Pontiggia Luca, Klar Agnieszka, Böttcher-Haberzeth Sophie, Biedermann Thomas, Meuli Martin, Reichmann Ernst
Tissue Biology Research Unit, University Children's Hospital Zurich, Zurich, Switzerland.
Pediatr Surg Int. 2013 Mar;29(3):249-56. doi: 10.1007/s00383-013-3268-x. Epub 2013 Feb 3.
Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3-4 weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality.
Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6 days), and the culture time of keratinocytes (3, 6, and 12 days) before transplantation of DESS on nude rats. Additionally, the impact of the air-liquid interface culture during 3 days before transplantation was investigated. 3 weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina.
Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6 days. The minimal culture time of keratinocytes on hydrogels was also 6 days. The air-liquid interface culture did not improve graft quality.
By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12 days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skipped.
体外生成的自体真皮 - 表皮皮肤替代物（DESS）是临床实践中治疗全层皮肤缺损的一种有前景的治疗手段。对于急性患者而言，一个严重的缺点是其生产时间相对较长，为3 - 4周。通过本实验研究，我们旨在减少DESS的生产时间，同时不影响其质量。
改变DESS构建的两个体外步骤：成纤维细胞在水凝胶中的预培养时间（1天、3天和6天），以及在将DESS移植到裸鼠之前角质形成细胞的培养时间（3天、6天和12天）。此外，还研究了移植前3天进行气液界面培养的影响。移植3周后，评估宏观外观并制作组织切片，以分析表皮和真皮的结构与厚度、表皮的分层以及基膜的存在情况。
成纤维细胞预培养6天时可获得最佳的DESS形成。角质形成细胞在水凝胶上的最短培养时间也是6天。气液界面培养并未提高移植物质量。
通过优化我们的体外培养条件，有可能将DESS的生产时间从21天大幅缩短至12天。然而，真皮替代物中成纤维细胞的预培养以及移植前角质形成细胞的增殖对于表皮的平衡成熟仍然至关重要，不能完全省略。
