Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

Fabio      Bucchieri; Alberto      Fucarino; Antonella      Marino Gammazza; Alessandro      Pitruzzella; Vito      Marciano; Carlo      Paderni; Viviana      De Caro; Maria      Gabriella Siragusa; Lorenzo      Lo Muzio; Stephen      T Holgate; Donna      E Davies; Felicia      Farina; Giovanni      Zummo; Yasusei      Kudo; Italo      Libero Giannola; Giuseppina      Campisi

doi:10.2174/138161212803307482

Abstract

Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an artificial extracellular matrix (MatrigelTM), allowing the interactions required for proper differentiation of the various citotypes which form the mucosal layer. Biopsies of human oral mucosa, in fragments of about 0.5 mm3, were placed onto 6.5mm Transwells, covered with MatrigelTM and grown in a specific culture medium. A gradual formation of an architectural structure similar to that of the in vivo oral mucosa was observed. Transmission electron and confocal microscopy were employed to characterize the newly developed model: the cell components (keratinocytes and fibroblasts) differentiated properly within the outgrowth and reconstituted, in vitro, the physiological structure of the human oral mucosa, including a stratified non-keratinized squamous layer composed of four different layers, a proper basal membrane and a lamina propria where fibroblasts produce ECM. Moreover, keratinocytes expressed CK5, CK13, CK19 and E-cadherin, whereas fibroblasts expressed collagen type I and IV, laminin and fibronectin.

3D outgrowths could be considered a valid alternative to animal models, and provide useful information for researchers interested in studying the responses of the human oral mucosa to locally delivered drugs or other exogenous treatments.

Keywords: Tissue-engineering, human oral mucosa, 3D outgrowth model, keratinocytes, fibroblasts, transmission electron microscopy, immunofluorescence, immunogold, extracellular matrix (ECM), lamina propria.