灌注去细胞方法制备猪主动脉瓣去细胞支架

Perfusion decellularization method for the fabrication of decellularized porcine aortic valve scaffold

目的 观察灌注去细胞方法快速制备猪主动脉瓣去细胞支架的可行性,并研究其有效性及对细胞外基质的影响.方法 利用生物反应器、以0.1％十二烷基磺酸钠对猪主动脉带瓣管道进行灌注去细胞,并与普通振荡去细胞方法进行对比.行4’,6-二脒基-2-苯基吲哚（DAPI）染色和脱氧核糖核酸（DNA）含量检测判断去细胞的效果,行组织学染色观察去细胞方法对细胞外基质结构的影响,行胶原蛋白及弹性蛋白含量检测评估去细胞方法对细胞外基质成分的影响.结果 DAPI染色显示灌注去细胞10 h和普通振荡去细胞60h均可完全去除猪主动脉瓣的细胞核成分.灌注去细胞组瓣膜的残余DNA含量[（2.48 ±0.64） ng/mg]与普通振荡去细胞组[（2.25±0.74） ng/mg]差异无统计学意义（P＞O.05）.组织学染色显示两种去细胞方法较好地保留了猪主动脉瓣的3层结构,但灌注去细胞组瓣膜的胶原纤维排列更为紧密,弹性纤维结构更为清晰.灌注去细胞组瓣膜的胶原含量[（528.02 ±57.18） μg/mg]与普通振荡去细胞组[（511.93 ±67.10） μg/mg）]差异无统计学意义（P＞0.05）.灌注去细胞组的弹性蛋白含量[（83.72 ±4.35） μg/mg]显著高于普通振荡去细胞组[（72.70±6.04） μg/mg,P＜ 0.05].结论 灌注去细胞方法用于制备猪主动脉瓣去细胞支架是一种快速、有效且对细胞外基质损伤较小的方法.

Objective To investigate the feasibility and effectivity of perfusion decellularization method for the fabrication of decellularized porcine aortic valve scaffold and its effects on extracellular matrix.Methods Porcine aortic valves were decellularized with a perfusion decellularization protocol using 0.1% sodium dodecyl sulfonate in a bioreactor,or decellularized with a common decellularization protocol using 0.1% sodium dodecyl sulfonate with constant shaking.4＇,6-diamidino-2-phenylindole （DAPI） staining and resident deoxyribonucleic acid （DNA） determination were used to assess decellularization effectiveness of decellularization methods.Histologic stainings were performed to test the effect of decellularization methods on the structure of extracellular matrix.Quantitative biochemistry of collagen and elastin were used to examine the impact of decellularization methods on the extracellular matrix contents.Results Both perfusion decellularization protocol for 10 h and common decellularization protocol for 60 h could removed all cells and nucleus completely as indicated by DAPI staining.DNA determination showed that resident DNA content of valve decellularized with perfusion decellularization protocol [（2.48 ± 0.64） ng/mg] was not significantly different from that of valve decellularized with common decellularization protocol [（2.25 ± 0.74） ng/mg].As indicated by histological staining,the tri-layered structures in the valves decellularized with both protocols were well preserved.However,the collagen and elastin fibres were more tight and intact in valves decellularized with perfusion decellularization protocol.The collagen content of valve decellularized with perfusion decellularization protocol [（528.02 ± 57.18） μg/mg] was not significantly different from that of valve decellularized with common decellularization protocol [（511.93 ± 67.10） μg/mg].The elastin content in the valve decellularized with perfusion decellularization protocol [（83.72 ± 4.35） μg/mg] was significantly higher than that in valve decellularized with common decellularization protocol [（72.70 ± 6.04） μg/mg,P 〈 0.05].Conclusion Perfusion decellularization method could be considered as a fast and effective decellularization method with minimal damage to extracellular matrix for the fabrication of decellularized porcine aortic valve scaffold.