Study Of Osteogenesis In Bone Defect Around Implant And Cell Signal Pathway Of RhBMP2/7

BMP2/7heterodimers were more effective than BMP homodimers in bone formation. And BMP2/7heterodimers don't have the side effect of BMP homodimers. BMP2/7heterodimers were promising in bone regeneration.The aim of the present research was to study the characteristic and mechanism of rhBMP2/7heterodimers in induing bone formation.Part1Materials and Methods:8-mm-diameter defects were created on the frontal skull of18minipigs. Collagen sponges with low-dose (30ng/ml) rhBMP2/7heterodimer, rhBMP2or rhBMP7homodimer were adopted and titanium implants were centrally implanted. New bone formation and the expression of type I collagen (Coll), alkaline phosphatase (ALP),osteocalcin (OCN) were evaluated after2,3, and6weeks of implantation.Results:RhBMP2/7resulted in significantly higher new bone areas percentage in the defect region than rhBMP2and rhBMP7(P<0.05). Immunohistochemical staining of Coll, ALP and OCN was stronger in rhBMP2/7group than rhBMP2, rhBMP7and control group (P<0.05).Conclusions:Based on the findings obtained, it could be concluded that rhBMP2/7heterodimer is a stronger inducer of osteoblastogenesis and could be applied at low dose to reduce the cost and side effects of rhBMP homodimers. Part2Materials and Methods:The same bone defect animal model in part one was used. Tartrate-resistant acid phosphatase(TRAP) stain was applied to examine the osteoclast in new bone. Expression of osteoprotrgerin (OPG),receptor activator of nuclear factor-KB ligand (RANKL) were evaluated after2,3, and6weeks of implantation.Results:The amount of osteoclasts in rhBMP2/7group was less than rhBMP2, rhBMP7and control group after2,3weeks of implantation. Immunohistochemical staining of OPG was stronger in BMP2/7group than rhBMP2, rhBMP7and control group (P<0.05). Immunohistochemical staining of RANKL was weaker in rhBMP2/7group than rhBMP2> rhBMP7and control group (P<0.05).Conclusions:The activation of osteoclast was weaker in BMP2/7group than rhBMP2, rhBMP7. RhBMP2/7can affect the OPG/RANKL/RANK system and bone remodeling.Part3Materials and Methods:Animal experiment:The same bone defect animal model in part one was used. Expressions of smadl,smad4,smad7and runx2were evaluated after2,3, and6weeks of implantation. Cell experiment:Preosteoblast cell line (MC3T3-E1) was cultured with stimulation of purified rhBMP2,rhBMP7homodimer and rhBMP2/7heterodimer. The gene expressions of Smadl,Smad4,Smad7and Runx2were detected by real-time RT-PCR analysis and normalized by P-actin gene expression.Results:Animal experiment:There was no obviously difference in expression of Smadl and Smad7among four experiment groups (P<0.05) in3and6weeks. There was no obviously difference in expression of Smad4among four experiment groups in2 and3weeks(P<0.05). The expression of Runx2in rhBMP2/7group was stronger than rhBMP2and rhBMP7(P<0.05).Cell experiment:In1st day, the gene expression of Smadl was weaker than rhBMP2but stronger than rhBMP7in the concentration period(0-150ng/ml)(P<0.05). The gene expression of Smad4was weaker in rhBMP2/7group than rhBMP2(P<0.05). The gene expression of Smad7and Runx2was weaker in rhBMP2/7group than rhBMP2,but stronger than rhBMP7(P<0.05). In4th day, the gene expression of Smadl in rhBMP2/7group was weaker than rhBMP2but stronger than rhBMP7in the concentration period(25-100ng/ml). The gene expression of Smad4was stronger in rhBMP2/7group than rhBMP2(P<0.05). The gene expression of Smad7was stronger in rhBMP2/7group than rhBMP2and rhBMP7(P<0.05) in the concentration period(0-50ng/ml). The gene expression of Runx2was stronger in rhBMP2/7group than rhBMP2and rhBMP7(P<0.05).Conclusions:In vitro and vivo experiment comparing with rhBMP2and rhBMP7, rhBMP2/7group showed unconspicuous acceleration with Smadl1, Smad4,Smad7and Runx2. We presumed that RhBMP2/7may increase the activation of Smadl,Smad4and decrease activation of Smad7in the phosphorylation of BMP-Smad Cell signaling pathway.