| ObjectiveIn this study,using biphasic calcium phosphate of biological activity ceramics (Biphasic calcium phosphate,BCP) as the substrate,through compounding nano-hydroxyapatite(Nano-Hydroxyapatite,Nano-HA) and chitosan(Chitosan,Cs) to make out nano hydroxyapatite/chitosan/biphasic calcium phosphate composite material(Nano-HA/Cs/BCP),again loading recombinant human bone morphogenetic protein 2(Recombinant human bone morphogenetic protein 2,rhBMP-2) to make out recombinant human bone morphogenetic protein 2/nano-hydroxyapatite/chitosan / biphasic calcium phosphate composite material(rhBMP-2/Nano-HA/Cs/BCP).Then building muscular bag model in the erector spinae of rabbit,to evaluate the internal biocompatibility of the two composite materials,mainly on the influence of the blood system,liver and kidney,and local organization;furthermore,to observe ectopic bone formation in 16 weeks,to find out the ectopic osteogenesisoste,osteoconductivity and oinductivity of them,and thus to guide future research.Methods48 Japanese white rabbits were randomly divided into A group and B group,24 rabbits each group,embedded Nano-HA/Cs/BCP material(A material) into muscular bag of erector spinae formed in group A,and embedded rhBMP-2/Nano-HA/ Cs/BCP material(B material) in group B.Before operation,6 rabbits in each group were randomly selected to take routine blood test,detect C-reactive protein(CRP),liver and kidney function in the blood,which results used as normal reference value; 2,4,8and16 weeks after operation,rabbits were inspected the same way with preoperation,then compared and analyzed by statistics the results of two groups on the same period,preoperation and postoperation of each group.And the wound healing and general condition of all the experimental animals were observed after operation;4,8,12,16 weeks,postoperative X-rays were taken to observe the embedded material,and 6 rabbits were killed every time to obtain the liver and kidney specimens,and specimens of materials together with surrounding tissue for gross and optical microscope observation.Results1.Laboratory ExaminationAll the results of laboratory examination compared and analyzed by statistics between two groups in the same period,and between preoperative and postoperative each group showed no significant difference(P＞0.05).2.Gross ObservationThe diets and activities of rabbits after operation were normal;the wound healed in normal;and the liver and kidney had no obviously abnormal.A group:during the whole observation period,materials were closely integrated with the surrounding muscle tissue;the muscle had no congestion and edema,no infection and necrosis;and the interface between muscle and material revealed no significant fiber wall formation;surrounding and internal material had no cartilage and bone formation.4 weeks after operation,the surface of material was not smooth, pores enlarged,and a small amount of blood vessels and soft tissue grew into the pores which distributed in the peripheral material.8 weeks after operation,the surface and internal material were covered with blood vessels and soft tissue,pores enlarged significantly.12 and 16 weeks after operation,the surface of material was covered by new vascular,and the internal pores were filled with a large number of blood vessels and soft tissue.B group:during the whole observation period,,materials were closely integrated with the surrounding muscle tissue;the muscle had no congestion and edema,no infection and necrosis;and the interface between muscle and material revealed no significant fiber wall formation.4 week after operation,a large number of blood vessels and soft tissues grew into the surface and peripheral pores of material,and less was distributed in center of materials.8 weeks after operation,a large number of blood vessels and soft tissue were distributed in the increasing surface pores of the material;and the internal pores of material were filled by intensive blood vessels;the number of pore was increased,and the size was enlarged.12 weeks after operation, the edge of the materials appeared crushed powder;internal porosity increased significantly and were full of new blood vessels and soft tissue.16 weeks after operation,the edge of the materials had a small amount of bone,and the texture was hard.3.Histological ObservationLiver and kidney:both of them were normal.A group:4 weeks after operation,fibroblasts,new blood vessels and fibrous tissue were observed in the muscle and interface;muscle cells were slightly shrinking, and the main inflammatory cell were lymphocytes and macrophages.8 weeks after operation,the interface formed a thin layer of fiber membrane,including a small amount of dense connective tissue and fibroblasts;a large number of blood vessels and fibrous tissue were in the material;and a small amount of macrophage was found in the material.12 weeks after operation,the fiber membrane in the interface became thinner;the internal and peripheral pores of materials were distributed by a large number of blood vessels and fibrous tissue;and the macrophages and fibroblasts were in the material;16 weeks after operation,fiber membrane was thinne and became incomplete,and the enlarged pores were filled with blood vessels and fibrous tissue.no bone,osteoblasts and chondroblastoma were found in the whole experiment.B group:muscle tissues were normal during the 16 weeks.4 weeks after operation,the interface formed a thin and incomplete layer of fiber membrane, fibroblast,angiogenesis and loose connective tissue were in the muscle;a small amount of lymphocytes and macrophage were found;fibrous tissue and blood vessels grew into the peripheral and surface pores.8 weeks after operation,fiber membrane had no significant changes,and fiber proliferation in the muscle decreased;there were no significant infiltration of inflammatory cell;a large number of capillaries and collagen fibers were in the material;12 weeks after operation,the fiber membrane became significantly thinner,a lot of fibrous tissue and blood vessels grew into the surface pores,no fibrosis were seen in the muscle,a small amount of cartilage formed in the peripheral materials,and cartilage lacuna,and chondroblastoma were found;16 weeks after operation,the edge of materials formed a small amount of osteoid and bone.4.Imaging Observation4,8,12 and16 weeks after operation,X-ray showed no significant change in the two material profile,but in the two materials had gradual emergence of translucent and the material density reduced gradually;16 weeks after operation,local marginal area of B material appeared high density shadow.Conlusions1.Two kinds of composite material implanted in rabbits for 16 weeks did not cause significant local tissue necrosis,local inflammatory reaction and rejection;the major blood components,inflammatory index,liver and kidney function index analyzed by statistics between the relults of preoperation and postoperation,showed no statistical significance;and observation of liver and kidneyed had no obvious abnormalities;all results above indicated two materials showed good biocompatibility in the blood system,local tissue,liver and kidney.2.B material(rhBMP-2/Nano-HA/Cs/BCP) embedded in the erector spinae of rabbit formed a small amount of bone in the16th week,which indicated A material loaded low dose of rhBMP-2(10μg) showed certain osteoconductivity and osteogenesis,and rhBMP-2 showed certain osteoinductivity.3.A material(Nano-HA/Cs/BCP) embedded in the erector spinae of rabbit showed no bone formation during 16 weeks,which indicated the material did not show osteoinductivity and osteogenesis during the time.4.In order to fully evaluate the in vivo biocompatibility of the composite materials,the long-term effects of the material and its degradation products on the local tissue and the whole body still need further study.5.A material(Nano-HA/Cs/BCP) as an ideal carrier of rhBMP-2 still need to improve and optimize.
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