Retro-Engineered Dental-Follicle-Precursor-Cells Enhance In-Vitro Osteogenic Commitment and Improve Bone Repair

Objectives: One of the main characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into multiple mature cell lines, including osteoblasts. This peculiarity makes such cells particularly suitable for therapeutic use in regenerative medicine, including bone regeneration. MSCs must undergo a state of de-differentiation, which consists of a down-regulation of linear-specific genes, before to differentiating towards a specific cellular lineage. Once the differentiation has occurred, the differentiated cell can be again retro-differentiated to the stem state, and then again differentiated: the objective of the research is to evaluate the osteogenic potential of retro-engineered Dental-follicle-precursor-cells (DFPCs) in bone regeneration. Methods: DFPCs have been isolated and grown, as previously described in literature. We compared the osteogenic commitment of DFPCs plated in osteogenic medium for 30days (Osteo-DFPCs) with retro-engineered DFPCs (Rediff- DFPCs). To retro-differentiated DFPCs, we first cultured DFPCs in an osteogenic medium for 10days; in the following 10days we plated osteo-induced DFPCs in a basal-medium to achieve de-differentiation. Finally, the de-differentiated DFPCs were once again plated for 10days in osteogenic medium. To assess if Rediff-DFPCs and Osteo-DFPCs were functional osteoblasts, and if any significative difference were observable among these two types of osteoblasts, we performed alizarin-red assay and quantitative analysis of gene expression (qRT-PCR) of osteo-specific markers (ALP, OPN, BSP and RUNX-2) to both Rediff-DFPCs and Osteo-DFPCs groups. Results: Interestingly, the expression of OPN, BSP and RUNX-2 mRNA was significantly increased in Rediff-DFPCs, compared to Osteo-DFPCs. Alyzarin-red staining assay confirmed a significantly higher mineralized matrix production in the Rediff-DFPCs, compared to Osteo-DFPCs. Conclusions: The strategy to retro-engineer dental-derived-stem-cells is characterized by accessible MSCs sources, easy in-vitro manipulation and a surprising commitment towards osteogenic lineage. All these skills make this specific protocol a feasible approach to bone tissue engineering and repair, for dental and maxillofacial applications