Purpose

SIRT1 has been suggested to play protective roles against osteoarthritis (OA) progression. The purpose of this study was to investigate the effects of intraperitoneal and intra-articular administration of SIRT1 activator SRT2104, which can be used in human, on OA progression.

Methods and Materials

OA was induced by destabilization of the medial meniscus in the knee joints of 12-week-old CL57BL/6J mice. Mice were divided into 3 groups; Control group (10% DMSO in isotonic saline, i.p.). SRT2104 intraperitoneal injection group (SRTi.p.; 25 mg/kg). SRT2104 intra-articular injection group (SRTi.a.; 17 ng/kg). Tissues were harvested at 4, 8, 12 and 16 weeks after surgery. OA progression was evaluated using the Osteoarthritis Research Society International (OARSI) score. The production of SIRT1, type 2 collagen, MMP-13, ADAMTS-5, cleaved caspase 3, PARP p85, acetylated NF-kB p65, IL-1b and IL-6 were examined by immunohistochemistry. For in vitro experiments, mouse primary epiphyseal chondrocytes were cultured with or without 10 ng/ml IL-1b for 24h, and then treated with or without 2.0 µM SRT2104. The expression of Sirt1, Col2a1, Mmp-3, Mmp-13 and Adamts-5 were examined by Real-time PCR.

Results

The OARSI score was significantly lower in the SRTi.p. group and the SRTi.a. group compared with that in the control group at 8 and 12 weeks, while there was no statistically significant difference between the SRTi.p. group and SRTi.a. group (Figure 1). The immunohistochemical analysis showed that Sirt1 and type 2 collagen were significantly more detected while MMP-13, ADAMTS-5, cleaved caspase 3, PARP p85, acetylated NF-kB p65, IL-1b and IL-6 were significantly decreased in the SRTi.p. and SRTi.a. groups compared with the control group (Figure 2). The treatment with SRT2104 significantly increased the expression of Sirt1 and Col2a1 while decreased Mmp3 and Mmp13.

Conclusion

Both intraperitoneal and intra-articular injection of SRT2104 attenuated OA progression in the mouse OA model. SRT2104 could be a new treatment for OA.

Purpose

Osteoarthritis (OA) is the most prevalent degenerative disease in musculoskeletal system. However, no convincing disease-modifying OA drugs (DMOADs) have received regulatory approval, suggesting that none have convincingly disease-modifying efficacy. Intact cartilage ECM was essential to maintain cartilage function. Thus, modulating cartilage ECM generation represented as a promising approach to treat OA.

Methods and Materials

In the present study, we screened for 2320 compounds using chondrogenesis induction system of ATDC5. We then evaluated the protective effects of compound using OA chondrocyte, OA cartilage explants, and rat OA model developed by ACLT. The molecular target of compound was tested through the experiments in vitro, such as RNA sequencing, RT-PCR, and immunofluorescence analysis et al.

Results

Here we describe BNTA, a small molecule with ECM modulatory properties. BNTA promotes generation of ECM components in cultured chondrocytes isolated from individuals with osteoarthritis. In human osteoarthritic cartilage explants, BNTA treatment stimulates expression of ECM components while suppressing inflammatory mediators. Intra-articular injection of BNTA delays the disease progression in a trauma-induced rat model of osteoarthritis. Furthermore, we identify superoxide dismutase 3 (SOD3) as a mediator of BNTA activity. BNTA induces SOD3 expression and superoxide anion elimination in osteoarthritic chondrocyte culture, and ectopic SOD3 expression recapitulates the effect of BNTA on ECM biosynthesis.

Conclusion

(1) In this study, we reported a novel candidate of DMOAD, BNTA, which significantly enhanced anabolic metabolism in OA chondrocytes, and rescued the decrease of main ECM structural molecules in OA cartilage explants and OA cartilage tissue developed by ACLT in rats.

(2) It was confirmed that BNTA exerted its effect by upregulation of SOD3, which protected chondrocytes by catalyzing the dismutation reaction of superoxide anions.

(3) Upregulation of SOD3 by BNTA or SOD3 plasmid prominently enhanced the anabolism of OA chondrocytes and regulated cartilage ECM synthesis, which identified SOD3 as a promising new drug target for OA.

Purpose

Therapeutic solutions aiming to stimulate endogenous repair of osteochondral defects by using scaffolds or hydrogels are emerging. To achieve good integrative repair, mesenchymal stromal cells from the underlying bone marrow (BMSCs) should migrate into the hydrogel/scaffold and deposit extracellular matrix. Osteochondral defects are often accompanied by synovial inflammation. We investigated how synovial inflammation influences BMSC migration, and whether modulation of inflammation improves migration.

Methods and Materials

Osteoarthritic synovial tissue explants were cultured with/without 1 µM triamcinolone acetonide (TAA) for 24 hours to obtain synovium conditioned medium (SCM). The effect of 6 SCM donors on migration of passage 3 BMSCs was examined in a boyden chamber assay. Inflammation of the synovial explants was assessed with gene expression analysis and flow cytometry of synovial macrophages. Human peripheral blood monocytes were stimulated with TNF-α/IFN-γ towards pro-inflammatory macrophages, with IL-4 towards repair macrophages, and with IL-10 towards anti-inflammatory macrophages and their conditioned medium was used to assess BMSC migration on a collagen gel.

Results

SCM resulted in a donor dependent increase in MSC migration. Modulation of synovial inflammation with TAA significantly decreased expression of TNFA, IL1B and IL6, genes associated with inflammation, and increased gene expression of CD163, associated with anti-inflammatory macrophages, in synovial tissue explants. The percentage of CD14+CD80+(p<0.001) or CD14+CD86+(p<0.001) pro-inflammatory macrophages was lower in TAA-treated samples, whereas the percentage of CD14+CD163+ [KS1] anti-inflammatory macrophages was higher(p<0.001) than without TAA. Modulation of synovial inflammation with TAA resulted in a 1.5-fold increase(p<0.01) in migration. Moreover, BMSC migration in collagen gel increased 2.5-fold(p<0.001) in response to medium conditioned by repair macrophages, and 2.3-fold(p<0.01) by anti-inflammatory macrophages. Migration was unaffected by pro-inflammatory macrophage CM.

Conclusion

Decreased synovial inflammation increased BMSC migration. Modulation of inflammation, and macrophage phenotype in synovium using TAA seems promising to enhance BMSC migration. This knowledge could be used in approaches stimulating endogenous repair of osteochondral defects.

Purpose

Osteoarthritis (OA) is a progressive joint disease characterized by loss of articular cartilage and synovial inflammation accompanied by pain and disability. Synovial macrophages avidly engulf cartilage fragments and release inflammatory cytokines that exaggerates the osteoarthritic process and severity of disease. However, molecular mechanisms by which cartilage fragments triggers cellular responses are unclear and remain to be investigated. Therefore, the current study aims at analyzing molecular response of macrophages to cartilage fragments as step towards identifying molecular candidates for potential therapies.

Methods and Materials

Bone marrow derived macrophages and femoral head cartilages were isolated from C57/B6 mice. Cartilage fragments were prepared using optimal procedure for obtaining fragments similar to these isolated from synovial fluid of OA patients. Macrophages were co-cultured with cartilage fragments for 24h, and then harvested for RNA extraction and RNA sequencing. Data were analyzed for sequence alignment, transcript quantification, mapping and annotating. Significantly expressed genes with p-value ≤ 0.01 were subjected to gene ontology and pathway enrichment analyses.

Results

A total 153 genes were differentially upregulated, and 110 genes were down-regulated in response to cartilage fragments (Fig. 1). GO enrichment analysis revealed that the upregulated genes were mainly categorized into 11, 7, and 11 GO terms for cellular components, molecular function and biological process (p ≤ 0.001), respectively. The most significantly enriched terms included extracellular exosome, scavenger receptor activity, and innate immune response. (Fig. 2) Upregulated genes in response to cartilage fragments were enriched in 8 KEGG pathways including TNF-a signaling pathway, and toll-like receptor signaling. Moreover, ATF2, STAT3, ESR1, and NFKB1 were the most significantly enriched transcriptional factors in this response.

Conclusion

Bioinformatic analysis revealed that stimulated macrophages elicited common gene expression signature for inflammation, including activation of toll-like receptors, and TNF-α and NFKB signaling. Such data broaden our knowledge of the immunological response of macrophages to cartilage fragments and their role in osteoarthritic process.

Purpose

Cartilage repair takes place in a joint environment, with elevated levels of proinflammatory cytokines. The current work builds on our promising findings and demonstrates that cLIUS, when applied at cell resonant frequency of 5 MHz that maximizes cLIUS-induced bioeffects, promotes cartilage repair in a pro-inflammatory environment (IL6 and TNFα) by inhibiting the expression of catabolic factors and by promoting the chondrocytic phenotype.

Methods and Materials

8-mm bovine osteochondral explants with 4-mm cylindrical incisions were used. The incised explants were maintained under cLIUS stimulation (5 MHz, (14 kPa, 20 minutes, 4 times per day) in the cLIUS-assisted bioreactor as shown in Fig-1 Non-stimulated explants served as controls. Explant specimens were evaluated as shown in Fig-1.

Results

Histological assessment of the explants (Fig.2) displayed closed gaps and maintenance of tissue continuity in LIUS treated explants (panel IV) whereas visible gaps remained in non LIUS-treated controls (panel I). Cartilage-to-cartilage bonding was also observed in osteochondral explants exposed to IL6 or TNFa and treated with cLIUS (panels V and VI). Fissure or a cleft was observed in osteochondral explants exposed to IL6 or TNFa and not exposed to cLIUS (panels II and III). Loss of glycosaminoglycan (GAG) from the matrix was visible in explants treated with cytokines and not treated with cLIUS (Fig. 2, panel B/C). As expected, exposure of chondrocytes to pro-inflammatory cytokines resulted in over-expression of MMP13 and ADAMTS4/5, known matrix metalloproteinases and catabolic agents (Fig. 3A-B). Interestingly, cLIUS was observed to suppress the expression of MMP13 and ADAMTS4/5in osteochondral specimens exposed to IL6 and TNFa (Fig. 3A/B); while increasing the expression of TIMP1, an inhibitor of metalloproteinases (Fig. 3C).

Conclusion

Our work is expected to have broad translational importance in therapies that seek to employ LIUS in both regeneration and rehabilitation, and the knowledge gained will advance solutions to address the needs of persons with impaired joint function.

Purpose

Introduction: Mature articular cartilage displays poor intrinsic healing and its degradation is the major hallmark of osteoarthritis (OA). TGF-β is a multifunctional cytokine that plays a critical role in cartilage repair and maintenance. Aberrant TGF- β signaling in chondrocytes has been strongly implicated in the pathogenesis of OA. Our group has previously reported CD109 as a novel TGF-β co-receptor and shown that CD109 is a potent negative regulator of TGF-β signaling in the skin.

Objective: The proposed study is aimed at identifying whether manipulation of CD109 expression levels modulates the balance between TGF-β signaling via ALK1 versus ALK5 signaling pathways and regulates ECM protein expression in vivo in articular chondrocytes.

Methods and Materials

Methods: Articular cartilage tissue was collected and primary chondrocytes were isolated from CD109 KO and wild-type mice. TGF-β signaling components were analyzed in isolated chondrocytes or cartilage tissue by determining ALK5 versus ALK1 levels and Smad2/3 versus Smad1/5 levels using Western blot. Chondrocyte function was determined by evaluating the expression of, collagen type II, aggrecan, collagenase (MMP-13) and aggrecanase (ADAMTS-5), at the protein and mRNA levels by Western blot, real time PCR or immunocytochemistry (ICC).

Results

Results: CD109 KO mice markedly enhance ALK5 levels and promote expression of collagen type II, aggrecan expression in articular chondrocytes in comparison with wild-type mice. On the other hand, that loss of CD109 expression results in decreasing of ALK1 levels and inhibiting MMP13 and ADAMTS5 expression in CD109 KO mice chondrocytes. Moreover, histological results indicates that the collagen content in CD109 KO mice articular cartilage is increased significantly in comparison with wild-type mice.

Conclusion

Conclusion: Our findings suggest that CD109 differentially regulates TGF-β signaling pathways and inhibits ECM protein production while promoting proteases expression in articular chondrocytes. We conclude that CD109 may play an important role in maintaining cartilage function and integrity.

Purpose

Osteoarthritis (OA) is a widespread whole-joint disease affected by many factors and is a major challenge for human beings. Compared with other OA treatments, exercise therapy can effectively relieve joint pain and improve joint dysfunction, but it does not completely hinder the progress of OA. In addition, the mechanism of exercise therapy is not clear^[1]. Research shows that intervention in OA-related signaling pathways can prevent progression of OA^[2,3]. In this study, we combined exercise therapy with related signaling pathway interventions in order to achieve the goal of treating OA.

1. Roos EM et al. Nature Reviews Rheumatology, 2016, 12(2): 92.

2. Chen L X et al. 2008, 16(2): 174-184.

3. Yazici Y et al. Osteoarthritis cartilage, 2017, 25(10): 1598-1606.

Methods and Materials

Rats underwent DMM surgery in their right and left knee and were assigned to either the sedentary group or walking group (n=6/group). Rats were treated with intra-articular signaling pathway activator or inhibitor or vehicle. Animals in the walking group were subjected to treadmill exercise 7 days after surgery, which included walking for 3 different intensity parameters for 4 and 12week(s). Subchondral bone and cartilage changeswere evaluated by gait analysis, micro-CT analysis, histological analysis, and biochemistry analysis.

Results

The results showed that the stride length step width and step speed of the rats in the sedentary group, low intensity running, high intensity running group injected with Wnt activator in the joint cavity were larger than those in the control group.

we found low intensity runing can supress the progress of osteoarthritis.

Conclusion

Exercise therapy plus Wnt signaling pathway activators are better for relieving OA in rats than in exercise and administration groups alone.

Purpose

This study investigated the biological function of microRNA-195(miR-195) and the molecular mechanism in osteoarthritis(OA).

Methods and Materials

The knee cartilage tissues obtained from traumatic amputees (n=10) and OA patients (n=10). Real-time PCR and Western blot were used to detect the expression of miR-195 and Smad3 from cartilage. Human chondrocytes were transfected with the miR-195 mimic, the miR-195 inhibitor, or the control, respectively. The expression level of Smad3 , type II collagen (Col II) were detected by Real-time PCR 24h later or by Western blot 48h later. We constructed a Smad3 3'-UTR-luciferase reporter and examined the effect of miR-195 on Smad3 3'-UTR luciferase activity by dual luciferase reporter gene assay in 293T cells. To further understand whether Smad3 mediates the downstream effects of miR-195, human chondrocytes were transfected with the miR-195 inhibitor with and without siRNA-Smad3 (siSmad3). The study was approved by the Ethics Committee of The Second Hospital of ShanXi Medical University.

Results

miR-195 levels were significantly upregulated in OA tissue (P<0.05) while Smad3 mRNA/protein expression was substantially decreased in OA tissue (P<0.05). Consistently, upregulated miR-195 strongly inhibited Col II expression, while downregulated miR-195 significantly increased Col II expression (P<0.05). Furthermore, it has been demonstrated that Smad3 is a novel target of miR-195 using dual luciferase reporter assay. Most importantly, miR-195 mediated the changes of Col II were substantially attenuated by siSmad3 treatment(P<0.05).

Conclusion

Our study identifies that miR-195 is up-regulated in OA cartilage, while the content of Smad3 is decreased in OA cartilage. Smad3 is a novel target of miR-195. We further demonstrated that Smad3 is involved in the progress of miR-195 mediated OA cartilage degeneration.

Purpose

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, synovial inflammation and osteophyte formation. The disability of joint is associated with a loss of the highly sulfated proteoglycans and an increase of apoptotic chondrocytes in cartilage matrix. The continued mechanical stress on the affected cartilage leads to production of cartilage fragments that are known to provoke the production of macrophages pro-inflammatory cytokines and chondrocyte catabolic factors. A better understanding of the molecular response of chondrocytes elicited by inflammatory macrophages phagocytizing cartilage fragments may provide essential clues for development of novel therapeutic agents. To this end, we examined the gene profile of chondrocytes stimulated with inflamed macrophages by cartilage fragments.

Methods and Materials

Femoral head cartilage of C57/B6 mice were separated and crushed for preparing cartilage fragments or enzymatically treated for isolation of chondrocytes. Macrophages were prepared from the same mice by culturing bone marrow-derived cells with growth factors. Thereafter, chondrocytes were co-cultured with macrophages stimulated by cartilage fragments using transwell culture system for 48 h. RNA-Seq analysis was performed for chondrocyte RNA, and data were subjected for gene ontology and pathway analyses.

Results

A total 340 transcripts were significantly upregulated in these chondrocytes (Fig. 1A) and were enriched in multiple biological processes including ossification, bone mineralization and connective tissue development (Fig. 1B). Major functional pathways involved in this response were ECM-receptor interaction, spliceosome, and protein digestion and absorption (Fig. 1C).

Conclusion

Our ongoing work is to validate results using cartilage ex vivo models. These results showed the pathological impact of inflamed macrophages on the function of chondrocytes and progression of the disease. Our results suggest that macrophages activated by cartilage fragments might be involved in the endochondral ossification signals occurred in progressive OA.

Purpose

Osteoarthritis (OA) is a common debilitating joint disorder, there’s still no available disease-modifying drug for OA currently. TGF-β-activated kinase 1 (TAK1) is a key intermediate in immune-related signal transduction induced by TGF-β and proinflammatory stimuli such as IL-1β and TNF-α. However, the role of TAK1 in OA pathogenesis and the therapeutic efficiency of TAK1 inhibition for OA remains to be elucidated. This study aims to clarify the function of TAK1 in OA process and its mechanism, and systematically elucidate the delaying effect of targeted TAK1 inhibition towards OA.

Methods and Materials

The contribution of TAK1 to OA pathogenesis was investigated by intra-articular injection of TAK1-encoding adenovirus in rats. The suppressive function of TAK1 inhibitor 5Z-7 on NF-κB, JNK and p38 MAPK pathway activation in both chondrocytes and synoviocytes was determined by western blot and luciferase reporter assay. 5Z-7-induced expression changes of extracellular matrix (ECM)-related genes were detected by real-time PCR. The protective effect of 5Z-7 against OA progression was evaluated in a post-traumatic OA rat model.

Results

Intra-articular injection of Ad-Tak1 significantly induced inflammation-related cartilage destruction in rat joints. TAK1 inhibition by 5Z-7 significantly blocked NF-κB, JNK and p38 pathway activation induced by IL-1β in normal chondrocytes and synoviocytes, and effectively reduced basal activation level of the above pathways in OA chondrocytes and synoviocytes. 5Z-7 also significantly downregulated the expression of a series of matrix-degrading enzymes and inflammatory cytokines, while upregulated the expression of ECM proteins, which are all crucial components in OA. Furthermore, 5Z-7 also ameliorated ECM loss in OA cartilage explants. More importantly, the application of 5Z-7 significantly protected against OA-related pathological changes in a rat model of OA.

Conclusion

Our findings have provided the first in vivo evidence that TAK1 contributes to OA by disrupting cartilage homeostasis, thus represents an ideal target for OA treatment, with 5Z-7 as a candidate therapeutic.