Advanced 3D Models Cultured to Investigate Mesenchymal Stromal Cells of the Human Dental Follicle

The human dental follicle (hDF) contains the developing tooth and is involved in regulating tooth maturation and eruption. To investigate the mesenchymal stromal cells of the dental follicle, 2 three-dimensional (3D) culture models were used, based on a dynamic bioreactor: the Rotary Cell Culture System (RCCS) and the 3D culture of precursor cells isolated from follicular tissue (human dental follicle cells [hDFCs]). The hDFCs were obtained from impacted third molars of 20 patients. Two 3D culture models were tested. In the first model, intact hDF explants were cultured in 3D conditions, preserving the original tissue architecture; they were studied using histomorphological and molecular analyses. The second model involved the 3D culture of hDFCs, which were characterized to evaluate their multipotency in terms of differentiation capability. Of the biomarkers known to characterize hDFCs, hDF precursors were selected for our study. The immunophenotype and in situ immunocytochemistry were evaluated for markers CD44, CD90, CD146, CD105, CD31, CD34, and CD45 Ag. The results show that the conditions provided by the RCCS preserve the original organizational architecture of the cells. The 3D conditions of the model enhanced differentiation in response to adipogenic, osteogenic, and chondrogenic inductive growth media. The immunophenotype and the immunocytochemistry showed generally high expression of CD90, CD44, and CD105, while CD146 expression was more restricted to approximate to 30% of cells. No expression was observed for CD31, CD34, and CD45 Ags. Two 3D tissue- and cell-based ex vivo models of the hDF supported the long-term maintenance of hDF-specific cell phenotypes and their ability to recapitulate typical cellular differentiation states. As such, these ex vivo models could be used to study the physiopathology of human odontogenesis. In addition, in a therapeutic context, they could be used to examine the role of specific chemical signals (e.g., new therapeutic agents) in the processes of dental tissue repair and regeneration.