Liu Po, Qi Shaohai, Shu Bin, Xie Julin, Xu Yingbin, Liu Xusheng
No. 1 Department of Surgery, Tangxia Hospital of Dongguan, Dongguan Guangdong, 523721, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):135-40.
To investigate the impact of dermal papillary cells on vascularization of tissue engineered skin substitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix.
Human foreskins from routine circumcisions were collected to separate epidermal cells by using dispase with trypsinogen. Collagen type IV was used to isolate epidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenase I from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seeding epidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the control group) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skin defects (1 cm x 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks after grafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate the microvessel density at 2 and 4 weeks after grafting.
Those adhesion cells by collagen type IV coexpressed Keratin 19 and beta1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressing high levels of a-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), than that in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with large epithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells. At 2 and 4 weeks after grafting, the microvessel density was (38.56 +/- 2.49)/mm2 and (49.12 +/- 2.39)/mm2 in experimental group and was (25.16 +/- 3.73)/mm2 and (36.26 +/- 3.24)/mm2 in control group respectively, showing significant differences between 2 groups (P < 0.01).
Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization, which promotes epidermis formation and improves the grafting survival rate.
探讨真皮乳头细胞对由表皮干细胞和异体脱细胞真皮基质组成的组织工程皮肤替代物血管化的影响。
收集常规包皮环切术的人包皮，用 dispase 加胰蛋白酶原分离表皮细胞。用 IV 型胶原从第 2 和第 3 代角质形成细胞中分离表皮干细胞。采用 I 型胶原酶消化法从胎儿头皮分离真皮乳头，并在常规成纤维细胞培养基中培养。将表皮干细胞接种于异体脱细胞真皮乳头侧构建组织工程皮肤替代物，实验组在网状侧接种真皮乳头细胞，对照组不接种。将两种复合皮肤替代物用于覆盖 BALB/C-nu 裸鼠背部的皮肤缺损（大小为 1 cm×1 cm，n = 30）。移植后 2 周记录移植成活率。采用 HE 染色和免疫组化方法检测移植后 2 周和 4 周 CD31 的表达并计算微血管密度。
IV 型胶原黏附的细胞共表达角蛋白 19 和β1 整合素，表明这些细胞为表皮干细胞。培养的真皮乳头细胞通过高表达α-平滑肌肌动蛋白得以鉴定。实验组移植成活率（28/30，93.3%）显著高于对照组（24/30，80.0%）。HE 染色显示，实验组移植复合皮肤中的上皮层为 12 层，上皮细胞大；对照组上皮层为 4 - 6 层，上皮细胞小。移植后 2 周和 4 周，实验组微血管密度分别为(38.56 ± 2.49)/mm²和(49.12 ± 2.39)/mm²，对照组分别为(25.16 ± 3.73)/mm²和(36.26 ± 3.24)/mm²，两组间差异有统计学意义（P < 0.01）。
在组织工程皮肤替代物中添加真皮乳头细胞可增强血管化，促进表皮形成，提高移植成活率。
