Rapidly progressive osteoarthritis of the hip (RPOH) is an unusual subset of osteoarthritis. It is characterized by rapid joint space loss, chondroly­sis, and sometimes marked femoral head and acetabular destruction as a late finding. The exact pathogenetic mechanism is unknown. Potential causes of RPOH include subchondral insufficiency fracture resulting from osteoporosis, increasing posterior pelvic tilt as a mechanical factor, and high serum levels of matrix metalloproteinase (MMP)-3 as biological factors. This study was aimed to identify some markers that associate with the destructive process of RPOH by analyzing the proposed pathological factors of the disease, MMP-3, pelvic tilt, and osteoporosis. Of female patients who visited our hospital with hip pain from 2012 through 2018, this study enrolled female patients with sufficient clinical records including the onset of hip pain, age and body mass index (BMI) at the onset, a series of radiographs during the period of >12 months from the onset of hip pain, and hematological data of MMP-3 and C-reactive protein (CRP). We found the hip joints of 31 patients meet the diagnostic criteria of RPOH, chondrolysis >two mm in one year, or 50% joint space narrowing in one year. Those patients were classified into two groups, 17 and 14 patients with and without subsequent femoral head destruction in one year shown by computed tomography, respectively. Serum MMP-3 and CRP were measured with blood samples within one year after the hip pain onset. The cortical thickness index (CTI) as an indicator of osteoporosis and pelvic tilt parameters were evaluated on the initial anteroposterior radiograph of the hip. These factors were statistically compared between the two groups. This study excluded male patients because RPOH occurs mainly in elderly females and the reference intervals of MMP-3 are different between males and females. There was no difference in age at onset or bone mass index between the RPOH patients with and without subsequent femoral head destruction. Serum levels of MMP-3 were significantly higher in the RPOH patients with the destruction (152.1 ± 108.9 ng/ml) than those without the destruction (66.8 ± 27.9 ng/ml) (P = 0.005 by Mann-Whitney test). We also found increased CRP in the patients with femoral head destruction (0.725 ± 1.44 mg/dl) compared with those without the destruction (0.178 ± 0.187 mg/dl) (P = 0.032 by Mann-Whitney test). No difference in the duration between the hip pain onset and the blood examination was found between the two groups. There was no significant difference in CTI or pelvic tilt between the two groups. The pathological condition that may increase serum MMP-3 and CRP could be involved in femoral head destruction after chondrolysis of the hip in patients with RPOH

Osteoarthritis (OA) is a multifactorial debilitating disease that affects over four million Canadians. Although the mechanism(s) of OA onset is unclear, the biological outcome is cartilage degradation. Cartilage degradation is typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II) – partly due to the up-regulation of catabolic enzymes - aggrecanases a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). There is currently no treatment that will prevent or repair joint damage, and current medications are aimed mostly at pain management. When pain becomes unmanageable arthroplastic surgery is often performed. Interest has developed over the presence of calcium crystals in the synovial fluid of OA patients, as they have been shown to activate synovial fibroblasts inducing the expression of catabolic agents. We recently discovered elevated levels of free calcium in the synovial fluid of OA patients and raised the question on its role in cartilage degeneration. Articular cartilage was isolated from 5 donors undergoing total hip replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase and expanded in DMEM supplemented with 10% heat-inactivated FBS. OA and normal human articular chondrocytes (PromoCell, Heidelberg, Germany) were transferred to 6-well plates in culture medium containing various concentrations of calcium (0.5, 1, 2.5, and 5 mM CaCl2), and IL-1β. Cartilage explants were prepared from the same donors and included cartilage with the cortical bone approximately 1 cm2 in dimension. Bovine articular cartilage explants (10 months) were used as a control. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. Immunohistochemistry was performed on cartilage explants to measure expression of Col X, MMP-13, and alkaline phosphatase. The sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of cartilage was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay, and aggregan fragmentation was determined by Western blotting using antibody targeted to its G1 domain. Western blotting was also performed on cell lysate from both OA and normal chondrocytes to measure aggrecan, Col II, MMP-3 and −13, ADAMTS-4 and −5. Ca2+ significantly decreased the proteoglycan content of the cartilage explants as determined by the DMMB assay. The presence of aggrecan and Col II also decreased as a function of calcium, in both the human OA and bovine cartilage explants. When normal and OA chondrocytes were cultured in medium supplemented with increasing concentrations of calcium (0.5–5 mM Ca2+), aggrecan and Col II expression decreased dose-dependently. Surprisingly, increasing Ca2+ did not induce the release of MMP-3, and −13, or ADAMTS-4 and-5 in conditioned media from OA and normal chondrocytes. Interestingly, inhibition of the extracellular calcium-sensing receptor CaSR) reversed the effects of calcium on matrix protein synthesis. We provide evidence that Ca2+ may play a direct role in cartilage degradation by regulating the expression of aggrecan and Col II through activation of CaSR

Introduction. Osteoarthritis (OA) has historically been thought of as a degenerative joint disease, but inflammation and angiogenesis are increasingly being recognised as contributing to the pathogenesis, symptoms and progression of OA. b-dystroglycan (b-DG) is a pivotal element of the transmembrane adhesion molecule involved in cell-extracellular matrix adhesion and angiogenesis. Matrix metalloproteinases (MMPs) are the main enzymes responsible for cartilage extracellular matrix breakdown and are also implicated in both angiogenesis and b-DG degradation in a number of malignancies. We aimed to investigate the expression and localisation of b-DG and MMP-3, -9, and -13 within cartilage, synovium and synovial fluid and establish their roles in the pathogenesis of OA. Methods. Following ethical committee approval, cartilage, synovium and synovial fluid were obtained from the hip joints of 5 osteoarthritic (patients undergoing total hip replacement) and 5 control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for b-DG expression using Western Blotting and for the distribution of b-DG, MMP-3, -9, and -13 using immunohistochemistry on paraffin embedded tissue. Results. Whilst no significant expression of b-DG was found in cartilage or synovial fluid, b-DG was expressed in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Moreover, b-DG was expressed in endothelium of blood vessels of OA synovium, but not in the normal endothelium. In the endothelium of osteoarthritic synovial blood vessels, b-DG co-localised with MMP -3 and -9. Discussion. Our results demonstrate that b-DG does not act as a cell adhesion molecule binding chondrocytes to the ECM. However, specific immunolocalisation of b-DG within endothelium of inflamed OA blood vessels suggests that b-DG may play a role in angiogenesis associated with OA. Its co-localisation with MMP-3 and -9, previously reported to also have pro-angiogenic roles, may be linked. Further research is required to understand these roles more fully

Introduction. Matrix metalloproteinases (MMP) play a key role in cartilage degradation in osteoarthritis. Statins are a potential suppressor of MMPs. The aim of this research was to assess the efficacy of Pravastatin in suppressing MMP gene and protein expression in an in vitro model. Methods. We stimulated normal human chondrocytes with IL-1b for 6 hours to induce MMP expression and then treated with Pravastatin (1, 5 & 10 mM) for a further 18 hours. Cells stimulated with IL-1b but not treated with Pravastatin served as controls. Real-time PCR was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Staistical analysis was performed using ANOVA. Results. MMP-3 and -9 mRNA expression was reduced at all concentrations tested with a statistically significant trends in reduction (p=0.002 and < 0.001 respectively). Analaysis of culture supernatants revealed that Pravastatin treatment led to a reduction in total MMP activity but not to a statistically significant degree (p=0.07). Conclusion. We conclude that treatment with Pravastatin of stimulated human chondrocytes leads to a down regulation of selected MMP genes and a reduction in MMP enzyme activity. Our results are further evidence that statins may have a role to play in the treatment of osteoarthritis and other disorders of cartilage degradation

Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response

Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly

Introduction. Some patients complain ingrown pain or discomfort after implanting Co-Cr conventional endprosthesis of the hip. Some of this complaint may be attributable for effect on cartilage metabolism. It have been reported that ceramic is bioinert for biological tissue. On the other hand, metal including cobalt-chrome (Co-Cr) have some detrimental effect on biological tissue. However, there is no report concerning acetabular cartilage metabolism after hip endprosthesis implantation. In the present study, we hypothesized that ceramic head have small detrimental effect on cartilage cell metabolism. Specific aim of the study is to compare the protein level of inflammation related cytokines, amount of hyaluronic acid (HA) in culture media, and cartilage mRNA expression in organ culture model of hip end prosthesis implanted using ceramic head and Co-Cr head. Materials and Methods. Six acetabulum of 3 matured crossbred pig (average weight: 36 +/− 3.6kg) was retrieved. Animal experiment was performed under the rules of ethical committee of animal experiment. Average diameter of pig acetabulum was 26.3 +/− 0.6 mm. Just after sacrifice, mechanical loading using Instron testing machine with 26mm diameter of Co-Cr in right hip and Ceramic heads in left hip was performed in culture media. Ten thousand cycles of cyclic compression and rotation load (1.5kN to 0.15kN of compression and 12 degrees of rotation) to cartilage was applied at 1Hz (Figure 1). Culture media was analyzed for protein levels of inflammation related cytokines and amount of HA. Relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from acetabular cartilage was performed as previously reported using specific primer sets for type II collagen, aggrecan, TNF-alpha, Interleukine-1 and 6, and MMP-1, 3, 13. Results. IL-1 beta protein level from culture media was significantly higher in Co-Cr than that in Ceramic (155+/−25.2 pg/ml vs. 86.3+/−9.6 pg/ml respectively). MMP-3 protein level had tendency to be higher in culture media from Co-Cr than that from Ceramic (16.3+/−10.6 ng/ml vs. 10.0+/−0.1 ng/ml respectively, p<0.05), however there was no significant difference. There were no significant differences of protein levels from culture media in MMP-1, IL-1a, and TNF between two groups. Amount of HA from culture media of Co-Cr group was significantly higher than that from Ceramic group (337+/−38.4 mg/ml versus 257+/−11.1 mg/ml respectively, p<0.05). Type II collagen mRNA expression was 3 times higher in Ceramic group than that in Co-Cr group. IL-1 beta mRNA expression was 4 times higher in Co-Cr group than that in Ceramic group. Other gene expression had no significant differences. Discussion. The present study showed that Co-Cr affects cartilage metabolism than Ceramic. Co-Cr group had higher protein level and mRNA expression of inflammation related cytokine, IL-1 beta, and higher HA. Concerning the mRNA expression from cartilage, type II collagen was significantly higher in Ceramic group. It has been reported that HA level is high in osteoarthritic joint. These report and our results showed that ceramic head have small detrimental effect on cartilage cell metabolism. There are limitations of the present study. Firstly, the sample size is small. Secondly, we did not evaluate synovial membrane metabolism

The purpose of this study was to profile the mRNA expression for the 23 known matrix metalloproteinases (MMPs), 4 tissue inhibitor of metalloproteinases (TIMPs) and 19 ADAMTSs (a disintegrin and metalloproteinase with thrombospontin motif) in Dupuytren's Disease and normal palmar fascia. Dupuytren's Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. The MMPs and ADAMTSs are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs and their inhibition by the TIMPs underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinomata, whereas an imbalance towards inhibition causes e.g. fibrosis. A group of patients with end-stage gastric carcinoma was treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of carcinoma advancement; a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD