作为压疮填充物的脱细胞真皮基质生物特性研究

Study on the Biological Characteristics of Acellular Dermal Matrix as Pressure Ulcer Filler

在前期同种异体脱细胞真皮基质的研究基础上,对脱细胞真皮基质作为填充物治疗压疮的生物特性进行了研究。采用γ射线灭菌人体皮肤,质量分数0.05%Trypsin酶/EDTA处理制备真皮脱细胞基质,并观察真皮基质微观结构等物理特性变化;苏木精-伊红(hematoxylin eosin,HE)染色观察基质脱细胞效果;把稳定表达荧光Lifeact的细胞接种到真皮基质表面上观察细胞的黏附行为,把人体成纤维细胞(human fibroblasts,HFB)接种到基质真皮表面,使用CCK-8检测基质对细胞生长的毒性,采用DAPI染色观察细胞在基质中的长入情况。实验结果显示:脱细胞真皮基质微观结构与正常皮肤相似,基质的压缩弹性模量为(12.59±5.50)MPa,与对照组材料(6.75±2.20)MPa比较,差异无统计学意义(P>0.05);孔隙率与表观密度均适于细胞长入基质内部;HE染色发现细胞脱除效果良好,基质中无明显可见的细胞及细胞碎片;HFB与基质共培养1 d、4 d、7 d后和仅HFB与培养液组相比,发现基质对HFB生长无影响,表明基质无细胞毒性;共培养3 d细胞数不断增加,9 d可见DAPI染色的HFB向基质厚度方向长入,HFB充满厚度方向。皮肤组织经过γ射线灭菌、质量分数0.05%Trypsin酶/EDTA处理后的脱细胞真皮基质作为组织填充物是治疗压疮可靠的修复手段,值得临床推广。

On the basis of previous studies on allogeneic acellular dermal matrix,the biological characteristics of acellular dermal matrix were investigated as filler in the treatment of pressure ulcers.Human skin was sterilized byγ-ray,and then treated with 0.05%Trypsin enzyme/EDTA to prepare dermal acellular matrix.The changes of physical properties of dermal matrix,such as microstructure,were observed.Hematoxylin eosin(HE)staining was used to observe the decellulatory effect of matrix.Human fibroblasts(HFB)were inoculated into the dermal surface of the matrix to test the toxicity of the matrix on cell growth with CCK-8,and DAPI staining was used to observe the growth of cells in the matrix.The experimental result shows:the microstructure and compressive modulus of acellular dermal matrix are similar to these of normal skin,and no significant difference enists between the compressive elastic modulus of matrix(12.59±5.50)MPa and the control material(6.75±2.20)MPa,(P>0.05).HE staining results show that the cells are removed effectively,and no cells or cell fragments are visible in the matrix.After 1,4,and 7 days co-culture of HFB with substrate,the substrate has no effect on the growth of HFB compared with HFB with medium alone,indicating that the substrate has no cytotoxicity.After 3 days of co-culture,the number of cells increases continuously.After co-cul-ture for 9 days,DAPI-stained HFB grows towards the thickness of the matrix and fills the matrix along thickness.The acellular dermal matrix treated byγ-ray sterilization and 0.05%Trypsin enzyme/EDTA is a reliable repair method for treating pressure ulcers,which is worthy of clinical promotion.