Immune Responses In Irradiated Bone Allografts

Objective: Bone graft is the most common tissue transplantation in human. Allo geneic bone graft is different from solid organ transplantation. Generally, the host immune rejection induced by allogeneic bone graft does not cause serious consequences of life-threatening, but it causes extension of the inflammatory response, delayed healing of the graft and cartilage degradation, which affect the outcome of treatment. In this study, we observed the expression of osteogeneic indicators and immune factors in mice allogeneic bone graft systematically and dynamically, which parallel with the host immune response to transplantation, and analysed some clinical cases to study bone formation and immune response in bone allograft patients. By these methods, we try to provide the basis for establishing cytokines qualitative and quantitative indicators and methods, which can be used to predict and diagnose bone allograft rejection.Methods: Experimental mice model: Get bilateral humerus, femur and tibia from Kunming mice, get Freeze-drying skimmed decalcificated bone, useγray for sterilization, implant the bones into anterior tibial muscles of 15 Kunming mice. Sacrificed the mice 1 week,2 weeks,3 weeks,4 weeks,5 weeks after implantation, remove implanted bone and surrounding muscles. Get three other Kunming mice which did not get bone implanted as the control group, cut anterior tibial muscle.①Use Elisa method to analyze the level of serum osteocalcin (BGP).②Measured stimulation index of spleen lymphocytes by 3H-TdR incorporation method.③Make paraffin sections of cut tissue, administer HE staining and immunohistochemistry staining of IL-1β,IL-6,IL-10 . Use western blot method to analyze the expression of IL-1β,IL-6 and IL-10.④Use PCR to analyze the level of TNF mRNA at transplantation site. Clinical cases: Get serum of seven patients who received bone allograft, and serum of seven normal people as control, use radioimmunoassay to analyze the level of serum BGP and TNF.Results: Experimental mice model:①One week after irradiated bone implantation, serum osteocalcin rised to 48.8ng/ml, and one week later it decreased to normal. ②After receiving irradiated massive bone allograft, stimulation index of spleen lymphocyte gradually decreased in 5 weeks.③Observe the expression of inflammatory suppressing cytokine IL-10 at implanting site after bone allograft for the first time. IL-10 increased gradually with time and reached peak at 4 weeks after implantation. As time grows, IL-1βand IL-6 in immunoreaction region increased gradually, reached peak at 3 weeks, and then decreased.④The level of TNFαmRNA at transplantation site increased gradually, reached peak at 3 weeks, and then decreased. Clinical cases: Serum osteocalcin and tumor necrosis factor in bone allograft patients were higher than in normal people.Conclusion:1)One week after implanting irradiated bone, serum osteocalcin increased, and then decreased, reflecting the active bone formation function of osteoblasts at the early stage of ectopic bone formation. 2)Stimulation index of spleen lymphocyte decreased after implanting irradiated allograft bone.3)After implanting irradiated allograft, inflammatory cytokine IL-1β,IL-6 and TNFαat implantation site increased after implantation, reached peak at 3 weeks and then decreased.4) Inflammatory suppressing cytokine IL-10 continued to express in immunoreaction region, the level of IL-10 at implantation site reached peak at 4 weeks after implantation and then decreased. The expression of inflammatory suppressing cytokines and pro-inflammatory factor show a time sequence.5) Serum osteocalcin and tumor necrosis factor levels in bone allograft patients were higher than in normal people. Serum osteocalcin and tumor necrosis factor levels were negatively correlated.In short, BGP is the important bone formation indicator in bone allograft, which increased in the early stage after bone allograft. Inflammatory cytokines IL-1β, IL-6, TNFαand inflammatory suppressing cytokine IL-10 are important indicators for the host immune response after the bone allograft. Serum osteocalcin and tumor necrosis factor levels showed a weak negative correlation.