脂肪干细胞复合脱细胞真皮基质用于声带注射的实验研究

目的 探索脂肪干细胞(adipose-derived stem cell,ASC)与脱细胞真皮基质微粒(micronized acellular dermal matrix,MADM)复合培养后用于声带内注射的可行性.方法 体外分离培养兔ASC,取第3代细胞,用细胞膜红色荧光探针(DiI)标记,并与已制备好的MADM复合培养,形成细胞和材料复合体,应用荧光显微镜、扫描电镜了解ASC黏附材料情况,四唑类化合物3-(4,5-dimethylthizazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium,inner salt(MTS)比色法检测细胞增殖情况.ASC-MADM复合体培养3 d后与适量胶原混合后注入异体兔单侧声带内,术后2、4、8周内镜观察,并分别处死动物,检测ASC在声带中存活、分布情况.结果 MADM所含胶原纤维松散、多孔隙,具有良好三维结构;ASC可黏附其生长并能不断增殖,与没有复合MADM的对照组比较细胞增殖水平差异有统计学意义(P值均＜0.05或＜0.01).ASC-MADM复合体注入兔声带肌后均存活良好,连续内镜观察、冰冻切片、HE染色显示声带局部无不良反应,ASC-MADM复合体在兔声带内基本无炎症细胞浸润,ASC可在声带内存活8周.结论 MADM是较理想的细胞支架材料,ASC能黏附MADM生长并增殖.ASC-MADM复合体异体动物声带内注射可存活且无不良反应.

Abstract：

Objective To explore the feasibility of adipose-derived stem cells (ASC) combined with micronized acellular dermal matrix (MADM) for vocal cord injection. Methods The adipose-deprived stem cells were harvested from rabbit adipose tissue in vitro. The 3rd generation of ASC was labeled with DiI ( 1, 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) and cultured with MADM to form a complex. The adhesion of ASC to MADM was observed by fluorescence microscope and electron microscope.The proliferation of ASC on MADM was evaluated by 3-(4, 5-dimethylthizazol-2-yl)-5-( 3-carboxy methoxyphenyl)-2-(4-sulfonyl )-2H- tetrazolium, inner salt (MTS). Three days after the culture, the complex was mixed with appropriate amount of collagen, and then injected into the unilateral vocal cord of the rabbit. The animals were sacrificed 2, 4, 8 weeks after injection, the survival time and distribution of ASC in vocal fold were tested, and the responses of vocal cord to ASC-MADM and the degradation of MADM were observed. Results The ASC adhered to MADM and grew well (P ＜0. 05 or ＜0. 01 ), showing good compatibility with MADM in vocal cord tissue. The complex of ASC-MADM could be injected into the rabbit vocal cords, while no adverse reactions was observed in the vocal cord by endoscope, frozen section and HE staining. ASC could survive for 8 weeks in vocal cords, and no inflammatory cell infiltration was observed.Conclusions MADM is an ideal scaffold material and shows perfect compatibility with ASC which can adhere and proliferate well on it. The complex of ASC-MADM can be injected into the vocal cord and can survive. There is no adverse reaction in vocal cords.

Experimental study on adipose-derived stem cells combined with acellular dermal matrix particles for vocal fold injection

Objective To explore the feasibility of adipose-derived stem cells (ASC) combined with micronized acellular dermal matrix (MADM) for vocal cord injection. Methods The adipose-deprived stem cells were harvested from rabbit adipose tissue in vitro. The 3rd generation of ASC was labeled with DiI ( 1, 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) and cultured with MADM to form a complex. The adhesion of ASC to MADM was observed by fluorescence microscope and electron microscope.The proliferation of ASC on MADM was evaluated by 3-(4, 5-dimethylthizazol-2-yl)-5-( 3-carboxy methoxyphenyl)-2-(4-sulfonyl )-2H- tetrazolium, inner salt (MTS). Three days after the culture, the complex was mixed with appropriate amount of collagen, and then injected into the unilateral vocal cord of the rabbit. The animals were sacrificed 2, 4, 8 weeks after injection, the survival time and distribution of ASC in vocal fold were tested, and the responses of vocal cord to ASC-MADM and the degradation of MADM were observed. Results The ASC adhered to MADM and grew well (P ＜0. 05 or ＜0. 01 ), showing good compatibility with MADM in vocal cord tissue. The complex of ASC-MADM could be injected into the rabbit vocal cords, while no adverse reactions was observed in the vocal cord by endoscope, frozen section and HE staining. ASC could survive for 8 weeks in vocal cords, and no inflammatory cell infiltration was observed.Conclusions MADM is an ideal scaffold material and shows perfect compatibility with ASC which can adhere and proliferate well on it. The complex of ASC-MADM can be injected into the vocal cord and can survive. There is no adverse reaction in vocal cords.