Department of Plastic and Hand Surgery and Laboratory for Tissue Engineering and Regenerative Medicine, University Hopsital Elrangen, Friedrich Alexander University Erlangen-Nuernberg, Erlangen, Germany.
Orthopedic and Trauma Surgery Center, Lörrach, Germany.
Med Sci Monit. 2019 Sep 6;25:6702-6710. doi: 10.12659/MSM.915649.
BACKGROUND Skin replacement by means of cultured epithelial keratinocytes is a well-accepted method. However, several clinical drawbacks of sheet autografts (CEA - cultured epithelial autografts) have stimulated various efforts to optimize cell culture and cell delivery. Recent developments include use of cell monolayers instead of a fully differentiated epithelium, as well as use of various biomaterials to grow and transport the cultured cells. To optimize the transfer of human keratinocytes directly to the recipient wound bed, we used an "upside-down" technique, delivering cultured cells directly to the wound with the carrier material on top. MATERIAL AND METHODS Subconfluent second-passage human keratinocyte monolayers on esterified hyaluronic acid membranes (KHAMC - Keratinocyte-Hyaluronic-Acid-Membrane-Composites) were transplanted either as upside-down grafts or as upside-up grafts onto standardized full-thickness wounds in athymic nude mice versus controls with the cell-free membrane alone. RESULTS In the upside-down group, 14 days after grafting, a multi-layered, differentiating epidermis was found, whereas the wounds in the upside-up group and in the control group were not completely closed up to day 21. Persistence of human keratinocytes was shown in the upside-down group only, from day 7 until day 35 after grafting. CONCLUSIONS This study confirms that upside-down grafting of subconfluent monolayers of serum-free cultured human keratinocytes on esterified hyaluronic acid membranes is a suitable means to transfer actively proliferative keratinocytes, and reduces wound contraction. Compared to standard grafting protocols of cultured epithelium, such as CEA sheet grafts, it is easier to apply, does not need enzymatic detachment of cells from the culture dish, and limits the number of production steps required.
通过培养的上皮角质形成细胞进行皮肤替代是一种被广泛接受的方法。然而，片状自体移植物（CEA-培养的上皮自体移植物）的一些临床缺点刺激了各种努力来优化细胞培养和细胞输送。最近的发展包括使用单层细胞而不是完全分化的上皮，以及使用各种生物材料来培养和输送培养的细胞。为了优化将人类角质形成细胞直接转移到受体伤口床，我们使用了“倒置”技术，将培养的细胞直接用载体材料输送到伤口上。
在酯化透明质酸膜（KHAMC-角质形成细胞-透明质酸-膜-复合材料）上的亚汇合第二代人角质形成细胞单层被移植为倒置移植物或正置移植物，分别置于无细胞的膜上，与对照组的标准全层伤口进行比较。
在倒置组中，移植后 14 天，发现了多层分化的表皮，而正置组和对照组的伤口直到第 21 天仍未完全闭合。仅在倒置组中，从移植后第 7 天到第 35 天，才显示出人角质形成细胞的持续存在。
这项研究证实，将无血清培养的人角质形成细胞的亚汇合单层倒置移植到酯化透明质酸膜上是一种转移活跃增殖的角质形成细胞的合适方法，并减少了伤口收缩。与 CEA 片状移植物等标准移植物培养方案相比，它更容易应用，不需要从培养皿中酶解细胞，并且限制了所需的生产步骤数量。
