The aim of this experiment was to study the osteogenesis in vivo of allogenic osteoblast combined culture with calcium phosphate composites. The osteoblasts were obtained by enzymatic digestion of periosteum from fibula subcultured to 13 generations, the cells were combined culture with hydroxyapatite and biphasic calcium phosphate. Subseguently, the composite was implanted into rabbits subcutaneously or intramuscularly. The blank material was implanted in the contralateral side as control. Four weeks later, all animals were sacrificed. All the implants were examined by gross observation, histological examination and EDXA. The results showed: 1. obvious ingrowth of connective tissue with very little inflammatory reaction; 2. new bone formation in the composites with deposit of Ca and P on the surface of osteoblast, but none in the blank materials; 3. no significant difference of new bone formation between the different sites of implantation or different materials, but those implanted intramuscularly had lamellae form of new bone while those implanted subcutaneously had only mineralization of extracellular matrix. The conclusion were: 1. the composites are biocompatible with prior osteogenesis property; 2. periosteal-derived allogenic osteoblasts obatined by enzymatic digestion could survive following implantation with bioactivity; 3. rich blood supply might be advantageous to new bone formation and its maturation.
OBJECTIVE: To determine an optimal co-culture ratio of the rabbit periosteal osteoblasts (RPOB) and rabbit renal vascular endothelial cells(RRVEC) without direct contact for future study of bone tissue engineering. METHODS: RPOB and RRVEC in the ratios of 1:0(control group), 2:1(group 1), 1:1(group 2) and 1:2(group 3) were co-cultured by six well plates and cell inserts. Four days later, the proliferation of RPOB and RRVEC were examined through cell count. Differentiated cell function was assessed by alkaline phosphatase (ALP) activity assay and 3H proline incorporation assay. RESULTS: When RPOB and RRVEC were indirectly co-cultured, the proliferation of RPOB and 3H proline incorporation was higher in group 1 than in the other experimental groups and control group (P lt; 0.05). ALP activity of RPOB was higher in group 1 than in control group and group 3 (P lt; 0.05), but there was no significant difference between group 1 and group 2 (P gt; 0.05). CONCLUSION: These results suggest that RPOB and RRVEC co-cultured in a ratio of 2:1 is optimal for future study of bone tissue engineering.
Objective To explore the effectiveness and mechanism of pure platelet-rich plasma (P-PRP) on osteochondral injury of talus. Methods Thirty-six patients with osteochondral injury of talus selected between January 2014 and October 2017 according to criteria were randomly divided into control group (group A), leukocyte PRP (L-PRP) group (group B), and P-PRP group (group C), with 12 cases in each group. There was no significant difference in gender, age, disease duration, and Hepple classification among the three groups (P>0.05). Patients in the groups B and C were injected with 2.5 mL L-PRP or P-PRP at the bone graft site, respectively. Patients in the group A were not injected with any drugs. The American Orthopaedic Foot and Ankle Society (AOFAS) score and visual analogue scale (VAS) score were used to evaluate the effectiveness before operation and at 3, 6, and 12 months after operation. Study on the therapeutic mechanism of P-PRP: MC3T3-E1 cells were randomly divided into control group (group A), L-PRP group (group B), and P-PRP group (group C). Groups B and C were cultured with culture medium containing 5% L-PRP or P-PRP respectively. Group A was cultured with PBS of the same content. MTT assay was used to detect cell proliferation; ELISA was used to detect the content of matrix metalloprotein 9 (MMP-9) protein in supernatant; alkaline phosphatase (ALP) activity was measured; and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of osteopontin (OPN), collagen type Ⅰ, and MMP-9 in cells. Western blot was used to detect the expression of MMP-9 in supernatant and phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), and phosphorylated c-Jun (p-c-Jun) in cells. ResultsAll patients were followed up 13-25 months, with an average of 18 months. No complication such as wound infection and internal fixation failure occurred. MRI showed that the degree of injury was similar between the three groups before operation, and patients in the three groups all recovered at 6 months after operation. Moreover, group C was superior to groups A and B. Compared with preoperation, AOFAS scores and VAS scores in the three groups were all significantly improved at each time point after operation (P<0.05). AOFAS score of group C was significantly higher than that of groups A and B at 3, 6, and 12 months after operation (P<0.05); there was no significant difference in VAS score between the three groups (P>0.05). Study on the therapeutic mechanism of P-PRP: The absorbance (A) value, ALP activity, the relative mRNA expression of OPN and collagen type Ⅰ in group C were significantly higher than those in groups A and B (P<0.05), and those in group B were significantly higher than those in group A (P<0.05). The relative expression of MMP-9 protein and mRNA and the content of MMP-9 protein detected by ELISA in group B were significantly higher than those in groups A and C, while those in group C were significantly lower than those in group A (P<0.05). Western blot detection showed that the relative expression of PI3K, pAKT, and p-c-Jun protein in group B was significantly higher than those in groups A and C (P<0.05), but there was no significant difference between groups A and C (P>0.05). Conclusion P-PRP is superior to L-PRP for osteochondral injury of talus, which may be related to the inhibition of PI3K/AKT/AP-1 signaling pathway in the osteoblast, thereby reducing the secretion of MMP-9.
Objective To review the research progress of exosomes (EXOs) derived from different cells in the treatment of osteoporosis (OP). Methods Recent relevant literature about EXOs for OP therapy was extensively reviewed. And the related mechanism and clinical application prospect of EXOs derived from different cells in OP therapy were summarized and analyzed. Results EXOs derived from various cells, including bone marrow mesenchymal stem cells, osteoblasts, osteoclasts, osteocytes, and endothelial cells, et al, can participate in many links in the process of bone remodeling, and their mechanisms involve the regulation of proliferation and differentiation of bone-related cells, the promotion of vascular regeneration and immune regulation, and the suppression of inflammatory reactions. A variety of bioactive substances contained in EXOs are the basis of regulating the process of bone remodeling, and the combination of genetic engineering technology and EXOs-based drug delivery can further improve the therapeutic effect of OP. Conclusion EXOs derived from different cells have great therapeutic effects on OP, and have the advantages of low immunogenicity, high stability, strong targeting ability, and easy storage. EXOs has broad clinical application prospects and is expected to become a new strategy for OP treatment.
ObjectiveTo review the role and mechanism of protein factors in bone remodeling, and provides theoretical basis for further elucidating the pathogenesis and clinical treatment of bone-related diseases. MethodsThe relevant research results at home and abroad in recent years were extensively consulted, analyzed, and summarized. ResultsBone remodeling is an important physiological process to maintain bone homeostasis. Protein, as an important stimulator in bone remodeling, regulates the balance between bone resorption and bone formation. ConclusionAt present, the research on the mechanism of protein in bone remodeling is insufficient. Therefore, it is necessary to further study the specific time, process, and interaction network of protein in bone remodeling, and to confirm its mechanism in bone remodeling, so as to reveal and treat the pathogenesis of bone-related diseases.
ObjectiveTo summarize the research progress on the calcitonin gene-related peptide (CGRP) and receptor activator of nuclear factor κB (RANK)/receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) system during bone reconstruction to provide theoretical basis for further research on the prevention and treatment of bone-related diseases.MethodsThe relevant research results at home and abroad in recent years were analyzed and summarized.ResultsCGRP and RANK/RANKL/OPG system play important regulatory roles in the bone reconstruction.ConclusionAt present, the research on the mechanism of CGRP and RANK/RANKL/OPG system in bone reconstruction is insufficient. Therefore, it is necessary to study further on the process and interrelation of CGRP and RANK/RANKL/OPG system in bone reconstruction to confirm their mechanism, which will bring new ideas and methods for the treatment of bone related diseases in clinic.
ObjectiveTo explore the protective effects of sodium valproic acid (VPA) on oxidative stress injury of osteoblasts induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and its mechanism. Methods Osteoblasts were isolated from the skulls of 10 newborn Sprague Dawley rats and cultured by tissue block method, and the 1st generation cells were identified by alkaline phosphatase (ALP) and alizarin red staining. The 3rd generation osteoblasts were cultured with 2-18 μmol/L CCCP for 2-18 minutes, and cell counting kit 8 (CCK-8) was used to detect the cell survival rate. An appropriate inhibitory concentration and culture time were selected for the preparation of osteoblasts oxidative stress injury model based on half maximal concentration principle. The cells were cultured with 0.2- 2.0 mmol/mL VPA for 12-72 hours, and CCK-8 was used to detect cell activity, and appropriate concentration was selected for further treatment. The 3rd generation cells were randomly divided into 4 groups, including blank control group (normal cultured cells), CCCP group (the cells were cultured according to the selected appropriate CCCP concentration and culture time), VPA+CCCP group (the cells were pretreated according to the appropriate VAP concentration and culture time, and then cultured with CCCP), VPA+CCCP+ML385 group (the cells were pretreated with 10 μmol/L Nrf inhibitor ML385 for 2 hours before VPA treatment, and other treatments were the same as VPA+CCCP group). After the above treatment was complete, the cells of 4 groups were taken to detect oxidative stress indicators [reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA)], cell apoptosis rate, ALP/alizarin red staining, and the relative expressions of osteogenic related proteins [bone morphogenetic protein 2 (BMP-2), RUNX2], anti-apoptotic family protein (Bcl2), apoptotic core protein (Cleaved-Caspase-3, Bax), channel protein (Nrf2) by Western blot. Results The osteoblasts were successfully extracted. According to the results of CCK-8 assay, the oxidative stress injury model was established by 10 μmol/L CCCP cultured for 10 minutes and 0.8 mmol/mL VPA cultured for 24 hours was selected for subsequent experiments. Compared with blank control group, the activity and mineralization capacity of osteoblasts in CCCP group decreased, the contents of ROS and MDA increased, the activity of SOD decreased, and the apoptosis rate increased. Meanwhile, the relative expressions of BMP-2, RUNX2, and Bcl2 decreased, and the relative expressions of Cleaved-Caspase-3, Nrf2, and Bax increased. The differences were significant (P<0.05). After further VPA treatment, the oxidative stress damage of osteoblasts in VPA+CCCP group was relieved, and the above indexes showed a recovery trend (P<0.05). In VPA+CCCP+ML385 group, the above indexes showed an opposite trend (P<0.05), and the protective effects of VPA were reversed. Conclusion VPA can inhibit the CCCP-induced oxidative stress injury of osteoblasts and promote osteogenesis via Keap1/Nrf2/Are pathway.
ObjectiveTo discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells.MethodsThe 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection.ResultsAfter 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05).ConclusionPiezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.
ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
In this study, we aim to investigat the effect of microgravity on osteoblast differentiation in osteoblast-like cells (MC3T3-E1). In addition, we explored the response mechanism of nuclear factor-kappa B (NF-κB) signaling pathway to " zero-g” in MC3T3-E1 cells under the simulated microgravity conditions. MC3T3-E1 were cultured in conventional (CON) and simulated microgravity (SMG), respectively. Then, the expression of the related osteoblastic genes and the specific molecules in NF-κB signaling pathway were measured. The results showed that the mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and type Ⅰ collagen (CoL-Ⅰ) were dramatically decreased under the simulated microgravity. Meanwhile, the NF-κB inhibitor α (IκB-α) protein level was decreased and the expressions of phosphorylation of IκB-α (p-IκB-α), p65 and phosphorylation of p65 (p-p65) were significantly up-regulated in SMG group. In addition, the IL-6 content in SMG group was increased compared to CON. These results indicated that simulated microgravity could activate the NF-κB pathway to regulate MC3T3-E1 cells differentiation.