Objective Simvastatin has been reported to be effective on stimulation of bone formation. To investigate the effects of simvastatin on bone formation relative factors of proximal tibia trabecular bone and on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Fourty 1-week-old male Sprague Dawley rats were divided randomly into 2 groups, 20 rats per group. Rats in experimental group received subcutaneous injection of simvastatin [(5 mg/ (kg• d)], and the rats in control group received injection of normal sal ine at the same dose. The expressions of bone morphogenetic protein 2 (BMP-2), matrix metalloproteinase 13 (MMP-13), and vascular endothel ial growth factor (VEGF) of trabecular bone were analyzed in the tibia by immunohistochemical staining at 1 and 3 weeks after injection. BMSCs from the rat femur at 1 and 3 weeks after injection were cultured under condition of osteogenic induction. ALP staining wasperformed on the 14th day after culture; real-time fluorescent quantitative PCR was used to detect the mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 on the 21st day after culture; and von Kossa staining was performed on the 28th day after culture. Results There was no significant difference in the expressions of BMP-2, MMP-13, and VEGF betweenthe experimental group and control group at 1 and 3 weeks after injection (P gt; 0.05). There was no significant difference in the percentages of ALP positively-stained cells between the experimental group and the control group on the 14th day after culture (P gt; 0.05). The mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 in osteogenic differentiation-inducedBMSCs had also no significant difference between the experimental group and the control group at 1 and 3 weeks after culture (P gt; 0.05). No significant difference in biomineral ization was found between the experimental group and control group at 1 and 3 weeks after culture (P gt; 0.05). Conclusion Subcutaneous injection of simvastatin [(5 mg/(kg•d)] for 1 or 3 weekscan affect neither the expressions of bone formation relative factors of proximal tibia trabecular bone nor the osteogenic differentiation of the BMSCs.
【Abstract】 Objective To produce a new bone tissue engineered carrier through combination of xenograft bone (X)and sodium alginate (A) and to investigate the biological character of the cells in the carrier and the abil ity of bone-forming in vivo, so as to provide experimental evidence for a more effective carrier. Methods BMSCs were extracted from 2-week-old New Zealand rabbits and the BMSCs were induced by rhBMP-2 (1 × 10-8mol/L). The second generation of the induced BMSCs was combined with 1% (V/W) A by final concentration of 1 × 105/mL. After 4-day culture, cells in gel were investigated by HE staining. The second generation of the induced BMSCs was divided into the DMEM gel group and the DMEM containing 1% A group. They were seeded into 48 well-cultivated cell clusters by final concentration of 1 × 105/mL. Seven days later, the BMP-2 expressions of BMSCs in A and in commonly-cultivated cells were compared. The second generation of the induced BMSCs was mixed with 2% A DMEM at a final concentration of 1 × 1010/mL. Then it was compounded with the no antigen X under negativepressure. After 4 days, cells growth was observed under SEM. Twenty-four nude mice were randomly divided into 2 group s (n=12).The compound of BMSCs-A-X (experimental group) and BMSCs-X (control group) with BMSCs whose final concentrat ion was 1 × 1010/mL was implanted in muscles of nude mice. Bone formation of the compound was histologically evaluated by Image Analysis System 2 and 4 weeks after the operation, respectively. Results Cells suspended in A and grew plump. Cell division and nuclear fission were found. Under the microscope, normal prol iferation, many forming processes, larger nucleus, clear nucleolus and more nuclear fission could be seen. BMP-2 expression in the DMEM gel group was 44.10% ± 3.02% and in the DMEM containing 1% A group was 42.40% ± 4.83%. There was no statistically significant difference between the two groups (P gt; 0.05). A was compounded evenly in the micropore of X and cells suspended in A 3-dimensionally with matrix secretion. At 2 weeks after the implantation, according to Image Analysis System, the compound of BMSCs-A-X was 5.26% ± 0.24% of the totalarea and the cartilage-l ike tissue was 7.31% ± 0.32% in the experimental group; the compound of BMSCs-X was 2.16% ± 0.22% of the total area and the cartilage-l ike tissue was 2.31% ± 0.21% in the control group. There was statistically significant difference between the two groups (P lt; 0.05). At 4 weeks after the operation, the compound of BMSCs-A-X was 7.26% ± 0.26% of the total area and the cartilage-l ike tissue was 9.31% ± 0.31% in the experimental group; the compound of BMSCs-X was 2.26% ± 0.28% of the total area and the cartilage-l ike tissue was 3.31% ± 0.26% in the control group. There was statistically significant difference between the two groups (P lt; 0.05). Conclusion The new carrier compounding A and no antigen X conforms to the superstructural principle of tissue engineering, with maximum cells load. BMSCs behave well in the compound carrier with efficient bone formation in vivo.
Objective To observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
ObjectiveTo review the mechanism and research progress of signal ing pathways which play key roles in the regulation of osteoblast differentiation and bone formation. MethodsRecent articles about signal ing pathways of osteoblast differentiation and bone formation were reviewed and comprehensively analyzed. ResultsAt present, multi ple signaling pathways have been found to be involved in the regulation of osteoblast differentiation and bone formation, among which bone morphogenetic protein-Smads, Wnt/β-catenin, Notch, Hedgehog, and fibroblast growth factor signaling pathways may play the most important roles. Not only each pathway has a complex regulatory mechanism itself, but also contacts and impacts with each other, thus they formed a more compl icated and sophisticated regulatory network, and regulate together osteoblast differentiation and bone formation. However, the mechanisms in detail of those pathways are still not very clear, because the animal experiment techniques are not yet mature as well as the relevant cl inical trials were carried out not too much. ConclusionThe complete molecular mechanism of osteoblast differentiation and bone formation should be further investigated, so as to lay a theory foundation for preventing and treating the common bone diseases in cl inical which are involve in osteoblast differentiation and bone formation.
ObjectiveTo summarize the research progress of the effects and mechanisms of Hedgehog signaling pathway in regulating bone formation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). MethodsThe related literature concerning the regulations and mechanism of Hedgehog signaling pathway in osteogenic differentiation of BMSCs and bone formation in vivo, in vitro, and ex vivo studies in recent years was analyzed and summarized. ResultsThe in vitro studies indicate that Hedgehog signaling pathway can promote osteogenic differentiation of BMSCs via activation of key molecules Smoothened (Smo) and Gli1 which are downstream of Hedgehog signaling, and Hedgehog signaling can activate mTORC2-Akt signaling by upregulation of insulin-like growth factor which has similar effects. Hedgehog signaling regulates osteoblast differentiation via activation of Hh-Smo-Ptch1-Gli signaling pathway and inhibition of Hh-Gαi-RhoA stress fibre signaling. Hedgehog signaling can regulate key molecules of osteogenesis Runx2 for promoting osteogenic differentiation and matrix mineralization by synergism of bone morphogenetic protein and Wnt signaling, and promotes bone formation and repair and healing for bone defect and bone graft model in vivo. ConclusionHedgehog signaling can regulate bone formation and osteogenic differentiation of BMSCs via activation of Hedgehog signaling and other signaling pathways. Hedgehog signaling pathway may be a potential target for developing treatment for bone related diseases of osteoporosis and fracture healing disorders.