As high incidences of tendinopathies are observed particularly in those who intensively use their tendons, we assume that pathological changes are caused, at least partially, by mechanical overload. This has led to the so-called overload hypothesis, explaining the development of tendinopathies by structural failure resulting from excessive load. At the same time, tendon loading is an important part in tendon rehabilitation. Currently, exercise treatment approaches such as eccentric training or heavy load resistance training are widely applied in tendinopathy rehabilitation, with good clinical results such as an improvement in function and a reduction in pain. Particularly those rehabilitative approaches which impose high strains on the tendon may induce an adaptation of the tendon's mechanical properties such as increased tendon stiffness. An increased tendon stiffness is often interpreted as desirable, as it may protect the tendon from overloading and thus prevent future strain injuries. However, the tendinopathic tendon is not necessarily less stiff than the tendon in the contralateral leg and an improvement in tendon stiffness is not necessarily accompanied by an improvement in tendon pain or function. In addition, metabolic factors, resulting e.g. in low-level systemic inflammation, may contribute to pathological tendon tissue changes and are not necessarily affected by an exercise program, while nutritional interventions or dietary supplements may potentially affect tendon cell metabolism. Indeed, dietary supplements have been introduced as an additional therapeutic approach in the treatment of tendinopathies in recent years, and their positive curative effects have been reported for both the general population and athletes. In the management of tendinopathies, it may thus be advisable if therapeutic approaches aim to address both tendon mechanics and tendon metabolism for better treatment effectiveness and a sustainable improvement in pain and function

Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation. We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness. We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis. Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2). Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Intervertebral disc degeneration (IDD) is a major cause of low back pain, which affects 80% of the adult population at least once in their life. The pathophysiological conditions underlying IDD are still poorly understood. Genetic makeup, aging, smoking, physical inactivity and mechanical overloading, especially due to obesity, are among the strongest risk factors involved. Moreover, IDD is often associated with chronic inflammation within disc tissues, which increases matrix breakdown, glycosaminoglycan (GAG) loss and cell death. This micro-inflammatory environment is typical of several metabolic disorders, including diabetes mellitus (DM). As the etiopathogenesis of IDD in diabetic subjects remains scarcely understood, we hypothesised that this may be driven by a DM-induced inflammation leading to a combination of reduced GAG levels, decreased proteoglycan synthesis and increased matrix breakdown within the disc. The objective of the study was to investigate the pathogenesis of IDD in a murine model of type 1 DM (T1DM), namely non-obese diabetic (NOD) mouse. Total disc glycosaminoglycan (GAG) content, proteoglycan synthesis, aggrecan fragmentation mediated by matrix metalloproteinases (MMPs) and a Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), glucose transporter (mGLUT1) gene expression and apoptosis (TUNEL assay) were assessed in NOD mice and wild-type euglycemic control mice. Spinal structural and molecular changes were analysed by micro-computed tomography (mCT), histological staining (Safranin-O and fast green) and quantitative immunofluorescence (anti-ADAMTS-4 and 5 antibodies). Statistical analysis was conducted considering the average of 35 samples ± standard error for each measurement, with 95% confidence intervals calculated to determine statistical significance (p-value < 0.05). IVDs of NOD mice showed increased disc apoptosis (p < 0.05) and higher aggrecan fragmentation mediated by ADAMTS (p < 0.05). However, ADAMTS-4 and −5 did not appear to be involved in this process. The total GAG content normalized to DNA and PG synthesis showed no statistically significant alterations, as well as Safranin O staining. Although not significantly, NOD mice showed reduced glucose uptake. In addition, the vertebral structure of NOD mice at mCT seemed not to be altered. These data demonstrate that DM may contribute to IDD by increasing aggrecan degradation and promoting cell apoptosis, which may represent early indicators of the involvement of DM in the pathogenesis of IDD

Osteoarthritis (OA) is the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of hypertrophic markers known from the growth plate. Although physiological mechanical loading can support cartilage formation and maintenance, mechanical overload represents one major risk factor for OA development. To date, little is known on how an OA-like hypertrophic chondrocyte phenotype alters the response of cartilage tissue to mechanical loading. The aim of this study was to investigate whether a hypertrophic phenotype change of chondrocytes affects the response to physiological mechanical loading and to reveal differences compared to normal control cartilage. Cartilage replacement tissue was generated using human articular chondrocytes (normal control cartilage, n=3–5) or human mesenchymal stromal cells which develop a hypertrophic phenotype similar to the one observed in OA (OA cartilage model, n=3–6). Cells were seeded in a collagen type I/III carrier and attached to a beta-TCP bone replacement phase, building an osteochondral unit for simulation of natural conditions. After 21 and 35 days of chondrogenic (re)differentiation, a single physiological mechanical compression episode (1 Hz, 25 %, 3 h) was applied, imitating three hours of normal walking in ten-minute intervals. Proteoglycan and collagen synthesis, gene expression and activation of signaling pathways were assessed. Cartilage replacement tissue of both groups had similar proteoglycan and collagen type II content as well as hardness properties. During (re)differentiation, both cell types showed a comparable upregulation of the chondrogenic marker genes COL2A1 and ACAN. As expected, hypertrophic marker genes (COL10A1, ALPL, MEF2C, IBSP) were only upregulated in the OA cartilage model. Mechanotransduction in both tissues was confirmed by load-induced activation of pERK1/2 signaling. While the 3 h loading episode significantly increased proteoglycan synthesis in normal control cartilage at day 35, the same protocol resulted in a suppression of proteoglycan and collagen synthesis in the OA cartilage model, which was accompanied by a downregulation of COL2A1 gene expression. In addition, hypertrophic marker genes COL10A1, ALPL and IBSP were significantly reduced after loading. Along lower load-induced SOX9 mRNA and protein stimulation in the OA cartilage tissue, a weaker induction of mechanosensitive BMP2, BMP6, FOS and FOSB gene expression was observed. While stable cartilage showed anabolic effects after physiological loading, the hypertrophic chondrocytes reacted with a reduced extracellular matrix synthesis. This could be explained by a lower mechanoinduction of the BMP signaling cascade and insufficient SOX9 stimulation. Progressive OA development could thus be influenced by a reduced mechanocompetence of osteoarthritic chondrocytes

Summary Statement. An organ culture experiment was simulated to explore the mechanisms that can link cell death to mechanical overload in the intervertebral disc. Coupling cell nutrition and tissue deformations led to altered metabolic transport that largely explained cell viability measurements. Introduction. Part of intervertebral disc (IVD) maintenance relies on limited nutrient availability to the cells and on mechanical loads, but effective implication of these two factors is difficult to quantify. Theoretical models have helped to understand the link between solute transport and cell nutrition in deforming IVD, but omitted the direct link between tissue mechanics and cell metabolism. Hence, we explored numerically the relation between disc mechanics and cell death in relation to an organ culture experiment. Methods. A finite element model of a caudal bovine IVD was created to reproduce an organ culture experiment. All subtissues were modelled, and coupled to cell metabolism in two ways: (i) mechanical strains and metabolic reactions were simply coupled to the diffusions of oxygen, lactate and glucose through a mechano-transport algorithm (IND model). (ii), a hypermetabolism model based on in vitro data involved a 30% increase in glucose consumption by the cells, activated either as a Step or as a Gaussian function over 15% strain (DIR model). Exponential decays of cell density occurred below 0.5 mM of glucose and/or below pH 6.78. Concentrations of 21 kPa oxygen and 4.5 mM glucose were imposed at the boundary, and a combination of 0.2 MPa compression and 10° bending was applied over 7 days. Results. The highest hypermetabolic response was given by the Step activation. For all models, cell death mostly occurred in the compressed area of the flexed IVD, and steady-state cell viability was reached in about two days of load. In the outer annulus fibrosus (AF), the DIR model with Step activation led to increased cell death, in line with the cell viability measured in vitro. In the inner AF, all cell viability results matched the reported measurements. Discussion/Conclusion. This study focused on elucidating the links between mechanical stimulation and cell survival in the IVD, and simulation of nutrition issues allowed reproducing the results of an organ culture experiment. Results suggest that mechano-regulated metabolism can play a significant role in the nutrition-related cell death. Truly, the IND model gave both low glucose and low pH, and altered metabolic transport represented the main cell death mechanism. Yet, the role of hypermetabolism was increased nearby the nutrient supply at the outer AF, meaning that cell death could occur, even in regions where nutrient supply seems ensured by short diffusion distances. Though further mechanistic developments must be considered, this novel mechano-regulated metabolism model permits mechano-transport models to be used to explore important interactions between tissue biophysics and multiphysics. In particular, the extracellular matrix degradation along degeneration and cell death can be coupled to the poromechanical parameters introduced, e.g. initial porosity and osmotic pressure values that largely depend on the proteoglycan concentration

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.