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OBJECTIVES@#This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO).@*METHODS@#The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.@*RESULTS@#Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05).@*CONCLUSIONS@#Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.
Subject(s)
Graphite/pharmacology , Titanium/pharmacology , Haversian System , Macrophages , Cell Differentiation , Oxides/pharmacology , Surface PropertiesABSTRACT
Objective:To investigate the combined effect of fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation in rats.Methods:SD rats were divided into four groups by the method of random number table, namely normal nutrition group, low nutrition treatment group, fluoride exposure group and co-treatment of fluoride and low nutrition group according to 2 × 2 factorial experimental design, 8 rats in each group, half male and half female. Five months after the experiment, immunohistochemistry was used to test the expression levels of femoral alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL). Analysis of variance of factorial design was used to determine the interaction between fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation.Results:The immunohistochemical results of bone tissue showed that there were significant differences in the expression levels of osteogenesis differentiation markers ALP and Runx2 between different groups ( F = 25.98, 17.77, P < 0.001). Compared with normal nutrition group (0.005 2 ± 0.002 7, 0.003 1 ± 0.001 4), the expression levels of ALP and Runx2 in fluoride exposure group were higher (0.019 5 ± 0.005 0, 0.014 4 ± 0.004 4, P < 0.05). There was no significant difference between low nutrition treatment group (0.002 6 ± 0.001 8, 0.004 4 ± 0.003 2) and co-treatment of fluoride and low nutrition group (0.003 6 ± 0.000 7, 0.002 9 ± 0.000 8, P > 0.05). The expression levels of ALP and Runx2 in co-treatment of fluoride and low nutrition group were lower than those of fluoride exposure group ( P < 0.05). There were significant differences in the expression level osteoclastic differentiation marker of RANKL and the ratio of RANKL/OPG ( F = 10.50, 31.05, P < 0.001). Among them, the RANKL/OPG ratio (0.115 3 ± 0.039 5) in fluoride exposure group was lower than that in normal nutrition group (1.426 3 ± 0.777 2), and the RANKL expression level and RANKL/OPG ratio (0.019 5 ± 0.007 7, 7.258 7 ± 3.674 3) in co-treatment of fluoride and low nutrition group were higher than those in normal nutrition group (0.004 4 ± 0.002 5, 1.426 3 ± 0.777 2, P < 0.05). However, there was no significant difference in the RANKL expression level and RANKL/OPG ratio (0.004 0 ± 0.001 9, 2.022 3 ± 0.753 7) in low nutrition treatment group ( P > 0.05). The expression level of RANKL and the ratio of RANKL/OPG in the co-treatment of fluoride and low nutrition group were higher than those in low nutrition treatment group and fluoride exposure group ( P < 0.05). The 2 × 2 analysis of variance of factorial design showed that fluoride exposure and low nutrition had interaction on ALP, Runx2, RANKL expression levels and RANKL/OPG ratio ( F = 4.38, 19.39, 22.12, 108.00, P < 0.05), antagonistic effect on ALP and Runx2 expression, synergistic effect on RANKL expression and RANKL/OPG ratio. Conclusions:In rat bone tissue, fluoride exposure promotes osteogenesis differentiation, inhibits osteoclastic differentiation dominated by active osteogenic function. The interaction between fluoride and low nutrition on osteogenesis and osteoclastic differentiation is antagonistic osteogenesis differentiation and synergistic promotion of osteoclastic differentiation. Normal nutrition conditions are material basis of osteogenesis differentiation, and low nutrition is the inducement of enhanced osteoclastic differentiation.
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Osteoprotegerin (OPG), secreted by osteoblasts, is a marker of bone turnover. OPG can inhibit osteoclastic differentiation by binding receptor activator of nuclear factor-κB ligand (RANKL). In this study, we found that rutaecarpine (RUT) had the up-regulating OPG activity, and it could significantly increase OPG protein levels in both mouse embryonic osteogenic precursor MC3T3-E1 and human osteosarcoma U-2OS cells. Osteoblastogenic differentiation calcified nodules staining results showed that RUT significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Osteoclastic differentiation tartrate resistant acid phosphatase (TRAP) staining results showed that RUT obviously inhibited the osteoclast differentiation of mouse macrophages RAW264.7 induced by RANKL. In vivo studies showed that low-dose RUT group (5 mg·kg-1·day-1) and high-dose RUT group (45 mg·kg-1·day-1) treatments for 3 months significantly increased bone density in ovariectomized (OVX) rats; calcein double labeling experiment and toluidine blue staining results indicated that low-dose RUT group promoted bone formation and decreased bone loss in vivo; immunohistochemistry results showed that low-dose RUT group increased the expression of OPG in rat femur. All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. In summary, this study demonstrated that RUT could up-regulate OPG expression and had promoting osteoblastic differentiation and inhibiting osteoclastic differentiation effects in vitro and in vivo.
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Objective To determine the effect of ostecytic TGF-β/Smad4 signaling on osteoblastic and osteoclastic differentiation in bone marrow stromal cells (BMSCs).Methods Mice with osteocytic TGF-β/Smad4 conditional knock down (Smad4ot CKD) were generated as previously by crossing DMP1-8kb-Cre mice with Smad4lox(ex8)/lox(ex8) mice.The osteocytes were isolated from tibial and femoral diaphysis and co-cultured with wild-type BMSCs.ALP staining, Alizarin red staining and TRAP staining were performed to show osteoblastic and osteoclastic differentiation.Then, their marker genes were detected by qPCR and proteins measured by Western blot.ResultsThe expression of Runx2 and Osterix were reduced in smad4 CKDot co-cultured with BMSCs compared with controls(P<0.01).Similarly, the specific markers of osteoblastic differentiation were decreased (P<0.01).Additionally, the expression of RANKL was not significantly changed in with BMSCs.However, OPG was highly expressed incontrol group compared with smad4 CKD in co-cultured group (P<0.05).Thus, the radio of RANKL/OPG was significantly reduced (P<0.05).Furthermore, the expression of RANK was inhibited.Conclusions The terminally-differentiated osteocytes are the cells regulating bone metabolism, while down-regulation of osteocytic-TGF-β/Smad4 inhibits BMSC osteoblastic and osteoclastic differentiation.