Study On Nano-phosphate Artificial Bone Scaffolds Manufacture And Bioactivity Evaluation In Vivo And Vitro

Objective:It is very difficulty to surgrons that repair the large defect in maxilla and mandible in clinic. Traditional biological methods of bone-defect management including autografting and allografting bone and metal implantation are limited in clinical use. Nano-phosphate artificial bone scaffolds with higher bioactivity and biocompatibility were manufactured according to biomimetic and self-organization mechanism, then they were evaluated in vivo and vitro in the study.Methods:Raw collagen was extracted from bovine tendon in acetic acid, then by salting out, dialysis and lyophilization , pure collagen scaffold was acquired. Methods including ultraviolet spectrophotography ,amino acid analysis, histology observtion .scanning electron microscope were used to evaluate the pure collagen. According to biomimetic and self-organization mechanism , nano-phosphate artificial bone scaffolds were manufactured with two kinds nano phosphate(nano-hydroxyapatite and nano-tribasic calcium phosphate) and pure collagen . Generally observation, scanning electron microscope, Porosity measurement, X ray diffraction were used to analyze two scaffolds. We obtained rabbit bone marrow stromal cells from rabbit thighbone,then these cells were proliferated and induced to rabbit osteoblasts. Cell morphology,akaline phosphatase activity detect, Von-Kossa stainand and flow cytometer were utilized to identify rabbit osteoblasts characteristic.These cells were inoculated into two nano - scaffolds. Control groups included particles hydroxyapatite/collagen artificial compounded bone scaffold and pure collagen scaffold. We evaluated bioactivity and biocompatibility of four scaffolds by invert microscope and scanningelectron microscope. In vivo test , model of rabbit skull defect was created firstly, thus, four kinds scaffolds were embeds in rabbit skull defect, one vacuity as control. The vivo study involved 8 rabbits, merely scaffolds were embed in 4 rabbits, the rest were osteoblast culture scaffolds. In 2,4,8,12 weeks, rabbits were sacrificed in sequence and skull defect repair speciments were obtained for digital X ray measurement and histology observation.Result:The pure collagen characteristics included about 225nm absorbency, no tryp-tophan, about 24.85% aminoacetic acid. Two kinds of nano-phosphate artificial bone scaffolds form was similar to sponge, the pore size was 100-300um, the ratio of porosity was above 90%, and inner structure was homothetic to the nature bone . The cells induced from bone marrow stromal cell had osteoblast characteristics such as positive stain of akaline phosphatase and Von-Kossa and classic osteoblast shape. The cell cycle and DNA analysis showed that the percentage of G0+G1 and G2+S of primary osteoblast was 89.3%, 10.7% respectively. No cytotoxicity was found in osteoblast- scaffold combined culture in vitro and no acute or chronic immunologic rejection in vivo. Two nano- phosphate repaired the rabbit skull defect completely about 12 weeks,but particles hydroxyapatite/collagen compounded artificial bone scaffold or collagen scaffold could not in same time. Osteoblast combined scaffolds were better than merely scaffolds on osteogenesis in the same period. Pure collagen was coin- cidence with type I collagen. Pure collagen block had good strcture to form bone scaffold. Rabbit bone marrow stromal cells could be cultured .proliferated and induced to osteoblast, suited to act as seed for bone tissue engineering. Two kinds nano- phosphate artificial bone scaffolds structure was homothetic to nature bone structure, and had good bioactivity , biocompatibility, osteogenesis and fitting degradation.