Cryopreserved lip mucosa tissue derived keratinocytes can fabricate tissue engineered palatal mucosa equivalent

Clinical application of tissue engineered palatal mucosa is hampered by unavailability of suitable oral keratinocytes as seeding cells. The aim of this study is to fabricate a tissue engineered palatal mucosa equivalent from the oral keratinocytes which cultured from cryopreserved lip mucosa tissues. Abundant lip mucosa tissues during cheilorrhaphy were firstly cryopreserved in liquid nitrogen for four to six months, and then recovered to culture oral keratinocytes for the fabrication of oral mucosa equivalent. In the control groups, oral keratinocytes cultured from fresh lip mucosa, fresh palate mucosa, and cryopreserved palate mucosa were used to fabricate oral mucosa equivalents. Attachment rate of the oral keratinocytes derived from cryopreserved lip mucosa was lower than that of the keratinocytes from fresh lip mucosa samples, however, the cell cycle distribution of oral keratinocytes cultured from all four groups of mucosa samples were similar. Histologically, the fabricated mucosa equivalents from these four groups had four- to six epithelial layers, the basal cells were cubic and the outmost cells were flatten with narrow nuclei which paralleled to the surface of the dermal matrix. Additionally, Ki-67 positive stained cells were mainly located in the basal layer of the epithelium of these equivalents. These characteristics disclosed that the oral mucosa equivalent cultured from the cryopreserved lip mucosa tissue was not different with the equivalents from other groups and similar to the native palate mucosa tissue. It suggested that the cryopreserved lip mucosa tissues could be used for the construction of palatal mucosal equivalent for clinical application.