Heterotopic Ossification Formation Proteomics Preliminary Study Of The Process Of Dynamic Change

Purpose:To develop a rabbit model of heterotopic ossification by surgical method and evaluate radiologic,histologic and serologic changes,which are determinates to decide whether the animal model is suitable for studying a stable process of ectopic bone formation in muscle.Methods:This experiment was carried out on eighty hips of forty New Zealand White rabbits,which were divided into four groups corresponding to different surgical methods.Heterotopic ossification was probably induced bilaterally using surgical procedures to enter the medullary canal of femoral bone,infuse muscle tissue with bone marrow and reserve blood supply to injured muscle tissue.Six rabbits of each group were made a radiograph four months,eight months and twelve months postoperatively.Heterotopic ossification was graded according to a modification of the scale of Brooker et al.The mean grade,the interobserver and intraobserver reliability,and the significance(P＜0.05) of the differences between the groups were evaluated.Two rabbits of each group were killed for histological examination every four weeks after operations.Results:The induction rate of HO(the scale of Brooker grade 2 or higher) and levels of HO increased gradually until twelve months postoperatively.Groups with bone marrow infusion had a higher induction rate of HO(the scale of Brooker grade 2 or higher) than the groups without bone marrow infusion in the three different time points postoperatively.The differences between Groups with blood supply reservation and groups without blood supply reservation did not approach significance.The group with bone marrow infusion and blood supply reservation had higher grade HO than the group without the two factors in each time point(four-month,eight-month and twelve-months after surgery). Serum alkaline phosphatase level in each group peaked at postoperative four-month stage,decreased moderately at postoperative eight-month and twelve-month stage.The decrease rate of group with two factors was less than group without the two factors.Histological findings demonstrated process of ectopic bone formation in group with the two factors at three time points was more continuous and stable than other groups.Conclusion:We demonstrated that HO can be induced by using surgical procedures to enter the medullary canal of femoral bone,infuse muscle tissue with bone marrow and reserve blood supply to injured muscle tissue.This animal model could be used for evaluation of ectopic bone formation at different stage.Bone marrow infusion played chief role in promoting HO. Blood supply reservation had a synergistic effect on the process of ectopic bone formation.The level of serum alkaline phosphatase could be an indicator to judge severity of high-grade HO and possibility of ectopic bone proliferation. Purpose:To prevent loss and degradation of total protein of bone,maintain concentration of protein extraction especially the low molecular proteins and reduce concentration of metal ion in protein extraction,we develop a modified method to extract total proteins in bone tissue,which is feasible for proteomic analysis.Method:We optimized the traditional protocol including three basic steps (grinding,dialyzing and acetone precipitation).Normal New Zealand White rabbit bone tissue of proximal femoral shaft was divided into two parts.Protein extraction of one part was followed traditional protocol and the remains was extracted by another protocol including decalcification and ultrasound-concussion under 4℃.A comparison of the two protocols was evaluated by Bradford assay and MS compatible silver stain SDS-PAGE.Result:With a comparison of the traditional protocol,the modified one can reduce loss and degradation of proteins in extraction,strengthen protein extracting from the part difficult to dissolve,increase concentration of total protein extraction,decrease concentration of metal ion in solution,and enhance sharpness of separation especially low molecular proteins.Conclusion:Optimized protocol of bone tissue extraction is compatible to subsequent proteomic study based on 2DE technical platform better.