Preparation Of Sintered Bone Surface Modified And Bone Formation Investigation In Vitro And In Vivo

Part Ⅰ Preparation of sintered bone surface modified and a study of its physico-chemical property and its biocompatibilityObjective The aim of this study is to investigate a new method for preparing a biomimetic bone material-sintered bovine cancellous bone surface modified, and to improve its bioactivity as a tissue engineering bone.Methods The same size of the prepared sintered bovine cancellous bone was randomly divided into two groups, immersing in simulated body fluid (SBF) and1.5SBF, respectively. The three time points of soak time are7days,14days, and21days. After sintered bone were dried, the samples at each time point were sprayed gold, and then the surface morphology of sintered bone and surface mineralization composition were observed under scanning electron microscopy (SEM). By comparing the effect of surface modification of sintered bone materials, we chose the best ideal material and to study its pore size, and the rate of the porosity, and the compress and bend intensity. And the material and the sintered bone material without surface modification were compared. The two group materials at each time point under SEM were observed and analyzed. And then its biocompatibility was preliminarily evaluated. Results The surface of the material soaked in1.5SBF for7days showed that there was scattered apatite, and complete and uniform apatite surface layer in the14th day, and cluttered and cracked apatite surface layer in the21st day. But the surface apatite formed of sintered bone immersed in SBF for7days,14days, and21days were all a little. There was no significant difference in physico-chemical properties between sintered bone immersed in1.5SBF for14days and the sintered bone without surface modification. And its biocompatibility was good.Conclusion The sintered bone material immersed in1.5SBF for14days showed the best effect of surface modification, and retaining the original physico-chemical properties of sintered bone, with good biocompatibility. Adhesion, proliferatin and osteodifferentiation of MC3T3-E1cells on sintered bone surface mineralization modified/BMP-2-related peptide biomaterialObjective In this research, a new bone tissue engineering scaffold material, sintered bone surface mineralization modified/BMP-2-related peptide P24composite was constructed. The effect of composite to the adhesion, proliferation and osteodifferentiation of MC3T3-E1cells were investigated.Methods Sintered bone was modified to improve its osteoconductivity and osteoinductivity. The peptide P24was introduced into the prepared sintered bone surface mineralization modified. And sintered bone surface mineralization modified/BMP-2-related peptide P24composite was synthesized. Then its in vitro release kinetics was then measured. The biomaterials were divided into three groups:Group A (sintered bone surface mineralization modified/BMP-2-related peptide P24composite); Group B (sintered bone surface mineralization modified) and Group C (sintered bone). MC3T3-E1cells were cultured on three kinds of material respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1cells were measured by MTT assay and scanning electron microscope (SEM). And the measure of alkaline phosphatase (ALP) activity and ALP staining and calcium tubercle staining (alizarin red staining) were performed to assess the differentiation of cells into osteoblasts.Results The in vitro experiment showed that BMP-2-related peptide P24had a slow release characteristics on the surface of sintered bone surface mineralization modified.The adhesive ratio measure and MTT assay and SEM observed suggested that adhesion and proliferation of MC3T3-E1cells on the surface of sintered bone surface mineralization modified/BMP-2-related peptide P24composite is much higher than Group B and Group C (P<0.05). The ALP activity of MC3T3-E1cells in sintered bone surface mineralization modified/P24composite group were significant higher than in sintered bone surface mineralization modified group and sintered bone group. And in the assessment of ALP staining and calcium tubercle staining (alizarin red staining), positive staining was expressed firstly in Group A.Conclusion The sintered bone surface mineralization modified/BMP-2-related peptide P24composite material was confirmed to improve the adhesion rate and proliferation of MC3T3-E1cells, and maintained their morphology. The sintered bone surface mineralization modified/P24composite material was an ideal carrier material, which made for the osteogenic activity of P24. Part ⅢExperimental study on ectopic osteogenesis of a BMP-2-related peptide/type Ⅰ collagen complexObjective To study the ectopic osteogenetic capacity of synthesis BMP-2-related peptide/type Ⅰ collagen from rat rail support by animal experiment.Methods To extract type Ⅰ collagen from rat rail by oneself and then to observe its structure after freeze-dried under low-vacuum electron microscopy. SD rats were divided into two groups (control group, n=6; experiment group, n=18). Pure type Ⅰ collagen from rat rail was implanted as control group, while the BMP-2-related peptide/type Ⅰ collagen from rat rail complex was implanted as experiment group. Twenty-four SD rats were gained to establish models of quadriceps femoris myo-bag that the right side thigh of each rat making2cm opening and divided into2groups with6in each group, implanting the above material in the myo-bag separately. The rats were made the radioactivity inspection (X-ray, CT) separately at the3,6th week after implantation. At the6th week, all rats were killed and tissue response was observed histologically (HE and Von Kossa staining).Results The diameter of the pores of type Ⅰ collagen from rat rail after freeze drying is appropriate, and that of the BMP-2-related peptide/type Ⅰ collagen from rat rail complex changes not obviously, and it is one kind of ideal carrier. At the3,6th week after implantation, radioactivity inspection showed that experimental group had the obvious calcification shade formation. At the6th week, the scope became bigger than that of the3rd week, but the control group was not the osteogenesis phenomenon. At the6th week, histology observation indicated experimental group have osteoblast and massive new bone formation around the implants, but the control group only saw inflammatory cells.Conclusion Type Ⅰ collagen from rat rail is an ideal scaffold material for bone tissue engineering. BMP-2-related peptide complexed with rat rail collagen can induce ectopic osteogenesis more effectively. Part Ⅳ Repair of rabbit radial bone defects using sintered bone surface mineralization modified combined with BMP-2-related peptideObjective To observe the feasibility and efficacy of repair of rabbit radial bone defects using sintered bone surface mineralization modified combined with BMP-2-related peptide.Methods36New Zealand white rabbits (male or female) were randomly divided into three groups. A1.0mm rabbit radial bone defect was created in the left middle diaphysis in all rabbits. Three kinds of materials were used respectively:Group A (BMP-2-related peptide P24/sintered bone surface mineralization modified composite); Group B (sintered bone surface mineralization modified) and Group C (sintered bone). The three groups were observed by scanning electron microscopy (SEM) pre-surgery, respectively. All animals were not fixed post-surgery. Radiographic examination (X-ray and3D-CT) and histological examination (HE staining and Masson staining) were taken to evaluate the bone repair capability of each kind of sintered bone materials.Results SEM showed that three groups of materials retained the natural pore structure and the pore size was200μm～850μm. White flocculent or fibrous active peptide was observed on the surface of sintered bone surface mineralization modified in Group A. And complete and uniform apatite surface layer was observed on the surface of sintered bone in Group B. X-ray examination showed:At4th week, in group A, callus around the implant material was abundant, and the boundary between bone callus and host bone became obscure; callus around the implant material was less in group B; the boundary between bone callus and host bone was clear and no significant callus formed in group C. At8th week, in group A, the boundary between bone callus and host bone became more obscure, bone defect almost disappeared and a large number of bone callus formed; callus around the implant material increased in group B; the boundary between bone callus and host bone was obscure in group C. At12th week, in group A, implanted materials and host bone united naturally, callus began shaping and the canal recanalizated; bone defect almost disappeared in group B; bone defects became obscure in group C. At different postoperative time points, the X-ray scores on the GroupA were significantly higher than GroupB and Group C (P<0.05).3D-CT at12weeks post-surgery showed that bone defect was repaired on the whole, the boundary between new bone and host bone was no obvious in group A; bone defect was not completely repaired in group B; bone defect was partially repaired in group C. Histological examination (HE staining) results showed the score of Group A was significantly higher than that of Group B and Group C (P<0.05). At12 weeks post-surgery, Masson staining results showed a large number of new bone and lamellar bone formed, irregular bone marrow cavity and a large number of fibrous tissue was visible in group A; new bone formation in group B was less than that of group A; bone defects are filled with a lot of fibrous tissue, less new bone formed, and marrow cavity was closed in group C.Conclusion This biomaterials sintered bone surface mineralization modified/P24is an ideal scaffold for bone tissue engineering, which could trigger typical endochondral bone formation and improve repair of bone defect.