The Experimental Studies On Biphasic Calcium Phosphate Nanocomposite As The Scaffold In Bone Tissue Engineering

Large amounts of bone grafts are frequently used to reconstruct the maxillofacial bone defects resulted from tumor resection , trauma and congenital disease.Autograft and allograft bones are often used, but both of them have limitations. To avoid the potential risks, artificial substitutes including bioceramics, polymers, alloys, and composite biomaterials have been studied, Among which calcium phosphate ceramics, e.g. HA and [3-TCP have been extensively studied for their good biocompatibility and osteoconductive properties.Biphasic calcium phosphate nanocomposite is a new kind of biomaterial. In our experiments, the Biphasic calcium phosphate nanocomposite were prepared and we investigated their components, characteristics, osteogenic ability and biocompatibility and explored its application as the scaffold in bone tissue engineering.At first, TEM , XRD, and SEM were used to characterize the scaffolds. The experimental results showed that the Hydroxyapatite nanoparticles were in shape of short rods with a length of 50 ~ 80nm and a diameter of 20~40nm. XRD showed that the Biphasic calcium phosphate nanocomposites were composed of HA and TCP. SEM showed that the pore size was 100-400um.The average bending strength of scaffold was 4.6 Mpa and the compression strength of scaffold was 8.5 Mpa .The porosity of the scaffold was 60%-80% .The above characteristic surface configuration meets the needs of bone tissue scaffolds.Then the hemolysis test, hemopexis test, acute toxicity test and subcutaneous implant test were induced to evaluate biocompatibility of the NanoBCP. Results show: The material had no hemolytic activities and did not influence the function of hemopexis. The material were found no toxicity by animal test. Subcutaneous implant test revealed the fibrous tissue could grow into the material and the material had no irritation to subcutaneous tissue . Only in some specimens a minimal inflammatory response was observed due to surgery injury and partly degradation of the material.The NanoBCP scaffold had good biocompatibility.In order to evaluate the cellular biocompatibility of the NanoBCP scaffold , we get BMSCs obtained from SD rat bone marrow, which were induced and proliferated in vitro after their osteoblast phenotype were verified , BMSCs were seeded onto prepared porous Nano BCP (Experiment group) and BCP (Control group). The cell adhension were evaluated by scanning electron microscope and reverse phase difference microscope. Synthesis of ALP and OC were detected and cell cycle were detected by FCM too. Results show BMSCs could fully attach to and extend on Experiment and Control group. The differentiation between BMSCs and osteoblastic phenotype was demonstrated by the positive staining of mineralized node, ALP and collagen I . FCM shows that the Osteoblasts cell cycle was obviously affected by when they are cultured withscaffolds. The cell number decreased in GO/G1 phase but increased in S > G2/M phase and the PI increased , on the contrary, the cell number of apoptosis in experiment group was less than that in the control group. Synthesis of AL P , OC in experiment group is more than that in the control group when they are cultured with BMSCs. Result demonstrated experiment group were superior to Control group in adhension, proliferative abilities and osteogenic activity.In order to understand the adhesion characteristic between cell and scaffold , and get some theory data. In our experiment, SD rats bone marrow stromal cells (BMSCs) that had been induced and proliferated were seeded onto prepared porous NanoBCP (Experiment group) and BCP (Control group) . Their adhesion situation were analyzed by reverse phase difference microscope and scanning electron microscope ,and the initial optimal cell seeding density between new pattern porous NanoBCP was investigated by MTT automated colormetric microassay method .Results shows the cell number in Experiment group was more than that in Control group. The most appropriate seeding density between them was 2.0 xlO6/ml. The maximal number which BMSC could adhere to porous NanoBCP was 3.2x 107/cm3.In order to understand osteogenic capability of NanoBCP loaded with BMSCs, our study use two methods, one is the ectopic osteogenesis, the other is osteogenesis in situ. In Ectopic osteogenesis experiment , the BMSCs were isolated , expanded and induced in vitro and seeded in the NanoBCP and BCP scaffold. The scaffold/BMSCs composites were implanted subcutaneously into the backs of nude mice. Implantation of the NanoBCP alone was used as control. At 8 weeks after implantation, the specimenswere harvested and histologic observation showed much more newborn bone had formed in the surface and pores of the NanoBCP. RT-PCR showed Col-lmRNA and OCmRNA express positive well in the NanoBCP/ BMSCs group. The NanoBCP/ BMSCs have better osteogenic capability than the BCP/ BMSCs.In the osteogenesis in situ experiment, NanoBCP was used as bone tissue engineering scaffold for repairing of calvarial defects in order to observe the osteogenesis when loaded with BMSCs.The NanoBCP/BMSCs composites were implanted into the calvarial defects of rat. Implantation of the NanoBCP alone served as controls. At 16 weeks after implantation the newly formed tissue engineering bone were integrated with the calvarial bone in experiment group. The NanoBCP /BMSCs composites can repair calvarial defects effectively.The porous NanoBCP as bone tissue engineering scaffolds had good cell biocompatibility, when it was loaded with BMSCs, new bone formation could be observed. This study suggested that the NanoBCP could be a promising scaffold material for bone tissue engineering.