Development of Refolding Process to Obtain Active Recombinant Human Bone Morphogenetic Protein-2 and its Osteogenic Efficacy on Oral Stem Cells

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED₅₀ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

Double-layer nanoparticles loaded with rhBMP-2 and SDF-1:Preparation,characterization and drug re-lease in vitro / 实用口腔医学杂志

Objective:To construct double-layered controlled release system containing SDF-1 and rhBMP-2 molecules and to study the release profile of the system in vitro.Methods:The polylactic acid/chitosan(PLA/CS)nanoparticles were prepared with “emulsification-solution evaporation method”,the preparation parameters were determined by orthogonal test design.The particle size was observed by nanoparticle size analyzer,the morphology of the nanoparticles was observed with electron microscope.Then rhBMP-2 and SDF-1 were loaded into the nanoparticles in the process of emulsification,the loading efficiency and encapsulation efficiency were calculated and in vitro release was observed.Results:The double-layer nanoparticles showed spherical geometry,smooth surface and complete separation. The average particle size of the nanoparticles was (542.33 ±14.38)nm;The drug loading and incorporation efficiency of rhBMP-2 were (82.41 ±1.05)% and (24.67 ±0.43)ng/mg,those of rhBMP-2 were (75.58 ±0.84)% and (22.63 ±0.41)ng/mg,respectively. The release time of the drug from the system sustained over at least 30 days,the release profile of both drugs showed “biphasic release”. The cumulative release rate of SDF-1 and rhBMP-2 was 72.85% and 91.01% in 30 days respectively.Conclusion:The SDF-1 and rh-BMP-2 loaded PLA/CS nanoparticles have excellent morphology,high entrapment and good sustained-release in vitro.

Treatment of femoral neck fracture using modified cannulated screw with injectable BMP release system / 中国矫形外科杂志

[Objective]To treat the femoral neck fracture using cannulated screw and BMP release system in animals,and to evaluate the possibility of treating femoral neck fracture and provide experimental basis for clinical application.[Method]Eighteen mongeral were used in this study.The model of bilateral femoral neck dislocation fracture was established.The control side was fixed using cannulated screw,and the experiment side was fixed with cannulated screw and injectable BMP release system.At 4,8,12 weeks,6 animals were sacrificed at one time point respectively.Results were obtained through histology,radiography,scintimetry and gross observation.[Result]The fracture line was vague at four weeks,and at eight weeks the fracture line almost disappeared.It was healed completely at twelve weeks.Radiological study showed that the healing of the fracture in experimental side was better than that of the control side.There was the same result of observation in histology.[Conclusion]The cannulated screw combined with BMP used in the experiment is effective and feasible.It may not only provide strong internal fixation but also infuse growth factor into site of fracture.It would accelerate the reconstructing of the vascular supply to the femoral head after the fracture and promote the restoration of bone.

Effect of human bone morphogenetic protein-2 on vascular endothelial growth factor expression in fetal mouse osteoblasts / 中华创伤骨科杂志

Objective To investigate effects of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) on vascular endothelial growth factor (VEGF) expression in fetal mouse osteoblasts.Methods Calvaria osteoblasts of fetal mice of 19 days were cultured.The effects of rhBMP-2 at different doses and different action times on VEGF expression patterns in fetal mouse osteoblasts were observed with reverse transcrip- tion-polymerase chain reaction (RT-PCR) and Western blot staining.Results In the present study,RT-PCR detected a steady expression of VEGF mRNA in the control fetal mouse osteoblasts.The levels of VEGF mRNA increased in an apparent biphasic manner with a maximum stimulation (about 2-fold above the control,P＜0.05 ) in both VEGF mRNA species observed at 300 ng/mL of rbBMP-2.After 48 h of rhBMP-2 treatment,the VEGF mRNA levels approached those in the control.The VEGF mRNA levels appeared to be biphasic in rhBMP-2-treated cultures,showing peak induction at 3 and 24 h and remaining elevated at 48 b.Compared with the individual control value at each time point,an apparent maximum increase (about 2.5-fold above the control,P＜0.05) occurred at 6 h.The second peak induction,about 2-fold above that in the control,occurred at about 36 h.Conclusions The expression of VEGF mRNA is steady in the control fetal mouse osteoblasts.RhBMP-2 can promote the expression of VEGF in dose-dependent and time-dependent manners.

The effect of rhBMP-2 on the osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts in vitro / 대한치주과학회지

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts(control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. beta-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.