制备去细胞髓核支架最佳条件的初步优化研究

Primary research on optimal method for rat nucleus pulposus decellularization scaffold

目的探讨机械振荡和去污剂浓度对髓核去细胞支架形态学的影响,建立一种髓核的优化脱细胞方法。方法取成年兔髓核分成A、B、C(含C1/C2/C3子组)、D、E组,并在不同震荡频率下用Triton X-100和脱氧胆酸钠处理。振荡频率分别为0 r/min(A组,n=5)、80 r/min(B组,n=5)、130 r/min(C1、C2、C3组,n1=n2=n3=5)、180r/min(D组,n=5)以及对照组(E组,n=5),其中C组中3个子组(C1/C2/C3)的去污剂的浓度分别为1%、3%、5%,其余组均为3%。对所得标本行HE染色和扫描电镜观察,比较其细胞清除情况和基质形态学变化。结果 A、B及C1组残留大量脱核的髓核细胞胞体,C2、C3及D组细胞被彻底清除。所得支架呈一种无细胞的三维网状结构,但C3和D组纤维结构出现明显破坏。结论合适的机械振荡频率及去污剂浓度在脱去髓核细胞的同时还能较好地保存髓核内天然的基质纤维和三维空间结构。

Objective To investigate the effects of mechanical vibration and extracting agent concentration on the morphology of the acellular scaffold for the nucleus pulposns（NP）, and to establish an optimal method for NP decellularization. Methods The segments of rabbit NP were divided randomly into 5 groups and subjected to mechanical vibration at the frequency 0 r/min （Group A, n =5）, 80 r/rain （Group B, n =5）, 130 r/min （Group C1 ,C2,（3, nl =n2 = n3 =5）, 180 r/rain（Group D, n =5） and control group （Group E, n =5） respectively. In Group C, 3 subgroup （Group C1/C2/（3, n =5） were divided randomly and concentration of ex~＇acting （TritonX-100 and sodium deoxyeholate） were 1%, 3%, 5% in sequence, and in other groups concentration of extrac- ting was 3%. The specimens were observed microscopically with HE staining, and the ultrastructure was observed using scanning electron microscope. Resttlts In Group A, B ,C1 the NP specimens contained numerous ceils or cell nucleus matter, and all the NP cells or cell nucleus matters were eliminated in Group C2, （3,D after treatnnt with TritonX-100 and sodium deoxycholate. In Group （3 and D, however, obvious disruption of the matrix fiber occurred in the acellular NP. Conclusion All the NP cells can be removed by chemical extraction with Triton X-100 and sodium deoxycholate. Mechanical vibration at suitable frequency and appropriate concentration of chemical extraction preserve the3-dimensional structure of ＆e matrix fibers.