In Vitro Development of a Mucocutaneous Junction for Lip Reconstruction

Abstract

Purpose: We present a straightforward and reproducible technique to create, in vitro, a construct containing a mucocutaneous junction (MCJ) with a transitional zone (vermilion) for fabrication of a microvascular prelaminated flap for use in lip reconstruction.

Materials and methods: Cultured primary human skin keratinocytes and oral mucosal epithelial cells at premixed ratios of 50% skin cells to 50% oral cells, 25% skin cells to 75% oral cells, and 75% skin cells to 25% oral cells were grown on an AlloDerm dermal equivalent (LifeCell, Branchburg, NJ) to create an MCJ equivalent with a lip or transitional zone (vermilion) using a novel 3-dimensional (3D) culture device with a barrier to separate co-cultured skin and oral cells. The 3 different cell ratios were compared by staining for the following specific differentiation markers to define the different areas of skin and mucosal keratinocytes: filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3.

Results: Immunohistochemical results showed that MCJ equivalents seeded with premixed cells were similar to the differentiation patterns of tissue-engineered 3D cultures using 100% oral mucosal epithelial cells or skin keratinocytes. The engineered MCJ-equivalent constructs, grown in the 3D device specifically constructed with a cell-free gap at the barrier site, formed a transitional zone (vermilion) at the barrier site with intermingling of the skin and oral keratinocytes. The results showed different and unique expression patterns of filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3 by those cells migrating into the gap, which were similar to those seen in human lip tissue. This pattern was not seen in MCJ equivalents created using premixed skin and oral cells.

Conclusions: Using a device to separately co-culture human oral and skin keratinocytes to allow the cells to migrate into a cell-free zone resulted in phenotypic expression closer to what is seen in native tissue, in comparison to premixing the skin and oral cells before seeding.