Wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of RANL in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particletreated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis

The development of a representative human, in vitro OA model could deepen understanding of disease mechanisms. Our research aimed to reprogram healthy and OA-derived synoviocytes to induced pluripotent stem cells (iPSCs), thereby generating a novel OA in vitro model. Comparison between the two models shall enable research into underlying processes with potential for clinical translation. A meta-analysis of OA synovial biomarkers was conducted, identifying up to thirteen relevant pathophysiology-related factors, including, amongst others, IL-13, IL-10, IL-6, PIICP, and HA, with PIICP demonstrating the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001). With these findings in mind, human fibroblast-like synoviocytes (HFLS) from healthy and OA patients were transduced using Sendai viral reprogramming. Two clones for each of the resulting iPSC lines were expanded and preliminarily analysed in triplicate by ICC and RT-qPCR for pluripotency characteristics. Healthy HFLS-derived and OA-HFLS-derived iPSC (UoS-B and UoS-C lines, respectively) were generated, indicating successful reprogramming. Morphological observations demonstrated typical iPSC appearance, and ICC confirmed presence of pluripotency markers Tra-1-60, Oct3/4 and Nanog. Expression of Oct3/4, Nanog and Sox2 were confirmed by RT-qPCR with OA-iPSC lines expressing higher levels of all markers compared to non-OA iPSC. In particular, expression of Oct3/4 and Sox2 was 3.5 fold and 4.6 fold higher (p <0.001) in OA-iPSCs (UoS-C) vs. non-OA iPSCs (UoS-B), respectively. Sendai virus clearance was confirmed by passage 4. The successfully obtained OA and non-OA iPSCs can be differentiated towards mesenchymal lineages, including chondrocyte and bone progenitor cells, enabling phenotypic comparison and biomarker analysis as identified in meta-analysis. Cell bank dissemination of these cell lines could deepen further in vitro OA research, with potential impact for clinical translation via the identification of novel cellular and molecular targets

We describe a model which can be used for in vitro biocompatibility assays of biomaterials. We studied the in vitro response of human osteoarthritis or rheumatoid arthritis fibroblast-like synoviocytes to Al. 2. O. 3. or ZrO. 2. particles by analyzing the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) and the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase pathways. Our results show that, in these cells and under our experimental conditions, Al. 2. O. 3. and ZrO. 2. did not significantly modify the synthesis of IL-1 and IL-6 or the metabolism of arachidonic acid

Cryotherapy is often applied after injuries of synovial joints. Although positive clinical effects on periarticular swelling and pain are well known, the effects on molecular processes of cartilage and synovial cells remained largely unknown so far. Therefore, the hypothesis was tested that hypothermia alleviates the synovial reaction and prevents chondrocyte death as well as cartilage destructive processes after blunt trauma. Human articular cartilage and synovial tissue was obtained with informed consent from patients undergoing knee joint replacement. Cartilage explants from macroscopically intact cartilage were impacted by a drop-tower apparatus with defined energy (0.59J) and cultivated for 24h or 7d at following temperature conditions: 2h, 16h or throughout at 27°C and afterwards or throughout at 37°C. Furthermore, human fibroblast-like synoviocytes (FLS) were stimulated with conditioned medium from traumatized cartilage (t-CM) and cultivated as indicated above up to 4d. Effects of hypothermia were evaluated by live/dead assay, gene expression (RQ-PCR), and type II collagen synthesis/cleavage as well as release of MMP-2, MMP-13 and IL-6 on protein level (ELISA, gelatin zymography). Statistical analysis was performed by 2-way ANOVA. The experimental study was performed in the research laboratory of the Orthopedic Department, University Hospital Ulm, Germany. Hypothermic treatment significantly improved chondrocyte viability 7d after blunt cartilage trauma (2h: p=0.016; 16h: p=0.036; throughout: p=0.039). 2h posttraumatic hypothermia attenuated expression of MMP-13 (m-RNA: p=0.012; protein: p=0.024). While type II collagen synthesis was significantly increased after 16h hypothermia, MMP-13 expression (mRNA: p=0.003; protein: p<0.001) and subsequent cleavage of type II collagen (p=0.049) were inhibited. Continuous hypothermia for 7d further significantly suppressed MMP release (proMMP-2, active MMP-2 and MMP-13) and type II collagen breakdown. On day 4 t-CM stimulated FLS revealed significantly suppressed gene expression of matrix-destructive enzymes (16h: ADAMTS-4; throughout: ADAMTS-4, MMP-3, MMP-13) and by trend reduced IL-6 expression in case of 16h or continuous hypothermia. Overall, hypothermia for only 2h and/or 16h after blunt cartilage trauma exhibited significant cell- and matrix-protective effects and promoted anabolic activity of surviving chondrocytes. Expression of matrix-destructive enzymes by FLS stimulated with Danger Associated Molecular Patterns (DAMPs) released from traumatized cartilage was attenuated by more prolonged hypothermia. These findings suggest that an optimized cryotherapy management after cartilage trauma might have the potential to ameliorate early molecular processes usually associated with the pathogenesis of posttraumatic osteoarthritis

Summary. PCA-III, a phosphocitrate analog, acts not only as a potent calcification inhibitor but also as a protective agent for extracellular matrices. PCA-III has potential as a disease-modifying drug in the treatment of primary osteoarthritis and posttraumatic osteoarthritis in humans. Introduction. Phosphocitrate (PC) inhibits the development of primary osteoarthritis (OA) in Hartley guineas pigs but not menisectomy-induced OA in rabbits (1). We sought to examine the molecular mechanisms underlying the disease-modifying activity of PC, and evaluate the effect of PCA-III, a PC analog (PCA), on the development of primary and secondary OA. Patients & Methods. Meniscal explant and microarray. OA menisci were obtained from OA patients undergoing joint replacement surgery. OA meniscal explants were cultured in medium containing PC (three wells) and medium without PC (three wells). Total RNA was extracted from the explant, and subjected to microarray analysis. RT-PCR. OA fibroblast-like synoviocytes were treated with basic calcium phosphate (BCP) crystals in the absence or presence of PCA-III. RNAs were extracted, and subjected to semi-quantitative RT-PCR to examine the expression of MMP1 and IL-1b. Micromass culture. A droplet of OA chondrocyte suspension was placed in each well of a 24-well plate. After placing all droplets, the wells were fed with chondrogenesis medium with PCA-III (five wells) and without PCA-III (five wells). The production of proteoglycans was examined by alcian blue staining. Animal treatment. The first group of Hartley guinea pigs (n=5) received injections of PCA-III and the second group received injections of saline as control. Two months later, partial-menisectomy surgery was performed on the right knee of all guinea pigs. After the surgery, injections of PCA-III and saline were resumed. All animal were euthanatised four months later, and both knees were examined. Results. PC inhibited the expression of many genes classified into the molecular function group of MMP activity. Of the 23 genes classified into MMP activity, the expression of 16 genes, including CPM, ADAM28, MMP7, MMP10, MMP1, MMP3, ADAMTS5, ADAMTS1, and ADAMTS9, was inhibited. In contrast, the expression of many genes classified into the molecular function group of extracellular matrix structural constituents, was induced by PC, including COL2A1, COL11A1, COL1A1 and ACAN. PC also inhibited the expression of numerous genes classified into the biological process of inflammatory response (data not shown). PCA-III, similar to PC, inhibited BCP crystals-induced expression of MMP1 and IL-1b). In addition, PC-III strongly stimulated the production of proteoglycans by OA chondrocytes while inhibiting calcium deposition (not shown). Microscopic examination of the Indian ink stained medial tibia plateau of the left knees (non-surgery knee) of the guinea pigs indicated that PCA-III inhibited the development of primary OA in the Hartley guinea pigs. Microscopic examination also indicated that PCA-III inhibited the development of partial-menisectomy-induced OA or posttraumatic OA in the post-operative knees. Discussion/Conclusion. PC is thought to act as a potential structural disease-modifying drug for crystal-associated OA by inhibiting crystal deposition within the OA joints. However, PC and its analogs are not only potent calcification inhibitors, but also protective agents for extracellular matrices. Our findings indicate that PCA-III has potential as a disease-modifying drug for both human crystal-associated OA and posttraumatic OA