Molecular evaluation of the BMP2/DLX3 induced osteogenic differentiation in dental follicle cells (DFCs)

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Precursor cells of the human dental follicle (DFCs) are the precursors of the periodontium and have the endogenous potential to differentiate into cementoblasts, alveolar osteoblasts and fibroblasts of the periodontal ligament. Due to their high differentiation capability, DFCs provide the promising potential for use in regenerative therapies of craniofacial bone tissue and probably also for the treatment of peripheral bone diseases. However, the molecular mechanisms of differentiation into (alveolar) osteoblasts are still inadequately examined despite much progress during the last years, and need to be further understood before. It was already shown that the initiation of osteogenic differentiation in DFCs is mainly controlled by BMP2 and its induction of the transcription factor DLX3, which leads to the expression of further differentiation markers. Besides, activation of protein kinase A is involved via the phosphorylation und therefore stabilization of β-Catenin, which is the key protein of the canonical WNT signaling pathway. Expression of DLX3 is enhanced after binding of β-Catenin to the TCF/LEF promotor. In contrast, the non-canonical WNT signaling pathway via WNT5A mostly affects proliferation and viability of DFCs and does only indirectly influence differentiation. Furthermore, previous results of the project showed that later differentiation phases until mineralization mainly depend on the regulation of protein kinase C (PKC) and protein kinase B (also called AKT). In addition, both kinases as well as WNT5A and DLX3 affect viability of DFCs. Further preliminary studies showed that increased oxidative stress and a decreased expression of the hypoxia-induced protein HIF-1α are observed during differentiation as well as a significantly increased expression of mitochondrial markers during middle and late periods of differentiation. The preliminary results lead to the new working hypothesis that oxidative stress increases during osteogenic differentiation of DFCs which needs to be neutralized by OSD (oxidative stress defense) proteins – the expression of which might be regulated e.g. via PKC or Akt – in order to facilitate successful osteogenic differentiation. The oxidative stress could originate mainly from increased oxidative phosphorylation in the mitochondria, which enables the energy consuming processes of matrix secretion and mineralization. Hence, the first part of the project is intended to investigate the role of oxidative stress, viability and energy metabolism during osteogenic differentiation, while the second part aims to evaluate the involvement of PKC and Akt. The results of this project will further extend the molecular knowledge about DFCs and contribute to create a fundament for the use of the cells in regenerative therapies of (craniofacial) bone tissue.