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We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and β-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1β in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1β, as well as the nuclear translocation of NF-κB and β-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of β-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.

We investigated the roles of low-density lipoprotein receptor-related protein (LRP) 4 and its ligand Agrin in the pathophysiology of cartilage degeneration. Immunohistochemical analysis of human normal articular cartilage and cartilage tissues from patients with osteoarthritis (OA) obtained during surgery of the knee joint showed marked LRP4 expression in the early stages of OA, which then decreased with cartilage degeneration, whereas Agrin was consistently increased with cartilage degeneration. In normal human articular chondrocytes (NHACs), mild cyclic tensile strain (CTS) (0.5 Hz, 5% elongation, 2 h) increased the expression of LRP4 and aggrecan (ACAN), while intense CTS (0.5 Hz, 10% elongation, 6 h) increased the expression of Agrin without affecting LRP4 expression. Treatment with recombinant human (rh) Agrin downregulated the mRNA expression of LRP4 and ACAN, but upregulated the expression of LRP5/6, SRY-box transcription factor 9 (SOX9), Runt-related transcription factor 2 (RUNX2), and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4). Immunocytochemistry and Western blot analysis showed that rhAgrin treatment upregulated the expression of β-catenin and SOX9. Agrin knockdown by siAGRN transfection partially reduced the nuclear protein expression of β-catenin, which was increased with intense CTS. LRP4 knockdown by siLRP4 transfection increased the expression of LRP5/6, SOX9, RUNX2, ADAMTS-4, and Agrin. These results suggested that intense CTS increases the expression of Agrin, which might interfere with the role of LRP4 in the inhibition of LRP5/6 and their downstream β-catenin signaling, leading to cartilage degeneration.

The osteo-immunomodulatory properties of biomaterials play an important role in the outcomes of bone regeneration. Graphene oxide (GO) has been widely applied in many research fields due to its unique properties. However, the immunomodulatory properties of GO as a biomaterial for bone tissue engineering are still unclear. In this study, we evaluated the Inflammatory response of RAW264.7 cells influenced by GO. Then the osteogenic differentiation of BMSCs, and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) by stimulation with GO/RAW 264.7-conditioned culture medium were accessed. We also further investi gated the possible mechanisms underlying the osteo- and angio-immunomodulatory effects of GO. Our results showed that GO stimulates the secretion of oncostatin M, tumor necrosis factor alpha and other factors through the nuclear factor-κB pathway. GO/RAW264.7-conditioned medium promoted the osteogenic differentiation of BMSCs, stimulated upregulation of the HUVECs of vascular-related receptors, and promoted their tube formation in vitro. In conclusion, our research shows that GO, as a biomaterial, can induce the formation of a beneficial osteo-immunomodulatory environment and is a promising biomaterial for bone tissue engineering.

The migration of mesenchymal stem cells (MSCs) and osteogenic differentiation occupy an important role in fracture healing. High mobility group box 1 (HMGB1), a widely distributed inflammatory factor in fractures, has been confirmed to act as a chemoattractant to MSCs. To investigate the effect of HMGB1 on MSC migration and the underlying mechanism, the synthesis of MSC chemokines, and the consequent activation of signaling pathways following HMGB1 stimulation, were evaluated. A Quantibody® array was performed to determine which chemokines were secreted from MSCs with or without treatment with HMGB1. The results indicated differential chemokine synthesis by MSCs following treatment with HMGB1, including that of CCL4 and CCL13. In addition, the Ras-associated protein-1 (Rap1) signaling pathway was markedly activated in the HMGB1-treated groups, suggesting that HMGB1 may enhance the migrational ability of MSCs via Rap1 activation. Furthermore, HMGB1 was able to promote the secretion of various chemokines derived from MSCs, which would, in turn, increase the mobility of MSCs. Taken together, these results provide a mechanistic basis for developing novel approaches to promote fracture healing.

High mobility group box 1 (HMGB1) epxression has been found in the inflammatory microenvironment of fractures. It is well known that HMGB1 acts as a chemoattractant for mesenchymal stem cells (MSCs); however, the effects of HMGB1 on MSC migration and osteoblastic differentiation, and the signaling pathways involved in these effects, have not yet been elucidated. In this study, we aimed to investigate these effects, as well as the signaling mechanisms involved, using in vitro models. We found that HMGB1, in varying concentrations, promoted the osteoblastic differentiation of MSCs, the synthesis of receptor for advanced glycation end products (RAGE) and Toll-like receptor (TLR)2/4, and the activation of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Subsequently, we cultured the MSCs in the appropriate concentration of HMGB1, and determined the signaling pathways involved in the effects of HMGB1 on MSC migration and differentiation, using receptor neutralizing antibodies and signaling pathway inhibitors. From the results of this study, we concluded that HMGB1 promotes MSC migration through the activation of the p38 MAPK signaling pathway, and also promotes MSC differentiation by binding to TLR2/4 and activating the p38 MAPK signaling pathway. These findings elucidate the mechanisms underlying the effects of HMGB1 in the fracture microenvironment, which may provide a theoretical basis for the development of improved clinical treatments for fractures.

High mobility group box 1 (HMGB1) protein has been previously been detected in the inflammatory microenvironment of bone fractures. It is well known that HMGB1 acts as a chemoattractant to mesenchymal stem cells (MSCs). In the present study, the effects of HMGB1 on cytokine secretion from MSCs were determined, and the molecular mechanisms underlying these effects of HMGB1 on osteogenic differentiation were elucidated. To detect cytokine secretion, antibody array assays were performed, which demonstrated that HGMB1 induced the differential secretion of cytokines that are predominantly associated with cell development, regulation of growth and cell migration, stress responses, and immune system functions. Moreover, the secretion of epidermal growth factor receptor (EGFR) was significantly upregulated by HMGB1. The EGFR-activated Ras/MAPK pathway regulates the osteogenic differentiation of MSCs. These results suggested that HMGB1 enhances the secretion of various cytokines by MSCs and promotes osteogenic differentiation via the Ras/MAPK signaling pathway. The present study may provide a theoretical basis for the development of novel techniques for the treatment of bone fractures in the future.

The long-term survival of osteosarcoma has remained unchanged in the last several decades. Immunotherapy is proved to be a promising therapeutic strategy against osteosarcoma, especially for those with metastasis. Our previous study explored the sensibilization of zoledronate (ZOL) in γδ T cell-mediated cytotoxicity against osteosarcoma, but we have not yet elucidated the specific mechanism. Besides, high concentration is required to achieve these effects, whereas plasma ZOL concentration declines rapidly in the circulation. Valproic acid (VPA), a histone deacetylase inhibitor commonly used as the antiepileptic drug, has attracted much attention due to its synergistic antitumor efficacy with chemotherapy or immunotherapy. Here, we demonstrated that VPA combined with ZOL revealed the synergistic effect in enhancing antitumor efficacy of γδ T cells against osteosarcoma cells. This enhancement was mainly TCR-mediated and largely dependent on granule exocytose pathway. Of note, our findings indicated that ZOL sensitized osteosarcoma cells to γδ T cells by increasing the accumulation of the mevalonate pathway intermediates, which could be facilitated by VPA. We also found that this combination had similar effects on primary osteosarcoma cells. All the results suggested that VPA combined with ZOL could reduce the dose required to achieve a significant antitumor effect of γδ T cells, promoting it to be a novel therapy against osteosarcoma.