2.1. IGFs and Their Receptor Systems

IGF family consists of IGF-1, IGF-2, their receptors (IGF1R, IGF2R), and six IGFBPs 1-6 [11]. Binding to IGF1R triggers receptor dimerization and autophosphorylation of β-subunit tyrosine residues, which activates the classical downstream pathways: (i) PI3K/AKT pathway: promotes cell survival and metabolism regulation by phosphorylating AKT, which plays a central role in enamel-forming cells’ energy supply; and (ii) MAPK/ERK pathway: regulates the expression of cell cycle proteins (e.g., cyclin D1) to drive enamel-forming cells’ proliferation [12, 13]. Notably, the functional divergence between IGF-1 and IGF-2 may stem from differences in their receptor binding modes. Although both rely primarily on the IGF1R for signaling, IGF-2 has more than 100-fold higher affinity for the IGF2R (i.e., cation-independent mannose-6-phosphate receptor) than IGF-1 [14]. This property suggests that at specific stages of tooth germ development (e.g., enamel degradation), IGF-2 may remove locally excessive IGFs through IGF2R-mediated endocytosis, forming a “self-braking” mechanism for dynamic homeostasis. In addition, glycosylation modifications of IGFBPs may confer spatiotemporal-specific regulatory capabilities: mass spectrometry analysis showed that IGFBP5 secreted by enamel-forming cells exhibits a unique O-GlcNAc modification during the bell-shaped phase, and this posttranslational modification may regulate the bioavailability of IGF-1 by altering its ability to bind to the extracellular matrix. This hypothesis can be tested in organoid models by constructing glycosylation site mutants [12].

2.2. Dynamic Expression Patterns in Tooth Germ Development

By double fluorescence staining technique, the researchers revealed the spatiotemporal specificity of IGFs in dental embryo development: bud stage to cap stage: IGF-1 and HSP70 expression in mouse molar dental epithelium was higher than that in the mesenchyme, suggesting that they dominate epithelial-mesenchymal interactions; bell stage: the peak expression of IGF-1 in enamel precursor cells were synchronized with the degradation of enamel nodes, while the expression of HSP70 in this stage less coincided with the disintegration of primary enamel nodules; IGF-2 was highly expressed mainly in enamel-forming vessels, while PTEN was seen to be mild to moderately expressed in papillae and some enamel organs. IGF-1R expression was ubiquitous and showed strong intensity throughout the enamel organs. In contrast, IGF-2R expression has a rather irregular pattern in enamel organs and dental papillae [15–17].

When the tooth tissue is affected by trauma or caries, IGFs have been proved to be involved in the formation of tertiary dentin and the defensive activities of the tooth, which can promote the formation and recruitment of odontoblast-like cells and the formation of dental mineralized tissue [9]. This whole process is closely related to the ability of odontoblasts and ameloblasts derived from cranial nerve crest to maintain the persistence of embryonic programs. d’Aquino et al. [18] found that neural crest-derived cells (DFC) co-express early neural progenitor cell markers and vascular endothelial growth factor receptors, including Brn3a and flk-1, and retain embryonic markers and transcription factors, such as Oct-4, nanog, TRA1-60, and TRA-1-80-1. And can form neurons, osteoblasts, adipocytes, and other cell types after being stimulated by relevant differentiation signals, which is also an important basis for odontoblasts and ameloblasts to participate in dental cell events at birth or adulthood.

The spatial and temporal expression profiles of IGFs revealed that they may be involved in the regulation of “dual signaling centers” in the formation of dental embryonic patterns: high expression of IGF-2 in early enamel junctions for the early proliferation and differentiation of primary progenitor cells, and the expression of IGF-1 in the late enamel cell layer overlapping with HSP70 signaling. We hypothesize that IGF-2 may initiate the “initiation wave” of apical morphogenesis by activating the primary signaling of enamel nodule cells, while IGF-1 ensures the regular arrangement of the enamel column structure by maintaining the homeostasis of the enamel precursor cell pool in the subsequent stages.

3.1. Promote the Proliferation and Differentiation of Enamel-Forming Cells and the Establishment of Cell Polarity

Scholars found that GHR and IGF1 proteins were detected in both incisors and molars of rat embryos and were highly expressed in dental epithelial cells, suggesting that the GH/IGF axis plays a regulatory role in their value-added differentiation process and that treatment with GH in vitro promotes the further expression of IGF1 proteins in the epithelial cells and promotes epithelial cell proliferation and differentiation [19–21]. From the bud stage to the capitulum stage, IGF-1 was expressed in the dental plate and inner enamel cell layer, and the epithelial cells proliferated rapidly in this stage, and with the entry into the campanulate stage, the expression of IGF-1 declined, and was gradually confined to the dental plate area, while stellate reticulation cells appeared, which signaled the formation of the stellate layer. In the late campanulate stage, IGF-1 is only expressed in the apical region and cervical ring of the future teeth [22, 23], while IGF-2 is expressed in the epithelial tissues throughout the process, and forms a negative feedback mechanism with PTEN to regulate the proliferation and differentiation of enamel cells [24, 25].

The polarity-building process of enamel cells, in which the nucleus is located away from the basement membrane and the organelles are abundant and located in the near-basement membrane direction, is extremely important for the differentiation and maturation of enamel cells, as well as for their functional perfection. The polarity establishment process of enamel cells is mainly related to RhoA, Rac1, and CDC42 of the Rho family of proteins, which regulate the polymerization of the actin microfilament backbone through the rho-associated coiled-coil containing kinases (ROCK) signaling pathway, thus affecting the polarity and morphology of the cells [26]; Rac1 expression pattern during rat molar development is similar to that of RhoA, with strong positive expression during dental embryo morphogenesis and enamel cell differentiation, and enhanced expression of Rac1 in polarized enamel cells, which is mainly expressed in the cytoplasm away from the side of the basement membrane with a punctate distribution, but the specific pathway mechanism needs to be further investigated; CDC42 showed a highly dynamic spatiotemporal expression pattern in developing enamel-forming organelles, and was strongly expressed in the outer enamel epithelium, the stellate reticular layer and the middle layer. It has been found that mice with epithelial cell-specific knockout of CDC42 have thinner enamel, chalky incisors, easily abraded molar teeth after eruption, reduced microhardness and shear resistance compared with the normal group, and disorganized enamel column structure as shown by scanning electron microscopy. CDC42 and Rac1 can interact with laminin-10/11-integrin α6β4 (laminin- 10/11-integrin α6β4), which regulates cell polarity and affects the size and shape of the dental embryo through the phosphatidylinositol 3-kinase-CDC42/Rac pathway [27, 28]. Some studies have shown that IGF-1 can be involved in the RhoA/ROCK signaling pathway, activating cancer cells to promote cell scattering and invasion [29], and can also activate the up-regulation of P13K through the IGF-1/IGF-1R-PI3K/Akt pathway [30], which can indirectly affect the process of enamel cell polarization, and the specific mechanisms need to be further investigated.

3.2. Regulation of Enamel-Specific Gene Expression

Some researchers placed the first molar teeth of mice in Petri dishes containing IGF-1 and IGF-2 and reverse transcribed the extracts at 6, 12, and 18 days, respectively, and found that the concentrations of enamelogenic and enamel-forming proteins, mrana, were increased, suggesting that IGF-1 and IGF-2 can promote the synthesis of enamel-associated proteins [31]; at the same time, it was found that activation of IGF1R recruited the histone acetyltransferase p300 to the Runx2 promoter region, which increased the level of H3K27ac modification and in turn upregulated the transcription of the gene coding for the amelogenin protein. The spatial proximity of the Runx2 binding site to the topologically associated domain (TAD), where the amelogenin gene is located was increased after IGF-1 stimulation [32–34]. This suggests that IGFs may regulate the amelogenin gene cluster through three-dimensional genome remodeling, altering the chromatin spatial conformation and collaborating with multiple distal regulatory elements to drive amelogenin expression.

4.1. Promote Calcium and Phosphorus Deposition

It has been found that IGFs regulate mineralization kinetics through multiple dimensions: (i) Ion transport: IGF-1 promotes the translocation of TRP channels located in the intracellular pool to the plasma membrane, thereby enabling Ca²⁺ inward flow, and we hypothesize that this mechanism promotes calcium ion deposition in enamel and regulates cellular activity [35, 36]; and (ii) matrix regulation: matrix metalloproteinases are involved in the modification, degradation, and clearance of enamel matrix and enamel progenitor proteins, which is extremely important to the formation of enamel, studies have shown that IGF-1 and IGF-2 can activate matrix metalloproteinases through PI3K/AKT pathway, and we speculate that this mechanism can regulate enamel formation [37, 38]. The regulation of mineralization kinetics by IGFs may follow the “ion-substrate double-lock model”: on the one hand, IGF-1 and IGF-2 activate enamel metalloproteinases through TRP, and on the other hand, it protects the enamel matrix scaffold by regulating MMP-20, which synergistically ensures the deposition of hydroxyapatite along specific orientations.

4.2. Regulation of pH Homeostasis

It has been found that IGF-1 can regulate intracellular PH homeostasis and inhibit its acid efflux under hypoxic conditions, which is mainly achieved through the maintained mitochondria-derived PI-3 kinase-dependent pathway downstream of ROS [39]. Meanwhile, in the acidosis experiment in rats, the acidosis indicator HCO₃- of the experimental group treated using IGF-1 changed from 20.4 ± 0.4 mM to 22.0 ± 0.5 mM, suggesting that it has a positive effect on the regulation of intracellular PH homeostasis [40]. Carbonic anhydrase (CA) can regulate PH homeostasis by generating hydrogen ions and bicarbonate ions. CA IX has four structural domains, which are proteoglycan-like, catalytic, transmembrane, and intracellular structural domains, among which the intracellular structural domain can participate in the negative feedback regulation of aerobic glycolysis by the PI3K/AKT pathway, and we hypothesized that IGF-1 may affect cellular PH homeostasis through this pathway [41] (Figure 1).