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.
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 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.
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.
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.
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 study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
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.
Objective To evaluate the bone regenerative potential of reconbinant human bone morphogenetic protein 2(rhBMP-2) / collagen on adult rat calvarial bone. Methods A tight subperiosteal pocket was produced under both sides ofthe temporal muscle in rats. rhBMP-2 / collagen was implanted in one side and collagen alone was implanted in the other side as control. The rats were sacrificed 2, 4 and 8 weeks after operation. The specimen was harvested and examined histologically. For morphometric analysis, the thickness of the temporal bone of both sides was measured and compared. Results The rhBMP-2 / collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage production. The ossification process in the rhBMP-2 / collagen occurred directly through bone formation, similar to intramembranous ossification. Conclusion rhBMP-2 / collagen is an effective material as a biological onlay implant.
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.
