Establishment of a Mouse Degenerative Model of Patellar Tendinopathy with Upregulation of Inflammation.

There is no mouse model of patellar tendinopathy. This study aimed to establish a mouse inflammatory and degenerative patellar tendon injury model, which will facilitate research on patellar tendinopathy using advanced molecular tools including transgenic models. Collagenase at different doses (low dose (LD), medium dose (MD), high dose (HD)) or saline was injected over the mouse patellar tendon. At weeks 1, 2, 4, and 8 post-injection, the tendons were harvested for histology and further examined by micro-computed tomography (microCT) imaging at week 8. The optimal dose group and the saline group were further evaluated by immunohistochemical staining, gait pattern, and biomechanical properties. The histopathological score increased dose-dependently post-collagenase injection. Ectopic mineralization was observed and increased with collagenase dose. The LD group was selected for further analysis. The expression of IL-10, TNF-α, and MMP-1 significantly increased post-injection. The changes of limb idleness index (ΔLII) compared to preinjury state were significantly higher, while the ultimate load, stiffness, ultimate stress, and maximum Young's modulus were significantly lower in the LD group compared to the saline group. A mouse inflammatory degenerative model of patellar tendon injury resembling tendinopathy was established as indicated by the dose-dependent increase in tendon histopathology, ectopic calcification, decrease in biomechanical properties, and pain-associated gait changes.

Increased Risk of Concomitant Meniscal Injuries in Adolescents With Elevated Body Mass Index After Anterior Cruciate Ligament Tear: A Systematic Review.

PURPOSE: To investigate existing studies examining the association between body mass index (BMI) and outcomes of anterior cruciate ligament reconstruction (ACLR) in adolescent patients. METHODS: A literature search was conducted on PubMed and Embase. Studies examining associations between BMI and outcomes after ACLR in adolescents were included. Quality assessment was performed. Data on patient age, sex, study design, time of follow-up, sample size, graft type, concomitant injuries (meniscal injury, surgical procedures), clinical outcomes (revision ACLR, postoperative weight gain, post-traumatic osteoarthritis [PTOA], range of motion [ROM]), and functional outcome (muscle strength) were extracted. RESULTS: Eleven papers of Levels II-IV evidence were included. Five studies found positive correlations between BMI and risk of concomitant meniscal injuries. Two of them reported young patients with elevated BMI having 1.6 times greater odds of requiring meniscectomy (P < .01) and 1.031 times greater odds of requiring concomitant surgeries (P = .011). One study showed significant positive association of postoperative weight gain by time (r = 0.28, P < .01), with smaller increase in the overweight and obese groups compared with the normal-weight group. One study demonstrated greater cartilage breakdown in young patients with overweight and obesity postsurgery, contributing to PTOA (r = 0.42, P = .009). There was no clinically important difference in postoperative ROM and muscle strength. Four studies reviewed the association between BMI and revision ACLR risk, but results were heterogeneous and a firm conclusion cannot be drawn. CONCLUSIONS: Adolescents with elevated BMI are more likely to have concomitant meniscal injuries and surgical procedures after ACL tear. There is some weak evidence of the association of elevated BMI with PTOA and slight postoperative weight gain post-ACLR. There may not be any clinically significant association of obesity with post-operative muscle strength and ROM, and current studies are inconclusive regarding the impact of BMI on revision ACLR risk. LEVEL OF EVIDENCE: Level IV, systematic review of Level II-IV studies.

Roles of Oxidative Stress in Acute Tendon Injury and Degenerative Tendinopathy-A Target for Intervention.

Both acute and chronic tendon injuries are disabling sports medicine problems with no effective treatment at present. Sustained oxidative stress has been suggested as the major factor contributing to fibrosis and adhesion after acute tendon injury as well as pathological changes of degenerative tendinopathy. Numerous in vitro and in vivo studies have shown that the inhibition of oxidative stress can promote the tenogenic differentiation of tendon stem/progenitor cells, reduce tissue fibrosis and augment tendon repair. This review aims to systematically review the literature and summarize the clinical and pre-clinical evidence about the potential relationship of oxidative stress and tendon disorders. The literature in PubMed was searched using appropriate keywords. A total of 81 original pre-clinical and clinical articles directly related to the effects of oxidative stress and the activators or inhibitors of oxidative stress on the tendon were reviewed and included in this review article. The potential sources and mechanisms of oxidative stress in these debilitating tendon disorders is summarized. The anti-oxidative therapies that have been examined in the clinical and pre-clinical settings to reduce tendon fibrosis and adhesion or promote healing in tendinopathy are reviewed. The future research direction is also discussed.

Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model.

BACKGROUND AIMS: Treatment of tendon-derived stem cells (TDSCs) with connective tissue growth factor (CTGF) and ascorbic acid promoted their tenogenic differentiation. We investigated the effects of TDSCs pre-treated with CTGF and ascorbic acid on tendon repair in a patellar tendon window injury rat model. METHODS: Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16. RESULTS: The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical test. The fibrils in the treated TDSC group showed better alignment and larger size compared with those in the control group at week 8 (P = 0.004). There was lower risk of ectopic mineralization after transplantation of treated or untreated TDSCs (all P ≤ 0.050). The transplanted cells proliferated and could be detected in the window wound up to weeks 2 to 4 and week 8 for the untreated and treated TDSC groups, respectively. CONCLUSIONS: The transplantation of TDSCs promoted tendon repair up to week 16, with CTGF and ascorbic acid pre-treatment showing the best results up to week 8. Pre-treatment of TDSCs with CTGF and ascorbic acid may be used to further enhance the rate and quality of tendon repair after injury.

Peri-tunnel bone loss: does it affect early tendon graft to bone tunnel healing after ACL reconstruction?

PURPOSE: The clinical relevance and mechanisms of local bone loss early post-anterior cruciate ligament (ACL) reconstruction remain unclear. The early spatial and temporal changes of peri-tunnel bone, its molecular mechanisms and its relationships with graft-bone tunnel healing were investigated in a 12-week-old rat model. METHODS: At various times, the reconstructed ACL complex was harvested for vivaCT imaging, biomechanical test, histology and immunohistochemical staining of CD68+ cells (a monocyte-macrophage lineage marker), MMP1 and MMP13. RESULTS: The peri-tunnel bone resorbed simultaneously with improvement of graft-bone tunnel healing. There were 30.1 ± 17.4, 46.8 ± 10.5 and 81.5 ± 12.3 % loss of peri-tunnel BMD as well as 43.2 ± 21.7, 78.7 ± 8.5 and 92.4 ± 17.7 % loss of peri-tunnel bone volume/total volume (BV/TV) at week 6 at the distal femur, epiphysis and metaphysis of tibia, respectively. MMP1, MMP13 and CD68+ cells were expressed at the graft-bone tunnel interface and peri-tunnel bone and increased with time post-reconstruction at the tibia. The ultimate load and stiffness of the healing complex positively correlated with tibial tunnel bone formation and negatively correlated with tibial peri-tunnel bone. Tunnel BV/TV at the tibial metaphysis and epiphysis showed the highest correlation with ultimate load (ρ = 0.591; p = 0.001) and stiffness (ρ = 0.427; p = 0.026) of the complex, respectively. CONCLUSION: There was time-dependent loss of peri-tunnel bone early post-reconstruction, with the greatest loss occurring at the tibial metaphysis. This was consistent with high expression of MMP1, MMP13 and CD68+ cells at the graft-bone tunnel interface and the peri-tunnel region. The significant loss of peri-tunnel bone, though not critically affecting early tunnel healing, suggested the need to protect the knee joint early post-reconstruction.

Vitamin D as an intervention for improving quadriceps muscle strength in patients after anterior cruciate ligament reconstruction: study protocol for a randomized double-blinded, placebo-controlled clinical trial.

BACKGROUND: The goal of anterior cruciate ligament reconstruction (ACLR) is to restore the preinjury level of knee function to return to play (RTP). However, even after completing the rehabilitation programme, some patients may have persistent quadriceps muscle weakness affecting knee function which ultimately leads to a failure in returning to play. Vitamin D has been long recognized for its musculoskeletal effects. Vitamin D deficiency may impair muscle strength recovery after ACLR. Correcting vitamin D levels may improve muscle strength. METHODS: This is a double-blinded, randomized controlled trial to investigate the effects of vitamin D supplementation during the post-operative period on quadriceps muscle strength in anterior cruciate ligament (ACL)-injured patients. Patients aged 18-50 with serum vitamin D < 20 ng/ml, unilateral ACL injury, > 90% deficit in total quadriceps muscle volume on the involved leg compared with uninvolved leg, Tegner score 7 + , and no previous knee injury/surgery will be recruited. To assess patient improvement, we will perform isokinetic and isometric muscle assessments, ultrasound imaging for quadriceps thickness, self-reported outcomes, KT-1000 for knee laxity, biomechanical analysis, and Xtreme CT for bone mineral density. To investigate the effect of vitamin D status on quadriceps strength, blood serum samples will be taken before and after intervention. DISCUSSION: Patients with low vitamin D levels had greater quadriceps fibre cross-sectional area loss and impaired muscle strength recovery after ACL. The proposed study will provide scientific support for using vitamin D supplementation to improve quadriceps strength recovery after ACLR. TRIAL REGISTRATION: ClinicalTrials.gov NCT05174611. Registered on 28 November 2021.

Identity of tendon stem cells--how much do we know?

Tendon stem cells are multi-potent adult stem cells with broad differentiation plasticity that render them of great importance in cell-based therapies for the repair of tendons. We called them tendon-derived stem cells (TDSCs) to indicate the tissue origin from which the stem cells were isolated in vitro. Based on the work of other sources of MSCs and specific work on TDSCs, some properties of TDSCs have been characterized / implicated in vitro. Despite these findings, tendon stem cells remained controversial cells. This was because MSCs residing in different organs, although very similar, were not identical cells. There is evidence of differences in stem cell-related properties and functions related to tissue origins. Similar to other stem cells, tendon stem cells were identified and characterized in vitro. Their in vivo identities, niche (both anatomical locations and regulators) and roles in tendons were less understood. This review aims to summarize the current evidence of the possible anatomical locations and niche signals regulating the functions of tendon stem cells in vivo. The possible roles of tendon stem cells in tendon healing and non-healing are presented. Finally, the potential strategies for understanding the in vivo identity of tendon stem cells are discussed.

Histopathological changes in tendinopathy--potential roles of BMPs?

The pathogenesis of tendinopathy remains unclear. Chondro-osteogenic BMPs such as BMP-2, BMP-4 and BMP-7 have been reported in clinical samples and animal models of tendinopathy. As chondrocyte-like cells and ossified deposits have been observed in both clinical samples and animal models of failed tendon healing tendinopathy, chondro-osteogenic BMPs might contribute to tissue metaplasia and other histopathological changes in tendinopathy. In this review I have summarized the current evidence supporting the roles of chondro-osteogenic BMPs in the histopathological changes of tendinopathy. The potential targets, effects and sources of these BMPs are discussed. I have also provided directions for future studies about the potential roles of BMPs in the pathogenesis of tendinopathy. Better understanding of the roles of these BMPs in the histopathological changes of tendinopathy could provide new options for the prevention and treatment of this disabling tendon disorder.

Expression of Wnt pathway mediators in metaplasic tissue in animal model and clinical samples of tendinopathy.

OBJECTIVE: Tissue metaplasia is observed in both ossified failed healing animal model and clinical samples of tendinopathy. The Wnt signalling pathway plays a vital role in pathological calcification. We hypothesized that the Wnt signalling pathway might contribute to tissue metaplasia and failed healing in tendinopathy. This study aimed to examine the spatial-temporal expression of Wnt pathway mediators in an ossified failed tendon healing animal model and clinical samples of tendinopathy. The effect of Wnt3a on the osteogenic differentiation of tendon-derived stem cells (TDSCs) was also examined. METHODS: Ossified failed tendon healing was induced by the injection of collagenase into the patellar tendon of rats. At various times the tendons were harvested for immunohistochemical staining of Wnt3a, ß-catenin, Lrp5 and Tcf1. Patellar tendon samples were obtained from 13 patients with patellar tendinopathy (11 unossified and 2 ossified) and 10 controls. Immunohistochemical staining of Wnt3a, ß-catenin, Lrp5 and Tcf1 was similarly performed. Rat patellar TDSCs were treated with Wnt3a. The osteogenic differentiation of TDSCs was examined by ALP activity, alizarin red S staining and mRNA expression of osteogenic markers. RESULTS: There was increased expression of Wnt3a, ß-catenin, Lrp5 and Tcf1 in the healing fibroblast-like cells, chondrocyte-like cells and ossified deposits in the animal model and in some clinical samples of tendinopathy. Wnt3a increased ALP activity, calcium nodule formation and expression of osteogenic markers in TDSCs. CONCLUSION: Activation of the Wnt signalling pathway and its effect on TDSCs might contribute to tissue metaplasia and failed healing in some cases of tendinopathy.

Bioactive Decellularized Tendon-Derived Stem Cell Sheet for Promoting Graft Healing After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Stem cell sheets provide a scaffold-free option for the promotion of graft healing after anterior cruciate ligament reconstruction (ACLR). However, cell viability, stability, and potential uncontrolled actions create challenges for clinical translation. The decellularization of cell sheets may overcome these problems as studies have shown that the natural extracellular matrix of stem cells is bioactive and can promote tissue repair. HYPOTHESIS: The decellularized tendon-derived stem cell (dTDSC) sheet can promote graft healing after ACLR. STUDY DESIGN: Controlled laboratory study. METHODS: An optimized decellularization protocol was developed to decellularize the TDSC sheets. A total of 64 Sprague-Dawley rats underwent ACLR with or without the dTDSC sheet wrapping the tendon graft (n = 32/group). At 2 and 6 weeks after surgery, graft healing was assessed by micro-computed tomography, histology, and biomechanical testing. The accumulation of iNOS+ and CD206+ cells and the expression of metalloproteinase 1 (MMP-1), MMP-13, and tissue inhibitor of metalloprotease 1 (TIMP-1) were assessed by immunohistochemistry. RESULTS: The decellularization was successful, with the removal of 98.4% nucleic acid while preserving the collagenous proteins and bioactive factors. The expression of bone morphogenetic protein 2 (BMP-2) and VEGF in the dTDSC sheet was comparable with the TDSC sheet (P > .05). Micro-computed tomography showed significantly more tunnel bone formation in the dTDSC sheet group. The dTDSC sheet group demonstrated better graft osteointegration and higher integrity of graft midsubstance with significantly higher ultimate failure load (16.58 ± 7.24 vs 8.93 ± 2.45 N; P = .002) and stiffness (11.97 ± 5.21 vs 6.73 ± 2.20 N/mm; P = .027). Significantly fewer iNOS+ cells but more CD206+ cells, as well as lower MMP-1 and MMP-13 but higher TIMP-1 expression, were detected at the tendon-bone interface and graft midsubstance in the dTDSC sheet group. CONCLUSION: An optimized decellularization protocol for producing bioactive dTDSC sheets was developed. Wrapping tendon graft with a dTDSC sheet promoted graft healing after ACLR, likely via enhancing bone formation and angiogenesis by BMP-2 and VEGF, modulating macrophage polarization and MMP/TIMP expression, and physically protecting the tendon graft. CLINICAL RELEVANCE: dTDSC sheets alleviate the quality control and safety concerns of cell transplantation and can be used as a cell-free alternative for the promotion of graft healing in ACLR.

Preparation and biomedical applications of artificial cells.

Artificial cells have received much attention in recent years as cell mimics with typical biological functions that can be adapted for therapeutic and diagnostic applications, as well as having an unlimited supply. Although remarkable progress has been made to construct complex multifunctional artificial cells, there are still significant differences between artificial cells and natural cells. It is therefore important to understand the techniques and challenges for the fabrication of artificial cells and their applications for further technological advancement. The key concepts of top-down and bottom-up methods for preparing artificial cells are summarized, and the advantages and disadvantages of the bottom-up methods are compared and critically discussed in this review. Potential applications of artificial cells as drug carriers (microcapsules), as signaling regulators for coordinating cellular communication and as bioreactors for biomolecule fabrication, are further discussed. The challenges and future trends for the development of artificial cells simulating the real activities of natural cells are finally described.

Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 (Adgrf1).

Background: Recent research has shown that the adhesion G protein-coupled receptor F1 (Adgrf1; also known as GPR110; PGR19; KPG_012; hGPCR36) is an oncogene. The evidence is mainly based on high expression of Adgrf1 in numerous cancer types, and knockdown Adgrf1 can reduce the cell migration, invasion, and proliferation. Adgrf1 is, however, mostly expressed in the liver of healthy individuals. The function of Adgrf1 in liver has not been revealed. Interestingly, expression level of hepatic Adgrf1 is dramatically decreased in obese subjects. Here, the research examined whether Adgrf1 has a role in liver metabolism. Methods: We used recombinant adeno-associated virus-mediated gene delivery system, and antisense oligonucleotide was used to manipulate the hepatic Adgrf1 expression level in diet-induced obese mice to investigate the role of Adgrf1 in hepatic steatosis. The clinical relevance was examined using transcriptome profiling and archived biopsy specimens of liver tissues from non-alcoholic fatty liver disease (NAFLD) patients with different degree of fatty liver. Results: The expression of Adgrf1 in the liver was directly correlated to fat content in the livers of both obese mice and NAFLD patients. Stearoyl-coA desaturase 1 (Scd1), a crucial enzyme in hepatic de novo lipogenesis, was identified as a downstream target of Adgrf1 by RNA-sequencing analysis. Treatment with the liver-specific Scd1 inhibitor MK8245 and specific shRNAs against Scd1 in primary hepatocytes improved the hepatic steatosis of Adgrf1-overexpressing mice and lipid profile of hepatocytes, respectively. Conclusions: These results indicate Adgrf1 regulates hepatic lipid metabolism through controlling the expression of Scd1. Downregulation of Adgrf1 expression can potentially serve as a protective mechanism to stop the overaccumulation of fat in the liver in obese subjects. Overall, the above findings not only reveal a new mechanism regulating the progression of NAFLD, but also proposed a novel therapeutic approach to combat NAFLD by targeting Adgrf1. Funding: This work was supported by the National Natural Science Foundation of China (81870586), Area of Excellence (AoE/M-707/18), and General Research Fund (15101520) to CMW, and the National Natural Science Foundation of China (82270941, 81974117) to SJ.

Being overweight or obese increases the risk of developing numerous medical conditions including non-alcoholic fatty liver disease (NAFLD), where excess fat accumulates in the liver. NAFLD is a major global health issue affecting about 25% of the world's population and, if left untreated, can lead to liver inflammation as well as serious complications such as type 2 diabetes, heart disease, and liver cancer. Currently, there are no medications which specifically treat NFALD. Instead, only medications which help to manage the associated health complications are available. Therefore, a better understanding of NFALD is required to help to develop new strategies for diagnosing and treating the progression of this disease. A family of proteins known as GPCRs have crucial roles in regulating various bodily processes and are therefore commonly targeted for the treatment of disease. By identifying the GPCRs specifically involved in liver fat accumulation, new treatments for NFALD could be identified. Previous studies identified a GPCR known as Adgrf1 that is mainly found in liver cells, but its role remained unclear. To investigate the function of Adgrf1 in the liver, Wu et al. studied obese mice and human patients with NAFLD. The experiments showed that elevated levels of Adgrf1 in human and mouse livers led to increased fat accumulation. On the other hand, livers with lower levels of Adgrf1 exhibited reduced fat levels. A technique called RNA sequencing revealed that Adgrf1 induces expression of enzymes involved in fat synthesis, including a key regulator called Scd1. Treating mice with high levels of liver fat with molecules that inhibit Scd1 decreased the symptoms of Adgrf1-mediated fatty liver disease. These findings suggest therapies that decrease the levels of Adgrf1 may help to stop too much fat accumulating in the liver of human patients who are at risk of developing NAFLD. Further research is needed to confirm the effectiveness and safety of targeting Adgrf1 in humans and to develop suitable candidate drugs for the task.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1ß Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1ß and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway.

Chronic tendinopathy is a tendon disorder that is common in athletes and individuals whose tendons are subjected to repetitive strain injuries. The presence of ossification worsened the clinical manifestation of the disorder. The change of tendon loading due to mechanical overload, compression, or disuse have been implicated as the possible etiologies, but the pathological mechanisms of tendinopathy remain unclear. In this study, we demonstrated that ossification in tendon tissue might be due to the osteogenesis of tendon-derived stem cells (TDSCs) induced by uniaxial mechanical tension (UMT) which mimics the mechanical loading in tendon. Rat TDSCs (rTDSCs) could be induced to differentiate into osteogenic lineage after treatment with 2% elongation UMT for 3 days as shown by the increased expression Runx2 mRNA and protein, Alpl mRNA, collagen type 1 alpha 1 (Col1a1) mRNA, ALP activity, and ALP cytochemical staining. RhoA, an osteogenesis regulator, was activated in rTDSCs upon UMT stimulation. Blockage of RhoA activity in rTDSCs by C3 toxin or ROCK activity, a downstream target of RhoA, by Y-27632 inhibited UMT-induced osteogenesis in rTDSCs. UMT up-regulated the mRNA expression of Wnt5a but not the other non-canonical Wnts. The inhibition of Wnt5a expression by siRNA abolished UMT-induced Runx2 mRNA expression and RhoA activation in rTDSCs and the inhibition of Runx2 expression could be rescued by addition of LPA, a RhoA activator. In conclusion, our results showed that UMT induced osteogenic differentiation of rTDSCs via the Wnt5a-RhoA pathway, which might contribute to ectopic ossification in tendon tissue due to mechanical loading.

Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy.

PURPOSE: The pathogenesis of patellar tendinopathy remains unclear. Expression of BMP-2/-4/-7 was reported in an ossified failed tendon healing animal model of patellar tendinopathy. This study aimed to investigate the expression of these chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. METHODS: Patellar tendon samples were collected from 16 consecutive patients with patellar tendinopathy and 16 consecutive controls undergoing anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft in the authors' hospital after getting their consent. The expression of BMP-2/-4/-7 was examined in all samples using immunohistochemistry. Ossification observed in two tendinopathy samples was characterized by histology, alizarin red S staining, alcian blue staining, TRAP staining and immunohistochemical staining of Sox9, osteopontin (OPN) and osteocalcin (OCN). RESULTS: Regions of hypo- and hyper-cellularity and vascularity, with loss of crimp structure of collagen matrix, were observed in patellar tendinopathy samples. Round cells and in some cases, cells with typical chondrocyte phenotype were observed. For the ossified tendinopathy samples with positive alizarin red S staining, OPN-positive and Sox9-positive chondrocyte-like cells in alcian blue-stained extracellular matrix, OCN-positive osteoblast-like cells and TRAP-positive multi-nucleated cells were observed around the ossified deposits. No expression of BMP-2/-4/-7 was observed in healthy patellar tendons. However, the expression of BMP-2/-4/-7 was observed in all patellar tendinopathy samples with or without ossification. CONCLUSIONS: Clinical samples of patellar tendinopathy showed ectopic expression of BMP-2/-4/-7. This was not evident in control samples from healthy patellar tendons. LEVEL OF EVIDENCE: Prognostic studies, Level III.

Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells.

PURPOSE: Surgical reattachment of tendon to bone often fails due to regeneration failure of the specialised tendon-bone junction (TBJ). The use of mesenchymal stem cells for TBJ regeneration has been reported with promising results. Tendon-derived stem cells (TDSCs) with high proliferative and multi-lineage differentiation potential have been isolated. As stem cells residing in tendons, TDSCs can be considered a new cell source for TBJ repair. Bone morphogenic protein 2 (BMP-2) is a potent osteogenic factor with roles in normal bone healing and pathological ectopic bone formation in soft tissues. The use of BMP-2 to promote TBJ repair has been well reported. This study aimed to compare TDSCs to the gold standard bone-marrow-derived mesenchymal stem cells (BMSCs) with respect to osteogenic response to BMP-2 in vitro. METHOD: The clonogenicity and multi-differentiation potential of TDSCs and BMSCs were identified by colony-forming-unit assay, osteogenic, adipogenic and chondrogenic differentiation assays. Their osteogenic response to BMP-2 in vitro was examined by alkaline phosphatase (ALP) cytochemical staining, ALP activity assay and Alizarin red S staining of calcium nodule formation. Messenger RNA (mRNA) and BMP receptor (types IA, IB and II) protein expression were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: Our results showed that both TDSCs and BMSCs exhibited stem cell properties, including clonogenicity and multi-differentiation potential. TDSCs expressed higher mRNA and protein levels of BMP receptors IA, IB and II. They also exhibited higher osteogenic differentiation with and without BMP-2 stimulation compared with BMSCs. CONCLUSIONS: TDSCs with/without BMP-2 might be an attractive source for TBJ repair compared with BMSCs.

Practical Considerations for Translating Mesenchymal Stromal Cell-Derived Extracellular Vesicles from Bench to Bed.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown potential for the treatment of tendon and ligament injuries. This approach can eliminate the need to transplant live cells to the human body, thereby reducing issues related to the maintenance of cell viability and stability and potential erroneous differentiation of transplanted cells to bone or tumor. Despite these advantages, there are practical issues that need to be considered for successful clinical application of MSC-EV-based products in the treatment of tendon and ligament injuries. This review aims to discuss the general and tissue-specific considerations for manufacturing MSC-EVs for clinical translation. Specifically, we will discuss Good Manufacturing Practice (GMP)-compliant manufacturing and quality control (parent cell source, culture conditions, concentration method, quantity, identity, purity and impurities, sterility, potency, reproducibility, storage and formulation), as well as safety and efficacy issues. Special considerations for applying MSC-EVs, such as their compatibility with arthroscopy for the treatment of tendon and ligament injuries, are also highlighted.

Continuous cyclic mechanical tension inhibited Runx2 expression in mesenchymal stem cells through RhoA-ERK1/2 pathway.

Tensile load is known to regulate the osteogenesis of mesenchymal stem cells (MSCs) and osteogenic progenitors; therefore it is widely used in clinical treatment and tissue engineering. Meanwhile, in vitro, both published studies and our lab data demonstrate that the application of intermittent tensile loading which stimulates cells several minutes or hours each day for several days has promoted the osteogenic differentiation of MSCs. Whereas, for clinic trails, it is important to know accurately how and how long mechanical tension should be applied. Hence, it is necessary to investigate different kinds of mechanical tension on osteogenesis of MSCs. Until now, during the osteogenesis, there has been no research on the effect of continuous cyclic mechanical tension (CCMT) which provides continuous stimulation throughout the study period. We firstly figure out CCMT inhibiting the expression of osteogenic genes such as key transcription factor Runx2. It is known that RhoA regulates cell differentiation in response to mechanical stimuli. MAPK signaling acts as a downstream effector of RhoA. So, we ask in MSCs, if CCMT regulates the osteogenic master gene Runx2 through RhoA-ERK1/2 pathway. And then, we find out there is a decrease in RhoA activity after CCMT stimulation. Pre-treatment of CCMT-loaded MSCs with LPA, a RhoA activator, restores ALP activity and significantly rescues Runx2 expression, while pre-treatment with C3 toxin, a RhoA inhibitor, further decreases the activity of ALP and down-regulates the expression of Runx2. Following results indicate that the inhibition of Runx2 expression after CCMT stimulation is mediated by RhoA-ERK1/2 pathway.

Mesenchymal Stem Cell-Derived Extracellular Vesicles for the Promotion of Tendon Repair - an Update of Literature.

Tendon injuries are prevalent in physical activities and sports. Tendon heals slowly after injuries. The results of conservative treatments and surgery are not satisfactory with high re-injury rate and scar tissue formation. The application of mesenchymal stem cells (MSCs) to the injured tendons was reported to promote tendon repair. Recent studies have suggested that MSCs supported tendon repair via the secretion of paracrine factors. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are produced and secreted by most eukaryotic cells. They carry a plethora of proteins, lipids, microRNA and mRNA which reprogram the recipient cells and are involved in multiple physiological and pathological processes. EVs were shown to promote tissue repair and mediate the healing effects of MSCs. In this review, I aim to review the recent literature on the promotion of tendon repair using EVs-derived from MSCs (MSC-EVs). The mechanisms underlying these actions are also reviewed and future research directions are discussed. Better understanding of the roles of MSC-EVs in tendon repair would offer a new treatment strategy to circumvent this devastating soft tissue disorder. Graphical Abstract.

Inflammatory mechanisms linking obesity and tendinopathy.

Chronic tendinopathy is a debilitating tendon disorder with disappointing treatment outcomes. This review focuses on the potential roles of chronic low-grade inflammation in promoting tendinopathy in obesity. A systematic literature search was performed to identify all clinical studies supporting the actions of obesity-associated inflammatory mediators in the development of tendinopathy. The mechanisms of obesity-induced chronic inflammation in adipose tissue are firstly reviewed. Common inflammatory mediators potentially linking obesity and the development of tendinopathy, and their association with mechanical overuse, are discussed, along with pre-clinical evidences and a systematic literature search on clinical studies. The potential contribution of local adipose tissues in the promotion of inflammation, pain and tendon degeneration is then discussed. The future research directions are proposed. TRANSLATIONAL POTENTIAL STATEMENT: Better understanding of the roles of obesity-associated inflammatory mediators on tendons will clarify the pathophysiological drivers of tendinopathy in patients with obesity and identify possible treatment targets. Further studies on the mechanisms of obesity-induced chronic inflammation on tendon are a promising direction for the treatment of tendinopathy.