Falanga Vincent, Butmarc Janet, Cha Jisun, Yufit Tatyana, Carson Polly
Department of Dermatology, Roger Williams Medical Center, Providence, Rhode Island 02908, and Boston University School of Medicine, Boston, Massachusetts, USA.
Tissue Eng. 2007 Jan;13(1):21-8. doi: 10.1089/ten.2006.0148.
A bilayered bioengineered living skin construct (LSC) consisting of viable human neonatal keratinocytes over a collagenous dermis seeded with dermal fibroblasts has been used extensively in difficult-to heal human wounds. Its biological properties include production of several mediators, cytokines, and growth factors and the ability to heal itself upon injury. In this study, we investigated the process of keratinocyte migration in LSC. At baseline, 6-mm punch biopsies of the construct were placed in serum-free medium (AIM-V) or Dulbecco's modified Eagle medium. At varying time points, the LSC samples were processed and analyzed using histology and immunohistochemistry. By 72 h, in a time-dependent manner, the overlying epidermis had migrated over and enveloped the entire underlying dermis, a process known as epiboly. Increasing concentrations of neutralizing antibodies to epidermal growth factor or interleukin-1 alpha down-regulated the extent of epiboly, as measured using computerized planimetry, but antibodies to transforming growth factor-beta 1 did not affect it. The consistent expression of laminin V, alpha3beta1 integrin, and vitronectin (epibolin) and its integrin receptor (alphavbeta5) characterized the tongue of migrating epidermis. Increasing concentrations of antibodies to vitronectin blocked the process of epiboly, as did antibodies to the alphavbeta5 integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. This process of epiboly provides novel mechanisms of action for bioengineered skin constructs.
一种双层生物工程化活皮肤构建体（LSC），由覆盖在接种了真皮成纤维细胞的胶原真皮上的有活力的人类新生儿角质形成细胞组成，已被广泛用于难以愈合的人类伤口。其生物学特性包括产生多种介质、细胞因子和生长因子，以及受伤后自我愈合的能力。在本研究中，我们调查了LSC中角质形成细胞迁移的过程。在基线时，将构建体的6毫米打孔活检组织置于无血清培养基（AIM-V）或杜氏改良 Eagle 培养基中。在不同时间点，对LSC样本进行处理，并使用组织学和免疫组织化学进行分析。到72小时时，上层表皮以时间依赖性方式迁移并包裹了整个下层真皮，这一过程称为外包。使用计算机图像分析测量，针对表皮生长因子或白细胞介素-1α的中和抗体浓度增加会下调外包的程度，但针对转化生长因子-β1的抗体则没有影响。层粘连蛋白V、α3β1整合素和玻连蛋白（表皮松解素）及其整合素受体（αvβ5）的持续表达是迁移表皮舌的特征。针对玻连蛋白的抗体浓度增加会阻断外包过程，介导玻连蛋白驱动的角质形成细胞运动的αvβ5整合素受体的抗体也会如此。这种外包过程为生物工程皮肤构建体提供了新的作用机制。
