged as a significant entrainment factor for cartilage and intervertebral disc circadian rhythms. Understanding the implications of exercise on connective tissue peripheral clocks holds promise for enhancing tissue health and disease prevention. Exercise-induced factors such as heat, glucocorticoid release, mechanical loading, and inter-tissue crosstalk may play pivotal roles in entraining the circadian rhythm of connective tissues. This mini review underscores the importance of elucidating the mechanisms through which exercise influences circadian rhythms in connective tissues to optimize ECM homeostasis. Leveraging exercise as a modulator of circadian rhythms in connective tissues may offer novel therapeutic approaches to physical training for preventing musculoskeletal disorders and enhancing recovery.

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts in vitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading in vivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

Distinct myofibre domains of the human myotendinous junction revealed by single-nucleus RNA sequencing.

The myotendinous junction (MTJ) is a specialized domain of the multinucleated myofibre that is faced with the challenge of maintaining robust cell-matrix contact with the tendon under high mechanical stress and strain. Here, we profiled 24,124 nuclei in semitendinosus muscle-tendon samples from three healthy males by using single-nucleus RNA sequencing (snRNA-seq), alongside spatial transcriptomics, to gain insight into the genes characterizing this specialization in humans. We identified a cluster of MTJ myonuclei represented by 47 enriched transcripts, of which the presence of ABI3BP, ABLIM1, ADAMTSL1, BICD1, CPM, FHOD3, FRAS1 and FREM2 was confirmed at the MTJ at the protein level in immunofluorescence assays. Four distinct subclusters of MTJ myonuclei were apparent, comprising two COL22A1-expressing subclusters and two subclusters lacking COL22A1 expression but with differing fibre type profiles characterized by expression of either MYH7 or MYH1 and/or MYH2. Our findings reveal distinct myonuclei profiles of the human MTJ, which represents a weak link in the musculoskeletal system that is selectively affected in pathological conditions ranging from muscle strains to muscular dystrophies.

Proteome profiles of intramuscular connective tissue: influence of aging and physical training.

Both aging and physical activity can influence the amount of intramuscular connective tissue in skeletal muscle, but the impact of these upon specific extracellular matrix (ECM) proteins in skeletal muscle is unknown. We investigated the proteome profile of intramuscular connective tissue by label-free proteomic analysis of cellular protein-depleted extracts from lateral gastrocnemius muscle of old (22-23 mo old) and middle-aged (11 mo old) male mice subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We hypothesized that aging is correlated with an increased amount of connective tissue proteins in skeletal muscle and that regular physical activity can counteract these age-related changes. We found that dominating cellular proteins were diminished in the urea/thiourea extract, which was therefore used for proteomics. Proteomic analysis identified 482 proteins and showed enrichment for ECM proteins. Statistical analysis revealed that the abundances of 86 proteins changed with age. Twenty-three of these differentially abundant proteins were identified as structural ECM proteins (e.g., collagens and laminins) and all of these were significantly more abundant with aging. No significant effect of training or interaction between training and advance in age was found for any proteins. Finally, we found a lower protein concentration in the urea/thiourea extracts from the old mice compared with the middle-aged mice. Our findings indicate that the intramuscular ECM solubility is affected by increased age but is not altered by physical training.NEW & NOTEWORTHY We investigated the impact of aging and exercise on extracellular matrix components of intramuscular connective tissue using proteomics. Middle-aged and old mice were subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We prepared extracts of extracellular matrix proteins depleted of cellular proteins. Our findings indicate that intramuscular connective tissue alters its soluble protein content with age but is unaffected by training.

Disruption of day-to-night changes in circadian gene expression with chronic tendinopathy.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 h apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night reduction in collagen I expression. KEY POINTS: Tendinopathy is a major clinical problem with unsolved pathogenesis. Previous work in mice has shown that a robust circadian rhythm is required for collagen homeostasis in tendons. The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue. Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues. We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy.

Tensile Loaded Tissue-Engineered Human Tendon Constructs Stimulate Myotube Formation.

Skeletal muscle possesses adaptability to mechanical loading and regenerative potential following muscle injury due to muscle stem cell activity. So far, it is known that muscle stem cell activity is supported by the roles of several interstitial cells within skeletal muscle in response to muscle damage. The adjacent tendon is also exposed to repetitive mechanical loading and possesses plasticity like skeletal muscle. However, the interplay between the skeletal muscle and adjacent tendon tissue has not been fully investigated. In this study, we tested whether factors released by three-dimensional engineered human tendon constructs in response to uniaxial tensile loading can stimulate the proliferation and differentiation of human-derived myogenic cells (myoblasts). Tendon constructs were subjected to repetitive mechanical loading (4% strain at 0.5 Hz for 4 h) and nonrepetitive loading (0% strain at 0 Hz for 4 h), and the conditioned media from mechanically loaded and nonmechanically loaded control constructs were applied to myoblasts. Immunofluorescence analysis revealed both an increase of myotube fusion index (≥5 nuclei within one desmin+ myotube) and the myotube diameter when conditioned medium from mechanically loaded tendon constructs was applied. Myostatin, myosin heavy chain 7, and AXIN2 gene expressions were downregulated in myotubes treated with conditioned medium from mechanically loaded tendon constructs. However, proliferative potential (number of Ki67+ and bromodeoxyuridine+ myoblasts) did not differ between the two groups. These results indicate that tendon fibroblasts enhance myotube formation by mechanical loading-induced factors. Our finding suggests that mechanical loading affects the signaling interplay between skeletal muscle and tendon tissue and is thus important for musculoskeletal tissue development and regeneration in humans. Impact statement The interplay between satellite cells and various types of resident cells within the skeletal muscle for muscle regeneration has been extensively studied. However, even though tendon tissue is located adjacent to skeletal muscle tissue and cells in these tissues are exposed to repetitive mechanical loading together, the interaction between muscle and tendon tissues for muscle regeneration remains to be elucidated. In this study, we report that the conditioned media from engineered human tendon tissues undergoing repetitive tensile mechanical loading enhanced myotube formation. Our in vitro findings extend the fundamental understanding of the crosstalk between adjacent tissues of the muscle-tendon unit.

Autophagy guards tendon homeostasis.

Tendons are vital collagen-dense specialized connective tissues transducing the force from skeletal muscle to the bone, thus enabling movement of the human body. Tendon cells adjust matrix turnover in response to physiological tissue loading and pathological overloading (tendinopathy). Nevertheless, the regulation of tendon matrix quality control is still poorly understood and the pathogenesis of tendinopathy is presently unsolved. Autophagy, the major mechanism of degradation and recycling of cellular components, plays a fundamental role in the homeostasis of several tissues. Here, we investigate the contribution of autophagy to human tendons' physiology, and we provide in vivo evidence that it is an active process in human tendon tissue. We show that selective autophagy of the endoplasmic reticulum (ER-phagy), regulates the secretion of type I procollagen (PC1), the major component of tendon extracellular matrix. Pharmacological activation of autophagy by inhibition of mTOR pathway alters the ultrastructural morphology of three-dimensional tissue-engineered tendons, shifting collagen fibrils size distribution. Moreover, autophagy induction negatively affects the biomechanical properties of the tissue-engineered tendons, causing a reduction in mechanical strength under tensile force. Overall, our results provide the first evidence that autophagy regulates tendon homeostasis by controlling PC1 quality control, thus potentially playing a role in the development of injured tendons.

Circadian regulation of protein cargo in extracellular vesicles.

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography-mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Mutual stimulatory signaling between human myogenic cells and rat cerebellar neurons.

Insight into the bidirectional signaling between primary human myogenic cells and neurons is lacking. For this purpose, human myogenic cells were derived from the semitendinosus and gracilis muscles of five healthy individuals and co-cultured with cerebellar granule neurons from two litters of 7-day-old Wistar rat pups, in muscle medium or neural medium, alongside monocultures of myogenic cells or neurons. RT-PCR was performed to determine human mRNA levels of GAPDH, Ki67, myogenin, and MUSK, and the acetylcholine receptor subtypes CHRNA1, CHRNB1, CHRNG, CHRND, and CHRNE, and rat mRNA levels of GAPDH, Fth1, Rack1, vimentin, Cdh13, and Ppp1r1a. Immunocytochemistry was used to evaluate neurite outgrowth (GAP43) in the presence and absence of myogenic cells. Co-culture with primary neurons lead to higher myogenic cell gene expression levels of GAPDH, myogenin, MUSK, CHRNA1, CHRNG, and CHRND, compared to myogenic cells cultured alone. It appeared that neurons preferentially attached to myotubes and that neurite outgrowth was enhanced when neurons were cultured with myogenic cells compared to monoculture. In neural medium, rat mRNA levels of GAPDH, vimentin, Cdh13, and Ppp1r1a were greater in co-culture, versus monoculture, whereas in muscle medium co-culture lead to lower levels of Fth1, Rack1, vimentin, and Cdh13 than monoculture. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells, providing support for an active role for both the neuron and the muscle cell in stimulating neurite growth and myogenesis. Bidirectional muscle nerve signaling.

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts.

BACKGROUND: Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. METHODS: Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. RESULTS: Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFß1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. CONCLUSIONS: Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract.

Comparison of Tenocyte Populations from the Core and Periphery of Equine Tendons.

Tendon is a highly organized, dense connective tissue that has been demonstrated to have very little turnover. In spite of the low turnover, tendon can grow in response to loading, which may take place primarily at the periphery. Tendon injuries and recurrence of injuries are common in both humans and animals in sports. It is unclear why some areas of the tendon are more susceptible to such injuries and whether this is due to intrinsic regional differences in extracellular matrix (ECM) production or tissue turnover. This study aimed to compare populations of tenocytes derived from the tendon core and periphery. Tenocytes were isolated from equine superficial digital flexor tendons (SDFTs), and the proliferation capacity was determined. ECM production was characterized by immuno- and histological staining and by liquid chromatography-mass spectrometry-based proteomics. Core and periphery SDFT cultures exhibited comparable proliferation rates and had very similar proteome profiles, but showed biological variation in collagen type I deposition. In conclusion, the intrinsic properties of tenocytes from different regions of the tendon are very similar, and other factors in the tissue may contribute to how specific areas respond to loading or injury.

Circadian control of the secretory pathway maintains collagen homeostasis.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

Importance of the circadian clock in tendon development.

Tendons are remarkable tissues that transmit force from muscle to bone during joint movement. They are remarkable because they withstand tensile forces that are orders of magnitude greater than can be withstood by isolated cells. The ability of the cells to survive is directly attributable to the stress shielding properties of the collagen-rich extracellular matrix of the tissue. A further remarkable feature is that the vast majority (>98%) of the collagen is never turned over; it is synthesized during embryonic through early adult development and persists for the lifetime of the person. How the collagen is synthesized, and importantly, how it is protected from fatigue failure for decades of countless loading cycles, remains a mystery. A recent discovery is that tendons are peripheral circadian clock tissues in which the expression of ~5% of the transcriptome is rhythmic during 24h. Evidence is emerging that a fraction of the total amount of collagen is synthesized and removed on a daily basis without being incorporated into the lifelong permanent collagen. This review provides some of the background, and summarizes the findings, of these latest discoveries. Detailed descriptions of tendon development, collagen synthesis and collagen fibrillogenesis can be found in excellent reviews (cited here) and will not be a major part of this review.

Investigating circadian clock gene expression in human tendon biopsies from acute exercise and immobilization studies.

PURPOSE: The discovery of musculoskeletal tissues, including muscle, tendons, and cartilage, as peripheral circadian clocks strongly implicates their role in tissue-specific homeostasis. Age-related dampening and misalignment of the tendon circadian rhythm and its outputs may be responsible for the decline in tendon homeostasis. It is unknown which entrainment signals are responsible for the synchronization of the tendon clock to the light-dark cycle. METHODS: We sought to examine any changes in the expression levels of core clock genes (BMAL1, CLOCK, PER2, CRY1, and NR1D1) in healthy human patellar tendon biopsies obtained from three different intervention studies: increased physical activity (leg kicks for 1 h) in young, reduced activity (2 weeks immobilization of one leg) in young, and in old tendons. RESULTS: The expression level of clock genes in human tendon in vivo was very low and a high variation between individuals was found. We were thus unable to detect any differences in core clock gene expression neither after acute exercise nor immobilization. CONCLUSIONS: We are unable to find evidence for an effect of exercise or immobilization on circadian clock gene expression in human tendon samples.

Synchronized mechanical oscillations at the cell-matrix interface in the formation of tensile tissue.

The formation of uniaxial fibrous tissues with defined viscoelastic properties implies the existence of an orchestrated mechanical interaction between the cytoskeleton and the extracellular matrix. This study addresses the nature of this interaction. The hypothesis is that this mechanical interplay underpins the mechanical development of the tissue. In embryonic tendon tissue, an early event in the development of a mechanically robust tissue is the interaction of the pointed tips of extracellular collagen fibrils with the fibroblast plasma membrane to form stable interface structures (fibripositors). Here, we used a fibroblast-generated tissue that is structurally and mechanically matched to embryonic tendon to demonstrate homeostasis of cell-derived and external strain-derived tension over repeated cycles of strain and relaxation. A cell-derived oscillatory tension component is evident in this matrix construct. This oscillatory tension involves synchronization of individual cell forces across the construct and is induced in each strain cycle by transient relaxation and transient tensioning of the tissue. The cell-derived tension along with the oscillatory component is absent in the presence of blebbistatin, which disrupts actinomyosin force generation of the cell. The time period of this oscillation (60-90 s) is well-defined in each tissue sample and matches a primary viscoelastic relaxation time. We hypothesize that this mechanical oscillation of fibroblasts with plasma membrane anchored collagen fibrils is a key factor in mechanical sensing and feedback regulation in the formation of tensile tissues.

Changes in S100 Proteins Identified in Healthy Skin following Electrical Stimulation: Relevance for Wound Healing.

OBJECTIVE: Targeted electrical energy applied to wounds has been shown to improve wound-healing rates. However, the mechanisms are poorly understood. The aim of this study was to identify genes that are responsive to electrical stimulation (ES) in healthy subjects with undamaged skin. METHODS: To achieve this objective, study authors used a small, noninvasive ES medical device to deliver a continuous, specific, set sequence of electrical energy impulses over a 48-hour period to the skin of healthy volunteers and compared resultant gene expression by microarray analysis. MAIN RESULTS: Application of this specific ES resulted in differential expression of 105 genes, the majority of which were down-regulated. Postmicroarray analyses revealed there was commonality with a small number of genes that have previously been shown to be up-regulated in skin wounds, including venous leg ulcers. CONCLUSIONS: The specific sequence of ES applied continuously for 48 hours to the skin of healthy patients has the effect of modifying expression in a number of identified genes. The identification of the differential expression in this subset of genes in healthy subjects provides new potential lines of scientific inquiry for identifying similar responses in subjects with slow or poorly healing wounds.

Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix.

Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D-3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed.

Matrix metalloproteinase 14 is required for fibrous tissue expansion.

Type I collagen-containing fibrils are major structural components of the extracellular matrix of vertebrate tissues, especially tendon, but how they are formed is not fully understood. MMP14 is a potent pericellular collagenase that can cleave type I collagen in vitro. In this study, we show that tendon development is arrested in Scleraxis-Cre::Mmp14 lox/lox mice that are unable to release collagen fibrils from plasma membrane fibripositors. In contrast to its role in collagen turnover in adult tissue, MMP14 promotes embryonic tissue formation by releasing collagen fibrils from the cell surface. Notably, the tendons grow to normal size and collagen fibril release from fibripositors occurs in Col-r/r mice that have a mutated collagen-I that is uncleavable by MMPs. Furthermore, fibronectin (not collagen-I) accumulates in the tendons of Mmp14-null mice. We propose a model for cell-regulated collagen fibril assembly during tendon development in which MMP14 cleaves a molecular bridge tethering collagen fibrils to the plasma membrane of fibripositors.