Determination of the molecular mechanism by which macrophages and γδ-T cells contribute to ZOL-induced ONJ.

OBJECTIVE: This study aims to explore the molecular mechanism of macrophages and γδ-T cells in the ZOL drug-induced osteonecrosis of jaws based on the IFN-γ involved osteoblast differentiation signaling pathway. RESULTS: The number and apoptotic rate of CD11b+Gr1hi cells and γδ-T cells in the ONJ group were significantly higher. The TNF-α, IL-1ß, IFN-γ, CCL3, CCL4, IL-12 and IL-13 levels were significantly higher in the ONJ group. The expression of CTSK and FGFR3 was lower in the ONJ group, but was higher in the NF-κB and ERBB2IP group. CONCLUSION: The proliferation of macrophages and γδ-T cells promote the inflammation in ZOL-induced jaw necrosis. METHODS: A total of 20 patients with osteonecrosis of the jaw from January 2016 to March 2018 were collected and assigned into the observation group, while 20 healthy subjects were assigned into the control group. Furthermore, 40 SD rats were selected and assigned into observation group, while 10 non-treatment SD rats were selected and assigned as controls. The distribution and proportion of CD11b+Gr1hi cells and γδ-T cells in the necrotic tissues of the jaw were analyzed. Then, the TNF-α, IL-1ß, IFN-γ, CCL3, CCL4, IL-12 and IL-13 levels were measured. Afterwards, the expression of CTSK, FGFR3, NF-κB and ERBB2IP in the necrotic tissues of the jaw in the animal models were analyzed.

Zoledronate inhibits the differentiation potential of adipose-derived stem cells into osteoblasts in repairing jaw necrosis.

OBJECTIVE: To explore the inhibitory effects of zoledronate (ZOL) on adipose-derived stem cells (ADSCs) into osteoblasts for repairing jaw necrosis. METHODS: ADSCs were induced to differentiate into osteoblasts. The differentiation characteristics of osteoblasts was observed under inverted microscope by alizarin red staining. The transwell assay was performed to evaluate the migration of ADSCs co-cultured with osteoblasts and divided into ZOL group treated with ZOL and N-ZOL group without ZOL treatment. The differentiation and proliferation characteristics of ADSCs differentiated osteoblasts were observed respectively. The expression of CTSK (Cathepsin K) and FGFR3 (Fibroblast growth factor receptor 3) in osteoblasts were analyzed by immunofluorescence and western blot. RESULTS: The differentiation degree and proliferation of ADSCs to osteoblasts in N-ZOL group were both higher than those in ZOL group. The migratory cell number in ADSCs differentiation in ZOL group was higher than that of N-ZOL group. The protein expression of CTSK and FGFR3 in ADSCs differentiated to osteoblasts in ZOL group was higher than that in N-ZOL group. CONCLUSION: The differentiation of ADSCs into osteoblasts is significantly inhibited by ZOL. Due to this reason, it may be difficult to achieve good results by ZOL induced ADSCs into osteoblasts in repairing jaw necrosis.

LncRNA TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the miR-134-5p/MBTD1 axis.

AIM: To explore the mechanism by which long non-coding RNA (lncRNA) TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the microRNA miR-134-5p/malignant brain tumour domain containing 1 (MBTD1) axis. RESULTS: The lncRNA TTN-AS1 was highly expressed in osteosarcoma and was associated with poor prognosis. The lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. MiR-134-5p targeted MBTD1, which was regulated by lncRNA TTN-AS1. MBTD1 was highly expressed in osteosarcoma and was associated with poor prognosis. MBTD1 promoted cell viability and inhibited apoptosis, and knockdown of MBTD1 reversed the cancer-promoting effects of lncRNA TTN-AS1. Downregulation of lncRNA TTN-AS1 reduced drug resistance. CONCLUSION: In osteosarcoma, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance. METHODS: The expression characteristics of genes in osteosarcoma patients were analysed using bioinformatics. Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect protein and RNA, respectively. A cell counting kit 8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis. The effects of lncRNA TTN-AS1 and MBTD1 on osteosarcoma in vivo were studied by using a tumour burden assay.

The importance of Src signaling in sarcoma.

Src is a tyrosine kinase that is of significance in tumor biology. The present review focuses on Src, its molecular structure, and role in cancer, in addition to its expression and function in sarcoma. In addition, the feasibility of Src as a potential drug target for the treatment of sarcoma is also discussed. Previous studies have suggested that Src has essential functions in cell proliferation, apoptosis, invasion, metastasis and the tumor microenvironment. Thus, it may be a potential target for cancer therapy. Src has been found to enhance proliferation, reduce apoptosis and promote metastasis in certain subtypes of sarcoma, including osteosarcoma, chondrosarcoma and Ewing's sarcoma. Furthermore, a number of novel effective therapeutic agents, such as SI-83, which target Src have been investigated in vitro and in vivo. Bosutinib and dasatinib, which inhibit Src, have been approved by the U.S. Food and Drug Administration for the treatment of chronic myelogenous leukemia. In addition, vandetanib is approved for the treatment of medullary thyroid cancer. Furthermore, the Src inhibitor, saracatinib, is currently in clinical trials for the treatment of a variety of solid tumors, including breast and lung cancers. Thus, Src is considered to be an important factor in sarcoma progression and may present a novel clinical therapeutic target. This review demonstrates the importance and clinical relevance of Src in sarcoma, and discusses a number of small molecular inhibitors of src kinase, such as dasatinib and sarcatinib, which are currently in clinical trials for the treatment of sarcoma patients.

AMD3100 reduces CXCR4-mediated survival and metastasis of osteosarcoma by inhibiting JNK and Akt, but not p38 or Erk1/2, pathways in in vitro and mouse experiments.

Osteosarcoma (OS) has an unfavorable prognosis and tends to metastasize to lung tissue. Although the CXCL12-CXCR4 axis appears to affect progression and metastasis in numerous tumors, its mechanism and downstream pathways in OS remain unclear. We used western blotting and flow cytometry to detect CXCR4 and CXCR7 expression in two OS cell lines (LM8 and Dunn). An MTT assay was used to evaluate the effects of CXCL12 and AMD3100, a specific CXCR4 antagonist, on cell viability. Flow cytometry was utilized to analyze changes in apoptosis induced by serum deprivation following treatment with CXCL12 and AMD3100. A Transwell assay was used to assess cell migration in response to CXCL12 and AMD3100. Western blotting was performed to identify the phosphorylation of signaling molecules (JNK, c-Jun, Akt, p38 and Erk1/2) and expression of caspase-3 and -8, and PARP. Mouse models were employed to evaluate AMD3100 inhibition of primary OS growth and lung metastasis in vivo. CXCR4 expression was detected in LM8 but not Dunn cells, and neither cell line expressed CXCR7. The addition of CXCL12 induced the survival and migration of serum-starved CXCR4+ LM8 cells activating JNK and Akt pathways, which were abrogated by adding AMD3100. However, similar results were not observed in CXCR4- Dunn cells. CXCL12 protected LM8, but not Dunn cells, from apoptosis induced by serum deprivation by suppressing PARP cleavage, which was partly reversed by AMD3100. In a mouse model, AMD3100 reduced primary tumor growth and lung metastasis compared with the controls. Thus, the CXCL12-CXCR4 axis regulated OS survival and metastasis through the JNK and Akt pathways, and blocking them with AMD3100 was found to be a potential OS treatment.

Monocyte chemotactic protein-1 promotes the proliferation and invasion of osteosarcoma cells and upregulates the expression of AKT.

Monocyte chemotactic protein­1 (MCP­1/CCL2) is an important immune factor, which may be important in cancer progression by promoting proliferation, invasion, metastasis and the tumor microenvironment. Previous studies have demonstrated that CCL2 affects the proliferation of osteosarcoma cells via the RANKL signaling pathway. However, the underlying mechanisms remain to be elucidated. To investigate the role of CCL2 in osteosarcoma cells, MTT, spheroid forming, wound healing and transwell assays were performed to examine the proliferation and invasion abilities of the osteosarcoma cells. It was revealed that the high-grade osteosarcoma cells exhibited increased expression levels of CCL2 compared with the low-grade osteosarcoma cells (P<0.001). Furthermore, knockdown of CCL2 decreased the proliferation and invasion abilities of the osteosarcoma cells (P<0.01). These results suggested that the expression of CCL2 is high in high-grade osteosarcoma cells and promotes the proliferation and invasion of osteosarcoma cells.

Hiporfin-mediated photodynamic therapy in preclinical treatment of osteosarcoma.

This study was carried out to investigate the anti-tumor effect and mechanism of hiporfin-mediated photodynamic therapy (hiporfin-PDT) in osteosarcoma. We found that hiporfin accumulated mainly in the cytoplasm of osteosarcoma cells in a time and concentration-dependent manner. Hiporfin-PDT inhibited the proliferation, induced apoptosis and produced cell cycle arrest at G2M in osteosarcoma cell lines. Hiporfin-PDT increased the expression of cleaved-caspase-3, cleaved PARP-1, Bax and RIP1 while it decreased the expression of Bcl-2; in addition, low concentration of hiporfin increased LC3 conversion. Furthermore, cell death caused by hiporfin-PDT could be rescued by Nec-1 but not by Z-VAD-FMK. Production of reactive oxygen species was increased after hiporfin-PDT. In vivo studies showed a significant decrease in tumor volume and weight after hiporfin-PDT in all three tumor mouse models investigated (subcutaneous and orthotopic). Histological analysis showed widespread cell apoptosis and necrosis after treatment. Immunohistochemistry also showed upregulation of cleaved-caspase-3 and downregulation of Bcl-2 after hiporfin-PDT. These results indicate that hiporfin-PDT exhibits a killing effect in osteosarcoma both in vitro and in vivo, which is associated with apoptosis and necroptosis, while autophagy plays a protective role. All these findings shed light on a potential future clinical use for hiporfin in the treatment of osteosarcoma.

Is fibrin sealant effective and safe in total knee arthroplasty? A meta-analysis of randomized trials.

The objective of this study was to evaluate the efficacy and safety of fibrin sealant in patients following total knee arthroplasty (TKA). A comprehensive literature search of the electronic databases PubMed, MEDLINE, Web of Science, and Cochrane Library for published randomized controlled trials (RCTs) was undertaken. The evidence base was critically appraised using a tool from the Cochrane Bone, Joint and Muscle Trauma Group. Eight RCTs involving 641 patients were included. The use of fibrin sealant significantly reduced postoperative drainage (weighted mean difference (WMD) -346, 95% confidence interval (CI) -496.29 to -197.54, P < 0.00001) and blood transfusions (risk ratio (RR) 0.47, 95% CI 0.35 to 0.63, P < 0.00001) and led to a significant improvement in the range of motion (WMD 16.59, 95% CI 6.92 to 26.25, P = 0.0008). However, using fibrin sealant did not significantly reduced total blood loss (WMD -305.25, 95% CI -679.44 to 68.95, P = 0.11). Regarding complications, there were no significant differences in any adverse events, fever, infection, or hematoma among the study groups. In conclusion, the present meta-analysis indicates that the use of fibrin sealant was effective and safe as a hemostatic therapy for patients with TKA.

All-arthroscopic versus mini-open repair of small to large sized rotator cuff tears: a meta-analysis of clinical outcomes.

PURPOSE: The purpose of this study was to compare clinical outcomes of patients with full-thickness small to large sized tears undergoing all-arthroscopic versus mini-open rotator cuff repair. METHOD: A literature search for electronic databases and references for eligible studies was conducted through Medline, Embase and Cochrane library between 1969 and 2013. RESULTS: A total of 12 comparative studies (n = 770 patients) were included. Pooled results showed: there were no differences in function outcome, pain scores, retear rate or the incidence of adhesive capsulitis between all arthroscopic and mini-open repair groups. CONCLUSIONS: There were no differences in outcomes between the arthroscopic and mini-open rotator cuff repair techniques, they should be considered alternative treatment options. LEVEL OF EVIDENCE: Level IV, Meta analysis.

CYC1 silencing sensitizes osteosarcoma cells to TRAIL-induced apoptosis.

AIMS: Osteosarcoma (OS) is an aggressive bone malignancy with poor prognosis. Many OS cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). In our previous study, we found that the serum level of cytochrome c1 (CYC1) is significantly higher in OS patients than in healthy subjects. Our aim was to investigate the effects of CYC1 silencing on TRAIL-induced apoptosis in human OS in vitro and in vivo along with the underlying mechanisms. METHODS: First, we determined the expression of CYC1 in human OS tumors and cell lines versus normal adjacent tissues and cell line. We then studied the effects of CYC1 silencing alone or in combination with TRAIL on OS cell growth and apoptosis in vitro and OS tumorigenesis in vivo. RESULTS: We found that CYC1 is overexpressed in human OS tissues and cell lines. CYC1 silencing by shRNA transfection inhibits proliferation, slightly induces apoptosis in human OS cells in vitro, and suppresses human OS tumor growth in a mouse xenograft model in vivo. Additionally, CYC1 silencing sensitizes OS to TRAIL-induced apoptosis in vitro and in vivo. Our results also showed that CYC1 silencing significantly reduces complex III activity and potentiates TRAIL-induced cytochrome c release and caspase-9 activation in OS cells, suggesting that CYC1 silencing acts via the mitochondria-dependent apoptotic pathway. CONCLUSION: Taken together, our results provide evidence that CYC1 plays an important role in OS tumorigenesis, and modulation of CYC1 may be an effective strategy to potentiate OS to apoptotic induction by TRAIL.

The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis.

BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers. METHODS: MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001). CONCLUSIONS: Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Detection of significant pathways in osteoporosis based on graph clustering.

Osteoporosis is the most common and serious skeletal disorder among the elderly, characterized by a low bone mineral density (BMD). Low bone mass in the elderly is highly dependent on their peak bone mass (PBM) as young adults. Circulating monocytes serve as early progenitors of osteoclasts and produce significant molecules for bone metabolism. An improved understanding of the biology and genetics of osteoclast differentiation at the pathway level is likely to be beneficial for the development of novel targeted approaches for osteoporosis. The objective of this study was to explore gene expression profiles comprehensively by grouping individual differentially expressed genes (DEGs) into gene sets and pathways using the graph clustering approach and Gene Ontology (GO) term enrichment analysis. The results indicated that the DEGs between high and low PBM samples were grouped into nine gene sets. The genes in clusters 1 and 8 (including GBP1, STAT1, CXCL10 and EIF2AK2) may be associated with osteoclast differentiation by the immune system response. The genes in clusters 2, 7 and 9 (including SOCS3, SOD2, ATF3, ADM EGR2 and BCL2A1) may be associated with osteoclast differentiation by responses to various stimuli. This study provides a number of candidate genes that warrant further investigation, including DDX60, HERC5, RSAD2, SIGLEC1, CMPK2, MX1, SEPING1, EPSTI1, C9orf72, PHLDA2, PFKFB3, PLEKHG2, ANKRD28, IL1RN and RNF19B.

[Effect of various concentrations of platelet-rich plasma on osteogenic differentiation of skeletal muscle-derived stem cells].

OBJECTIVE: To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. METHODS: Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindlimb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologous PRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, alizarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor alpha1 (Cbfalpha1) of osteogenic gene expression was tested by RT-PCR. RESULTS: The full blood PRP count and the platelet count in PRP was (3.06 +/- 0.46) x 10(5)/microL and (18.08 +/- 2.10) x 10(5)/microL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P < 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P < 0.05). Alizarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineralization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineralization rate (P < 0.05), the experimental group at 12.50% had a higher mineralization rate than other experimental groups (P < 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hours after culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. CONCLUSION: PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.