去细胞髓核支架材料制备方法的比较学研究

Comparison study on different methods for preparation of acellular nucleus pulposus scaffold

目的探索去细胞化天然髓核支架,以构建组织工程椎间盘修复脊柱退行性疾病。方法 1个月龄及1岁龄Beagle犬各4只,平均体质量分别为0.6kg、7.5kg。取结构完整椎间盘髓核组织各40块。随机分为4组,每组10块。分别采用化学萃取及酶消化2种方法分别处理Beagle犬椎间盘髓核组织,进行形态学和组织学比较研究,分析并比较不同方法及不同年龄髓核供体的去细胞效果及对基质结构的完整性。以噻唑蓝法进行间接接触细胞毒性实验检测去细胞髓核的细胞生物相容性。结果 1岁龄髓核去细胞处理后残余细胞分别为(4.32±4.15)个/高倍视野(化学萃取组)、(4.98±4.52)个/高倍视野(酶消化组),显著高于1个月龄髓核组,且结构完整性显著改善。化学萃取与酶消化2种方法制备的去细胞髓核浸提液在1周内的细胞毒性分级均为0级与1级。1个月龄髓核去细胞效果良好,但无论化学萃取或是酶消化法均对基质成分有严重破坏作用,不宜作为去细胞髓核支架的供体。结论酶消化法能够在去除犬1岁龄髓核细胞成分的同时更好地保留细胞外基质框架结构,无明显的细胞毒性,不影响细胞的正常功能;是制备去细胞髓核天然支架的良好方法。

Objective To compare the effect of two decellularizing methods for the preparation of acellular nucleus pulposus,and provide ideal scaffold for tissue engineered intervertebral disc which has potential application in treatment of disc degenerative diseases.Methods Integrated nucleus pulposus was harvested from 1-month-old（mean body weight 0.6 kg） and 1-year-old（mean body weight 7.5 kg） Beagle’s dogs,and 80 of complete structured nucleus organization were collected,which were divided into 4 groups（n = 10） and then decellularized by the method of chemical extraction（immersed in 30 mL/L Triton X-100 and 40 mL/L sodium deoxycholate） or by enzyme digestion（immersed in 0.5 % Trypsin followed by the mixture of 1 × 103 U/mL DNase and RNase） respectively.The degree of decellularization,demyelization and integrity of the scaffold construct were assessed by HE staining and scoring system.The cytocompatibility of acellular nucleus pulposus was measured by methyl-thiazolyl-tetrazolium（MTT） assay.Results The decellularizing effects of 1-month-old nucleus pulposus by the 2 methods were satisfactory,which were（4.32 ± 4.15） / high power field（group chemical extraction） and（4.98 ± 4.52） / high power field（group enzyme digestion）.But the scaffold construct were seriously damaged at the same time,and cytotoxicity classification in 1-week were grade 0 and grade 1,respectively.Nucleus pulposus cells of 1-year-old Beagle’s dogs disappeared by the method of enzyme digestion and maintenance of scaffolds were superior over that of chemical extraction method.Conclusion It is demonstrated that decellularized nucleus pulposus of 1-year-old Beagle’s dogs by enzyme digestion maintains normal function without obvious cell toxicity,so it could be a good candidate for natural acellular scaffold based tissue engineering of intervertebral disc.