To Antigen Heterogeneous Cancellous Bone And Rabbit Osteoblasts Histocompatibility Of Experimental Research

Objective:To observe the influence of xenoantigen expression and distribution on porcine cancellous bone matrix by different preparation methods and to study the histocompatibility changes of rabbit osteoblasts with porcine cancellous bone matrix prepared by different methods. For the clinical application of bone xenograft to repair bone defects and joint disease provide some experimental evidence.Methods:Use of the deep cryogenic frozen,the enzymatic digestion and combination of deep cryogenic frozen and enzymatic digestion preparation porcine cancellous bone matrix.The experiment was divided into group A、B、C、D,according to different bone matrix.Group A:fresh porcine bone matrix;Group B:deep cryogenic frozen bone matrix;Group C:enzymatic digestion bone matrix;Group D:combination of deep cryogenic frozen and enzymatic digestion bone matrix.The change of the histological charateristics of each group of matrix was detected by hematoxylin-eosin staining and the change of distribution and expression of α-Gal antigens,MHC-Ⅰ antigens and MHC-II antigens was detected by immunohistochemical.At the same time using inverted phase contrast microscope,optical microscope and scanning electron microscopy to obverse rabbit osteoblasts into the adhesion of the growth and proliferation of osteocytes in the surface after rabbit osteoblasts were cultured with bone matrix of group B、C、D respectively.Results:Heamatoxylin-eosin staining was observed by microscope that lacunae, osteocytes,osteoblasts and a large number of the fat network structure,containing bone marrow cells was widely distributed in fresh bone matrix.Deep cryogenic frozen bone matrix containing a large number of lacunae,a small amount of osteocytes, osteoblasts, bone marrow cells and fat network structure.Enzymatic digestion bone matrix containing a large number of lacunae,with a very small amount of osteocytes, osteoblasts,no fat in the bone marrow cavity. Combination of deep cryogenic frozen and enzymatic digestion bone matrix containing a large number of lacunae,and no osteocytes, osteoblasts,no fat network structure and bone marrow cells in the bone marrow cavity.Immunohistochemistry was observed by microscope that the a-Gal antigens,MHC-Ⅰ antigens were widely distributed around the bone marrow cells, osteocytes and osteoblasts in fresh bone matrix.While MHC-Ⅱ antigens distribution around the bone marrow cells only.Deep cryogenic frozen bone matrix showed the lacunae contained a small amount of osteocytes retained,cells surface distribution and expression of a-Gal antigens,MHC-I antigens and MHC-Ⅱ antigens.Enzymatic digestion bone matrix showed lacunae within a small amount of osteocytes retained,cells surface of a small amount of MHC-Ⅰ antigen and MHC-II antigen expression and distribution and no distribution and expression of the a-Gal antigen. Combination of deep cryogenic frozen and enzymatic digestion bone matrix showed no different from the positive expression of the antigens.Prepared by different methods of porcine cancellous bone material with rabbit osteoblasts cultured demonstrated that rabbit osteoblasts were survival in all bone matrix.The cellular compatibility was detected by electronic scanning microscope that combination of deep cryogenic frozen and enzymatic digestion bone matrix was a large number of cells growth on the surface of the bone matrix,adhesion into a sheet structure,cell morphology,polygonal or fusiform pseudopods intertwined reticular structure covering the pore surfaces,cell surface was spindle extension of enzymatic digestion bone matrix,merging into a piece of not more than. There was a small amount of cells growth on the surface of deep cryogenic frozen bone matrix,adhesion,cell spreading is not sufficient.Conclusion:The combined of deep cryogenic frozen and enzymatic digestion method can effectively removal of the xenoantigen,the deep cryogenic frozen method alone can significantly reduce the xenoantigen distribution of porcine cancellous bone,but can not be completely effective removal of the major xenoantigen,pure enzymatic digestion method can significantly reduce the a-Gal antigens distribution in the porcine cancellous bone,but for the MHC-I antigen and MHC-II antigen can hardly be effectively removed.From the result of rabbit osteoblasts were cultured with each of different preparation porcine cancellous bone material,the combined method of deep cryogenic frozen and enzymatic digestion preparation porcine cancellous bone materials in histocompatibility was better than the deep cryogenic frozen method alone and pure enzymatic digestion method of cancellous bone material.