Reduction of mechanical loading in tendons induces heterotopic ossification and activation of the ß-catenin signaling pathway.

BACKGROUND: Tendons are the force transferring tissue that enable joint movement. Excessive mechanical loading is commonly considered as a primary factor causing tendinopathy, however, an increasing body of evidence supports the hypothesis that overloading creates microdamage of collagen fibers resulting in a localized decreased loading on the cell population within the damaged site. Heterotopic ossification is a complication of late stage tendinopathy, which can significantly affect the mechanical properties and homeostasis of the tendon. Here, we the examine the effect of mechanical underloading on tendon ossification and investigate its underlying molecular mechanism. METHOD: Rabbit Achilles tendons were dissected and cultured in an underloading environment (3% cyclic tensile stain,0.25 â€‹Hz, 8 â€‹h/day) for either 10, 15 or 20 days. Using isolated tendon-derived stem cells (TDSCs) 3D constructs were generated, cultured and subjected to an underloading environment for 6 days. Histological assessments were performed to evaluate the structure of the 3D constructs; qPCR and immunohistochemistry were employed to study TDSC differentiation and the ß-catenin signal pathway was investigated by Western blotting. Mechanical testing was used to determine ability of the tendon to withstand force generation. RESULT: Tendons cultured for extended times in an environment of underloading showed progressive heterotopic ossification and a reduction in biomechanical strength. qPCR revealed that 3D TDSCs constructs cultured in an underloading environment exhibited increased expression of several osteogenic genes: these include RUNX2, ALP and osteocalcin in comparison to tenogenic differentiation markers (scleraxis and tenomodulin). Immunohistochemical analysis further confirmed high osteocalcin production in 3D TDSCs constructs subject to underloading. Western blotting of TDSC constructs revealed that ß-catenin accumulation and translocation were associated with an increase in phosphorylation at Ser552 and decrease phosphorylation at Ser33. CONCLUSION: These findings unveil a potential mechanism for heterotopic ossification in tendinopathy due to the underloading of TDSCs at the damage sites, and also that ß-catenin could be a potential target for treating heterotopic ossification in tendons. THE TRANSLATIONAL POTENTIAL: Tendon heterotopic ossification detrimentally affect quality of life especially for those who has atheletic career. This study reveals the possible mechanism of heterotpic ossification in tendon related to mechanical loading. This study provided the possible to develop a mechanical stimulation protocol for preventive and therapeutic purpose for tendon heterotopic ossification.

Intramuscular injection of Botox causes tendon atrophy by induction of senescence of tendon-derived stem cells.

BACKGROUND: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. HYPOTHESIS: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). STUDY DESIGN: Controlled laboratory study METHODS: A total of 36 mice were randomly divided into 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into the right vastus lateralis of quadriceps muscles either with Botox (to induce mechanical stress deprivation of the patellar tendon) or with normal saline as a control. At 2 weeks post-injection, animals were euthanized prior to tissues being harvested for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity or the presence of senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, the PTEN/AKT-mediated cell senescence pathway was evaluated in TDSCs from both groups. RESULTS: Two weeks after Botox injection, patellar tendons displayed several atrophic features including tissue volume reduction, collagen fibre misalignment and increased degradation. A colony formation assay revealed a significantly reduced number of colony forming units of TDSCs in the Botox-injected group compared to controls. Multipotent differentiation capacities of TDSCs were also diminished after Botox injection. To examine if mechanically deprived TDSC are capable of forming tendon tissue, we used an isolated bioreactor system to culture tendon constructs using TDSC. These results showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of the signalling pathway revealed that injection of Botox into quadriceps muscles causes PTEN/AKT-mediated cell senescence of TDSCs. CONCLUSION: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. CLINICAL RELEVANCE: Intramuscular Botox injection for tendinopathy or tendon injury could result in adverse effects in human tendons and evaluation of its long-term efficacy is warranted.

Subchondral bone deterioration in femoral heads in patients with osteoarthritis secondary to hip dysplasia: A case-control study.

OBJECTIVES: Residual hip dysplasia is the most common underlying condition leading to secondary osteoarthritis (OA) of the hip. Subchondral bone alterations in OA secondary to hip dysplasia (HD-OA) are poorly investigated. The aim of the present study was to analyse the microarchitecture, bone remodelling and pathological alterations of subchondral bone in femoral heads from patients with HD-OA. METHODS: Subchondral bone specimens were extracted from both weight-bearing and non-weight-bearing regions of femoral heads from 20 patients with HD-OA and 20 patients with osteoporotic femoral neck fracture, during hip replacement surgery. Micro-CT and histological examination were performed to assess the microarchitecture and histopathological changes. RESULTS: The weight-bearing subchondral bone showed significantly more sclerotic microarchitecture and higher bone remodelling level in HD-OA as compared with osteoporosis. In the non-weight-bearing region, the two diseases shared similar microarchitectural characteristics, but higher bone remodelling level was detected in HD-OA. Distinct regional differences were observed in HD-OA, whereas the two regions exhibited similar characteristics in osteoporosis. In addition, HD-OA displayed more serious pathological alterations, including subchondral bone cyst, metaplastic cartilaginous tissue, bone marrow oedema and fibrous tissue, especially in the weight-bearing region. CONCLUSIONS: Osteoarthritic deteriorations of subchondral bone induced by hip dysplasia spread throughout the whole joint, but exhibit region-dependent variations, with the weight-bearing region more seriously affected. Biomechanical stress might exert a pivotal impact on subchondral bone homeostasis in hip dysplasia. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The histomorphometric findings in the project indicate an early intervention for the development of hip dysplasia in clinic.

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients' life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

Pathogenesis and clinical management of obesity-related knee osteoarthritis: Impact of mechanical loading.

Obesity-related osteoarthritis (OA) is a complex, multifactorial condition that can cause significant impact on patients' quality of life. Whilst chronic inflammation, adipocytokines and metabolic factors are considered to be important pathogenic factors in obesity related OA, there has been limited investigation into the biomechanical impact of obesity on OA development. This review aims to demonstrate that mechanical factors are the major pathological cause of obesity-related OA. The effect of obesity on pathological changes to the osteochondral unit and surrounding connective tissues in OA is summarized, as well as the impact of obesity-related excessive and abnormal joint loading, concomitant joint malalignment and muscle weakness. An integrated therapeutic strategy based on this multi-factorial presentation is presented, to assist in the management of obesity related OA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Despite the high prevalence of obesity-related OA, there is no specific guideline available for obesity-related OA management. In this review, we demonstrated the pathological changes of obesity-related OA and summarized the impact of biomechanical factors by proposing a hypothetical model of obesity-related OA change. Therapeutic strategies based on adjusting abnormal mechanical effects are presented to assist in the management of obesity-related OA.

Horizontal fissuring at the osteochondral interface: a novel and unique pathological feature in patients with obesity-related osteoarthritis.

OBJECTIVES: Obesity is a well-recognised risk factor for osteoarthritis (OA). Our aim is to characterise body mass index (BMI)-associated pathological changes in the osteochondral unit and determine if obesity is the major causal antecedent of early joint replacement in patients with OA. METHODS: We analysed the correlation between BMI and the age at which patients undergo total knee replacement (TKR) in 41 023 patients from the Australian Orthopaedic Association National Joint Replacement Registry. We then investigated the effect of BMI on pathological changes of the tibia plateau of knee joint in a representative subset of the registry. RESULTS: 57.58% of patients in Australia who had TKR were obese. Patients with overweight, obese class I & II or obese class III received a TKR 1.89, 4.48 and 8.08 years earlier than patients with normal weight, respectively. Microscopic examination revealed that horizontal fissuring at the osteochondral interface was the major pathological feature of obesity-related OA. The frequency of horizontal fissure was strongly associated with increased BMI in the predominant compartment. An increase in one unit of BMI (1 kg/m2) increased the odds of horizontal fissures by 14.7%. 84.4% of the horizontal fissures were attributable to obesity. Reduced cartilage degradation and alteration of subchondral bone microstructure were also associated with increased BMI. CONCLUSIONS: The key pathological feature in OA patients with obesity is horizontal fissuring at the osteochondral unit interface. Obesity is strongly associated with a younger age of first TKR, which may be a result of horizontal fissures.

Correction: Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes.

[This corrects the article DOI: 10.18632/oncotarget.9034.].

Fabrication of a silver nanoparticle-coated collagen membrane with anti-bacterial and anti-inflammatory activities for guided bone regeneration.

Alveolar bone loss is a common problem that affects dental implant placement. A barrier between the bone substitute and gingiva that can prevent fibro-tissue ingrowth, bacterial infection and induce bone formation is a key factor in improving the success of alveolar ridge reconstruction. This study aims to develop a bioactive collagen barrier material for guided bone regeneration, that is coupled with anti-bacterial and anti-inflammatory properties. We have evaluated two silver coating methods and found controllable and precise coating achieved by sonication compared with sputtering. The optimized AgNP-coated collagen membrane exhibited excellent anti-bacterial effects against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) with limited cellular toxicity. It also displayed effective anti-inflammatory effects by reducing the expression and release of inflammatory cytokines including IL-6 and TNF-alpha. Additionally, AgNP-coated collagen membranes were able to induce osteogenic differentiation of mesenchymal stem cells that guide bone regeneration. These findings demonstrate the potential application of AgNP-coated collagen membranes to prevent infection after bone graft introduction in alveolar ridge reconstruction.

Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes.

Osteocytes comprising over 90% of the bone cell population are highly susceptible to the adverse effects of glucocorticoids (GC) administration. Here we observed that Dexamethasone (Dex) induces a robust cytoskeleton rearrangement and decreases Cx43 protein expression in osteocyte-like MLO-Y4 cells. Using a Dmp1Cre-mT/mG osteocyte ex vivo culture system, we found significant shortening of dendritic processes in primary osteocytes following Dex administration. Loss of dendritic processes is a consequence of reduced Cx43 connectivity upon Dex induced autophagy in both RFP-GFP-LC3B transfected MLO-Y4 cells and primary calvarial osteocytes from LC3GFP transgenic mice. Upon the induction of autophagy by Dex, Cx43 was internalized into autophagosome/autolysosomes and degraded by autophagy. The degradation was attenuated following lysosomal inhibition using chloroquine (CLQ) and suppression of autophagy by Atg5 silencing. Inhibition Akt-mTORC1 signaling by Dex induces autophagy subsequently resulting in Cx43 degradation.Activation of Akt phosphorylation by IGF-1 attenuated Dex induced autophagy and degradation of Cx43. Together, we demonstrated that GC impair osteocyte cell-cell connectivity via autophagy mediated degradation of Cx43 through inhibition of the Akt-mTORC1 signaling. This may account for the deleterious effect of GC-induced bone loss.