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.
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.
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.
Bone remodeling requires an intimate cross-talk between osteoclasts and osteoblasts and is tightly coordinated with regulatory proteins that interact through complex autocrine/paracrine processes. Osteocytes, bone lining cells, osteomacs and vascular endothelial cells also regulate bone remodeling in the basic multicellular unit (BMU) via cell signaling networks of ligand-receptor complexes. In addition, through secreted and membrane-bound factors in the bone microenvironment, T and B lymphocytes mediate bone homeostasis for osteoimmunology. Osteoporosis and other bone diseases occur because multicellular communication within the BMU is disrupted. This review focuses on the roles of the cells in the BMU and the interaction between these cells and the factors involved in regulating bone remodeling at the cellular level. Understanding the process of bone remodeling and related genes could help us to lay the foundation for drug development against bone diseases.
ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.
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 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.
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.
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.