2.2.2. Dentinogenesis

Dentin is an acellular calcified tissue consisting of 70% hydroxyapatite, 20% organic phase, and 10% water by weight. Dentin formation is executed by odontoblasts (or dentinoblasts), which are cells of mesenchymal origin. During dentinogenesis, odontoblasts migrate towards dental pulp and deposit collagen types I, III, and V, proteoglycans, and other matrix proteins, which provide the nucleation base for hydroxyapatite crystals. Besides scaffold-mediated mineralization, minerals precipitation and cell-derived matrix vesicles-driven mineralization occur during various stages of dentinogenesis [12]. After dentin synthesis is complete, odontoblasts remain beneath it with tiny cellular projections called odontoblast processes protruding into the microscopic channels in the dentin (Figure 1A). These

projections are involved in detecting environmental stimuli (pH, cytokines, inflammatory mediators, and other signaling molecules) by odontoblasts, which can be mobilized for dentin regeneration in a case of damage. Thus, dentin possesses a limited capacity for regeneration [5,12,71,76]. Therefore, finding the appropriate cell source and differentiation strategy for dentin regeneration is of crucial importance. Thus far, dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), AT-MSCs, bone marrow-derived MSCs (BM-MSCs), and iPSCs have been shown to have the dentinogenic potential [25,80,81,90,101–103].

**Figure 2.** Major signaling cascades involved in amelogenesis, odontogenesis, and cementogenesis. (**A**) Signaling pathways modulating amelogenesis with TGF-β superfamily ligands (BMP2 and TGF-β1/2/3) playing the major role in matrix protein and metalloproteinases feedback-regulation and Runx2 being an important transcription factor. (**B**) Central signaling cascades of odontogenesis are depicted. The TGF-β superfamily ligands (BMP2/4 and TGFβs) regulate many odontogenic genes with ERK1/2 as convergence point and Klk4-Osx as important transcription factor tandem. (**C**) Major cementogenesis-related signaling cascades with Osx as the central transcription factor being regulated via Wnt/β-catenin in a feedback-loop. Ameloblast-derived products (LRAP and amelogenin) were shown modulate key cementogenic gene expression in vitro.

Shh is secreted by an epithelial cell layer, the zone of amelogenesis initiation, and serves as a paracrine differentiation signal for odontogenic cells [23,30]. It is later secreted by dentinoblasts during dentinogenesis and dental pulp stem cells (DPSCs), suggesting its autocrine function in odontogenic di fferentiation and dental pulp stem cell niche maintenance [30]. Amelogenin, secreted by ameloblasts, also participates in odontogenic di fferentiation of DPSCs by upregulating dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) expression via the ERK1/2 and p38 pathways [104]. A similar e ffect could be achieved by the application of leptin: DSPP and DMP1 expression and ERK1/2, p38, and c-Jun N-terminal kinase (JNK) phosphorylation levels were markedly increased in leptin-treated DPCs [25,105]. Moreover, leptin application in the induced pulp cavity in rats leads to increased dentin formation during reparative dentinogenesis [106].

FGF exerts a time-dependent e ffect on dental-pulp derived odontoblast precursors. Transient exposure to FGF2 during the proliferation phase is beneficial for odontogenesis while no such e ffect is achieved upon constitutive FGF application until the maturation phase. FGF2 induces DSPP and DMP1 expression, which is also mediated via ERK1/2 pathway activation. Moreover, the agonistic e ffect on BMP2 and Wnt signaling during early odontogenesis were noted in cells treated with FGF2 [26,27].

BMP/TGF-β signaling is important during early odontogenesis, where it activates SMADs and regulates Msx-1/2 transcription factors expression, as well as in di fferentiated odontoblasts, for matrix gene expression [101,107–109]. BMP2 positively regulates odontogenic di fferentiation of stem cells from exfoliated deciduous teeth (SHEDs) by promoting the expression of DSPP, DMP1, and matrix extracellular phosphoglycoprotein (MEPE) [80]. BMP2 knock-out in dental mesenchyme results in dentin deposition and microstructure abnormalities indicating its pivotal non-redundant role in early dentinogenesis [28,107], while BMP2 together with BMP4 have redundant functions in mature odontoblasts where they regulate DSPP, DMP1, bone sialoprotein (BSP) and collagen type I alpha-1 (Col1a1) expression [108]. Smad4, the intracellular component downstream of BMP/TGF-β signaling, is also necessary for DSPP, Col1a1, and osteocalcin (OCN) expression and proper odontoblast maturation. If Smad4 is ablated, dentin formation is largely impaired and does not reach normal thickness in mice [29].

Wnt ligands are involved in odontoblast di fferentiation from mesenchymal precursor cells during the early stages of tooth development and later regulate dentin matrix deposition. It is proposed that at early stages of tooth development some Wnt ligands exert e ffects via the canonical Wnt/β-catenin signaling cascade and support odontoblast precursor cells stemness, while other Wnt ligands expressed at later developmental stages activate non-canonical pathways and promote the migration, proliferation, and mineralization of odontoblast precursors during dentinogenesis [31,83,110]. Experiments in vitro demonstrated that Wnt7b stimulates the expression of Runx2 and the key dentin matrix proteins DSPP, DMP1, and Col1a1 via ERK1/2-mediated activation during dentinogenesis [83]. Wnt7b can activate canonical Wnt/β-catenin, but also the JNK cascade, thus promoting cellular migration and odontogenic di fferentiation [31]. Notably, activation of Wnt/β-catenin signaling by inhibition of GSK3β is beneficial for reparative dentine formation during cavity repair [111].

Purinergic signaling mediated by adenosine receptors (P1 receptors, ARs) and purine receptors (P2X and P2Y) was also shown to play an important role in odontogenic di fferentiation of human DPSCs. P2 receptor activation by ATP promotes the expression of DSPP, DMP1 and mineralization of DPSCs via rapid phosphorylation of ERK1/2 [32]. Treatment of DPSCs with P1 receptor agonists in combination with ATP further improved odontogenesis by contributing to the upregulation of DSPP (mediated by A2BR and A3R) and DMP1 (via A1R and A2BR) and increased mineralization (via A1R and A2BR) [81]. Intracellular molecular events of P1 and P2 receptors agonistic action remain to be elucidated, but ERK1/2 is likely involved, at least partially, in the purinergic receptor-mediated odontogenic di fferentiation of DPCs as is the case with several other di fferentiations regulated by purinergic signaling.

Aside from the importance of activation of ERK1/2 and its downstream targets resulting in the expression of key dentin matrix genes, Tao and colleagues outlined Krüppel-like factor 4 (Klf4) as a major transcription factor regulating odontogenesis [33]. Klf4 induces TGF-β secretion, which together with BMPs positively regulates DMP1, the major dentin matrix protein expression. Moreover, Klf4

regulates odontogenesis-related gene expression temporally by interacting with histone deacetylase 3 (HDAC3) during early phases of odontoblastogenesis where it represses the expression of *osterix* and *DSPP*, while at later stages, when paired up with P300, it promotes their expression [33]. Osterix is a master-regulator of many structural genes of dentin and also of odontoblasts including DSPP, DMP1, nestin, and alkaline phosphatase (ALP) [34].

Studies regarding odontoblast differentiation outline the importance of signaling pathways and their interactions alike noted to be important for ameloblast differentiation with ERK1/2 being a convergence point for several signaling cascades involved in odontogenic differentiation of dental mesenchymal cells. Recently identified Klk-Osx transcriptional tandem, p38 and JNK are important in dentin structural genes regulation and odontoblast function (Figure 2B). Several dentinogenesis-promoting molecules (listed in Table 1) were already tested in vivo and shown promising results.
