Experimental Study Of Distribution And Removal Of Xenogenous Targeted Antigen On Porcine Bone Tissue

Objective: (1) To investigate the distribution of xenogenous targeted antigen on porcine bone tissue. (2) To study the change of xenogenous antigen,biomechanical and physical properties, the porcine bone tissues were processed with three different methods. (3) To investigate the cellular compatibility of xenogenous bone scaffold materials. (4) To observe the immunological rejection and the turnover of the bone graft, the porcine bone tissues with different methods was implanted into the rabbits.Methods: (1) Distribution ofα-Gal,MHC-â… and MHC-â…¡antigen on porcine bone tissue were observed by immunohistochemistry. (2) Physical properties and heteroantigens of porcine bone were detected: The experiment was divided into group A,B,C,D according to different materials. Group A: porcine bone was processed with defatting and partial deproteinization; Group B: porcine bone was preserved in liqiud nitrogen;Group C: porcine bone was preserved in liqiud nitrogen and was digested by enzyme; Group D: fresh porcine bone. The change of expression of heteroantigen was detected by immunohistochemistry, and the microstructure and biomechanical properties of different groups were observed by Electronic Scanning and INSTRON 8874. (3) Cellular compatibility of xenogenous bone scaffold materials: Osteoblasts of rabbit were cultured with the bone scaffold materials of group A, group B, group C,respectively. Osteoblasts in 1×10⁷/ml density were cultured with the three materials for 7 days. And the adhesion and growth of osteoblasts were observed with phase microscope and electronic scanning microscope, the cell cycle was analyzed with flow cytometer. The growth of osteoblasts were observed on 1,3,5,7 day were detected by MTT assay. (4) The immune reaction of bone xenotransplantaion: The experiment was divided into group A,B,C,D,E,F according to different materials. Group A:porcine bone was processed with defatting and partial deproteinization;Group B: porcine bone was preserved in liqiud nitrogen; Group C: porcine bone was preserved in liqiud nitrogen and was digested with enzyme;Group D: fresh porcine bone; Group E: bone atuograft; Group F: control group. The bone scaffold materials were implanted into the bilaterial spinal muscles of rabbits. At 1,2,4 weeks postoperatively, the examination of CD4⁺,CD8⁺,CD4⁺/CD8⁺ in blood was detected by flow cytometer, and the levels of IL-2 and IL-4 in blood were analyzed by quantitative ELISA assay.The infiltration of lymphocytes and macrophages surrounding soft tissue of bone graft were also observed in order to evaluation biocompatibility of bone graft. (5) The effect of repairing bone defect with different processed porcine bone scaffold materials: Sixty New Zealand white rabbits were chosen, and the radial critical bone defects were made for 15mm long. The experiment was randomly divided into group A,B,C,D,E,F according to different materials, and each group details was equal to part (4). The bone defects were grafted by xenogenous bone materials derived from different groups. Groups of rabbits were sacrificed and the specimens were procured at 6,12,24 weeks after surgery for examination X-rays photography and histology evaluation. The ability of repaii'ing bone defect of each group was evaluated by Lane-Sandhux radiographic scoring. To study the bone defect repairing and reconstruction, new bone formation and substitute, the 5μm paraffin slices were obtained and were stained by hematoxylin-eosin and Masson method.Results:(1)α-Gal,MHC-â… xenogenous antigens were extensively observed on the surface of bone marrow cells,osteocytes, osteoblasts and Harversian canals, and the MHC-â…¡antigen mainly expressed on the bone marrow cells.(2) Change of physical property and xenogenous antigens: No positive antigen expression in group A and group C, a few positive xenogenous antigens expression were detected in group B, while many positive xenogenous antigens expression were detected in group D. The results of pore size showed that group A＞group C＞group B＞group D, there was no significant difference between group B and group D (P＞0.05), and there was significant difference among the other groups (P＜0.05); The results of porosity showed that group A＞group C＞group B＞group D, there was no significant difference between group A and group C, group B and group D (P＞0.05). Biomechanical analysis demonstrated that the maximum load in axial compression test, group D＞group C＞group B＞group A, the minimum load was observed in group A and it was significantly inferior than the other groups (P＜0.05), there was no significantly difference among group B,group C and group D (P＞0.05).(3) The results of cellular compatibility of xenogenous bone scaffold materials demonstrated that rabbit osteoblasts were survival in all groups, and the cell proliferation reached the peak at the fifth day. The cellular compatibility was detected by electronic scanning microscope and MTT assay, the results showed that group C＞group A＞group B. Compared to group B, the xenogenous bone materials in group A and group C had little influence on the G₀/G₁ stage and S stage of the rabbit osteoblasts.(4) CD4⁺, CD8⁺ T lymphocytes gradually increased in 2 weeks after operation. The CD4⁺/CD8⁺ ratio reached the peak at 1 week after operation, then decreased gradually. The detection of CD4⁺/CD8⁺ ratio at 2 weeks postoperatively, there was no significant difference among group A,group C and group E, while the CD4⁺/CD8⁺ ratio reached higher level. Evaluation of IL-2 and IL-4 in serum by ELISA assay, the highest level" of IL-2 and IL-4 was detected in group D at 1 week after operation, the lowest level was in group F. Compared to group A and group C, the level of IL-2 and IL-4 were increased obviously in group B at different intervals after operation (P＜0.05). There was medium dose of lymphocytes infiltration surrounding the soft tissue of bone graft in group B, and there was few lymphocytes infiltration in group A and group C.(5) According to the Lane-Sandhux radiographic scoring and the histological evaluation, the ability of reconstruction of radial defect was followed, group E＞group C＞group A＞group B＞group D＞group F.Conclusions:(1)α-Gal,MHC-â… xenogenous antigens were extensively observed on the surface of bone marrow cells,osteocytes,osteoblasts and Harversian canals, and the MHC-â…¡antigen mainly expressed on the surface of bone marrow cells.(2) The expression of xenogenous antigen was decreased obviously in group B, but there was still some of positive expression of antigen on the porcine bone tissues. The xenogenous antigen was effectively deleted in group A and group C.(3) The results of pore size showed that group A＞group C＞group B, the pore size of porcine bone materials derived these groups could meet the requirement of osteoblasts ingrowth, but porosity of group B was lowest, which maybe hinder the ingrowth of osteoblast. The xenogenous bone materials had the poor biomechanical performance in group A. Compared to the normal fresh porcine bone tissue, there was no significantly influence on the biomechanical performance in group B and group C.(4) The cellular biocompatibility showed that group C＞group A＞group B.(5) Immunological rejection of bone xenograft occurred during 2 weeks after operation, the host immunological response was lightly in group A and group C, and there was obviously immunological rejection in group B.(6) The ability of reconstruction of radial bone defect demonstrated that group C＞group A＞group B.