Matrix therapy is a newly coined name emphasizing the importance of the extracellular matrix in regenerative medicine. Heparan sulfates (HS) are key elements of the extracellular matrix (ECM) scaffold which store and protect most growth factors/cytokines controlling the cell migration and differentiation required for healing processes. We have engineered biodegradable nano-polymers (alpha 1–6 polyglucose carboxymethyl sulfate) mimicking (RGTA®) to replace destroyed HS in the damaged ECM scaffolding and to protect cytokines produced by healthy neighbouring cells, thereby restoring the ECM microenvironment and tissue homeostasis and, if needed, provide a homing niche for cell therapy. This matrix therapy approach has considerably improved the quality of healing in various animal models, including muscle and tendon, with reduction or absence of fibrosis resulting in a regeneration process. Over 50 000 patients have been treated in the last years for skin and corneal wounds with dedicated products based on this technology. A randomized controlled trial was performed on 22 racing French Standardbred Trotters (ST) horses to evaluate the efficacy of another polymer, OTR4131 Equitend®, to treat tendinopathies. We evaluated the effect versus placebo on acute superficial digital flexor tendonitis over 4 months by clinical and ultrasonographic measures and their racing performances followed up over the 2 years after treatment. A significant reduction on tendon cross section area was measured in treated animals, racing was 2–3 times more often than placebo, with 3.3 times fewer recurrences and pre-injury performance level was maintained. This study may pave the way for development in humans.

Introduction. Tendinopathies represent a significant health burden, causing inflammation, pain, and reducing quality of life. The pivotal role of macrophages (Mφ) characterized by their ability to differentiate into proinflammatory (M1) or anti-inflammatory (M2) phenotypes depending on the microenvironment, has gained significant interest in tissue inflammation research. Additionally, existing literature states that the interplay between tenocytes and immune cells during inflammation involves unidentified soluble factors (SF). This study aimed to investigate the effect of extracellular vesicles (EVs) and SF derived from polarized Mφ on tendon cells to provide deeper insights of their potential therapeutic applications in the context of inflammation. Method. Human monocytes were isolated from blood donor buffy coats and differentiated into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, EVs were isolated from the conditioned media from polarized Mφ and comprehensively characterized. In parallel, the elution media containing SF was collected. Furthermore, the EVs and SF were released independently onto tenocytes from human donors, previously induced with IL-1β to simulate an inflammatory environment. Finally, mRNA levels of tendon-related markers were evaluated by qPCR after the exposure to these EVs and SF. Result. Notably, the study found that the viability of the cells was not affected by the exposure to EVs nor SF, indicating their potential safety for therapeutic use. Moreover, the mRNA content of tendon-derived cells was evaluated following exposure to Mφ-EVs and SF revealing alterations in gene expression. Interestingly, a significant increase in the expression of tenomodulin was observed in tendon cells treated with Mφ-EVs. Conclusion. These results mark a significant advancement in understanding the interplay between Mφ and tenocytes at a molecular level. To fully understand the underlying causes of Mφ-EVs effects, and its potential clinical application in tendon inflammatory diseases, further comprehensive research is required. Acknowledgments. Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Tendinopathies represent the 45% of the musculoskeletal lesions and they are a big burden in clinics. Indeed, despite the relevant social impact, both the pathogenesis and the development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. Indeed, current treatment for tendinopathy are mainly symptomatic, and they present a high rate of pathology re-occurrence. In this contest, the development of an efficient in vivo model of acute tendinopathy, focused on the choice of the most appropriate species and strategy to induce the disease, would allow a better understanding of the pathology progression throughout its phases. Then, the purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies. 40 Sprague Dawley rats were randomly divided into two groups, treated by injection of collagenase type I within the Achilles tendon at 1 mg/mL (low dose, LD) or 3 mg/mL (high dose, HD). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days by H&E staining. Our results showed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater fatty degeneration and neovascularization with respect to the lower dose. These changes are time-dependent, thus resembling the tendinopathy development in humans. Indeed, the acute phase occurred from day 3 to day 15, while from day 15 to 45 it progressed towards the proliferative phase, displaying a degenerative appearance associated with a precocious remodeling process. The model represents a good balance between feasibility, in terms of reproducibility and costs, and similarity with the human disease. Moreover, the present model contributes to improve the knowledge about tendinopathy development, and then it could be useful to design further pre-clinical studies, in particular in order to test innovative treatments for tendinopathy

Introduction. Tendinopathies are among the most common musculoskeletal injuries. Nowadays, part of its diagnosis is established through subjective qualitative evaluation of 2D ultrasound (US). This enables limited diagnostic differentiation or therapeutic optimization and has limited added value to diagnosis in an earlier stage. It is generally accepted that extra diagnostic information can be obtained via strain evaluation. The accurate validation of strain estimation is challenging due to the lack of a ground-truth. Therefore we evaluate the repeatability of displacement and strain estimations in the longitudinal direction, using an easy, fast and interactive application to estimate local strain during dynamic loading of the tendon. Materials and Methods. One healthy volunteer laid in a prone position with the foot fixed to an isokinetic device. Three sets of passive movement between −10° plantarflexion and +10° dorsiflexion were performed and repeated the following day. During this, US images with a spatial resolution of 0.02mm × 0.09mm were acquired at a frame-rate of 100Hz. The US system used was the Vevo2100 with a MS250 linear array transducer with a center frequency of 20MHz. After image collection, consecutive pairs of 2D images were registered in a multi-resolution scheme, using an affine and b-spline transformation optimized by the minimization of the sum-of-squared differences, to obtain deformation vector fields. Lastly the interactive application allows local analysis of tissue displacement and strain within selected regions of interest. Mean and standard deviation of the intra- and inter-day relative differences were calculated. Results. The results show a mean intra-day relative difference of 13.71%±4.76% in displacement and of 16.29%±5.17% in strain. For inter-day comparison, the relative difference was 16.98%±14.62% in displacement and was 16%±13.51% in strain. Results show physiologically meaningful and similar strain tendencies when grouping proximal and distal regions. Discussion. This work shows promising preliminary data that suggest that with our method strain and deformation can be measured in a reproducible way using high-frequency US, with little effect of slight variations in acquisition conditions. This brings the application of US based strain estimation in clinical scenarios closer to reality. However, further tests are needed to confirm these conclusions

Introduction. Tendinopathies are debilitating and painful conditions. They are believed to result from repetitive overuse, which can create micro-damage that accumulates over time, and initiates a catabolic cell response. The aetiology of tendinopathy remains poorly understood, therefore the ideal treatment remains unclear. However, current data support the use of eccentric exercise as an effective treatment. In a previous study, we have shown that eccentric loading generates perturbations in the tendon at 10Hz, which is not present during other less effective loading regimes. Consequently, we hypothesis that 10Hz loading initiates an increased anabolic response in tenocytes, that can promote tendon repair. Materials and Methods. Human tenocytes from healthy hamstring tendons and tendinopathic Achilles tendons were derived by collagenase digest and outgrowth respectively. Tenocytes were seeded into 3D collagen gels. The gels were fixed in custom-made chambers and placed in an incubator for 24hrs whilst gene expression stabilised. After 24hrs, cyclic uniaxial strain at 1% ± 1% was applied to the cells, at either 1Hz (n=4) or 10Hz (n=4) using a Bose loading system. After 15 minutes of cyclic strain, the samples were maintained in chambers under 1% static strain for 24 hrs after which gene expression was characterised using RT-PCR. Results. In healthy cells, data showed an increase in expression of all analysed genes after loading (MMP1, MMP2, MMP13, COL1A1, COL3A1, COL5A1, ADAMTS5, IL6, IL8 and TIMP3). Furthermore, the increase in gene expression was larger in the higher frequency loading group, across all genes. Tendinopathic cells showed a more varied response, with upregulation of MMP1, MMP13, COL3A1, ADAMTS5, IL6 and IL8, and downregulation of COL1A1 and COL5A1. Once again, all changes were more pronounced in the higher frequency loading group. Discussion. These preliminary data suggest increased matrix turnover as a result of loading, particularly with high frequency loading in healthy tenocytes, whilst the profile of tendinopathic cells, may indicate an early healing response, where collagen type III is preferentially unregulated relative to types I and V. High frequency loading elicited a more pronounced cellular response, perhaps correlating with the improved repair seen with eccentric loading in vivo

Summary Statement. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Introduction. Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. We have previously investigated the regulation by mechanical strain of metalloproteinase gene expression in human tenocyte in a 3D collagen matrix. Integrins are important in cellular interaction with the ECM and are reported to mediate mechanotransduction in various non-tendon tissues. We have reported that TGFbeta activation is a key player in the regulation of metalloproteinases in response to mechanical load, which may be mediated by integrins. This project aims to investigate the effect of cyclic loading and TGFbeta stimulation on integrin expression by human tenocytes, in collagen and fibrin matrices. Methods. Human tenocytes were seeded at 1.5×106 cells/ml into collagen (rat tail type I, 1mg/ml) or fibrin (fibrinogen 6mg/ml, Thrombin 0.2u/ml) gels and stretched using a sinusoidal waveform of 0–5% at 1Hz using the Flexcell FX4000T(trade mark) system. Cultures were treated with or without 1ng/ml TGFbeta1 and load for 0–48 hours. Taqman Low density Array was used to asses a range of integrin, including ITGA1-6, ITGA10 and ITGA11 as well as ITGB1-5 (n=3). Results. In collagen cultures all integrins assayed were detectable (Ct < 35). ITGB1 was increased 2 fold with 48 hours of cyclic strain (p=0.006). ITGA6 and ITGA10 were decreased 1.4 and 2 fold with TGFbeta treatment after 24 hours (p=0.019, p=0.006). ITGA3 and ITGB3 were significantly decreased 7.6 and 8.3 fold with TGFbeta treatment after 48 hours (p=0.012, p=0.023). ITGA5 and ITGB1 showed similar responses with strain and TGFbeta, i.e. an increased trend. However, the other integrins showed a dissimilar response to strain and TGFbeta. Here we compare these responses to those in fibrin under the same conditions. Discussion. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Any differences in the cellular response to load in collagen and fibrin cultures, indicates that cellular interaction with the ECM is an important factor in the detection of load. Due to the differential regulation of some of the integrins with strain and TGFbeta, it appears that TGFbeta may not be responsible for the regulation of all integrins with strain. However this remains unconfirmed and may be explained by a temporal difference. Further analysis of how integrins are regulated in response to mechanical load and how this expression is translated to the protein level is required