Objective:To investigate the effects of exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation on the polarization of Raw264.7 macrophages and to elucidate the underlying mechanisms.Methods:PBS, uninduced BMSCs conditioned medium (CM), and osteogenic induction BMSCs CM for 7 d, 14 d, and 21 d were respectively added to Raw264.7 macrophages. After 48 h of treatment, Western blotting was used to detect and compare the expression of M1 markers of macrophages [inducible nitric oxide synthase (iNOS) and CD86] and M2 markers of macrophages [arginase-1 (ARG-1) and CD163] in each group. In addition, Raw264.7 macrophages were divided into three groups: the PBS group (only PBS added), the BMSCs-exo group (exosomes derived from uninduced BMSCs were added), and 7D-BMSCs-exo (exosomes derived from BMSCs were added after 7 days of osteogenic induction). The absorbance values of Raw264.7 cells in each group at 24 h, 48 h and 72 h were detected by an enzyme-linked immunosorbent assay (ELISA) reader. Western blotting was performed to assess changes of M1 or M2 marker proteins in Raw264.7 macrophages treated by exosome. The supernatants of the three groups of Raw264.7 macrophages were then co-cultured with BMSCs. Alizarin Red staining was used to quantify the formation of mineralized nodules, and alkaline phosphatase (ALP) staining was used to evaluate the osteogenic activity, and the expression levels of osteogenesis-related proteins Runx2, ALP, osteopontin (OPN), and osteocalcin (OCN) were detected. The migration ability of endothelial progenitor cells (EPCs) was detected by scratch assay for migration distance, and the angiogenesis ability was detected by in vitro tube formation assay for the number of vascular rings formed. The expressions of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF) were detected by Western blot. The activation of the MAPK signaling pathway was evaluated by measuring phosphorylated and total P38 and ERK1/2 levels.Results:Western blot analysis showed that CM from BMSCs after 7 days of osteogenic induction (7D-BMSCs-CM) induced the strongest M2 polarization in macrophages. Compared with the PBS group, 7D-BMSCs-CM induced the most significant polarization of Raw264.7 macrophages to the M2 type, and increased ARG-1 and CD163 expression to 1.36±0.09 and 1.69±0.09, respectively ( P<0.05), while decreasing iNOS and CD86 to 0.21±0.03 and 0.29±0.03 ( P<0.05). The absorbance values of macrophages in the 7D-BMSCs-exo group were significantly higher than those in the PBS group at 24 h, 48 h, and 72 h ( P<0.05). Compared with BMSCs-exo group, 7D-BMSCs-exo upregulated the expressions of CD163 and ARG-1 while inhibited the expressions of iNOS and CD86 ( P<0.05). Alizarin red staining showed enhanced mineral deposition and a higher degree of mineralization in the 7D-BMSCs-exo group, the staining intensity of ALP also increased simultaneously, the Western-blot results showed that the protein expressions of Runx2, ALP, OPN and OCN were respectively higher than those in the PBS group ( P<0.05). The results of the scratch assay showed that the relative migration distance of EPCs cells in the 7D-BMSCS-exo group at 24 h reached 2.30±0.05 μm, which was higher than that in the PBS group 1.10±0.02 μm ( P<0.05). The Tube formation experiment showed that the number of vascular rings in the EPCs group was higher than that in the PBS group (30.3±2.5 and 15.0±1.0, P<0.05), and the protein expressions of VEGF-A and PDGF were upregulated. Furthermore, the levels of phosphorylated P38 and ERK1/2 were significantly reduced in the 7D-BMSCs-exo group 0.40±0.06 and 0.25±0.06 compared with the PBS group ( P<0.05). Conclusions:Exosomes secreted by BMSCs during osteogenic differentiation promote the M2 polarization of Raw264.7 macrophages, with the most pronounced effect observed at the 7th day. M2-polarized macrophages, in turn, enhance the osteogenic differentiation of BMSCs and the angiogenic capacity of EPCs. These processes are closely associated with the suppression of the MAPK signaling pathway.

Objective:To investigate the effects of exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation on the polarization of Raw264.7 macrophages and to elucidate the underlying mechanisms.Methods:PBS, uninduced BMSCs conditioned medium (CM), and osteogenic induction BMSCs CM for 7 d, 14 d, and 21 d were respectively added to Raw264.7 macrophages. After 48 h of treatment, Western blotting was used to detect and compare the expression of M1 markers of macrophages [inducible nitric oxide synthase (iNOS) and CD86] and M2 markers of macrophages [arginase-1 (ARG-1) and CD163] in each group. In addition, Raw264.7 macrophages were divided into three groups: the PBS group (only PBS added), the BMSCs-exo group (exosomes derived from uninduced BMSCs were added), and 7D-BMSCs-exo (exosomes derived from BMSCs were added after 7 days of osteogenic induction). The absorbance values of Raw264.7 cells in each group at 24 h, 48 h and 72 h were detected by an enzyme-linked immunosorbent assay (ELISA) reader. Western blotting was performed to assess changes of M1 or M2 marker proteins in Raw264.7 macrophages treated by exosome. The supernatants of the three groups of Raw264.7 macrophages were then co-cultured with BMSCs. Alizarin Red staining was used to quantify the formation of mineralized nodules, and alkaline phosphatase (ALP) staining was used to evaluate the osteogenic activity, and the expression levels of osteogenesis-related proteins Runx2, ALP, osteopontin (OPN), and osteocalcin (OCN) were detected. The migration ability of endothelial progenitor cells (EPCs) was detected by scratch assay for migration distance, and the angiogenesis ability was detected by in vitro tube formation assay for the number of vascular rings formed. The expressions of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF) were detected by Western blot. The activation of the MAPK signaling pathway was evaluated by measuring phosphorylated and total P38 and ERK1/2 levels.Results:Western blot analysis showed that CM from BMSCs after 7 days of osteogenic induction (7D-BMSCs-CM) induced the strongest M2 polarization in macrophages. Compared with the PBS group, 7D-BMSCs-CM induced the most significant polarization of Raw264.7 macrophages to the M2 type, and increased ARG-1 and CD163 expression to 1.36±0.09 and 1.69±0.09, respectively ( P<0.05), while decreasing iNOS and CD86 to 0.21±0.03 and 0.29±0.03 ( P<0.05). The absorbance values of macrophages in the 7D-BMSCs-exo group were significantly higher than those in the PBS group at 24 h, 48 h, and 72 h ( P<0.05). Compared with BMSCs-exo group, 7D-BMSCs-exo upregulated the expressions of CD163 and ARG-1 while inhibited the expressions of iNOS and CD86 ( P<0.05). Alizarin red staining showed enhanced mineral deposition and a higher degree of mineralization in the 7D-BMSCs-exo group, the staining intensity of ALP also increased simultaneously, the Western-blot results showed that the protein expressions of Runx2, ALP, OPN and OCN were respectively higher than those in the PBS group ( P<0.05). The results of the scratch assay showed that the relative migration distance of EPCs cells in the 7D-BMSCS-exo group at 24 h reached 2.30±0.05 μm, which was higher than that in the PBS group 1.10±0.02 μm ( P<0.05). The Tube formation experiment showed that the number of vascular rings in the EPCs group was higher than that in the PBS group (30.3±2.5 and 15.0±1.0, P<0.05), and the protein expressions of VEGF-A and PDGF were upregulated. Furthermore, the levels of phosphorylated P38 and ERK1/2 were significantly reduced in the 7D-BMSCs-exo group 0.40±0.06 and 0.25±0.06 compared with the PBS group ( P<0.05). Conclusions:Exosomes secreted by BMSCs during osteogenic differentiation promote the M2 polarization of Raw264.7 macrophages, with the most pronounced effect observed at the 7th day. M2-polarized macrophages, in turn, enhance the osteogenic differentiation of BMSCs and the angiogenic capacity of EPCs. These processes are closely associated with the suppression of the MAPK signaling pathway.

Objective:To investigate the impact of peak pressure upon different bone densities during autonomous puncture by orthopedic robot on puncture path deviation and bone cement leakage.Methods:A spinal surgery robot system was designed for autonomous vertebral puncture and bone cement injection, and six porcine spine specimens were used for single-segment or double-segment simulated percutaneous vertebral augmentation surgery. The accuracy of puncture path (Gertzbein-Robbins grading), bone cement leakage classification, and peak bone drill pressure were measured to assess the accuracy of autonomous vertebral puncture and bone cement leakage in vertebral cortical and cancellous bone of different densities.Results:A total of 64 porcine vertebrae were simulated for puncture, among which 53 vertebrae were classified as Grade A, 8 as Grade B, and 3 as Grade C according to the Gertzbein-Robbins grading. The cortical bone pressure of Grade A vertebrae was 6.663±0.319 N which was lower than that of Grade B (8.348±0.418 N) and Grade C (11.500±0.600 N), with significant differences ( F=341.000, P<0.001). The cancellous bone pressure of Grade A, B, and C vertebrae were 3.660±0.317, 3.594±0.608, and 4.117±0.257 N, respectively, with no significant difference ( F=2.496, P=0.091). There were 40 cases of no leakage, 20 cases of Type I leakage (leakage into the surrounding vertebrae), and 3 cases of Type II leakage (leakage into the vertebral canal), with an overall leakage rate of 36% (23/64). The peak cortical bone pressure for no leakage, Type I, and Type II leakage was 6.638±0.301, 6.792±0.404, and 6.753±0.473 N, respectively, and the peak cancellous bone pressure was 3.634±0.279, 3.783±0.423, and 3.920±0.255 N, respectively, with no significant difference ( F=1.521, P=0.227; F=2.106, P=0.131). Conclusion:During the autonomous puncture process of the novel orthopedic robot, the accuracy of autonomous puncture path decreased when the puncture pressure through the cortical bone was high, and the probability of invading the pedicle increased. The puncture pressure of cortical and cancellous bone had no significant effect on the occurrence rate of bone cement leakage.

Objective:To investigate the effects of 5-aza-2′deoxycytidine(5-aza-dC),a DNA methyltransferase (DNMT)inhibitor, on the methylation status of the RECK gene and the invasion of salivary adenoid cystic carcinoma cell lines.Methods:Methylation-specific PCR,Western blot analysis and quantitative real-time PCR were used to investigate the methylation status of RECK gene and the expression of RECK mRNA and protein in SACC cell lines.The invasive ability of SACC cells was examined by transwell assay. Results:Promoter methylation was only found in ACC-Mcell line and not in ACC-2 cell line.Treatment of ACC-Mcells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of mRNA and pro-tein of RECK,suppressed ACC-Mcell invasive ability.Conclusion:5-aza-dC can inhibit ACC-Mcell invasion by reversal of hyperm-ethylation status of RECK gene.