Generación de nuevos modelos de mucosa oral humana masticatoria y de revestimiento con biomateriales funcionalizados y fuentes celulares alternativas para su uso en terapias avanzadas

Abstract

Disorders of the human oral mucosa, such as oral cancer, trauma and periodontal diseases, are very prevalent conditions today. The treatment of these pathologies depends to a great extent on the availability of healthy human oral mucosa that allows replacement therapy, something that makes their treatment difficult due to the scarcity of donors. In this sense, Tissue Engineering emerges as a great alternative for the treatment of these pathologies, since it is a branch of medicine whose basic objective is the elaboration of artificial tissues that allow the repair, restoration or even the improvement of the functions of damaged tissues and organs. In the present Doctoral Thesis, first, the optimization of the biomechanical properties of the fibrin-agarose biomaterial, previously developed by the Tissue Engineering group of the University of Granada, has been carried out with the aim of generating improved models of human oral mucosa by means of tissue engineering protocols. Specifically, five different types of agaroses have been studied, each at four different concentrations, on which the biomechanical properties were evaluated. Improved models of human oral mucosa would allow the clinical treatment of conditions affecting the human oral mucosa. However, biointegration is a major problem in the generation of these artificial tissues, which could be favored by the generation of substitutes with a higher vascularization potential. In this sense, in this Doctoral Thesis a second optimization for the improvement of biointegration processes has been carried out based on the incorporation of human mesenchymal stem cells (MSC) with vascularization capacity in the stroma of human oral mucosa substitutes, to generate improved models of prevascularized human oral mucosa. Specifically, MSC obtained from adipose tissue (ADSC), bone marrow (BMSC) and dental pulp (DPSC) were used. The three types of MSC were differentiated to endothelial lineage using inducing media. Once vascular differentiation was induced, the vascularization potential of MSCs was assessed before and after induction. Human umbilical vein endothelial cells (HUVEC) were used as a positive control. In vitro induction of MSCs to endothelial lineage was able to increase the expression of endothelial markers VEGF, CD31 and vWF, especially in bone marrow and dental pulp MSCs. The next step was to generate human oral mucosal surrogates using the previously optimized fibrin-agarose biomaterial by combining human oral mucosal fibroblasts (HFOM) and each type of MSC before and after vascular induction. Once generated, the enhanced human oral mucosa models were implanted into athymic mice for one week and the vascularization potential of each surrogate was assessed in vivo. In vivo implantation resulted in a significant increase in blood vessel formation in the intermediate zone between the artificial surrogate and host tissues, with significant expression of the vascular markers VEGF, CD31, vWF and CD34 compared to controls. As in the in vitro study, the value was higher in MSCs differentiated from BMSCs and DPSCs. Thus, the results obtained in this doctoral thesis represent a significant improvement in biomechanical and vascularization of the previously developed models. These improvements represent an advance for the potential clinical use of human oral mucosa generated by tissue engineering protocols. The preclinical characterization and analysis of the quality controls developed in this Doctoral Thesis constitute a fundamental step for its future clinical translation.