A Study Of Biological Safety Of Magnesium Phosphate Cement

Background:Trauma, tumor, surgery and other reasons nonunion often results in the absence of bone, osteoblasts difficult to climb over the gap and can not have the normal healing process, resulting eventually in nonunion. The body's own autogenous bone has a good bone induction, however, subject to the same kinds of bone and heterogeneous sources of supply constraints bone. Allogeneic bone-borne diseases there is the risk and incidence of immune rejection of the possibility of clinical application subject to certain restrictions. The use of PMMA (polymethylmethacrylate, PMMA) bone cement inside the implant through the enhancement of the surrounding cancellous bone to achieve the purpose of strengthening internal fixation. However, due to PMMA curing exothermic and can not be absorbed by the body and reshape impede fracture healing, these shortcomings significantly limit the scope of its clinical application. In recent years, as represented by the inorganic calcium phosphate artificial bone replacement materials research and development has grown considerably.East China University of Technology Professor Liu Changsheng and its partners in key projects of Shanghai Science and Technology Commission (98JC 14015) funded by the successful development of a new type that can absorb inorganic bone cement that is magnesium phosphate cement (Magnesium Phosphate Cement, MPC). Through the appraisal (02 G00 182) to reach the international advanced level of research and has been patented. Patents No. ZL01105373.9; PTC international patents: PTC/CN01/00282. In the city, funded by Science and Technology Commission (from September 2003 to September 2006, the surface of the project, numbered as follows: 034119904), preliminary studies have shown that:MPC bond strength significantly greater than the traditional calcium phosphate bone cement, can be small chip fracture in the non-weight-bearing area can be directly adhesion; materials, closely integrated with the surrounding bone, the interface does not appear significant accumulation of inflammatory cells and fibrous membrane, did not cause foreign body reaction, non-toxic internal organs, through preliminary experimental results are satisfied with the the implanted material, the material and its metabolites is an impact on the surrounding tissue, whether there is genetic toxicity, carcinogenicity and reproductive toxicity, especially on osteoblast proliferation and differentiation of speed of impact and whether the surrounding organization's cells caused by mutations in the role of further study. In addition, their in vivo would activate the cell inflammatory cytokines, triggering an abnormal immune reaction, there is no precise data, which will also be an element of the study.Objective:Since Magnesium phosphate cement(MPC) has been researched and developed,the preliminary studies have shown that the bond strength with MPC was significantly greater than the traditional calcium phosphate bone cement, a small chip fracture of the non-weight-bearing area can be directly bonding;Materials, closely integrated with the surrounding bone, the interface does not appear significant accumulation of inflammatory cells and fibrous membrane, did not cause body reaction, non-toxic internal organs. Biological characteristics of MPC, preliminary experimental results are satisfactory. However, new materials for the implant, this topic will examine whether the material and its metabolites have an impact on the surrounding tissue, especially on osteoblast proliferation and differentiation,produce mutagenic role in vivo of the surrounding tissue cells, would activate the cell inflammatory cytokines, triggering an abnormal immune reaction.in line with the requirements of biological safety, to test whether its biocompatibility is good or not.Method:According to research purposes, the subject is divided into three parts.1 Genetic toxicity test:(1)Ames test:Preparation of extract of MPC,Normal saline as a negative control, to take specific positive things for the positive group,Add a rat liver microsomal enzyme S9 conditions and without S9,to test the Mutagenicity Ratio(MR) of each group.To observe whether MPC extract will cause the reverse mutation.(2)Unscheduled DNA Synthesis, UDS test:using human peripheral blood, take different concentrations of MPC extract as a test group, normal saline as a negative control group, NiSO4 as positive group, RPMI 1640 cell culture medium added to hydroxyurea and 3H-TdR, After co-culture, shock, the termination of the reaction, cells were collected, washed, fixed, dehydrated; adding scintillation fluid, counting radioactivity measuring instrument do. Test results with radiation counts per minute, on behalf of DNA synthesis.(3) Micronucleus test:Taking the healthy mature male Kunming mice were divided into three groups, different concentrations of MPC extract as test group, normal saline as a negative control group, cyclophosphamide as a positive group, in mice, intraperitoneal injection of regular intervals of 24 hours, four times a dose method. Extraction of intra-bone marrow polychromatic erythrocytes, smear, fixation, staining, counting micronuclei under the microscope to calculate the percentage of micronuclei arise.2 In vitro cytotoxicity test:(1) Take the third generation of mouse osteoblasts, made cell suspension with different concentrations of MPC extract the experimental group, normal cell culture medium for the normal control group, observed 1 day,3 days,5 days, by adding MTT, and dimethyl sulfoxide (DMSO), using enzyme-linked immunosorbent assay instrument was measured at 570 nm absorbance and calculating the cell relative growth rate (RGR), and grade toxicity.(2) Take the third generation of mouse osteoblasts, made cell suspension with different concentrations of MPC extract for the trial group, phenol standard solution as the standard group, double-distilled water as a blank control group, normal 10% DMEM cell culture medium as negative the control group. After 1,3,5 days observation, after using alkaline phosphatase detection kit, the enzyme-linked immunosorbent detector were measured at 510 nm wavelength light absoiption value of phenol content in the calculation.3. Inflammatory cytokine expression tests:Take the third generation of mouse bone cells, producing cell suspension of different concentrations of MPC as a pilot group of extracts, cell culture medium as negative control, using IL-1β, IL-6, TNF-a antigen-antibody kit Determination of double-antibody sandwich made to observe the 1,3,5 days after the termination of the substrate Yexian color, microplate reader at 450 nm readings, drawing graph analysis.Results:1 In the Ames test, TA100, TA102, TA97, TA98 strain positive control group back to change the number of colonies exceeding two times higher than the negative control group (MR> 2); MPC dose groups extract of different concentrations and negative control of the colony average Group's ratio of less than 2 (MR<2); 2 UDS test, different concentrations of MPC leaching liquid radioactive counts per minute (CPM) values are concentration-dependent, with the increased concentration of leaching solution, CPM value also gradually increased; different concentrations of MPC extract the CPM values are much lower than the positive control group (different concentrations NiSO4) of the CMP value;3 Micronucleus test, the cyclophosphamide positive control group micronucleus rate 10 times higher than the negative group, with the MPC extract group of micronuclei values also differ by more than 10 times; different concentrations of MPC micronucleus rate, and negative control There were no significant differences; different concentrations of the micronucleus rate is not with the corresponding concentration gradient, and normal saline-negative group close to not cause polychromatic erythrocyte micronucleus rate of increase;4 MTT test, MPC test group of mice osteoblasts relative growth rate (RGR) in 1,3,5 days respectively,87.37%～115.13%,118.96%and 98.96%-88.46% 108.92%; cell toxicity grading are 0 or 1; different concentrations of MPC leaching solution had no effect on cell growth;5 ALP detection tests, with the extract concentration increased, ALP values decreased; different concentrations of MPC extract the value of the ALP as compared with the negative control group, no statistically significant differences, does not affect the bone cells in normal growth.6 Determination of inflammatory cytokine expression test, with the concentration of extract increased the expression of inflammatory factors there was no significant increase; different concentrations of MPC extract as compared with the negative control group, no statistically significant difference between the molecular level will not promote the expression of inflammatory factors.Conclusion:1 Magnesium phosphate cement does not cause Salmonella typhimurium reverse mutation to increase the amount does not cause genetic change;2 Magnesium phosphate cement leaching solution had no damage on the DNA synthesis;3 Magnesium phosphate cement for animal bone marrow erythrocyte micronucleus mutation does not produce effects on animals, the short term showed no mutagenic effect; 4 Magnesium phosphate cement on the osteoblast cells had no significant impact on the cells, no significant toxicity, have a good cell compatibility;5 Magnesium phosphate cement in the cellular and molecular level will not promote the expression of inflammatory factors, does not induce inflammatory response.