ObjectiveTo elucidate the role of interleukin-33 (IL-33) in glucocorticoid-induced osteonecrosis of the femoral head (ONFH) in mice, with particular emphasis on its effects on bone remodeling, inflammatory regulation, and fibrosis. Methods In vivo: Fifteen 9-week-old male C57BL/6J wild-type mice were randomly divided into a normal control group, an ONFH group, and an intervention group, with 5 mice in each group. A glucocorticoid-induced ONFH model was established in the ONFH group and intervention group using a combined administration of lipopolysaccharide and methylprednisolone. The intervention group received intraperitoneal injection of IL-33 for 4 consecutive days during the early stage of model establishment; the normal control group received saline injection at the same time points. General conditions of mice were observed during the experiment. Endogenous IL-33 and transmembrane ST2 (ST2 ligand, ST2L) expression in the femoral head was assessed via immunofluorescence, quantitative PCR (qPCR), and Western blot. Bone necrosis and fibrosis were evaluated using HE and Masson staining. Immunohistochemistry was performed to detect osteogenic markers [osteocalcin (OCN), osteopontin (OPN), Runt-related transcription factor 2 (Runx2)] and osteoclastic marker (receptor activator of nuclear factor-κB ligand, RANKL), while serum cytokine levels [tumor necrosis factor (TNF-α), IL-6, IL-1β, IL-4, IL-10] were quantified by ELISA. In vitro: Murine osteoblasts were divided into control group (DMEM+PBS), IL-33 group (DMEM+10 ng/mL IL-33), and IL-33+ST2L group (DMEM+10 ng/mL IL-33+1 μg/mL ST2L antibody). After corresponding treatment, cell proliferation was detected by EdU incorporation assay. Additional osteoblasts were subjected to osteogenic induction culture, and mineralization, and the expression of osteogenesis-related genes (Runx2, collagen type Ⅰ, OCN, and OPN) were assessed by using alkaline phosphatase (ALP) staining, Alizarin red staining, and qPCR, respectively. Results In vivo: All animals survived until the completion of the experiment. Mice in the intervention group and ONFH group showed restricted mobility. Compared with the normal control group, the expressions of IL-33 and ST2L significantly upregulated at both mRNA and protein levels (P<0.05). Exogenous IL-33 administration exacerbated, rather than ameliorated, trabecular destruction and fibrosis, with the intervention group showing significantly increased fibrosis area percentage and empty lacunae rate compared with the other two groups (P<0.05). Furthermore, IL-33 treatment further suppressed the expressions of osteogenic markers (Runx2, OCN, OPN) while significantly enhancing the expression of the osteoclastic marker (RANKL) (P<0.05). ELISA results showed that compared with the ONFH group, serum levels of pro-inflammatory cytokines (IL-4, IL-6, IL-1β) were significantly lower in the intervention group (P<0.05). In vitro: Compared with control group, IL-33 significantly impaired osteoblast proliferation and differentiation, as evidenced by reduced cell proliferation rate, decreased ALP activity, and reduced calcium nodule formation (P<0.05). The expression of osteogenesis-related genes was also suppressed, with significant differences between groups (P<0.05). ST2L blockade effectively reversed these IL-33-mediated suppressive effects, leading to significant recovery of osteoblast proliferation and differentiation (P<0.05). Notably, the mRNA expression levels of collagen typeⅠand OCN were restored to normal (P>0.05). ConclusionIL-33 exacerbates ONFH by impairing osteoblast viability and function and inhibiting bone regeneration. Targeting the IL-33/ST2L signaling axis may represent a promising novel therapeutic strategy for ONFH.
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.
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.
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.
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.
ObjectiveAfter using hyaluronic acid (HA) to modify curcumin (CUR), the effects of calcium phosphate cement (CPC) combined with HA/CUR on the proliferation and osteogenesis of osteoblasts were investigated.MethodsFirst, HA and CUR were esterified and covalently combined to prepare HA/CUR, and the characteristics were observed and the infrared spectrum was tested. Then, HA, CUR, and HA/CUR were mixed with CPC according to 5% (W/W) to prepare HA-CPC, CUR-CPC, and HA/CUR-CPC, respectively. Setting time detection, scanning electron microscope observation, injectable performance test, and compression strength test were conducted; and the CPC was used as a control. Osteoblasts were isolated and cultured from the skull of newborn Sprague Dawley rats, and the 2nd generation cells were cultured with the 4 types of bone cement, respectively. The effects of HA/CUR-CPC on the proliferation and osteogenesis of osteoblasts were estimated by the scanning electron microscopy observation, live/dead cell fluorescence staining, cell counting, osteopontin (OPN) immunofluorescence staining, alkaline phosphatase (ALP) staining,and alizarin red staining.ResultsInfrared spectroscopy test showed that HA and CUR successfully covalently combined. The HA/CUR-CPC group had no significant difference in initial setting time, final setting time, injectable rate, and compressive strength when compared with the other 3 groups (P>0.05); scanning electron microscope observation showed that HA/CUR was scattered on CPC surface. After co-culture of bone cement and osteoblasts, scanning electron microscopy observation showed that the osteoblasts, which had normal morphology and the growth characteristics of osteoblasts, clustered and adhered to HA/CUR-CPC. There was no significant difference in cell survival rate between HA/CUR-CPC group and other groups (P>0.05), and the number of cells significantly increased (P<0.05); the degrees of OPN immunofluorescence staining, ALP staining, and alizarin red staining were stronger than other groups.ConclusionHA/CUR-CPC has good biocompatibility and mechanical properties, which can promote the proliferation and osteogenesis of osteoblasts.
Objective To study the ectopic osteogenesis and vascularization ofthe tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2 D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC).Methods After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5×105/ml) and hUVEC (2.5×105/ml) were seeded at a ratio of 2∶1 onto the CHA scaffolds coated with 1,25-(OH)2 D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2 D3 (the control group). The scaffolds were culturedin vitro for 3 days, and then the scaffolds were implanted into the pockets that had beenmade on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with onecontrol engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed. Results The evaluationsby the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissuesand newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the newformed bones were 3.1±0.52 in the experimental group but2.30±0.59 in the control group at 8 weeks (Plt;0.05), and 4.63±0.55 vs. 3.53±0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74％±7.81％i n the experimental group but 19.52％±4.57% in the control group at 4 weeks (Plt;0.05), and 24.66％±7.38％ vs. 1784%±5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). Conclusion 1,25-(OH)2 D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.
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.
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.