Three dimensional oral mucosa models: development and applications

Animal experimentation has been extensively and for a long time applied in several research fields, but since 2011 it has been substantially limited by the Commission of the European Parliament to ensure people/animals safety and reduce research costs. To respond to these directives, many attempts have been focused on the development and validation of new in vitro 3D systems, bypassing the traditional 2D cell cultures. In this regard, diverse approaches to tissue-engineered bone and oral mucosa have been developed. Despite the promising premises and the cutting-edge results, the used 3D in vitro bone-oral mucosal models still lack interaction between the mucosal and the bone components. Therefore, this project aimed to create 3D models, entirely made with primary human cells (keratinocytes, fibroblasts, and osteoblasts), able to mimic the natural structure and interaction of bone and oral mucosa. In the present work, the regulatory role of the mesenchymal tissue onto epithelia was evaluated. The main results showed that that during the differentiation hMSC produce and secrete factors that induce the keratinization and the expression of the marker of differentiation CK10; in particular in the middle stage of differentiation (OB14). The proteomic analysis revealed that this effect can be ascribable to KGF secretion. This finding may impact the design of new implantable devices able to induce, alone, the epithelial growth and keratinization to improve implant graft avoiding epithelial graft linked to the morbidity of another zone. Moreover, we also showed that OM might have a pro-innervation effect, at least during the last stages of keratinocytes stratification. Finally, we obtained and characterized an innervated mucoperiosteal model that could open new in vitro frontiers for oral biomaterials validation as well as improve knowledge regarding the mesenchymal stem cells roles onto oral mucosa development.