不同退变程度人髓核脱细胞外基质支架的生物学特性研究

Study on the biological characteristics of human nucleus pulposus decellularized extracellular matrix scaffolds with different degrees of degeneration

中文关键词:  髓核  退变程度  髓核脱细胞外基质  组织工程

英文关键词:Nucleus pulposus  Degrees of degeneration  Nucleus pulposus extracellular matrix removal  Tissue engineering

中文摘要:

  【摘要】 目的：探索不同退变程度（Ⅱ、Ⅲ、Ⅳ度）人髓核脱细胞外基质（nucleus pulposus decellularized extracellular matrix，NP-dECM）支架的生物学特性，并选取出生物学特性最优的NP-dECM支架。方法：采用Ⅱ型胶原酶消化法提取人髓核间充质干细胞（nucleus pulposus mesenchymal stem cells，NPMSCs），镜下观察人NPMSCs贴壁、细胞形态及生长情况，行流式细胞术检测细胞表面标志物CD73、CD90、CD105、CD34、CD45、HLA-DR，并进行成脂、成骨、成软骨诱导分化，观察人NPMSCs的三系分化能力，以确定其为本研究所需要的种子细胞。采用物理、去污剂联合核酸酶法制备不同退变程度的人NP-dECM支架（分别为Ⅱ-D、Ⅲ-D、Ⅳ-D组）。通过肉眼观、DNA及细胞外基质（extracellular matrix，ECM）成分的定量和定性检测、扫描电镜等对比，评价不同退变程度的人NP-dECM支架脱细胞的效果。并运用CCK-8法和活/死细胞染色体外评估不同退变程度人NP-dECM支架的细胞相容性。结果：从髓核组织分离培养的人NPMSCs贴壁生长，细胞呈短梭形、纺锤形，第3代人NPMSCs高表达CD73、CD90、CD105等表面标志物，低表达CD34、CD45、HLA-DR等表面标志物，同时人NPMSCs具有成脂、成骨和成软骨分化能力，符合间充质干细胞标准。三种退变程度的人NP-dECM支架肉眼观形态保持完好，电镜下各组支架疏松多孔。HE染色、DAPI染色及DNA测定结果显示三种退变程度的人NP-dECM支架均有效去除了细胞成分。GAG和HYP定量及定性检测结果均显示ECM成分保留良好，GAG保留量从高到低依次为Ⅱ-D组＞Ⅲ-D组＞Ⅳ-D组，Ⅱ-D组和Ⅲ-D组HYP含量差异无统计学意义，均高于Ⅳ-D组。CCK-8及活/死细胞染色结果表明Ⅱ-D组、Ⅲ-D组和Ⅳ-D组均表现出良好的细胞相容性。结论：Ⅱ度退变的人髓核组织为制备人NP-dECM支架最佳选择。

英文摘要:

  【Abstract】 Objectives: To explore the biological characteristics of human nucleus pulposus decellularized extracellular matrix(NP-dECM) scaffolds with different degrees of degeneration(Ⅱ, Ⅲ, Ⅳ), and to select the NP-dECM scaffold with the best biological characteristics. Methods: Nucleus pulposus mesenchymal stem cells(NPMSCs) were isolated by collagenase type Ⅱ digestion method. Cell adhesion, cell morphology and growth of NPMSCs were observed under microscope. The cell surface markers CD73, CD90, CD105, CD34, CD45, HLA-DR were detected by flow cytometry, and the differentiation ability of NPMSCs was observed by adipogenic, osteogenic and chondrogenic induction, so as to confirm that they were the seed cells needed for this study. Human NP-dECM scaffolds with different degrees of degeneration were prepared by physical, stain remover and nuclease(group Ⅱ-D, Ⅲ-D and Ⅳ-D). The decellular effect of NP-dECM scaffold was evaluated by visual observation, quantitative and qualitative detection of DNA and extracellular matrix components, and SEM. The cytocompatibility of human NP-dECM scaffolds with different degrees of degeneration was evaluated using CCK-8 method and living/dead cells in vitro. Results: Human NPMSCs isolated and cultured from the nucleus pulposus tissue grew adherent, and the cells were short spindle shaped and spindle-shaped. The third passage of human NPMSCs expressed high levels of surface markers such as CD73, CD90 and CD105, and low levels of surface markers such as CD34, CD45 and HLA-DR. It met the criteria of mesenchymal stem cells. Human NP-dECM scaffolds with three degrees of degeneration remained intact, and the scaffolds of each group were loose and porous under electron microscope. HE staining, DAPI staining and DNA determination showed that the NP-dECM scaffold effectively removed cell components in the three degrees of degeneration. The quantitative and qualitative detection results of GAG and HYP showed that ECM was well retained, and the GAG retention was in the order of group Ⅱ-D > group Ⅲ-D > group Ⅳ-D from high to low. There was no statistical significance in HYP content between groups Ⅱ-D and Ⅲ-D, which was higher than that in group Ⅳ-D. The results of CCK-8 and live/dead cell staining showed that groups Ⅱ-D, Ⅲ-D and Ⅳ-D showed good cytocompatibility. Conclusions: The Ⅱ degree degeneration of human nucleus pulposus tissue is the best choice for the preparation of human NP-dECM scaffold.