Aims. To evaluate inducing osteoarthritis (OA) by surgical destabilization of the medial meniscus (DMM) in mice with and without a stereomicroscope. Methods. Based on sample size calculation, 70 male C57BL/6 mice were randomly assigned to three surgery groups: DMM aided by a stereomicroscope; DMM by naked eye; or sham surgery. The group information was blinded to researchers. Mice underwent static weightbearing, von Frey test, and gait analysis at two-week intervals from eight to 16 weeks after surgery. Histological grade of OA was determined with the Osteoarthritis Research Society International (OARSI) scoring system. Results. Surgical DMM with or without stereomicroscope led to decrease in the mean of weightbearing percentages (-20.64% vs -21.44%, p = 0.792) and paw withdrawal response thresholds (-21.35% vs -24.65%, p = 0.327) of the hind limbs. However, the coefficient of variation (CV) of weight-bearing percentages and paw withdrawal response thresholds in naked-eye group were significantly greater than that in the microscope group (19.82% vs 6.94%, p < 0.001; 21.85% vs 9.86%, p < 0.001). The gait analysis showed a similar pattern. Cartilage degeneration was observed in both DMM-surgery groups, evidenced by increased OARSI scores (summed score: 11.23 vs 11.43, p = 0.842), but the microscope group showed less variation in OARSI score than the naked-eye group (CV: 21.03% vs 32.44%; p = 0.032). Conclusion. Although surgical DMM aided by stereomicroscope is technically difficult, it produces a relatively more homogeneous OA model in terms of the discrete degree of pain behaviours and histopathological grading when compared with surgical DMM without stereomicroscope. Cite this article: Bone Joint Res 2022;11(8):518–527

Aims

Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown.

Introduction and Objective. Malunion after trauma can lead to coronal plane malalignment in the lower limb. The mechanical hypothesis suggests that this alters the load distribution in the knee joint and that that this increased load may predispose to compartmental arthritis. This is generally accepted in the orthopaedic community and serves as the basis guiding deformity correction after malunion as well as congenital or insidious onset malalignment. Much of the literature surrounding the contribution of lower limb alignment to arthritis comes from cohort studies of incident osteoarthritis. There has been a causation dilemma perpetuated in a number of studies - suggesting malalignment does not contribute to, but is instead a consequence of, compartmental arthritis. In this investigation the relationship between compartmental (medial or lateral) arthritis and coronal plane malalignment (varus or valgus) in patients with post traumatic unilateral limb deformity was examined. This represents a specific niche cohort of patients in which worsened compartmental knee arthritis after extra-articular injury must rationally be attributed to malalignment. Materials and Methods. The picture archiving system was searched to identify all 1160 long leg x ray films available at a major metropolitan trauma center over a 12-year period. Images were screened for inclusion and exclusion criteria, namely patients >10 years after traumatic long bone fracture without contralateral injury or arthroplasty to give 39 cases. Alignment was measured according to established surgical standards on long leg films by 3 independent reviewers, and arthritis scores Osteoarthritis Research Society International (OARSI) and Kellegren-Lawrence (KL) were recorded independently for each compartment of both knees. Malalignment was defined conservatively as mechanical axis deviation outside of 0–20 mm medial from centre of the knee, to give 27 patients. Comparison of mean compartmental arthritis score was performed for patients with varus and valgus malalignment, using Analysis of Variance and linear regression. Results. In knees with varus malalignment there was a greater mean arthritis score in the medial compartment compared to the contralateral knee, with OARSI scores 5.69 vs 3.86 (0.32, 3.35 95% CI; p<0.05) and KL 2.92 vs 1.92 (0.38, 1.62; p<0.005). There was a similar trend in valgus knees for the lateral compartment OARSI 2.98 vs 1.84 (CI −0.16, 2.42; p=0.1) and KL 1.76 vs 1.31 (CI −0.12, 1.01; p=0.17), but the evidence was not conclusive. OARSI arthritis score was significantly associated with absolute MAD (0.7/10mm MAD, p<0.0005) and Time (0.6/decade, p=0.01) in a linear regression model. Conclusions. Malalignment in the coronal plane is correlated with worsened arthritis scores in the medial compartment for varus deformity and may similarly result in worsened lateral compartment arthritis in valgus knees. These findings support the mechanical hypothesis that arthritis may be related to altered stress distribution at the knee, larger studies may provide further conclusive evidence

Introduction and Objective. Osteoarthritis (OA) is the most common inflammatory and degenerative joint disease. Mesenchymal Stromal Cells (MSCs), with their chondro-protective and immune-regulatory properties, have been considered as a new approach to treat OA. Considering the risk of cell leakage outside the articular space and the poor survival rate after intra-articular (IA) injection, we hypothesized that cell encapsulation in cytoprotective hydrogels could overcome these limitations and provide cells with a suitable 3D microenvironment supporting their biological activity. We previously generated micromolded alginate particles (diameter 150 μm) and demonstrated the long-term viability of microencapsulated MSCs isolated from human adipose tissue (hASCs). Encapsulated cells maintained their in vitro ability to sense and respond to a pro-inflammatory environment (IFN-γ/TNF-α or synovial fluids from OA patients) by secreting PGE. 2. , IDO, HGF and TGF-β. In this study, we evaluated the anti-OA efficacy of these microencapsulated hASCs in a post-traumatic OA model in rabbits. Materials and Methods. OA was surgically induced by anterior cruciate ligament transection (ACLT)-mediated destabilization of the right knee in rabbits (n=24). Eight weeks after surgery, destabilized joints were injected (IA, 26G needle) with 200 μL of either PBS, blank microparticles, non-encapsulated or microencapsulated cells (5×10. 5. cells). Six weeks after injection, rabbits were euthanized and all destabilized (right) and sham-operated (left contralateral) joints were dissected and analyzed for OA severity. Tibial subchondral bone histomorphometric parameters were measured by quantitative micro-computed tomography (micro-CT). Histological sections of samples were analyzed after Safranin-O staining and quantitatively assessed according to a modified Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemical detection of NITEGE was performed to assess the extracellular matrix degradation. Results. Micro-CT analysis of destabilized joints confirmed that the rabbit ACLT significantly affected the tibial subchondral bone architecture as early as eight weeks, as revealed by significant changes of the subchondral bone parameters of operated joints compared to the sham operated joints. In particular, destabilized joints exhibited a Bone Volume/Tissue Volume ratio (BV/TV) ranging from 53.4% to 56.6%, compared to a mean BV/TV of 65.4% for sham operated joints. All destabilized joints also exhibited a significantly increased modified OARSI score, ranging from 7.4±0.4 for those injected with encapsulated cells to 8.9±0.2 for those injected with PBS, as compared to 4.8±0.4 for sham-operated joints. Of interest, we identified a slight, while not significant, reduction of the severity of OA lesions after injection of microencapsulated cells using the modified OARSI scoring. Finally, semi-quantitative analysis of NITEGE immunostaining revealed a significant increase in all destabilized joints that were injected with PBS or blank microparticles, in comparison with sham ones. On the contrary, NITEGE immunostaining in destabilized joints that were injected with non-encapsulated or encapsulated hASC revealed a significant reduced NITEGE immunostaining, indicating a decreased matrix degradation. Conclusions. Our data suggest that the microencapsulated hASCs exerted their anti-OA properties after IA injection in rabbit knees, as evidenced by the tendency toward a reduced modified OARSI score, and most importantly a significant reduction in NITEGE immunostaining associated matrix degradation. Further studies are now warranted to investigate the anti-OA efficacy of microencapsulated hASCs in the long-term

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA. The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, Imaris. TM. and ImageJ software. There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion. These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening

Introduction. Osteoarthritis (OA) of the knee, a prevalently degenerative joint disorder provoked by articular cartilage loss, accounts for the leading cause of total knee arthroplasty. Autophagy is an indispensable intracellular event that maintains chondrocyte survival and metabolism. MicroRNAs are non-coding small RNAs participating in tissue morphogenesis, remodeling, and homeostasis. This study was undertaken to investigate the effect of microRNA-128 (miR-128) knockdown on the development of OA knees. Materials/Methods. Knee joints in rats were subjected to anterior cruciate ligament transection (ACLT) for inducing OA. Articular cartilage, synovium, and subchondral bone microarchitecture were assessed by OARSI scoring system, histomorphometry, and μCT imaging. Chondrocyte autophagy in terms of the expression of autophagic markers Atg4, Atg12, microtubule-associated protein 1 light chain 3 (LC3), and autophagosome formation was verified. Expression of microRNA, mRNA and signaling transduction were quantified with in situ hybridization, RT- quantitative PCR, and immunoblotting. Results. Chondrocytes in the affected knees showed weak expression of autophagic markers Atg4, Atg12, and LC3-II abundances in conjunction with significant increases in OARSI scores and a 2.5-fold elevation in miR-128 expression. The gain of miR-128 signaling in intact joints through intra-articular injection of miR-128 precursor resulted in 1.8–2.1-fold elevations in serum cartilage breakdown products CTX-II and COMP concentrations. miR-128 overexpression caused the joints to show evident chondrocyte apoptosis as evidenced by TUNEL staining concomitant with severe cartilage damage. Of note, antisense oligonucleotide knockdown of miR-128 (miR-128-AS) enabled the affected knee joints to show minor responses to the ACLT escalation of autophagy dysfunction in chondrocytes, cartilage breakdown histopathology, and OARSI scores. Administration with miR-128-AS also attenuated the ACLT-induced synovial membrane thickening, hyper-angiogenesis, and hypercellularity, which subsequently alleviated osteophyte accumulation, subchondral plate destruction, and trabecular microstructure loss. Conclusion. miR-128 signaling impairs chondrocyte autophagy, which ramps up chondrocyte apoptosis and OA knee development. This study highlights an emerging miR-128 knockdown strategy that sustains cartilage microarchitecture integrity and thereby delays OA knee pathogenesis

Introduction. Osteoarthritis (OA) causes pain, stiffness, and loss of function due to degenerative changes in joint cartilage and bone. In some forms of OA, exercise can alleviate symptoms by improving joint mobility and stability. However, excessive training after joint injury may have negative consequences for OA development. Sensory nerve fibers in joints release neuropeptides like alpha-calcitonin gene-related peptide (alpha-CGRP), potentially affecting OA progression. This study investigates the role of alpha-CGRP in OA pathogenesis under different exercise regimen in mice. Method. OA was induced in C57Bl/6J WT mice and alpha-CGRP KO mice via surgical destabilization of the medial meniscus (DMM) at 12 weeks of age (N=6). Treadmill exercise began 2 weeks post-surgery and was performed for 30 minutes, 5 days a week, for 2 or 6 weeks at intense (16 m/min, 15° incline) or moderate (10 m/min, 5° incline) levels. Histomorphometric assessment of cartilage degradation (OARSI scoring), serum cytokine analysis, immunohistochemistry, and nanoCT analysis were conducted. Result. OARSI scoring confirmed OA induction 4 weeks post-DMM surgery, with forced exercise exacerbating cartilage degradation regardless of intensity. No significant genotype-dependent differences were observed. Serum analysis revealed elevated cytokine levels associated with OA and inflammation in KO mice compared to WT mice 4 and 8 weeks post-surgery (VEGF-A, MCP-1, CXCL10, RANTES, MIP1-alpha, MIP1-beta, and RANKL). The observed effects were often exacerbated by intense exercise but rarely by DMM surgery. NanoCT analysis demonstrated increased sclerotic bone changes after 6 weeks of forced exercise in KO mice compared to WT mice. Conclusion. Our results suggest an OA promoting effect of exercise in early disease stages of posttraumatic OA. Intense exercise induced inflammatory processes correlated to increased cytokine levels in the serum that might exacerbate OA pathogenesis in later stages. The neuropeptide alpha-CGRP might play a role in protecting against these adverse effects

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development. Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining. Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production. Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis. PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks. PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A. Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA

Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed. Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3. Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2). Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects

Objectives. This study aimed to examine the effects of SRT1720, a potent SIRT1 activator, on osteoarthritis (OA) progression using an experimental OA model. Methods. Osteoarthritis was surgically induced by destabilization of the medial meniscus in eight-week-old C57BL/6 male mice. SRT1720 was administered intraperitoneally twice a week after surgery. Osteoarthritis progression was evaluated histologically using the Osteoarthritis Research Society International (OARSI) score at four, eight, 12 and 16 weeks. The expression of SIRT1, matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), cleaved caspase-3, PARP p85, and acetylated nuclear factor (NF)-κB p65 in cartilage was examined by immunohistochemistry. Synovitis was also evaluated histologically. Primary mouse epiphyseal chondrocytes were treated with SRT1720 in the presence or absence of interleukin 1 beta (IL-1β), and gene expression changes were examined by real-time polymerase chain reaction (PCR). Results. The OARSI score was significantly lower in mice treated with SRT1720 than in control mice at eight and 12 weeks associated with the decreased size of osteophytes at four and eight weeks. The delayed OA progression in the mice treated with SRT1720 was also associated with increased SIRT1-positive chondrocytes and decreased MMP-13-, ADAMTS-5-, cleaved caspase-3-, PARP p85-, and acetylated NF-κB p65-positive chondrocytes and decreased synovitis at four and eight weeks. SRT1720 treatment partially rescued the decreases in collagen type II alpha 1 (COL2A1) and aggrecan caused by IL-1β, while also reducing the induction of MMP-13 by IL-1β in vitro. Conclusion. The intraperitoneal injection of SRT1720 attenuated experimental OA progression in mice, indicating that SRT1720 could be a new therapeutic approach for OA. Cite this article: K. Nishida, T. Matsushita, K. Takayama, T. Tanaka, N. Miyaji, K. Ibaraki, D. Araki, N. Kanzaki, T. Matsumoto, R. Kuroda. Intraperitoneal injection of the SIRT1 activator SRT1720 attenuates the progression of experimental osteoarthritis in mice. Bone Joint Res 2018;7:252–262. DOI: 10.1302/2046-3758.73.BJR-2017-0227.R1

Mitochondrial dysfunction has been demonstrated in aging and osteoarthritic tissues. We investigated knee joints of prematurely aging mitochondrial DNA mutator mice (PolgD275A) to evaluate a relationship between mitochondrial dysfunction and osteoarthritis. Cartilage damage was evaluated using OARSI histopathology grading and osteoclast numbers were quantified by tartrate-resistant acid phosphatase staining in wild type, heterozygous and homozygous PolgD275A mice. Subchondral cortical plate and epiphyseal trabecular bone structures were determined by micro-computed tomography. Apoptosis in cartilage and subchondral bone tissues was studied using an indirect TUNEL method. Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type and heterozygous mutants. Subchondral osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). Wild type mice as well as hetero- and homozygous mutants displayed low-grade cartilage degeneration due to loss of cartilage proteoglycans. In contrast, chondrocyte hypertrophy was more abundant in the homozygous mice. There were no differences in chondrocyte apoptosis rates between groups. Prematurely ageing mtDNA mutator mice with or without further mechanic or metabolic stimuli might serve as a valuable model for further experimental studies on aging-induced osteoporotic OA phenotype

Introduction and Objective. Osteoarthristis (OA) has been associated with many genes and yet the genetic basis for this disease has never formally been established. Recent realization that epigenetic changes could be the underlying pathological mechanisms has helped to explain many complex multifactorial diseases with no clear genetic cause. We therefore asked whether epigenetics could also play a role in OA. We have previously shown that the DNA epigenetic modification, specifically the hydroxymethylation on cytosine (5hmC), undergoes a fivefold increase on OA-associated genes which are activated at OA onset. In this study, we further uncovered a set of 5hmC-mediated gene targets and their mechanistic link to OA progression. Materials and Methods. We surgically induced OA on 4 to 6 months old Tet1−/− mice (Tet1tm1.1Jae, the Jackson laboratory) and wild-type littermates by performing destabilization of the medial meniscus (DMM) surgery. Joints were collected for histological assessment through blinded grading with the OARSI scoring system. Human articular chondrocytes were harvested from OA cartilage samples obtained during total knee arthroplasty or from grossly normal cartilage pieces obtained during notchplasty or debridement from patients undergoing anterior cruciate ligament (ACL) reconstruction with no history of OA symptoms, under approved Human subjects Institutional Review Board protocols. Bioinformatic analyses of RNA-sequencing and CCGG sequencing (reduced representation 5hmC profiling) were performed to identify TET1 target genes associated with OA progression. Several measurements were used to assess the effect of TET1 ablation on the phenotype of mouse cartilage tissue and human chondrocytes including, histological evaluation, and quantitative bone assessment by micro-CT imaging and multiplex cytokine analyses in the serum of mice in vivo (mouse 39-plex assay) and in the supernatant of human chondrocyte cultures (human 62-plex assay). Results. We used a mouse model with surgically induced OA and found that OA onset was accompanied by a gain of ∼40,000 differentially hydroxymethylated sites prior the notable histological onset of the disease. We additionally revealed that these changes are mediated by the ten-eleven-translocation enzyme 1 (TET1), since Tet1−/− mice lost 98% of 5hmC sites upon OA induction. Remarkably, Tet1−/− mice were protected from OA development including degeneration of the cartilage surface and osteophyte formation. Silencing of TET1 expression in human OA chondrocytes reduced the expression in a set of genes, which may represent the pathological gene targets that exacerbate OA including MMP3 and MMP13 and several inflammatory cytokines. Therefore, our study reveals the unexpected beneficial role of TET1 inhibition in blocking OA progression. In fact, intra-articular injections of a dioxygenases’ inhibitor, 2 hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Furthermore, treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss, implicating a therapeutic potential of TET inhibition in OA patients. Conclusions. Collectively, our study not only demonstrate the role of TET1 in OA; the 5hmC-mediated gene targets acting on multiple OA pathways were identified and can be modulated as therapeutic intervention to treat OA

Osteoarthritis is a slowly progressive disease which includes the intervention of several cytokines and macrophage metalleinoproteinases reaction, leading to the degradation of the local cartilage but also having an impact on the serum acute phase proteins (APPs). Subsequently, biomarkers seem to be essential to estimate its progression and the need for any surgical intervention such as total arthroplasty, but also can be used as therapeutic agents. Recently, among APPs, fetuin-A drew attention regarding its possible anti-inflammatory role in animal models but also as a therapeutic agent in the inflammatory joint disease in clinical trials. The purpose of this study is to investigate the possible attenuating role of the intra-articular administration of Fetuin-A in post-traumatic induced secondary osteoarthritis in rats, and also its effect on the systematic levels of IL-2,4,7, BMPs 2,4,7, CRP and Fetuin-A. 30 male Sprague Dawley rats were separated in two groups where post-traumatic osteoarthritis was induced surgically by Anterior Cruciate Ligament Transection and the transection of the Medial Collateral Ligament of the right knee. In the Control Group, only surgical intervention took place. In Fetuin Group, along with the induction of osteoarthritis, a single dose of bovine fetuin was administrated intra-articularly intra-operatively in 5 and 8 weeks of the experimental protocol. Both groups were examined for 8 weeks. The levels of interleukins, bone morphogenetic proteins, Fetuin-A and C-Reactive Protein were evaluated by ELISA of peripheral blood in three time periods: preoperatively, 5 and 8 weeks post-operatively. Knee osteoarthritic lesions were classified according to Osteoarthritis Research Society International Grading System and Modified Mankin Score, by histologic examination. IL-2 levels were significantly decreased in the Fetuin Group. No statistical difference was signed on the levels of IL-7, BMP-2,4,7 and Fetuin-A between the two groups. CRP levels were significantly increased in the Fetuin Group in 5 weeks of the experiment. Fetuin Group signed better scores according to the OARSI classification system and Modified Mankin Score, without any statistical significance. Intra-articular administration of Fetuin-A restrictively affected the progression of post-traumatic arthritis in rats, as only the levels of IL-2 were decreased as well as limited osteoarthritic lesions were observed on the Fetuin Group

Purpose. It is well known that meniscus extrusion is associated with structural progression of knee OA. However, it is unknown whether medial meniscus extrusion promotes cartilage loss in specific femorotibial subregions, or whether it is associated with a increase in cartilage thickness loss throughout the entire femorotibial compartment. We applied quantitative MRI-based measurements of subregional cartilage thickness (change) and meniscus position, to address the above question in knees with and without radiographic joint space narrowing (JSN). Methods. 60 participants with unilateral medial OARSI JSN grade 1–3, and contralateral knee OARSI JSN grade 0 were drawn from the Osteoarthritis Initiative. Manual segmentation of the medial tibial and weight-bearing medial femoral cartilage was performed, using baseline and 1-year follow-up sagittal double echo steady-state (DESS) MRI, and proprietary software (Chondrometrics GmbH, Ainring, Germany). Segmentation of the entire medial meniscus was performed with the same software, using baseline coronal DESS images. Longitudinal cartilage loss was computed for 5 tibial (central, external, internal, anterior, posterior) and 3 femoral (central, external, internal) subregions. Meniscus position was determined as the % area of the entire meniscus extruding the tibial plateau medially and the distance between the external meniscus border and the tibial cartilage in an image located 4mm posterior to the central image (a location commonly used for semi-quantitative meniscus scoring). The relationship between meniscus position and cartilage loss was assessed using Pearson (r) correlation coefficients, for knees with JSN and without JSN. Results. The percentage of knees showing a quantitative value of >3mm medial meniscus extrusion was 50% in JSN knees, and only 12% in noJSN knees. The 1-year cartilage loss in the medial femorotibial compartment was 74±182µm (2.0%) in JSN knees, and 26±120µm (0.8%) in noJSN knees. There was a significant correlation between cartilage loss throughout the entire femorotibial compartment (MFTC) and extrusion area in JSN knees but not for noJSN knees. Also, the extrusion distance measured 4mm posterior to the central slice was not significantly correlated with MFTC cartilage loss. The strongest (negative) correlation between meniscus position and subregional femorotibial cartilage loss (r=−0.36) was observed for the external medial tibia. In contrast, no significant relationship was seen in the central tibia. No significant relationship was found in other tibial subregions, except for the anterior medial tibia, but only in JSN knees (r=−0.27). Correlation coefficients for the femoral subregions were generally smaller than those for tibial subregions, with only the internal medial weight-bearing femur attaining statistical significance (r =−0.26). Conclusions. The current results show that the relationship between meniscus extrusion and cartilage loss differs substantially between femorotibial subregions. The correlation was strongest for the external medial tibia, a region that is physiologically covered by the medial meniscus. It was less for other tibial and femoral subregions, including the central medial tibia, a region that exhibited similar rates of cartilage loss as the external subregion. The findings suggest that external tibia may be particularly vulnerable to cartilage tissue loss once the meniscus extrudes and the surface is “exposed” to direct, non-physiological, cartilage-cartilage contact

Osteoarthritis (OA) is a chronic degenerative joint disease with cartilage degeneration, subchondral bone sclerosis, synovial inflammation and osteophyte formation. Sensory nerves play an important role in bone metabolism and in the progression of inflammation. This study explored the effects of capsaicin-induced sensory nerve denervation on OA progression in mice. This study was approved by the Institutional Animal Care and Use Committee. OA was induced via destabilization of the medial meniscus (DMM). Sensory denervation was induced by subcutaneous injection of capsaicin (90mg/kg) one week prior to DMM. One week after capsaicin injection, sensory denervation in the tibia was confirmed by immunofluorescent staining with calcitonin gene-related peptide (CGRP)-specific antibodies. Four weeks after DMM, micro-CT scans, histological analysis and RT-PCR tests were performed to evaluate OA progression. Statistical analysis was performed using SPSS 13. P values of less than 0.05 were considered statistically significant. Subcutaneous injection of capsaicin successfully induced tibial sensory denervation (n=3), which aggravated OA by increasing subchondral bone resorption. The Osteoarthritis Research Society International (OARSI) score of the capsaicin+DMM group (n=8) (11.81±2.92) was significantly higher (P=0.003) than the score of the vehicle+DMM group (n=8) (8.31±1.80). The BV/TV of the tibial subchondral bone in the capsaicin+DMM group (n=8) was 55.67%±3.08, which was significantly lower (P < 0 .001) than in the vehicle+DMM group (n=8) (86.22%±1.92). In addition, the level of expression of somatostatin in the capsaicin+DMM group (n=8) was lower than in the vehicle+DMM group (n=8) (P=0.007). Capsaicin-induced sensory denervation increased tibial subchondral bone resorption, reduced the expression of somatostatin and eventually exacerbated the existing cartilage degeneration in mice. Despite capsaicin is often used clinically to relieve OA pain, its safety is still controversial according to the OARSI guidelines for the non-surgical management of knee osteoarthritis. The findings of our study suggest that application of capsaicin, although effective in relieving pain, may accelerate the progression of existing OA

Knee joint distraction (KJD) is a joint-preserving treatment strategy for severe osteoarthritis (OA) that provides long-term clinical and structural improvement. Data from both human trials and animal models indicate clear cartilage regeneration from 6 months and onwards post-KJD. However, recent work showed that during distraction, the balance between catabolic and anabolic indicators is directed towards catabolism, as indicated by collagen type 2 markers, proteoglycan (PG) turnover and a catabolic transcription profile [unpublished data]. The focus of this study was to investigate the cartilage directly and 10 weeks after joint distraction in order to elucidate the shift from a catabolic to an anabolic cartilage state. Knee OA was induced bilaterally in 8 dogs according to the groove model. After 10 weeks of OA induction, all 8 animals received right knee joint distraction, employing the left knee as an OA control. After 8 weeks of distraction, 4 dogs were euthanized and after 10 weeks of follow-up the 4 other dogs. Macroscopic cartilage degeneration and synovial tissue inflammation was assessed using the OARSI canine scoring system. PG content was determined spectrometrically using Alcian Blue dye solution and the synthesis of newly formed PGs was determined using . 35. SO. 4. 2-. as a tracer, as was described before. Directly after KJD, macroscopic cartilage damage of the right tibial plateau was higher compared to the left OA control (OARSI score: 1.7±0.2 vs 0.6±0.3; p < 0.001). 10 weeks post-KJD this difference persisted (OARSI score: 1.4± 0.6 vs 0.6±0.3; p = 0.05). Directly after KJD, there was no difference in synovial inflammation between KJD and OA control (OARSI score: 1.4±0.5). At 10 weeks synovial inflammation increased significantly in the distracted knee (OARSI score: 2.1±0.3 vs 1.4±0.5; p < 0.05). Biochemical analysis of the tibia cartilage directly after KJD revealed a lower PG content (20.1±10.3 mg/g vs 23.7±11.7 mg/g). At 10 weeks post-KJD this difference in PG content was less (24.8±6.8 mg/g vs 25.4±7.8 mg/g). The PG synthesis rate directly after KJD appeared significantly lower vs. OA (1.4±0.6 nmol/h.g vs 5.9±4.4 nmol/h.g; p < 0.001)). However, 10 weeks post-KJD this difference was not detected (3.7±1.2 nmol/h.g vs 2.9±0.8 nmol/h.g), and the synthesis rate in the distracted knee was increased compared to directly after distraction (p < 0.01). Further in-depth investigation of the material is ongoing; these first results suggest that the shift from a catabolic to an anabolic state occurs within the first weeks after joint distraction, mostly reflected in the biochemical changes. As such, the post-distraction period seems to be essential in identifying key-players that support intrinsic cartilage repair

Our aim was to investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Gonarthrosis (AMG). Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H& E staining) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content. Real time PCR compared gene expression between areas T and N. There was a decrease in OARSI grade across the four areas, with progressively less fibrillation between areas T1, T2 and T3. Area N had an OARSI grade of 0 (normal). The GAG immunoassay showed decreased levels with increasing severity of cartilage damage (p< 0.0001). There was no significant difference in the Collagen II content or gene expression between areas. The Collagen I immunohistochemistry showed increased staining within chondrocyte pericellular areas in the undamaged region (N) and immunoassays showed that the Collagen I content of this macroscopically and histologically normal cartilage, was significantly higher than the damaged areas (p< 0.0001). Furthermore, real time PCR showed a significant increase in Collagen I expression in the macroscopically normal areas compared to the damaged areas (p=0.04). In AMG there are distinct areas, demonstrating progressive cartilage loss. We conclude that in this phenotype the Collagen I increase, in areas of macroscopically and histologically normal cartilage, may represent very early changes of the cartilage matrix within the osteoarthritic disease process. This may be able to be used as an assay of early disease and as a therapeutic target for disease modification or treatment

Aim: To investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Osteoarthritis of the Knee (AMOA). Methods: Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H& E staining) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content. Real time PCR compared gene expression between areas T and N. Results: There was a decrease in OARSI grade across the four areas, with progressively less fibrillation between areas T1, T2 and T3. Area N had an OARSI grade of 0 (normal). The GAG immunoassay showed decreased levels with increasing severity of cartilage damage (ANOVA P< 0.0001). There was no significant difference in the Collagen II content or gene expression between areas. The Collagen I immunohistochemistry showed increased staining within chondrocyte pericellular areas in the undamaged region (N) and immunoassays showed that the Collagen I content of this macroscopically and histologically normal cartilage, was significantly higher than the damaged areas (ANOVA P< 0.0001). Furthermore, real time PCR showed that there was a significant difference in Collagen I expression between the damaged and macroscopically normal areas (p=0.04). Conclusion: In AMOA there are distinct areas, demonstrating progressive cartilage loss. We conclude that in this phenotype the Collagen I increase, in areas of macroscopically and histologically normal cartilage, may represent very early changes of the cartilage matrix within the osteoarthritic disease process. This may be able to be used as an assay of early disease and as a therapeutic target for disease modification or treatment

The aim of this study was to investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Osteoarthritis of the Knee (AMOA). Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H& E staining) and immunohistochemical analysis (Type I and II Collagen). Immunoassays were undertaken for Collagens I and II and GAG content. Real time PCR compared gene expression between areas T and N. There was a decrease in OARSI grade across the four areas, with progressively less fibrillation between areas T1, T2 and T3. Area N had an OARSI grade of 0 (normal). The GAG immunoassay showed decreased levels with increasing severity of cartilage damage (ANOVA P< 0.0001). There was no significant difference in the Collagen II content or gene expression between areas. The Collagen I immunohistochemistry showed increased staining within chondrocyte territorial areas in the undamaged region (N) and immunoassays showed that the Collagen I content of this macroscopically and histologically normal cartilage, was significantly higher than the damaged areas (ANOVA P< 0.0001). Furthermore, real time PCR showed that there was a significant increase in Collagen I expression in the macroscopically normal areas (p=0.04). In AMOA there are distinct areas, demonstrating progressive cartilage loss. We conclude that in this phenotype the Collagen I increase, in areas of macroscopically and histologically normal cartilage, may represent very early changes of the cartilage matrix within the osteoarthritic disease process. This may be able to be used as an assay of early disease and as a therapeutic target for disease modification or treatment