The Influence Of Defatting On Immunogenicity And Osteogenicity Of Cancellous Bone Allograft

ObjectiveTo explore the influence of defatting on immunogenicity and osteogenicity of cancellous bone allograft and the possible mechanism.Methods1. Animal model establishment. 15 inbred Lewis rats and 15 inbred Fisher344 rats were killed. Proximal tibial and distal femoral metaphyses were resected as cancellous bone grafts respectively. 30 inbred Fisher344 rats as the transplant recipients were devided into 2 groups: group IT(Isotransplantation) and group AT(Allogeneic Transplantation). Bone grafts were transplanted into double femoral muscle pouches of recipients. 5 rats of each group were killed at the 2^nd,4^th,8^th week post-operation respectively. Antigen stimulated T-lymphocyte proliferation index and ratio of CD4⁺/CD8⁺ were tested and histological changes of transplanted grafts were observed.2. Bone allograft preparation. 60 inbred Lewis rats were killed. Proximal tibial and distal femoral metaphyses were resected as cancellous bone grafts. Grafts were divided into 4 groups (A,B,C and D) randomly which were treated by 4 different procedures respectively. Content of total lipid and total protein in grafts were measured. Network structures were observed by SEM.3. Immunogenicity detection. 48 inbred Fisher344 rats were divided into 4 groups (A,B,C and D) randomly according to different grouped grafts which were transplanted into double femoral muscle pouches of the recipients. 3 rats of each group were killed at the 1^st,2^nd,4^th,8^th week post-operation respectively. Proliferation index and ratio of CD4⁺/CD8⁺ were tested, levels of IL-2,INF-γ,IL-4 were measured and histological changes of transplanted grafts were observed. The correlation and regression analysis was performed between proliferation index,CD4⁺/CD8⁺ ratio at the 4^th week post-operation and the content of total lipid.4. Osteogenicity detection. 12 inbred Fisher344 rats were divided into 4 groups (A,B,C and D) randomly according to different grouped grafts which were transplanted into femoral metaphysis bone defects of the recipients. Two different fluorochrome dyes were given subcutaneously to the recipients on 14^th and 4^th day before sacrifice respectively. All rats were killed at 8^th week post-operation and X-ray examinations were performed. The experimental distal femoral metaphyses were harvested and nondecalcified sections were prepared. Sections were observed with a fluorescent microscope and bone histomorphometris were studied. To observe histological changes, sections were stained by Goldner and toluidine blue.Results1. At the 2^nd,4^th,8^th week post-operation, both proliferation index and ratio of CD4⁺/CD8⁺ were higher in group AT (Allogeneic Transplantation) than in group IT (Isotransplantation) (P<0.05) and reached the peak at the 4^th week. Histological observations showed less vascular and new bone formation in group AT at the 8^th week.2. Results of measurement showed content of total lipid in four grouped grafts were group A>group B>group C>group D and the difference between groups were statistically significant (P<0.05). There was no significant difference of protein content between groups (P>0.05). SEM observations showed network structures of bone trabecula in every graft were intact. Network structures in group D were very clear and no cells and tissue debris were seen.3. Proliferation index increased in the early phase and reached the peak at the 4^th week post-operation. Level of proliferation index in four groups were gradiently decreased from group A to group D. At the 4^th week post-operation, difference of proliferation index were statistically significant between groups ?(P<0.05 ) , except between group C and group D (P>0.05 ) . The changing trend of ratio of CD4⁺/CD8⁺ was similar with that of proliferation index. Level of IL-2 and INF-γincreased in early phase and reached the peak at 4^th week post-operation. But the differences of level of IL-2,INF-γ,IL-4 were not statistically significant between groups at any time post-operation. Histological observations showed inflammatory cells infiltrated in early phase post-operation were gradiently decreased from group A to group D. At the 4 week post-operation, histological observations showed neovascularization in group D. At the 8^th week post-operation, more new bone formation was observed in group D. The correlation and regression analysis showed significant positive correlation between proliferation index,CD4⁺/CD8⁺ ratio at the 4^th week post-operation and the content of total lipid (P<0.05).4. At the 8^th week post-operation, X-ray examinations showed margins of bone defects were obscure and grafts were fusing with recipents' bone in all groups. Fluorescent microscopy observations showed double colored parallel lines were around margins of trabecula. Mineral Apposition Rate (MAR) in group D were biggest and there was significant difference between group D and other groups. Both Goldner and toluidine blue staining observations showed more new bone formation in group D.Conclusion1. Animal model designed by us could be used to study the immunogenicity of cancellous bone allografts.2. Content of total lipid could be one indicator of determining the immunogenicity of cancellous bone allografts.3. Defatting could decrease immunogenicity and increase osteogenicity of cancellous bone allografts.4. Both ultrasonic cleaning and chemical method could remove the lipid but not the protein of cancellous bone. Procedure which combines ultrasonic cleaning with chemical method could defat the grafts more thoroughly and decrease immunogenicity significantly.