Huber Birgit, Link Antonia, Linke Kirstin, Gehrke Sandra A, Winnefeld Marc, Kluger Petra J
1 Institute of Interfacial Process Engineering and Plasma Technology, University of Stuttgart , Stuttgart, Germany .
2 Reutlingen University , School of Applied Chemistry, Reutlingen, Germany .
Tissue Eng Part C Methods. 2016 Aug;22(8):756-64. doi: 10.1089/ten.TEC.2016.0141. Epub 2016 Jul 21.
Large, deep full-thickness skin wounds from high-graded burns or trauma are not able to reepithelialize sufficiently, resulting in scar formation, mobility limitations, and cosmetic deformities. In this study, in vitro-constructed tissue replacements are needed. Furthermore, such full-skin equivalents would be helpful as in vivo-like test systems for toxicity, cosmetic, and pharmaceutical testing. Up to date, no skin equivalent is available containing the underlying subcutaneous fatty tissue. In this study, we composed a full-skin equivalent and evaluated three different media for the coculture of mature adipocytes, fibroblasts, and keratinocytes. Therefore, adipocyte medium was supplemented with ascorbyl-2-phosphate and calcium chloride, which are important for successful epidermal stratification (Air medium). This medium was further supplemented with two commercially available factor combinations often used for the in vitro culture of keratinocytes (Air-HKGS and Air-KGM medium). We showed that in all media, keratinocytes differentiated successfully to build a stratified epidermal layer and expressed cytokeratin 10 and 14. Perilipin A-positive adipocytes could be found in all tissue models for up to 14 days, whereas adipocytes in the Air-HKGS and Air-KGM medium seemed to be smaller. Adipocytes in all tissue models were able to release adipocyte-specific factors, whereas the supplementation of keratinocyte-specific factors had a slightly negative effect on adipocyte functionality. The permeability of the epidermis of all models was comparable since they were able to withstand a deep penetration of cytotoxic Triton X in the same manner. Taken together, we were able to compose functional three-layered full-skin equivalents by using the Air medium.
由重度烧伤或创伤导致的大面积、深度全层皮肤伤口无法充分重新上皮化，从而导致疤痕形成、活动受限和外观畸形。在本研究中，需要体外构建的组织替代物。此外，这种全皮肤等效物作为用于毒性、化妆品和药物测试的类似体内的测试系统将很有帮助。迄今为止，尚无包含下层皮下脂肪组织的皮肤等效物。在本研究中，我们构建了一种全皮肤等效物，并评估了三种不同的培养基用于成熟脂肪细胞、成纤维细胞和角质形成细胞的共培养。因此，脂肪细胞培养基补充了对成功的表皮分层很重要的抗坏血酸 -2- 磷酸酯和氯化钙（空气培养基）。该培养基进一步补充了两种常用于角质形成细胞体外培养的市售因子组合（空气 -HKGS 培养基和空气 -KGM 培养基）。我们表明，在所有培养基中，角质形成细胞均成功分化以形成分层的表皮层，并表达细胞角蛋白 10 和 14。在所有组织模型中均可发现脂联素 A 阳性脂肪细胞，持续长达 14 天，而在空气 -HKGS 培养基和空气 -KGM 培养基中的脂肪细胞似乎较小。所有组织模型中的脂肪细胞都能够释放脂肪细胞特异性因子，而补充角质形成细胞特异性因子对脂肪细胞功能有轻微的负面影响。所有模型的表皮通透性相当，因为它们能够以相同的方式抵御细胞毒性曲拉通 X 的深度渗透。综上所述，我们能够使用空气培养基构建功能性的三层全皮肤等效物。
