Objective To review the regulation and mechanism of the microRNAs (miRNAs) in the bone and cartilage tissue. Methods Recent l iterature concerning the regulation and mechanism of the miRNAs in the bone and cartilage tissue was extensively reviewed, summarized, and analyzed. Results Recently miRNAs is a hot topic in the bone and cartilage tissue. More and more materials show its important regulatory role in osteogenesis and cartilage growth andregeneration, but the definite mechanisms have not been clear yet. Conclusion The study on miRNAs of bone and cartilage tissue can provide a new access to understanding the degenerative osteoarthritic diseases.
Objective To observe the effect of recombinant human osteoprotegerin (rhOPG) on osteoclasts which were stimulated by polyethylene particles and to investigate the feasibil ity of applying rhOPG for the prosthetic aseptic looseness. Methods The osteoclasts were isolated from the long bones of 5 New Zealand rabbits born within 24 hours, weighing 80-100 g, male or female, and were plated into the 24-well coversl ips (10 mm × 10 mm) and bone sl ices (8 mm × 8 mm × 50 μm) at the density of 1 × 105/mL. Based on the different concentration and density of rhOPG and polyethylene particles, the plates of culture were divided into 3 groups: the group with polyethylene particles of 1 × 109/mL (polyethylene group), the group co-cultured with polyethylene particles of 1 × 109/mL and rhOPG of 100 ng/mL (polyethylene/rhOPG group) and the control group . The glass cover sl ips and bone sl ices were exposed to HE, toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining at 1, 3, 5 and 7 days, and TRAP positive multinucleated cells and bone resorption tips were counted. Scanning electron microscope was used to observe the pits of bone resorption. Results The osteoclast was rose-red when exposed to TRAP staining. For the number of the TRAP-positive osteoclasts, the polyethylene group witnessed an obviouse increase compared with the control group and the polyethylene/rhOPG group after 5 and 7 days of culture (P lt; 0.05). And no significant difference between the control group and the polyethylene/rhOPG group was evident (P gt; 0.05). The pits of bone resorption was blue-purple when exposed to toluidine blue staining. For the number of bone resorption pits in the bone sl ice, significant difference was evident between the polyethylene group and the control group after 5 and 7 days of culture (P lt; 0.05), and there was significant different between the polyethylene/rhOPG group and the polyethylene group 1, 3, 5 and 7 days after culture (P lt; 0.05). Conclusion rhOPG could inhibit the stimulated effect of polyethylene particles on osteoclasts, and might be used to prevent the prosthetic aseptic looseness after artificial joint substitution.
Osteoblasts were cultured and isolated from a piece of tibial pettiosteum of four New-Zealandrabbits. After subeultured,these cells Were incubatd in vitro with tritiated thvmidine for 36 hoursand then these labeled cells were implanted in the subeutaneous layer of the defects of the auriclarcartilage and the radial bone, After 2 weeks and 4 weeks respectively, these rabbits were killed andthe spoimens were obtained from the site where the cells had been transplanted. The transformation of these cells was observed by autoradiographic method. The results indicated that nearly all of the cultured cells were labeled. After 2 weeks, it was observed that many labeled osteoblasts were in different stages of differentiation, some were beried by extracellular matrix and resembled osteocyte, thers were differentiated into chondrocyte-like cell. In addition, some labeled osteoblasts were congregated in the form of multinucleated osteoclast. After 4 weeks , in the subcutaneous layer the labeled osteoblasts were changed to osteoid tissue and in the defect of the auricular crtilage these cells transformed into chondritic tissue; moreover, those labeled osteoblsts which had been implanted into the radial defect had differentiated into typical bone tissue. The results of this research indicated that the osteoblasts isolated from the periosteum if reimplanted to the same donor might be possible to repair the bone and cartilage defects.
ObjectiveTo review the research progress focused on the effects of strontium ranelate (SR) on osteoarthritis. MethodsThe relevant literature about the effects and mechanism of SR intervening osteoarthritis in recent years was extensively reviewed and comprehensively analyzed. ResultsSR not only could improve the microenvironment of bone metabolism in articular cartilage with osteoarthritis, promote activity of osteoblasts, and inhibit activity of osteoclasts, but also could adjust the expression of key proteases which affect cartilage formation, and therefore it has a potential protective effect on subchondral bone during the progression of osteoarthritis cartilage. ConclusionSR is expected to become a drug of osteoarthritis disease remission, but further studies are needed to clarify the mechanism of SR in osteoarthritis, and finally confirm the best application dosage of SR in osteoarthritis treatment.
ObjectiveTo review the recent research progress of the regulatory mechanisms of osteoclast (OC) formation and functions. MethodsThe related literature on OC formation, activation, and functions was reviewed, analyzed, and summarized. ResultsMacrophage colony stimulating factor and receptor activator of nuclear factor-κB ligand are essential cytokines which regulate all aspects of OC formation and functions. In additional to mediating bone resorption, OCs also partici pate in regulation of osteoblast formation and immune responses. ConclusionThe researches on the regulation of OC formation and functions have further revealed the destruction mechanisms of various kinds of bone diseases, which will facil itate to find more suitable biological targets for cl inical therapy.
ObjectiveTo review the osteoclasts (OC) function beyond bone resorption. MethodsThe related literature on OC function beyond bone resorption was reviewed, analyzed, and summarized. ResultsOC control the bone formation through releasing of matrix-derived growth factors, bidirectional cell-to-cell signals, and secreting OC-coupling factors, and play an important role in the niche formation, hematopoietic stem cells mobilization, and maintenance of its quantity and function;besides, OCs also regulate angiogenesis. ConclusionThese discoveries greatly enrich the current knowledge of OC function and open up an all-new research domain. However, the exact regulatory mechanism of OC affecting the hematopoiesis is still lack in-depth understood. Additionally, it remains to be elucidated how OC-coupling factors act on osteoblast lineage differentiation and how OC-induced angiogenesis participates in physiological and pathological processes. Unclosing the underlying mechanisms will facilitate providing scientific therapeutic strategies for treatment of many OC-related diseases.
ObjectiveTo explore whether FTY-720P could enhance the effect of allograft bone for bone defect repair by suppressing osteoclast formation and function. MethodAnimal experiment:Forty-eight New Zealand white rabbits were selected to establish the tibia defect model (1.5 cm in length) and were divided into 4 groups (n=12) . Defect was not repaired in group A, defect was repaired with allograft bone in group B, with autogenous fibula in group C, and with allograft bone and FTY-720P in group D. Lane-Sandhu scoring system and bone density examination were used to evaluate the effect at 2, 4, 8, and 12 weeks after operation. Cell experiment:Bone marrow-derived mononuclear phagocytes (BMMs) were harvested from 1-month-old Sprague Dawley rats and induced into osteoclasts with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL), then were identified with tartrate-resistant acid phosphatas (TRAP). According to different concentrations of FTY-720P before induction, experiment was divided into 0, 500, 600, 700, 800, 900, 1 000, and 1 500 ng/mL groups. The effect of FTY-720P was studied by counting the number of osteoclasts and the number of bone resorption lacunae made by osteoclasts. ResultsAnimal experiment:Lane-Sandhu score showed no significant difference between groups at 2 weeks after operation (P>0.05) , but the score was significantly better in groups C and D than groups A and B, and in group B than group A (P<0.05) . The bone density of group C was significantly greater than that of groups A, B, and D at 2 weeks after operation (P<0.05) , but no significant difference was found among groups A, B, and D (P>0.05) ; the bone density of groups B, C, and D was significantly greater than that of group A at 4, 8, and 12 weeks (P<0.05) , but no significant difference was shown among groups B, C, and D (P>0.05) . Cell experiment:BMMs could be induced into osteoclasts by the addition of M-CSF and RANKL, which could be proved by counting the number of the nuclear and TRAP staining. The osteoclasts were significantly more in 0, 500, 600, 700, 800, 900 ng/mL groups than 1 000 and 1 500 ng/mL groups (P<0.05) , in 0, 500, 600, and 700 ng/mL groups than 800 and 900 ng/mL groups (P<0.05) , in 0, 500, 600 ng/mL groups than 700 ng/mL group (P<0.05) ; and there was no significant difference between the other groups (P>0.05) . The number of bone resorption lacunae in 0, 500, 600, and 700 ng/mL groups was significantly higher than that in 800, 900, 1 000, and 1 500 ng/mL groups (P<0.05) , and it was significantly higher in 0, 500 and 600 ng/mL groups than 700 ng/mL group (P<0.05) , but difference was not significant between the other groups (P>0.05) . ConclusionsFTY-720P combined with allograft bone for bone defect repair can have the same effect to autogenous bone by means of inhibiting osteoclast formation and function, which reduces bone loss.
ObjectiveTo investigate the effect of Staphylococcal peptidoglycan (PGN-sa) on raw264.7 cells differentiating into osteoclasts. MethodsThere were 5 groups in the experiment: 100 ng/mL PGN-sa group, 200 ng/mL PGN-sa group, 400 ng/mL PGN-sa group, positive control group [100 ng/mL receptor activator of nuclear factor κB ligand (RANKL)], and blank control group (PBS). Raw264.7 cells were cultured with different concentrations of PGN-sa, RANKL, or PBS for 5 days, and then tartrate resistant acid phosphatase (TRAP) staining was used to detect the formation of osteoclast-like cells; Image-Pro Plus 6.0 software was used to detect the bone resorption areas of osteoclast-like cells; and MTT assay was used to observe the proliferation activity of raw264.7 cells. ResultsTRAP staining showed that PGN-sa and RANKL can induce raw264.7 cells to differentiate into osteoclast-like cells; different concentrations of PGNsa groups had more osteoclast-like cells formation than blank control group (P < 0.05), and the number of osteoclast-like cells significantly increased with the increase of PGN-sa concentrations (P < 0.05). Bone resorption cavity experiment showed that bone resorption cavities were obvious in different concentrations of PGN-sa groups and in positive control group, and the area of bone absorption cavities was increased with the increasing PGN-sa concentrations, showing significant difference between groups (P < 0.05). MTT assay showed that no significant difference was found in the absorbance (A) value between different concentrations of PGN-sa groups and blank control group, and between different concentrations of PGN-sa groups (P > 0.05). ConclusionPGN-sa can promote raw264.7 cells to differentiate into osteoclasts with bone resorption activity.
ObjectiveTo investigate the molecular mechanism of osteoclast differentiation induced by Staphylococcal peptidoglycan (PGN-sa). MethodsRaw264.7 cells were stimulated with PGN-sa and with PGN-sa+SC75741[a potent inhibitor of nuclear factor κB (NF-κB) activation] in a concentration of 200 ng/mL. The protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was tested at 0, 1, 2, and 3 days; the proteins related to osteoclast differentiation of extracellular regulated protein kinases (ERK), p38, c-Jun N-terminal kinase (JNK), NF-κB, inhibitor of NF-κB (IκB-α), Akt, and the phosphorylation forms of p38, ERK, JNK, Akt, NF-κB were measured at 0, 5, 10, 20, 40, and 60 minutes by Western blot. In addition, Raw264.7 cells were stimulated with PGN-sa in the concentrations of 100 ng/mL (group A), 200 ng/mL (group B), 400 ng/mL (group C), and with PBS (group D) for 1, 2, and 3 days; the expression levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and IL-6 were detected by ELISA. ResultsThe results of Western blot showed that the expression of NFATc1 increased gradually with time, showing significant difference between different time points (P<0.05). However, after SC75741 was added, the expression of NFATc1 was inhibited at 2 and 3 days, showing significant difference when compared with no addition of SC75741 (P<0.001). After stimulation of PGN-sa, the expression of IkB-α decreased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes. Meanwhile, the expression of p-NF-κB increased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes; and the expression of p-NF-κB at 5 minutes was significantly higher than that at 10 minutes (P<0.001). After the addition of SC75741, there was no change in the expressions of IκB-α and p-NF-κB, showing no significant difference between different time points P>0.05). Moreover, the expressions of ERK, p38, JNK, NF-κB, Akt, p-p38, p-ERK, p-JNK, and p-Akt showed no significant change between different time points P>0.05). ELISA results showed that there were no expressions of TNF-α and IL-1α in groups A-D at different time points. The expression of IL-6 had an increasing trend with time prolonged in each group, showing significant differences between different time points (P<0.05). Moreover, at 1 day after culture, the expression of IL-6 showed no significant difference among groups P>0.05). At 2 and 3 days after culture, the expression of IL-6 in groups A-C showed an increasing trend and was significantly higher than that in group D, showing significant difference among groups (P<0.05). ConclusionPGN-sa can promote osteoclast differentiation through NF-κB signaling pathway, and IL-6 may play a role in this process.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.