2.2.1. Amelogenesis

Tooth enamel formation or amelogenesis is the process of tooth enamel generation by ameloblasts, during which ameloblasts move towards the enamel surface and secrete proteins such as amelogenin, ameloblastin, and enameling. These proteins serve as scaffolds for calcium and phosphorus ions to be deposited on, thus guiding hydroxyapatite crystals aggregates—the enamel rods—generation. In this process, amelogenin and amelotin phosphorylation appears to be essential for correct enamel rod formation/organization [84–86]. The scaffolds are later degraded by matrix proteases and ameloblasts undergo apoptosis, which makes enamel the most mineralized acellular tissue in the human body, consisting of 95% hydroxyapatite crystals and 5% organic matter and water by weight [10,11]. Enamel is subjected to wear and tear throughout life. However, unlike other mineralized tissues of the human body, enamel cannot be regenerated due to its acellular nature. Although several cell sources were shown to have amelogenic capacity including keratinocyte stem cells, epithelial cell rests of Malassez (ERM) from periodontal ligament, odontogenic oral epithelial stem cells (OEpSCs), adipose tissue-derived mesenchymal stem cells (AT-MSCs), and iPSCs [87–92].

Since ameloblasts undergo apoptosis upon fulfilling their function of enamel production, studies of amelogenesis rely on in vitro models such as murine immortalized ameloblast-lineage cell (ALC) line [15], organotypic cultures, or rodent models. Many discoveries regarding ligands, their downstream transcriptional factors and responsive genes expressing core enamel proteins and matrix metalloproteinases were done using the mentioned ALC line. Shh, which is one of the major ligands expressed in the enamel knot during tooth morphogenesis, was shown to have a direct effect on the expression of the major enamel matrix proteins amelogenin and ameloblastin. The upregulation of these proteins is mediated by an activated glioma-associated transcription factor (Gli1) in the presence

of Shh [42,77]. Notably, Gli1 was proposed as a marker for selecting stem cells with the odontogenic potential for tooth regeneration [93,94]. Runx2 together with odontogenic ameloblast-associated protein (ODAM) regulates matrix metalloproteinase 20 (MMP20) expression, the key enamel matrix-degrading enzyme [43], and has an affinity for the *Wdr72* (gene coding for maturation-stage ameloblast-specific protein) promoter [44]. WDR72 is an intracellular protein abundant in ameloblasts during enamel maturation with a proposed function in amelogenin endocytosis [20].

Studies in dental organotypic cultures and transgenic mice also point out the importance of the mentioned pathways in dentogenesis. For example, Shh in combination with FGF8 was recognized as a stemness promoting ligans for ameloblast precursors (human skin fibroblasts) in a human-mouse chimeric tooth [87], while Runx2 was shown to have an affinity for the amelotin promoter and regulates its expression during the enamel maturation stage [95]. Regarding amelogenin turnover, a novel role of cytoplasmatic B-cell CLL/lymphoma 9 protein (Bcl9), its paralog B-cell lymphoma 9-like protein (Blc9l) and interaction partners Pygopus 1/2 (Pygo1/2) is proposed to play a role in amelogenin secretion [96].

Timely expression of β-catenin in dental tissues shapes tooth development by modulating various developmental signaling pathways, leading to the proper tooth number and morphology [45]. It was demonstrated in vitro that the β-catenin pathway, which is regulated by Wnt ligands, is involved in ameloblast polarity and motility [97]. Overactivation of the β-catenin pathway in the dental epithelium during the earliest stages of tooth development results in hyperdontia, and ablation—in tooth agenesis [98]—while, if overactive in postnatal ameloblasts, it causes poorly structured, softened enamel and its delayed formation [46]. Additionally, β-catenin overactivation downregulates enamel matrix metalloproteinases MMP20 and kallikrein 4 (Klk4), which are important in the removal of scaffolding proteins from maturing enamel [46]. An important regulator of Wnt/β-catenin pathway activity in ameloblasts is glycogen synthase kinase 3 beta (GSK3β) [99].

TGF-β superfamily ligands such as BMPs and TGF-βs are regulating enamel structural genes and matrix metalloproteinases expression. MMP20 in turn regulates TGF-β isoforms activity [47,100]. All three TGF-β isoforms induce Klk4 expression, while TGF-β1 and β2 induce amelotin expression [47]. TFG-β1 regulates Runx2 and its downstream target Wrd72 gene [44]. Thus, it appears that TGF-βs are key ligands involved in the regulation of enamel scaffolding protein removal and endocytosis during enamel mineralization. BMP knock-outs result in downregulated matrix proteins and metalloproteinase expressions. In detail, BMP2 knock-out reduced amelogenin, enamelin, MMP20, and Klk4 expression, similarly to double-knockout of BMP2 and -4, which resulted in a significant reduction of MMP20 and Klk4 in ameloblasts [21,22]. Metalloproteinase insufficiency is detrimental for the enamel structure since excessive protein content in enamel does not allow properly organized crystalline structure formation, making the enamel softer and less shear-resistant.

From the above-reviewed studies, it is evident that timely regulation of ligands known to be important for cell stemness maintenance and calcified tissue metabolism are the keys to structurally and morphologically correct enamel formation. Enamel integrity depends on proper enamel scaffolding protein deposition, phosphorylation state, and timely cleavage, which allow ameloblast migration and crystals deposition in an organized oriented pattern. The summary of the major signaling pathways involved in amelogenesis is schematized in Figure 2A and pathways modulators listed in Table 1.
