EDC交联结合化学萃取制备脱细胞脊髓支架及其生物学特性研究

EDC crosslinking and chemical extraction for preparation of spinal cord scaffolds in a rat model

[目的]采用EDC(碳化二亚胺)交联结合化学萃取法制备脱细胞脊髓支架,探讨脱细胞脊髓支架制备效率,分析支架的生物学特性,观察支架能否保留细胞外基质黏多糖成分。[方法]采用EDC交联结合化学萃取法(液氮结合42℃恒温水浴反复冻融6次+1%Triton X-100+1%脱氧胆酸钠+EDC交联+液氮结合42℃恒温水浴反复冻融6次+1%Triton X-100+1%脱氧胆酸钠)处理正常脊髓20根,得到脱细胞脊髓支架(支架组);对照于正常大鼠脊髓组织(对照组)。通过HE染色检验支架组脱细胞效果,统计脱细胞脊髓支架的等级评分"优"、"良"、"一般"、"差"的百分比;选取HE染色脱细胞彻底、评分为"优"的脊髓支架,DAPI染色验证其内部细胞残留情况的等级评分是否一致为"优";通过电镜扫描观察两组内部三维结构并分析其孔径;分析脊髓组织脱细胞处理前后的含水率、孔隙率、抗酶解性的变化;通过免疫组化的方法,观察两组材料细胞外基质中黏多糖分布情况。[结果]支架组通过HE染色观察到脱细胞彻底、评分为"优"的百分比为80%;HE染色观察评分为"优"支架经DAPI染色验证其内部细胞残留量评分也为"优";其三维结构完整,其孔径均值为9.07μm;含水率为(228.14±19.39)%;孔隙率为(71.82±2.10)%;在胰酶中,第20 h的酶解率平均为(35.904±1.911)%;免疫组化分析鉴定细胞外基质中包含一定的黏多糖。对照组中正常脊髓经HE染色和DAPI染色观察到大量细胞;三维网孔状结构,孔径均值为40.7μm;含水率和孔隙率分别为(109.32±12.44)%和(61.18±4.19)%;在胰酶中,第20 h的酶解率平均为(25.704±1.030)%;细胞外基质中包含大量的黏多糖成分。[结论]EDC交联结合化学萃取法制备的支架脱细胞彻底、制备效率高,具有三维网状结构、良好的含水率、空隙率、抗酶解性,一定程度上保留细胞外基质中黏多糖成分,符合组织工程学支架制备要求,为脊髓支架提供了一种新的选择。

[Objective] To observe preparation efficiency,biological characteristics and mucopolysaccharide composition of spinal cord scaffold by using EDC crosslinking and chemical extraction. [Method]At first,rat spinal cord was decellularized by freezing and thawing of liquid nitrogen and 42℃ water bath,as well as chemical extraction and EDC crosslinking. Two groups were set for comparison in this experiment,including untreated spinal cords as control group and accellular spinal cord scaffolds as accellular spinal cord group. HE and DAPI staining were used for observation of the accellular effects. The effects of accellular spinal cord scaffolds were graded as being ＂ excellent＂, ＂ good＂, ＂ fair＂, ＂ poor＂,and the excellent results from HE staining were verified whether was in line with the same result from DAPI staining. The microstructure and pore size from two groups were observed by using scanning electron microscopy and the water ratio. Porosity,degradation rate in trypsin enzyme solution were measured as well. With immunohistochemistrical method,mucopolysaccharide reserved composition were determined in two groups. [Result]80% excellent results from HE staining were noticed in accellular spinal cord group,clearly matching to the excellent results from DAPI. It’s microstructure was three- dimensional network with the average pore size of 9. 07 um,with water ratio of（ 228. 14 ± 19. 39） %,porosity of（ 61. 18 ± 4. 19）,and with degradation rate of 25. 704% in trypsin enzyme solution at 20 hours post- experimentally,meanwhile,with a certain amount of mucopolysaccharide reserved composition. Meanwhile,results of HE and DAPI staining from control group revealed a large number of cells in untreated spinal cord. The micro- structure included similar three- dimensional network,with the average pore size of 40. 7 um. But the normal spine cord had water ratio of（ 109. 32 ± 12. 44） %,porosity of（ 61. 18 ± 4. 19） %,trypsin enzyme degradation rate of 25. 704% at 20 hours,at the same time,with abundant mucopolysaccharide. [Conclusion]The methods of EDC crosslinking combined with chemical extraction has efficient effect for preparation of decellularized normal spine cord. This accellular spinal cord scaffold has favourable properties,such as satisfactory three- dimensional network structure,pore size,water ratio,porosity,trypsin enzyme degradation rate,and mucopolysaccharide reserved composition. So,it is a new and effect choice for preparation of spinal cord scaffolds.