Comparison Of Osteoinductivity And Immunogenicity Among Cyelosporine-impregnated Allograft Bone, Freeze-dried Bone Allograft And Homologous Bone

BACKGROUNDRepairing bone defect, especially larger section defect, has been one of the major problem for orthopedists for a long time. Solutions are mainly bone grafting and bone callus lengthening。For the bone graft material which mainly autologous bone, allograft bone, xenograft bone and four types of artificial bone.Among these, autogenous bone material has the advantage of higher engraft capacity, less immunologic reject response and quicker healing. But it also has limitations and complications that can not be ignored, such as limited resource, secondary damage; increased secondary infection that restricted its clinical application. Artificial bone material also has not been used extensively because of its poor inductivity, insufficient pores, small particles, higher resolvability, friability and lower pressure resistance. On the other hand, allograft bone material has been chose as the favored material more and more frequently.1880, Macewen conducted the first allograft bone transplantation in human history.1908, Lexer conducted the first allograft bone transplantation after a knee tumorectomy; 1915, "Bone Transplant Surgery" written by Albee was published. Since then, allograft bone material has become the most important part of orthopedic clinical work. It has convenient resource, unlimited shape and quantity. And it can facilitate quicker rebuilding the circulation of damaged internal environment around bone defect. It has better bone-formation and dynamic capability than artificial bone material, broader resource than autogenous bone material. Therefore, allograft bone material has been more and more extensively used in clinic on account of all these outstanding advantages and clinical value. However, every thing has two faces, allograft bone material also has some unresolved problems, such as immunologic rejection, secondary infection and delayed engrafting ext.. Engraftment of allograft bone is under influence of biologic and especially immune mechanisms. Immunologic rejection manifest as inflammatory cells infiltration around graft, new vessels' degeneration and occlusion, and it cause decrease of inductivity and conductivity, as well as comprehensively bone absorption, then delaying of engraftment, failing engraftment, osteoporosis and bone fracture. Still, allograft bone material is biologic material; it has certain immune antigenic characteristics. Immunologic rejection is the key element limiting the engraftment. So, milder immunologic rejection of allograft bone transplantation is the pivot issue to improve prognosis and engraftment. Till today, immunologic response is still one of the paramount subjects in transplantation area. Along the progressing research about material's biocompatibility and emerging issues of clinical application of material and equipment, people come to realize the importance of interaction between human immune system and biologic material. The biggest problem in early allograft bone transplantation is infectious disease and immunologic rejection. Rejection to graft is still the most important reason failing engraftment presently. Patient's immune system generates a series of immunologic response caused by foreign element which was transplanted into patient's body. Immunologic responses include inflammatory reaction, antibodies generating, complements activation, and all kinds of cytokines expression. Introducing immunologic evaluation to biologic material and medical equipment evaluation will provide necessary scientific evidence to reduce the risk in clinical application. Allograft bone material is one of the most frequently used graft in orthopedic, its immunogenicity and the effect of bone defect repairing with allograft bone material has obvious relativity.Earlier people had tried to use Sodium Mercurothiolate and other chemical methods to reduce allograft immunogenicity and sterilizing them, but to little avail. As the preparation process improvement, people gradually tend to use deep-cryogenic method, freeze-drying method and the y-ray irradiation to reduce the immunogenicity of allograft bone, in which y-ray irradiation has more powerful sterilization effect.After After several decades of practice,the bone bank all over the world has proved the above-mentioned method can reduce the immunogenicity of allogeneic bone and effectively reduces the result of bone graft due to the spread of disease risk, which is currently the most commonly used in allogeneic bone preparation methods. However, strictly speaking, the above method does not completely eliminate the immunogenicity of allogeneic bone, but after the above-mentioned method of treatment, allogeneic bone-forming properties and bio-mechanical properties of bone have been obvious damage. So in the clinical application,the effect of allograft bone to repair bone defects, in particular, to repair the effects of large bone defects still not satisfied. In view of this,the research group made new exploration of allogeneic bone preparation methods. In particular,we will compound cyclosporine in the process of preparation of allogeneic bone,which will a strong immune inhibition in the local. This approach will instead of deep hypothermia frozen, freeze-drying, and Y-ray irradiation in this respect. In order to better suppression of immune rejection,and reduce the damage of osteogenesis ability and mechanical properties of bone in the production process. In addition, we hope thatit will lower the side effects though the local application of cyclosporine.OBJECTIVEIn the preparation process, Cyelosporine-impregnated Allograft Bone need to be skimmed and Deproteinized.It will mitigated the immune prototype, but also caused damage to the BMP and other bio-active ingredients which may play an important role in the biological characteristics of bone. So it is necessary to evaluate the immunogenicity and induction of Cyelosporine-impregnated Allograft Bone.In this article,we will disscuss the possibility for the the clinical application of Cyelosporine-impregnated Allograft Bone which is combined cyclosporine A and sterilize by low-temperature plasma through the comparision among Cyelosporine-impregnated Allograft Bone,freeze-dried bone and Homologous bone in osteoinductivity and immunogenicity.METHODS1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.Separate the spleen cells of Balb/c mouse and dilute the cell original solution into a stock solution concentrations,2×105,1×105,0.5×105,0.25×105,0.125×105,0.0625×105cell/ml. In the 96-well culture plate add 200μl of cell suspension at different concentrations,each well repeat six times and set the zero holes.Method Alamarblue:Add 20μl Alamarblue for each well afther decking.Detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength) after cultured 0,2,4,6,20h respectively, In accordance with the instructions, using the formula to calculate the reduction rate Alamarblue. Method MTT:decking with method Alamarblue, Add 20μl MTT for each well.Add DMSO after cultured for 4h,then detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength). Precision Measurement: Group differences indicated by coefficient of variation CV. CV=group standard deviation of the data (s)/group of data within the average O.2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bonePreparation of Cyelosporine-impregnated Allograft Bone:separate the iliac bone of C57 mouse,make them particles,skimmed and deprotained combined cyclosporine A in solid dispersion method, then sterilized in low-temperature plasma.Preparation of freeze-dried bone:separate the iliac bone of C57 mouse, make them particles,rinsed with distilled water repeatedly freeze-dryed at freeze-drying machine,sterilized by y-ray irradiation.Preparation of Homologous bone:separate the iliac bone of Balb/c mouse, make them particles before operation, washed with normal saline repeatly.Preparation of Bone homogenate:take C57 mice and Balb/c mice limb bones, insed with distilled water repeatedly, dried and ground into bone meal,then add Cell culture medium.Preparation of Rectus femoris bag Mode:implant the bone particles into bilateral rectus femoris of Balb/c mice.Draw the materials at 2,4,6,8 weeks for alkaline phosphatase detect and MASSON staining.Operate the spleen cells of mice after 4 weeks,make a certain concentration of cell suspension,add the corresponding bone homogenate supernatant and Alamarblue indicator, detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength), calculate the stimulation index of each group. 3 Analysis the dates with SPSS 13.0, the experimental results Said using x±s,compare different means in variance analysis method, when p<0.05, the difference was statistically significant.RESULTS1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.When the concentration is between 0.0625×105～2×105cell/ml,the correlation between cell concentration and t the rate of reduction was not obvious at Oh (P>0.05) and 2h (P<0.05), the correlation between cell concentration and t the rate of reduction was significant relevance at 4h (P<0.001),6h (P<0.001),20h (P<0.001).Different cell concentrations with the culture time prolonged, reduction rate is also increased.20h maximum cell density reduction rate has yet reached 100%.Reduction rate of Alamarblue and OD value of MTT increases along with the cell concentration increased. The two pairs of correlation analysis, correlation coefficient r=0.998, P<0.05. There are two ways to positive linear correlation between the closely related and relevant.The CV (Alamarblue)=0.0152, CV (MTT)=0.0180 when Cell concentration is 2×105 cell/ml and the CV (Alamarblue)=0.024, CV (MTT)=0.068 when cell concentration is 0625×105 cell/ml.2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone2.1 Secondary lymphocyte stimulation testAnalysis the lymphocyte stimulation index of the groups after 2,4 weeks. using ANOVA of completely random design data. F=0.3228, P=0.7302> 0.05 between the Treatment groups after 2 weeks, differences between the three groups is not significant.4w, the deal between the two groups F= 253.6085, P<0.001 between the Treatment groups after 4 weeks. Differences between the three groups is significant. The difference among the three groups for further use Bonferroni test.The group of Cyelosporine-impregnated Allograft Bone and the group of Homologous bone has no significant difference in lymphocyte stimulation index.the group of freeze-dried bone and the group of Cyelosporine-impregnated Allograft Bone has significant difference. Freeze-dried group has a higher lymphocyte stimulation index.2.2 Detect alkaline phosphatase LocallyWe find that F values of different groups (F between groups=25.1807; P<0.001) and different time (F time=239.5745; P<0.001) are significant difference through muti-way classification ANOVA.Compare different means in one-way ANOVA,we find that the Alkaline phosphatase level of Cyelosporine-impregnated Allograft Bone group at 2,4,6,8w has statistical difference (F=76.9263, P<0.001). Alkaline phosphatase level of Homologous bone group at 2,4,6,8w has statistical difference (F=60.7506,P<0.001). Alkaline phosphatase level of Freeze-dried bone group at 2,4,6,8w has statistical difference (F=132.4105, P<0.001). Alkaline phosphatase level of different groups has no statistical difference at 2 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 4 w (F=9.0822, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 6 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 8 w (F=5.7737, P<0.05).2.1 Histological stainingThe amount of new bone of Cyelosporine-impregnated Allograft Bone group and Homologous bone group is higher than Freeze-dried group.Three groups were not found in inflammatory response around the implanted bone particles at different time points. CONCLUSION1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.Alamarblue method and MTT method are highly correlated. At high concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. At low concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. Demonstrate Alamarblue has no difference at stability and reproducibility with traditional MTT whenever the concentration is high and low.2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous boneIt is clear that the cyclosporine A around the bone particles depress the T-cell activation,and provide a good enciroment for the formation of new bone. So Cyelosporine-impregnated Allograft Bone has a stronger osteoinductive and a weaker immunogenicity.