Construction of Vascularized Oral Mucosa Equivalents Using a Layer-by-Layer Cell Coating Technology

There have been many advances in tissue engineering with respect to in vitro and in vivo models of oral mucosa equivalents (OMEs). To apply in vitro reconstructed oral mucosa models to regenerative medicine and alternatives to animal testing, it is necessary to develop the technology of reconstructing different types of oral tissues, such as control of epithelial differentiation and introduction of appendages. We previously reported that functional three-dimensional (3D) tissue models could be quickly constructed by using a layer-by-layer (LbL) cell coating technique that assembles extracellular matrix (ECM) nanofilms to a cell surface. In this study, 3D human OMEs composed of lamina-propria, keratinized or non-keratinized epithelium, and blood capillaries were constructed by using the LbL cell coating technology. Human oral mucosal fibroblasts (HOMFs) were coated with ECM nanofilms and accumulated for the construction of oral mucosal lamina-propria. To construct OMEs with keratinized or non-keratinized epithelium, human oral keratinocytes isolated from gingiva (human oral gingival keratinocytes: HOGKs) or human oral keratinocytes isolated from oral mucosa (human oral mucosal keratinocytes: HOMKs) were used in this study. We further studied the construction of epithelialized OMEs with density- and size-controlled blood capillary networks by using human umbilical vein endothelial cells (HUVECs). It was revealed that these constructions had barrier functions in accordance with their histological characterization. The OMEs with keratinization (K-OMEs) showed higher transepithelial electrical resistance (TEER) values compared with OMEs with non-keratinization (N-OMEs). The constructed epithelialized OMEs with blood capillaries are useful for in vitro/ex vivo research models and regenerative medicine as in oral tissue regeneration. The results suggest that OMEs with oral tissue appendages are more promising alternatives to animal testing and can be applied to the design of in vitro oral models that mimic human tissue organs.