Objective To investigate and compare the osteogenic potential of three kinds of calcium phosphate ceramic as carriers for recombinant human bone morphogenetic protein-2(rhBMP-2) in vivo.Methods BCPceramics (HA,TCP,HA/TCP) impregnated with rhBMP-2 (experimental groups) and without rhBMP-2(control groups) were implanted into 6 muscles pockets on the dorsum of 3month-old Wistar rabbits. The rabbits were sacrificed 2, 4 and 8 weeks after implantation and bone induction was estimated by alkaline phosphatase(ALP) activity measurement. The implants were also examined histologically and histomorphometrically by HE staining and computerized graphical analysis. Results The ALPactivity of implants withrhBMP-2 was higher than that of control groups(P＜0.05), but there was no difference between 2 and 4 weeks in experimental groups. In all experimental groups,theimplants exhibited that new bone formation increased with the lapse of time. The amount of new bone formation is more in -HA/rhBMP-2 group than in the other two group in the 2nd and 4th weeks, but there was no difference between them (P＞0.05).In the 8th week, the amount of bone formation was most in HA/TCP with -rhBMP-2, and was more than that in the 2nd and 4th weeks. Whereas in control groups, there was only fibrous connective tissue. Conclusion HA/TCP- is a good carriers of rhBMP-2 and can be used as bone substitutes clinically.
Objective To evaluate the osteogenesis of three bio-bone derived materials in repairing segmental bone defects. Methods Sixty Japanese rabbits were made 10 mm radius segmental defects and divided into 5 groups(groups A, B, C ,D and E,n=12). Composite fully deproteinised bone(CFDB, group A), partially deproteinised bone(PDPB, group B), partially decalcified bone(PDCB, group C), autogenous iliac bone graft(group D) and no implant(group E) were implanted into the radius segmental bone defects of rabbits. The specimens were examined after 4, 8, 12 and 24 weeks; the osteogenesis was evaluated through X-ray radiograph and undecalcified solid tissue histological examination.Results The border between the material and host’s bone was distinct after 4 weeks and blurred after 8 weeks; the density of partial edge of the material was similar to that of radii after 12 weeks. The medullary cavity of bone reopened in group B; the density of most defect area was similar to that of the host bone and there was a few high density shadow in group C; the density of most defect area was higher than that of host bone in group A after 24 weeks. There was no significant difference in radiograph scoring between groups A, B and C after 4 weeks and 8 weeks(P＞0.05); the scores of group B and C were higher than that of group A after 12 weeks(P＜0.05); and the scores were arranged as follow: group Dgt;group Bgt; group Cgt;group A after24 weeks(P＜0.05). Bone callusgrew toward defect area and new bone adhered to the material after 4 weeks and 8 weeks; more new bone formed, and the materials were absorbed and degraded with time. The quantity of bone formation was more in group D than in group B andin group B than in group C and in group C than in group A after 24 weeks(P＜0.05).Conclusion PDPB had good osteogenesis in repairing the segmental bone defect, PDCB was inferior to it, both PDPB and PDCB are fit to repair segmental bone defect. Both of them were inferior to autogenous bone.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
Ceramiclike xenogeneic bone (CXB) was obtained from the fresh bone of pig ribs being treated by physical and chemical methods to deprive of its organic substance. The CXB possessed the same natural porous network system as that of the human. The CXB was cultured with the bone marrow stromal cells of rabit. When the marrow cells had integrated with the CXB, thus a new material was obtained. (CXB-BM), and was implanted sacro-spinal muscle of rabbit. The specimens were observed under phase microscope, light microscope and electronic scanning microscope. The results showed that: at the 2nd week after the implantation of CBX-BM composite material there began the new bone formation, and the rate of bone formation was increased with time. There was evident new bone formation after 24 weeks. The process of the new bone formation were quite similar to the composite graft of HAP red autogenous and marrow, but the former degraded faster and formed typical cancellous structure earlier. There was no new bone formation when CXB was implanted alone in the control. Both the mechanism of osteogenetic potential and its clinical application were discussed.
OBJECTIVE To observe the osteogenesis of percutaneous autogenous bone marrow grafting in cicatricial bone defect, to seek a good method for treating fracture nonunion. METHODS Eighteen rabbits were adopted in this study. 1 cm bone defect model was made in each side of radius, 6 weeks later, 2 ml autogenous bone marrow was injected in the right radial bone defect as experimental group, 2 ml autogenous peripheral blood in the left side as control group. X-ray features, histologic changes, Ca and P content in the site of bone defect were studied in various times. Also 15 patients were treated clinically for the nonunion fracture, the average time from nonunion to bone marrow grafting was 13 months. RESULTS In experimental group, the increasing new bone tissue were observed in X-ray and histologic examination. While in control group, no osteogenesis was observed. Ca and P content of experimental group was higher than that of control group. For the 15 patients, 13 cases healed in 5-9 months, 2 cases failed. CONCLUSION Percutaneous autogenous bone marrow grafting is capable of osteogenesis in the cicatricial bone defects. It can be used in nonunion cases which are not fit for operation of bone grafting because of poor condition of the skin.
Objective To investigate the role of micro RNA-451 (miRNA-451) in promoting the osteogenesis of mesenchymal stem cells (MSCs) by targeting regulatory calcium binding protein 39 (CAB39). Methods pMIR-report and pRL-TK vectors were selected to identify the relationship between miRNA-451 and CAB39 by using dual-luciferase reporter assay. pre-miRNA-451 (group A), anti-miRNA-451 (group C), pre-miRNA negative control (group B), and anti-miRNA negative control (group D) were transfected into the C3H10T1/2 cells, respectively. Then, the cells were collected after osteogenic induction for 7 and 14 days. At 7 and 14 days, the real-time fluorescent quantitative PCR and Western blot assays were performed to detect the related osteogenetic biomarkers [Runx2 and alkaline phosphatase (ALP) mRNA] and expressions of CAB39 protein. At 14 days, the extracellular calcium deposition during the osteogenesis of MSCs was tested by Alizarin red staining method. Results CAB39 was the target gene of miRNA-451. At 7 and 14 days after osteogenic induction, the mRNA expressions of Runx2 and ALP in group A were significantly higher than those in group B (P lt; 0.05), and the expressions in group C was significantly lower than those in group D (P lt; 0.05). Furthermore, at 14 days after osteogenic induction, the protein expression of CAB39 in group A (0.55 ± 0.05) was significantly lower than that in group B (1.00 ± 0.07), and the protein expression in group C (1.21 ± 0.05) was significantly higher than that in group D (1.00 ± 0.04), all showing significant difference (P lt; 0.05). Finally, at 14 days after osteogenic induction, the extracellular calcium deposition in group A was obviously more than that in group B, and group C was downregulated when compared with group D. Conclusion miRNA-451 can promote the osteogenesis process of MSCs by downregulating the CAB39.
Objective To investigate the effect of simvastatin on inducing endothel ial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Methods Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 × 106 cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins(2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. Results All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% ± 4.07% in experimental group and 59.45% ± 5.43% in control group, showing significant difference (P lt; 0.05). Conclusion Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.
OBJECTIVE To study the bone formation and osteogenesis after transplantation of human periosteal mesenchymal stem cells(PMSC). METHODS Suspension of PMSC which obtained from cell culture of periosteal segments in vitro were injected into the backs of nude mice subcutaneously, and the fracture site of neck of femur in old person. RESULTS Subdermal nodules were observed by naked eyes after 11 days of transplantation. 4 weeks later, their anatomic diameter reached 2-7 mm(averaged 3.6 mm). It was proved that the subdermal nodules were trabecular ball trapped with fibrous tissue. The nodules were investigated by human special apoB gene with PCR, and the test of anti-human-tissue precipitin reaction(AHTPR). The results of PCR and AHTPR were positive reaction. There were no subdermal nodules formed in the sites of injection of frozen-melted PMSC or culture medium. The new callus in the sites of fracture were tested by PCR test, and two kinds of apoB gene products were detected. CONCLUSION The results indicated that the implanted PMSC could form new bone directly in nude mice, and the cells of donor and recipient all could form new bone.
ObjectiveTo compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition. MethodsBMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α(HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days. ResultsThe BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P < 0.05). At each time point, compared with group A, the ALP expression, HIF-1αprotein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P < 0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P < 0.05); compared with group C, the HIF-1αprotein relative expression was significantly up-regulated in group B (P < 0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C. ConclusionHypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.
Objective To review the progress, controversy and trend in the regulation and mechanism of the microRNAs (miRNAs) during the osteogenesis. Methods Recent l iterature concerning regulation and mechanism of the miRNAs during the osteogenesis was extensively reviewed, summarized and analyzed. Results Recently miRNAs was a hot topic for osteogenesis. More and more materials showed its important role in ossification, but its definite mechanism was notclear. Conclusion Osteogenesis can be strengthened by miRNAs technology, which has a bright future and may also provide the molecular mechanism. The study on miRNAs of osteogenesis can provide a model to analyze and compare the osteogenetic effects of novel drugs.