Introduction. We previously reported that disruption of TGFβ signaling in limb mesenchyme resulted in complete failure of tendon differentiation. Materials and Methods. To bypass this early function and study additional roles of TGFβ signaling in tendon development we disrupted TGFβ signaling in tenocytes after they assumed the tendon cell fate by using the tendon deletor ScxCre to target the floxed type2 TGFβ receptor. Results. Most mutant (Tgfbr2;ScxCre) pups appeared normal at birth but exhibited movement difficulties and splayed limbs by P3. ScxGFP signal revealed that tendon formation was not affected in CKO embryos. Nonetheless, three distinct tendon phenotypes were manifested later in development: (a) a single flexor tendon consistently snapped at late embryonic stage; whereas at post-natal stage, some tendons that appeared intact at birth were (b) eventually eliminated or (c) retained structural integrity with a substantial loss of the ScxGFP signal. Interestingly, the ScxGFP-negative cells also lost other tendon marker genes. Lineage tracing revealed that these cells were derived from Scx-expressing cells, suggesting a disruption of the tendon cell fate (dedifferentiation) but we found no evidence of transdifferentiation. Varying degrees of tendon degeneration were also seen in CKO pups, as indicated by disrupted collagen fibrils, septation of the tendon and altered epitenon. Another striking feature we identified in the Tgfbr2;ScxCre tendon phenotype was recruitment of new cells into the degenerating tendon. Finally, our data also indicates that the Tgfbr2f;ScxCre tendon phenotype is not due to a direct requirement for TGFβ signaling in tenocytes. Discussion. This analysis thus highlights an unexpected possibility for loss of differentiated characteristics in tenocytes as a key factor in a tendon degenerative process. We hypothesize that the tendon phenotypes may represent a disruption of cell-cell or cell-matrix interactions, and investigations are currently underway to test this hypothesis. Moreover, this is the first demonstration of active cell recruitment into a non-injured tendon that may be used to identify the origin and activation mechanisms for tendon stem/progenitor cells. Taken together, our findings reveal an essential and non-cell autonomous role of TGFβ signaling in maintenance of the tendon cell fate

Depletion of Scleraxis-lineage (ScxLin) cells in adult tendon recapitulates age-related decrements in cell density, ECM organization and composition. However, depletion of ScxLin cells improves tendon healing, relative to age-matched wildtype mice, while aging impairs healing. Therefore, we examined whether ScxLin depletion and aging result in comparable shifts in the tendon cell environment and defined the intrinsic programmatic shifts that occur with natural aging, to define the key regulators of age-related healing deficits.

ScxLin cells were depleted in 3M-old Scx-Cre+; Rosa-DTRF/+ mice via diphtheria toxin injections into the hindpaw. Rosa-DTRF/+ mice were used as wildtype (WT) controls. Tendons were harvested from 6M-old ScxLin depleted and WT mice, and 21-month-old (21M) C57Bl/6 mice (aged). FDL tendons (n=6) were harvested for single-cell RNAseq, pooled, collagenase digested, and sorted for single cell capture. Data was processed using Cell Ranger and then aligned to the annotated mouse genome (mm10). Filtering, unsupervised cell clustering, and differential gene expression (DEG) analysis were performed using Seurat.

Following integration and sub-clustering of the tenocyte populations, five distinct subpopulations were observed. In both ScxLin depletion and aging, ‘ECM synthesizers’ and ‘ECM organizers’ populations were lost, consistent with disruptions in tissue homeostasis and altered ECM composition. However, in ScxLin depleted mice retention of a ‘specialized ECM remodeler’ population was observed, while aging tendon cells demonstrated inflammatory skewing with retention of a ‘pro-inflammatory tenocyte population’. In addition, enrichment of genes associated with protein misfolding clearance were observed in aged tenocytes. Finally, a similar inflammatory skewing was observed in aged tendon-resident macrophages, with this skewing not observed in ScxLin depleted tendons.

These data suggest that loss of ‘ECM synthesizer’ populations underpins disruptions in tendon homeostasis. However, retention of ‘specialized remodelers’ promotes enhanced healing (ScxLin depletion), while inflammatory skewing may drive the impaired healing response in aged tendons.

There is a growing socio-economic need (i.e. “ageing society”) for effective and reproducible strategies to repair musculoskeletal tissue. In particular, acute tendon injury and chronic tendinopathies remain clinically challenging and novel treatment modalities are urgently needed. Tendons resemble a connective tissue rich in highly organized collagen fibers, displaying a remarkably high tensile strength. However, partly due to the low number of cells and their more or less avascular nature tendons heal relatively slowly. Ultimately, tendon regeneration encompasses the full restoration of the biological, biochemical and biomechanical properties, which are often impaired by endogenous healing cascades. Usually, a connective scar tissue forms at the injury site and the replaced tissue does not function adequately at high strain levels, increasing the chance of re-rupture. Despite significant advancements in tissue regeneration and engineering strategies, the clinical impact for the regeneration of tendon remains limited. For the development of novel methods to repair tendons we need to pin down the molecular and cellular mechanisms amenable to modulate endogenous (or exogenous) cell behaviour towards functional tissue regeneration. By comparing the gene expression profile of Achilles tendon tissue harvested from young-mature and old mice we demonstrate profound changes in the expression of ECM-related proteins and a previously unknown role of Secreted protein acidic and rich in cysteine (Sparc; also known as BM-40 or osteonectin) in tendons. Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties potentially drives adipogenic differentiation of tendon stem and progenitor cells (TDSPCs) and consequently lipid accretion in tendons. Generally, the fate of stem/ progenitor cells is largely determined by stimuli from the stem cell niche. In tendons, we describe a novel cellular barrier, most likely preventing the leakage of blood-borne products into the tendon proper. We propose that this “blood-tendon barrier” is part of the stem cell niche in tendons controlling TDSCP fate, preventing erroneous differentiation. By investigating the developmental programs driving tendon tissue formation and on the other hand the mechanisms contributing to the senescence of tendons, ultimately resulting in decreased quality of tendons in the elderly, novel targets for clinical intervention potentially can be discovered.

Tendon and ligament tissues are fascinating in their simplistic appearance of tissue architecture coupled with outstanding biomechanical properties. In the last decade, the mechanisms governing their development, degenerative disease progression and step-wise repair process are becoming better understood. In this talk, I will present an overview of our basic research work on these following points. (i) Tendon generation: I will discuss our finding on the role of growth and biomechanical factors influencing tendon stem/progenitor cells; (ii) Tendon degeneration: I will provide evidences how disturbed cell-cell and cell-matrix contacts are involved in loss of tissue integrity; (iii) Tendon regeneration: I will present in vivo data on the application and performance of various cell populations in tendon repair

Patellar tendinosis (PT) is common and can result in prolonged disability, especially in jumping athletes. Recently developed ultra-short-echo (UTE) MRI sequences allow for quantitative evaluation of tendon biostructure with T2* relaxometry. This study evaluated the relationships between changes over time (COT) in quantitative T2*-metrics, qualitative PT grades, and patient reported symptoms within 10 male basketball players from a single collegiate basketball team. All subjects completed weekly VISA-P symptomology questionnaires over the basketball season. Bilateral 3-Tesla MRIs (GE Healthcare) were obtained at pre- and post-season study visits. High-resolution, PD-weighted, FSE sequences were used to qualitatively grade PT. Quantitative T2*-metrics were evaluated using high-resolution, 3D, multi-echo, UTE-MRI sequences. Bilinear exponential fits of SI to corresponding echo time were used to calculate T2*-metrics. All qualitative and quantitative evaluations were region specific (proximal, middle, distal). Linear mixed effects models assessed associations of side and region with T2*-metrics. Spearman correlations evaluated relationships between outcome measures. Within and between study visits, significant side-to-side differences in T2*-metrics were found and were significantly impacted by leg dominance (p<0.05). Pre-season T2*-metrics correlated with COT in T2*-metrics, COT in T2*-metrics correlated with COT in qualitative PT grades, and post-season T2*-metrics correlated with max changes in VISA-P scores (ρ≥0.64). Quantitative T2*-metrics can detect PT and may be capable of predicting the onset of pathology. T2*-metrics could benefit the clinical management of PT: it is sensitive to changes in pathologic severity over time, and therefore can serve as a quantitative metric to guide treatment and evaluate intervention efficacy.

The rotator cuff tendinopathy is one of the most common shoulder problems leading to full-thickness rotator cuff tendon tear and, eventually, to degenerative arthritis. Recent research on rotator cuff tendon degeneration has focused on its relationship to cell death. The types of cell death known to be associated with rotator cuff tendon degeneration are apoptosis, necrosis, and autophagic cell death. The increased incidence of cell death in degenerative tendon tissue may affect the rates of collagen synthesis and repair, possibly weakening tendon tissue and increasing the risk of tendon rupture. The biomolecular mechanisms of the degenerative changes leading to apoptotic cell death in rotator cuff tenofibroblasts have been identified as oxidative-stress-related cascade mechanisms. Furthermore, apoptosis, necrosis, and autophagic cell death are all known to be mediated by oxidative stress, a condition in which ROS (reactive oxygen species) are overproduced. Lower levels of oxidative stress trigger apoptosis; higher levels mediate necrosis. Although the signaltransduction pathway leading to autophagy has not yet been fully established, ROS are known to be essential to autophagy. A neuronal theory regarding rotator cuff degeneration has been developed from the findings that glutamate, a neural transmitter, is present in increased concentrations in tendon tissues with tendinopathy and that it induces rat supraspinatus tendon cell death. Recent studies have reported that hypoxia involved in rotator cuff tendon degeneration. Because antioxidants are known to scavenge for intracellular ROS, some studies have been conducted to determine whether antioxidants can reduce cell death in rotator cuff tendon-origin fibroblasts. The first study reported that an antioxidant has the ability to reduce apoptosis in oxidative-stressed rotator cuff tenofibroblasts. The second study reported that antioxidants have both antiapoptotic effects and antinecrotic effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The third study reported that an antioxidant has antiautophagic-cell-death effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The fourth study reported that glutamate markedly increases cell death in rotator cuff tendonorigin fibroblasts. The glutamate-induced cytotoxic effects were reduced by an antioxidant, demonstrating its cytoprotective effects against glutamate-induced tenofibroblast cell death. The fifth study reported that hypoxia significantly increases intracellular ROS and apoptosis. The hypoxia-induced cytotoxic effects were markedly attenuated by antioxidants, demonstrating their cytoprotective effects against hypoxia-induced tenofibroblast cell death. In conclusion, antioxidants have cytoprotective effects on tenofibroblasts exposed in vitro to an oxidative stressor, a neurotransmitter, or hypoxia. These cytoprotective effects result from antiapoptotic, antinecrotic, and antiautophagic actions involving the inhibition of ROS formation. These findings suggest that antioxidants may have therapeutic potential for rotator cuff tendinopathy. Further studies must be conducted in order to apply these in vitro findings to clinical situations

Tendinopathy is a disease associated with pain and tendon degeneration, leading to a decreased range of motion and an increased risk of tendon rupture. The etiology of this frequent disease is still unknown. In other musculoskeletal tissues like cartilage and intervertebral discs, transient receptor potential channels (TRP- channels) were shown to play a major role in the progression of degeneration. Due to their responsiveness to a wide range of stimuli like temperature, pH, osmolarity and mechanical load, they are potentially relevant factors in tendon degeneration as well. We therefore hypothesize that TRP- channels are expressed in tendon cells and respond to degeneration inducing stimuli. By immunohistochemistry, qRT-PCR and western blot analyses, we found three TRP channel members, belonging to the vanilloid (TRPV), and ankyrin (TRPA) subfamily, respectively, to be expressed in healthy human tendon tissue as well as in rodent tendon, with expression being located to cells within the dense tendon proper, as well as to endotenon resident cells. In vitro-inflammatory and ex vivo-mechanical stimulation led to a significant upregulation of TRPA1 expression in tendon cells, which correlates well with the fact that TRPA1 is considered as mechanosensitive channel being sensitized by inflammatory mediators. This is the first description of TRP- channels in human and rodent tendon. As these channels are pharmacologically targetable by both agonists and antagonists, they may represent a promising target for novel treatments of tendinopathy

Though retear rates following rotator cuff repair are well established, we set out to review current literature to determine when early retears occurred (defined as <12m following surgery), and examine which pre- and post-operative variables might affect outcome. Pubmed, Medline, and CINAHL were searched for literature published from 2011 to 2021 using specific search terms. The inclusion criteria were studies reporting retear rates within 12 months of initial surgical repair. Exclusionary criteria were studies that included partial thickness tears, and studies that did not use imaging modalities within 12 months to assess for retears. PRISMA guidelines were followed, identifying a total of 10 papers. A combined total of 3372 shoulders included (Mean age 56 −67 years). The most common modality used to identify early retears were ultrasound scan and MRI. 6 of the 10 studies completed imaging at 0-3 months, 6 studies imaged at 3-6 months and 6 studies imaged at 6-12 months. Across all studies, there was a 17% early retear rate (574 patients). Of these, 13% occurred by 3 months, whilst the peak for retears occurred at 3-6 months (82%) and 5% occurred at 6-12 months. The risk of retear was higher in larger tears and extensive tendon degeneration. All studies apart from one documented a return to work/sport at 6 months post-operatively. Postoperative rehabilitation does not appear to alter retear rate, although data is limited with only 1 of 10 studies allowing active range of movement before 6 weeks. Retorn tendons had poorer functional outcomes compared to intact tendons at 12m following initial repair. The majority of early retears occur at 3-6 months and this time period should be prioritised both in rehabilitation protocols and future research. Age, tear size, and tendon degeneration were found to influence likelihood of early retears

Tendons mainly consist of collagen in order to withstand high tensile forces. Compared to other, high turnover tissues, cellularity and vascularity in tendons are low. Thus, the natural healing process of tendons takes long and can be problematic. In case of injury to the enthesis, the special transition from tendon over cartilage to bone is replaced by a fibrous scar tissue, which remains an unsolved problem in rotator cuff repair. To improve tendon healing, many different approaches have been described using scaffolds, stem cells, cytokines, blood products, gene therapy and others. Despite promising in vitro and in vivo results, translation to patient care is challenging. In clinics however, tendon auto- or allografts remain still first choice to augment tendon healing if needed. Therefore, it is important to understand natural tendon properties and natural tendon healing first. Like in other tissues, senescence of tenocytes seems to play an important role for tendon degeneration which is interestingly not age depended. Our in vivo healing studies have shown improved and accelerated healing by adding collagen type I, which is now used in clinics, for example for augmentation of rotator cuff repair. Certain cytokines, cells and scaffolds may further improve tendon healing but are not yet used routinely, mainly due to missing clinical data, regulatory issues and costs. In conclusion, the correct diagnosis and correct first line treatment of tendon injuries are important to avoid the necessity to biologically augment tendon healing. However, strategies to improve and accelerate tendon healing are still desirable. New treatment opportunities may arise with further advances in tendon engineering in the future

Introduction. Diabetes mellitus type 2 (DMT2) patients often develop Achilles tendon (AS) degeneration. The ZDF rat model is often used to study DMT2. Hence, this study investigated whether tenocytes isolated from diabetic and non diabetic ZDF rats respond differentially to normo- (NG) and hyperglycemic (HG) conditions in the presence of tumor necrosis (TNF)α. Method. AS tenocytes isolated from adult diabetic (fa/fa) or lean (fa/+) Zucker Diabetic Fatty (ZDF) rats were treated with 10 ng/mL TNFα either under NG or HG conditions (1 g/L versus 4.5 g/L glucose). Tendons were characterized histopathologically using Movin score. Tenocyte survival, metabolic activity, gene and/or protein expression of the main tendon extracellular matrix (ECM) component collagen type 1, the myofibroblast marker alpha smooth muscle actin (αSMA, Acta2), complement regulatory factors, the antioxidant defense enzyme heme oxygenase-1 (Hmox1), suppressors of cytokine signaling (Socs)1 and Soc3 were analyzed. Result. Tendons of diabetic rats showed significantly higher Movin score values suggesting tendon degeneration. Tenocyte vitality remained high, but metabolic activity was impaired by HG conditions, irrespectively of tenocyte origin. Higher amounts of αSMA were visualized in tendons/cells of diabetic rats or those exposed to TNFα. Collagen type 1 protein and gene expression was suppressed by TNFα (NG), but only in cells of non diabetic animals. The anaphylatoxin receptor C3aR was higher expressed in tenocytes from diabetic animals. CD46 was suppressed by TNFα (NG) in cells of diabetic rats. Hmox1, Socs1 and Socs3 were induced by HG, but only in tenocytes of diabetic rats (4 h). Conclusion. The response of tenocytes to TNFα depends on glucose supply and cell origin suggesting their irreversible impairment in DMT2

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to age-related injury. Tendons have poor healing capacity and a lack of effective treatments can lead to ongoing pain, reduced function and re-injury. It is therefore important to identify the mechanisms underpinning age-related tendinous changes in order to develop more effective treatments. Our recent single cell sequencing data has shown that tendon cell populations have extensive heterogeneity and cells housed in the tendon interfascicular matrix (IFM) are preferentially affected by ageing. There is, however, a lack of established surface markers for cell populations in tendon, limiting the capacity to isolate distinct cell populations and study their contribution to age-related tendon degeneration. Here, we investigate the presence of the cell surface proteins MET proto-oncogene (MET), integrin subunit alpha 10 (ITGA10), fibroblast activation protein alpha (FAP) and platelet derived growth factor receptor alpha (PDGFRA) in the equine SDFT cell populations and their co-localisation with known markers. Using Western blot we validated the specificity of selected antibodies in equine tissue before performing immunohistochemistry to establish the location of the respective proteins in the SDFT. We subsequently used double labelling immunofluorescence with the established mural cell marker desmin (DES) to distinguish between tenocyte and mural cell populations. In situ, MET, ITGA10, and FAP presence was found in cells throughout the tendon whereas PDGFRA was present in cells within the IFM. Double labelling immunofluorescence with the mural cell marker DES showed lack of co-localisation between PDGFRA and DES suggesting PDGFRA is labelling an IFM cell population distinct from those associated with blood vessels. PDGFRA is a promising target for the specific cell sorting of IFM-localised tenocytes, enabling their isolation and subsequent characterisation. Acknowledgments: The authors acknowledge the Biotechnology and Biological Sciences Research Council (BB/W007282/1) for funding this work

During aging, tendons demonstrate substantial disruptions in homeostasis, leading to impairments in structure-function. Impaired tendon function contributes to substantial declines quality of life during aging. Aged tendons are more likely to undergo spontaneous rupture, and the healing response following injury is impaired in aged tendons. Thus, there is a need to develop strategies to maintain tendon homeostasis and healing capacity through the lifespan. Tendon cell density sharply declines by ∼12 months of age in mice, and this low cell density is retained in geriatric tendons. Our data suggests that this decline in cellularity initiates a degenerative cascade due to insufficient production of the extracellular matrix (ECM) components needed to maintain tendon homeostasis. Thus, preventing this decline in tendon cellularity has great potential for maintaining tendon health. Single cell RNA sequencing analysis identifies two changes in the aged tendon cell environment. First, aged tendons primarily lose tenocytes that are associated with ECM biosynthesis functions. Second, the tenocytes that remain in aged tendons have disruptions in proteostasis and an increased pro-inflammatory phenotype, with these changes collectively termed ‘programmatic skewing'. To determine which of these changes drives homeostatic disruption, we developed a model of tenocyte depletion in young animals. This model decreases tendon cellularity to that of an aged tendon, including decreased biosynthetic tenocyte function, while age-related programmatic skewing is absent. Loss of biosynthetic tenocyte function in young tendons was sufficient to induce homeostatic disruption comparable to natural aging, including deficits in ECM organization, composition, and material quality, suggesting loss biosynthetic tenocytes as an initiator of tendon degeneration. In contrast, our data suggest that programmatic skewing underpins impaired healing in aged tendons. Indeed, despite similar declines in the tenocyte environment, middle-aged and young-depleted tendons mount a physiological healing response characterized by robust ECM synthesis and remodeling, while aged tendons heal with insufficient ECM

Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model. A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site. In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity. This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem

As we grow older, the risk of tendon degeneration and injuries increases, which can result in pain, disability, healthcare cost, and lost productivity. Even after surgical repair the results are often unsatisfactory. The cellular reasons for the differences in the healing potential, however, are not well studied. To get a deeper insight into the biological characteristics of tenocyte-like cells from different patient groups we established a biobank with material from over 150 human donors. The patients/donors suffered from rotator cuff tears and were operated to restore the function. A proportion of the isolated cells showed stem cell-like characteristics and was able to differentiate into the osteoblastic, chondrogenic and adipogenic linage. Investigating the differentiation potential of the cells with regard to donor characteristics, we were able to demonstrate that age, sex but also the “degeneration” has an impact of the cellular potential. A possibility to stimulate the cellular activity is the application of growth factors, as already clinically used for stimulation of bone healing. Therefore, the responsiveness of the cells to the growth factors Bone Morphogenetic protein-2/7 (BMP-2/7) was analysed in vitro. Independent of the donor characteristics, the cells responded to the BMP-stimulation by increased proliferation and collagen-1 synthesis. However, cells isolated from donors with high fatty infiltration of the muscle or older females were less responsive. Looking into the intracellular signalling pathway, the data showed that the BMP-signal is mainly mediated by the canonical-pathway with samd8 playing a major role. This basic research gives first information regarding the differences in tenocytes biology with respect to the donor and is important for the understanding of tendon regeneration and the future development of new treatment strategies

Objectives. Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß. Methods. Rotator cuff-derived cells were seeded under inflammatory stimulation conditions (with serum-free medium with 1 ng/ml IL-1ß for three hours), and then cultured in different media: serum-free (control group), serum-free + TA (0.1mg/ml) (TA group), serum-free + 10% PRP (PRP group), and serum-free + TA (0.1mg/ml) + 10% PRP (TA+PRP group). Cell morphology, cell viability, and expression of inflammatory and degenerative mediators were assessed. Results. Exposure to TA significantly decreased cell viability and changed the cell morphology; these effects were prevented by the simultaneous administration of PRP. Compared with the control group, expression levels of inflammatory genes and reactive oxygen species production were reduced in the TA, PRP, and TA+PRP groups. PRP significantly decreased the expression levels of degenerative marker genes. Conclusions. The combination of TA plus PRP exerts anti-inflammatory and anti-degenerative effects on rotator cuff-derived cells stimulated by IL-1ß. This combination has the potential to relieve the symptoms of rotator cuff injury. Cite this article: T. Muto, T. Kokubu, Y. Mifune, A. Inui, R. Sakata, Y. Harada, F. Takase, M. Kurosaka. Effects of platelet-rich plasma and triamcinolone acetonide on interleukin-1ß-stimulated human rotator cuff-derived cells. Bone Joint Res 2016;5:602–609. DOI: 10.1302/2046-3758.512.2000582

The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival. Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, functional tests were carried out by time-lapse recording gel contraction of four cell groups (Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC). Among the screened integrins we found that integrin a1 and a11 were significantly downregulated in A-TSPC with 3.8 and 5.6 folds, correspondingly. Therefore, to mimic the A-TSPC we carried out a gene KD of a1 and a11 in Y-TSPC. PCR and western blot clearly validated the efficient KD. Analyses of collagen contraction, revealed that Y-TSPC+a11KD significantly reduced collagen contractability comparable to A-TSPC. This indicated the indispensable role of this integrin in the signaling pathway of collagen matrix remodeling. In respect to integrin a1, we found that this receptor did not affect the contraction rate of Y-TSPC, which was similar to Y-TSPC+con. To our knowledge we have now identified for the first time the critical role of a11 integrin receptor in tendon collagen remodeling, and a follow up analysis of its exact downstream cascade is on the way. Future efforts in deciphering how tendon matrix makeover is regulated can lead to innovation in preventive strategies for tendon degeneration

Metabolic disorders are frequently associated with tendon degeneration and impaired healing after acute injury. However, the underlying cellular and molecular mechanisms remain largely unclear. We have previously shown that human and rat tendon cells responde to glucose stimulation in vitro by secretion of insulin. Therefore, we now hypothesize that nutritional glucose uptake affects tendon healing in a rat model. In female rats (n=30/group), unilateral full-thickness Achilles tendon defects were created. Immediately after surgery animals were either fed a glucose rich- or a control diet for up to 4 weeks. Gait analysis (Catwalk, Noldus) was performed at three time points. In addition, tendon thickness measurements, biomechanical testing and immunohistochemical analysis were conducted. Subsequently, gene expression analysis, comparing cDNA pools (n=5) prepared from repair tissues of both groups was performed. The repair tissues of the high glucose group were significantly thicker compared to the control group (p<0.001). The intermediate toe spread, an indicator of pain, were significantly improved in the high glucose group one and two weeks post surgery. Biomechanical analysis revealed that the repair tissues of the high glucose group were significantly stiffer (p<0.05) compared to the control group, no significant difference was detected for maximum tensile load…. The proportion of Ki67+ cells in the repair tissue was 3.3% in the control diet group and 9,8% in the high glucose group, indicating increased cell proliferation (p<0.001). Finally, gene expression analysis revealed the chondrogenic marker genes Collagen II, Aggrecan, COMP and SOX9 to be upregulated and genes involved in lipid metabolism like PPARgamma and Fabp2 to be downregulated in the glucose diet group. Here we show fort he first time that a high-glucose diet affects gait pattern and tendon biomechanics, influences tendon thickness and cell proliferation. Gene expression analysis reveals a regulation of chondrogenic as well as adipogenic marker genes. The molecular mechanisms underlying these effects on cells and extracellular matrix are currently under investigation, potentially revealing targets for developing a dietary intervention scheme to support tendon regeneration after trauma or tendon disease

Objectives. To investigate the appropriate dose and interval for the administration of triamcinolone acetonide (TA) in treating tendinopathy to avoid adverse effects such as tendon degeneration and rupture. Methods. Human rotator cuff-derived cells were cultured using three media: regular medium (control), regular medium with 0.1 mg/mL of TA (low TA group), and with 1.0 mg/mL of TA (high TA group). The cell morphology, apoptosis, and viability were assessed at designated time points. Results. In the low TA group, the cells became flattened and polygonal at seven days then returned to normal at 21 days. The cell apoptosis ratio and messenger ribonucleic acid expression of caspase-3, 7, 8, and 9 increased, and viability was reduced in the low and high groups at seven days. In the low TA group, apoptosis and viability returned to normal at 21 days, however, in the high TA group, the cell morphology, apoptosis ratio, caspase-3, 7, 8, and 9 and viability did not return by day 21. Re-administration was performed in the low TA group at 7-, 14-, and 21-day intervals, and cell viability did not return to the control level at the 7- and 14-day intervals. Conclusion. A 0.1 mg/mL dose of TA temporarily decreased cell viability and increased cell apoptosis, which was recovered at 21 days, however, 1 mg/mL of TA caused irreversible damage to cell morphology and viability. An interval > three weeks was needed to safely re-administer TA. These findings may help determine the appropriate dose and interval for TA injection therapy. Cite this article: Bone Joint Res 2014;3:328–34

Introduction. recent studies recognised metabolic abnormalities as additional factors in the development of rotator cuff (RC) tendinopathy. It has been hypothesised that the insertional area of this tendon is susceptible to degenerative changes due to intrinsic hypovascularization. The mechanisms underlying this process are not yet clear. In this study we attempted to confirm if larger lesions of the RC are related to impaired vasodilatatory response of the local circulation in conditions of “hemodynamic stress”. Patients & Methods. it was assumed that impaired vasal reaction to “hemodynamic stress” was a systemic condition. This phenomenon should therefore be not limited to the critical area of the tendon tear. Given this assumption post-ischemic vasodilation of brachial artery was studied through an echo-doppler (US) evaluation. 50 patients (mean 61 ± 4, range 50–65) all scheduled for surgical rotator cuff repair following a tendon tear, were enrolled. Three preoperative measurements of the brachial artery diameter before and after application of an ischemic band were collected. The size of the lesions was later assessed at the time of surgery. A statistical analysis was carried on to investigate the correlation between US assessment of brachial artery diameter and the corresponding size of the RC lesions. UCLA and ASES scores were also measured to assess clinical and functional outcomes. Results. Patients were classified into 4 groups according to Cofield's classification of tear size; respectively, 4 patients had massive lesions, 32 large, 10 medium and 4 had finally small lesions. The extent of the RC lesion showed an inverse correlation with the diameter of brachial artery after an ischemic stimulus: an increase in size of the lesion corresponded to lower mean post-ischemic diameter of the vessel (p <0.0001). UCLA and ASES data showed no statistically significant differences between the subgroups (p > 0.534). Discussion/Conclusion. It is not clear why the insertional area of tendons composing the RC is hypovascularised. We hypothesised there is an imbalance between local vasodilator and vasoconstrictor factors. The prevalence of vasoconstrictor substances determines a reduced post-ischemic vasodilation. The data presented provide the basis for the future identification of vascular impairment that could underlie the beginning of tendon degeneration in patients that are not yet affected by injury. This would be beneficial for effective prevention of this type of injury. An imbalance between vasodilator and vasoconstrictor factors could be the basis for vascular distress of RC eventually evolving into tendon lesions when other risk factors are associated. More specific vascular pathophysiological studies are however needed to further understand this mechanism and its potential in prevention of rotar cuff lesion

Introduction. The exact mechanisms leading to tendinopathies and tendon ruptures remain poorly understood while their occurrence is clearly associated with exercise. Overloading is thought to be a major factor contributing to the development of tendon pathologies. However, as animal studies have shown, heavy loading alone won't cause tendinopathies. It has been speculated, that malfunctioning adaptation or healing processes might be involved, triggering tendon tissue degeneration. By analysing the expression of the entirety of degrading enzymes (degradome) in pathological and non-pathological, strained and non-strained tendon tissue, the aim of this study was to identify common or opposite patterns in gene regulation. This approach may generate new targets for future studies. Materials and Methods. RNA was extracted from different tendon tissues: normal (n=7), tendinopathic (n=4) and ruptured (n=4) Achilles tendon; normal (n=4) and tendinopathic (n=4) posterior tibialis tendon; normal hamstrings tendon with or without subjection to static strain (n=4). The RNA was reverse transcribed, then pooled per group The expression of 538 protease genes was analysed using Taqman low-density array quantitative RT-PCR. To be considered relevant, changes had to be at least 4fold and measurable at a level below 36 Cts. Results. In general, there was little common regulation when exercised was compared with pathological tissue. The expression of PAMR1 and TNFαIP3 was upregulated with exercise (169-fold and 78-fold), Achilles tendinopathy (9724-fold and 7-fold) and Achilles tendon rupture (1809-fold and 10-fold), while DDI1, PSMB11 and PSH2 which were down-regulated with exercise were upregulated with Achilles pathology. Discussion. The newly found targets may deliver insights into the initiation and progression of tendon pathologies: PAMR1, a regeneration associated muscle protease which has been shown to be downregulated in Duchenne muscular dystrophy and upregulated in regenerating muscle fibers, might also be involved in tendon regeneration; TNFαIP3, which negatively regulates the NF-κB/pro-inflammatory pathway, could have anti-inflammatory function in tendon regeneration. PSMB11 and PSH2 are for the first time shown to be expressed in tendon and regulated in tendon pathology. Using this approach we were able to generate new targets and to add information on function, regulation and expression sites of recently identified proteins