不同脱脂方法对异种松质骨脱脂效果及免疫原性的影响

Effects of different defatting methods on degreasing effect and immunogenicity of xenogeneic cancellous bone

目的比较研究不同脱脂方法对异种松质骨脱脂效果及免疫原性的影响,以优选最佳脱脂工艺。方法将成年牛松质骨用4种脱脂方法处理,A组为化学法,B组为超声波清洗法,C组为超临界CO2萃取法,D组用纯化水冲洗(对照组)。观察处理后的异种松质骨大体结构,检测异种松质骨的总脂肪含量、总蛋白含量及力学强度,MTT法检测异种松质骨细胞毒性。将异种松质骨植入小鼠体内后2周检测各组脾脏中淋巴细胞增值率,以及血清IgG、IgM含量。结果A组与C组异种松质骨清洗后效果相当,但明显优于B组。A、B、C组极限载荷低于D组,且C组>A组>B组;A、B、C组总脂肪含量较D组明显降低,且C组<A组<B组;B组总蛋白含量高于A组与C组,差异有统计学意义(P<0.05)。A组与C组总蛋白含量差异无统计学意义(P>0.05)。MTT检测结果发现B组与C组异种松质骨无细胞毒性。B组淋巴细胞增值率与血清中IgG、IgM含量高于A组和C组,差异有统计学意义(P<0.05);而A组和C组淋巴细胞增值率与血清中IgG、IgM含量差异无统计学意义(P>0.05)。结论超临界CO2萃取法的脱脂效果与化学法相似,且生物相容性优于化学法,对异种骨力学性能无明显影响,是一种较优的脱脂方法。

Objective To compare effects of different degreasing processes on the degreasing effect and immunogenicity of xenogeneic bone and select the optimal degreasing process. Methods The heterogeneous cancellous bones were treated by four different processes, group A: chemical reagent soaking;group B: ultrasonic cleaning;group C: supercritical carbon dioxide extraction;group D: simple flushing of purified water (control group). The macrostructure after treatment was observed, the fat content and protein content after treatment were detected, the mechanical strength was measured by a mechanical testing machine, and the cytotoxicity of the four groups of materials was determined by MTT assay. The bone fragments obtained by different treatment methods were implanted into mice, and the samples were taken at 2 weeks after operation to detect the lymphocyte transformation rate in the spleen and the contents of serum immunoglobulin IgG and IgM. Results The effects of heterogeneous cancellous bone cleaning in group A and group C were comparable, but significantly better than group B. The ultimate load of group A, B and C was lower than that of group D, and C>A>B;the fat content of group A, B and C was lower than that of group D, and C<A<B;the protein content of group B was higher than that of group A and C, the difference was statistically significant (P <0.05). There was no significant difference in protein content between group A and group C (P > 0.05). MTT test results showed that the heterogeneous cancellous bone of group B and group C was not cytotoxic. The lymphocyte proliferation rate and serum IgG and IgM levels in group B were higher than those in group A and C was statistically significant (P <0.05). The difference of lymphocyte proliferation rate and serum IgG and IgM content between group A and group C was not statistically significant (P >0.05). Conclusion Supercritical carbon dioxide extraction is equivalent to chemical degreasing effect, better than physical method, safer and more economical than chemical method. It is safer and economical than chemical methods, and can be used as a better degreasing method in industrial production.