The body’s internal clock in tooth enamel formation

Tooth enamel has a distinct hierarchical microstructure known as enamel rods that are made up of hundreds of lath-like crystallites.³⁸ Interspersed between the enamel rod crystallites is the amorphous phase. There are two intergranular phases: magnesium amorphous calcium phosphate and mixed-phase iron oxide.³⁹ The amorphous phase of enamel controls the demineralization rate and renders it more resistant to acid attack. Hence, the existence of these two distinct phases in adult tooth enamel significantly contributes to its remarkable physical, mechanical and chemical properties, and is strongly suggestive that these may develop at different cyclic phases (i.e. day/night) of the circadian clock.

Other observed microstructures of mature tooth enamel are classified as incremental features, including the cross-striations, the intradian lines, the Retzius lines, the stress lines and the laminations⁴⁰ (Fig. 1), which are different classes of incremental lines, with some of these being more clearly observable after fluorochrome injections.^41–43 These structural features are closely related to the formation processes of teeth enamel and can therefore be regarded as its cyclical growth patterns,⁴² which is strongly suggestive of the role of the circadian clock in its development. The cross-striations are incremental lines with light and dark bands that cross the enamel prisms perpendicularly with a spacing interval of about 3–6 µm. These represent the daily secretory phases of enamel-forming cells, known as ameloblasts.⁴² The intradian lines are fine bands divided by cross-striations into two or three segments, representing a shorter enamel secretion period of less than 24 h.⁴⁰ However, the exact rhythmicity of the intradian lines remains obscure.⁴³ The Retzius lines are internal long-period features found in primate enamel, and the number of cross-striations between two successive Retzius lines indicates the interval of Retzius line formation.⁴⁴ The spacing interval of the Retzius lines has been reported to be between 4 and 150 μm and is observed to decrease from the center to the periphery.⁴⁵ The laminations are spaced similarly to the cross-striations and appear parallel to the Retzius lines, and these may also be found in aprismatic enamel as well. However the relationship between laminations and cross-striations remains unclear.^40,43,46

Apart from perturbations of circadian rhythms, any insult or disruption that occurs during enamel formation may also leave growth marks on mature enamel that are manifested as wider or darker bands, referred to as stress lines.⁴⁷ The neonatal line is one of the most prominent stress lines, which is evidence of sudden changes in the environment during the birth process.

Ameloblasts, or enamel-forming cells, are the key players in enamel formation and mineralization. The development and mineralization of enamel is a complex and precisely-orchestrated process that is tightly regulated by ameloblasts.⁴⁸ Amelogenesis is initiated when the ameloblasts secrete enamel matrix proteins (EMPs), forming a hydrated protein-rich matrix. Subsequently, this matrix is then proteolytically degraded and removed by ameloblasts, to facilitate enamel mineralization.⁴⁹ However, compared to bone which is a mineralized tissue with self-repair functions, mature enamel is acellular and hardly undergoes self-repair after damage.⁵⁰

The life cycle of ameloblasts includes the pre-maturation stage, secretory stage, transition stage, maturation stage and post-maturation stage (Fig. 2a). The secretory and maturation stages, in particular, are primarily responsible for enamel formation. The EMPs are secreted by the secretory stage ameloblasts (S-ABs), which play a crucial role in enamel development by facilitating its calcification. After undergoing the transition stage, the S-ABs differentiate into maturing ameloblasts (M-ABs), which help transport minerals, regulate enamel mineralization and modulate protein degradation and absorption.^50–52

During the maturation stage, ameloblasts cyclically alternate between two morphological phenotypes, which are known as ruffle-ended ameloblasts (RAs) and smooth-ended ameloblasts (SAs) respectively (Fig. 2b). The alternation between these two morphological phenotypes occurs 5 ~ 7 times.^53–55 In the case of rodent ameloblasts, the RA phase lasts for about 8.5 h, while the SA phase lasts for about 2 h.⁵⁶

It was observed that maturation-stage ameloblasts exhibited distinctively different subcellular localization and morphology of mitochondria, as compared to secretory-stage ameloblasts. During the secretory stage, mitochondria are mainly located between the cell nucleus and the proximal or basal pole.⁵⁷ Their shape is predominantly elongated and is variable in length.⁵⁷ As for the mitochondria of maturation stage ameloblasts, these are primarily located at the proximal pole within the infranuclear region or adjacent to the distal ruffled border, covering about half the way from the apical cytoplasm to the nucleus and continuing to the Golgi region,⁵⁴ which may contribute to ion movement.⁵⁸ The morphology of the mitochondria during the maturation-stage is mainly spheroidal-shaped, with some internal “helices” described as DNA structures.⁵⁷

Some studies have indicated that as the ameloblasts transition from the secretory to the maturation stage, the basal mitochondria increase in size and number, as well as tend to show inclusions such as helical structures.^59,60 A study based on HAT7 cells reported that RAs preferentially utilize OXPHOS for energy supply, while SAs rely on glycolysis-dominant energy metabolism.⁶¹ This could be because RAs require more ion transport, endocytosis and other functions, so their energy demand is higher than that of SAs. Some reports have suggested that the energy metabolic state is an important determinant of the RA/SA phenotype,⁶¹ as RA would undergo a transition to SA once its metabolism is exhausted.⁶² However, the regulatory mechanisms of the phenotype switch by maturation-stage ameloblasts need to be further elucidated.^63,64

Mature enamel contains very few organic materials such as proteins and peptides (less than 1%), whereas proteins make up the bulk of the developing enamel matrix. During the secretory stage, ameloblasts synthesize and secrete structural enamel matrix proteins (EMPs), including amelogenin (AMELX), ameloblastin (AMBN), enamelin (ENAM)^60,65,66 and various proteinases.⁶⁷ Among these up to 90% is AMELX,⁶⁸ which forms AMELX nanospheres through the utilization of its self-assembly properties. Aligned AMELX nanoribbons could act as a template for guided apatite crystal growth and serve as a precursor to enamel rods⁶⁹ (Fig. 3). Besides AMELX, other proteins also play crucial roles in amelogenesis. For example, AMBN is a key cell adhesion molecule during ameloblast development and differentiation,⁷⁰ while ENAM is essential for apatite nucleation.⁷¹

Various proteinases are also involved in amelogenesis. Enamelysin (Matrix metallopeptidase 20, MMP20) and kallikrein-related peptidase 4 (KLK4) are two key extracellular proteinases expressed during amelogenesis that cleave structural proteins and function in enamel maturation and mineralization.⁶⁵ MMP20 is expressed mainly during the secretory stage,^65,72,73 while KLK4 is predominantly expressed during the maturation stage.^72,74,75 These two proteinases degrade and remove EMPs preferentially and selectively from HAP in vitro.⁷⁶ The secretory profiles of most of these EMPs and proteinases follow a cyclical pattern,^19,77,78 which strongly indicate that the circadian clock is implicated in amelogenesis by modulating the secretion of these proteins by ameloblasts.

Maturation-stage ameloblasts facilitate ion transport towards the enamel layer.⁵¹ Passive and active pathways of ion transport are both present. The former is referred to as the passive paracellular/intercellular movement through “leaky” junctions^51,76 or the passive transcellular pathway; while the active transport pathways involve ion transporters, channels and pumps.^66,79,80 Ions from the circulation, such as Ca²⁺ and HCO₃^–, diffuse across the papillary layer and are actively transported into polarized ameloblasts via ion exchangers and pumps located at their basolateral membrane,^81,82 which are implicated in bicarbonate production (Car2), bicarbonate transport (Slc4a4) and enamel matrix endocytosis (Lamp1).⁶⁶ These various ion exchangers are known to be differently expressed during the light/dark cycle,⁷⁸ thus implicating the circadian clock also regulates ion transportation. Ca²⁺ is mainly transported actively through the store-operated Ca²⁺ entry (SOCE) channels. SOCE entails Ca²⁺ release from intracellular pools followed by Ca²⁺ entry. The Ca²⁺ release-activated Ca²⁺ (CRAC) channels,⁸³ which are primarily composed of ORAI calcium release-activated calcium modulator 1 (ORAI1), stromal interaction molecule 1 and 2 (STIM1 and STIM2), are the best-characterized SOCE channels and they are significantly upregulated in maturation stage ameloblasts. Intracellular [Ca²⁺] signaling plays an important role in both enamel mineralization and peripheral clock timekeeping,⁸⁴ therefore it could be an essential intermediary between the circadian clock and amelogenesis.

To date, much progress has already been made in the understanding of amelogenesis as well as the functional properties of mature enamel so far. However, partly due to enamel’s highly hierarchical structure and the complexity of its chemical composition which still needs to be further explored, there has been limited success in repairing enamel lesions or synthesizing enamel in vitro.^38,39

The autoregulatory cell-autonomous transcriptional-translational feedback loops (TTFLs) in mammals generate the molecular mechanisms of cellular circadian clocks.^30,85 This cellular clock system is made up of several genes, including the core clock genes circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1) which handle positive regulation, as well as period (PER1, PER2) and cryptochrome (CRY1, and CRY2) which handle negative regulation.^30,85,86 The general molecular mechanisms of TTFLs are illustrated in Fig. 4. CLOCK together with BMAL1 heterodimerize to form the CLOCK: BMAL1 heterodimers that bind directly to the regulatory elements (E-boxes) of a set of rhythmic genes encoding the repressor proteins period (Per1, Per2, Per3) and cryptochrome (Cry1, Cry2) to promote transcription.^86,87 Other clock proteins regulate the expression of Clock and Bmal1,⁸⁸ which is linked to three transcriptional autoregulatory feedback loops. The first feedback loop is made up of PER and CRY, and in mammals, PER heterodimerizes with CRY, inhibiting CLOCK: BMAL1 transcriptional activity and repressing their gene transcription.^87,89–91 The second feedback loop is composed of REV-ERB (encoded by the genes Nr1d1/Nr1d2 and Rora/b) and retinoic acid-related orphan receptor (ROR) proteins.^92,93 REV-ERB and ROR have opposite effects as they bind to retinoic acid response elements (RREs) on gene promoters, with REV-ERB inhibiting gene transcription, while ROR promotes gene transcription.^94–96 The target genes of REV-ERBs/RORs include BMAL1, CLOCK,⁹⁶and CRY1,⁹⁷ exerting both negative and positive effects on the expression of core clock genes and other clock-controlled genes (CCGs), especially metabolism-related genes.^98,99 REV-ERB gene expression is in turn regulated by CLOCK, BMAL1,⁹⁹ and PER2,¹⁰⁰ thus forming a feedback loop. Another feedback loop is related to D-site-binding protein (DBP)¹⁰¹ and nuclear factor interleukin-3 regulated (NFIL3),¹⁰² which dimerize and bind to D-box elements on target genes, including Per1/Per2,¹⁰³Nr1d1/Nr1d2, and Rora/Rorb. The timing of the PER/CRY negative feedback loop is also influenced by the DBP/NFIL3 loop.¹⁰⁴ Among these three loops, the REV-ERBs/ROR loop acts as a key output mediator, playing a key role in regulating genes related to metabolism,⁹⁸ while DBP/NFIL3 influences the circadian transcription of many other target genes and responds to cellular signals.^105,106

The binding of the CLOCK: BMAL1 complexes is also affected by other signaling molecules. For example, adenosylhomocysteinase (AHCY) is required for BMAL1 genome-wide recruitment to chromatin and subsequent circadian transcription,¹⁰⁶ which implies that the circadian clock system might have regulatory crosstalk with other physiological systems such as energy metabolism.¹⁰⁶ Additionally, TTFLs can be directly affected by extracellular stimuli via intracellular transduction by second messengers such as calcium and cAMP.^107,108 Calcium has been shown to act on TTFLs through the calcium/cAMP-dependent transcription factor (CREB).⁸⁴ Therefore, these three feedback loops have many complex interactions with the core clock genes and CCGs, as well as crosstalk with other systems that are essential for cellular activities.

BMAL1, CLOCK, PER1 and PER2 expression can be detected in dental tissues during tooth development and display regular oscillatory expression patterns in tooth germs.^19,78,109 Circadian clock gene expression could be influenced by photoperiod stimulus,¹¹⁰ with the expression levels of Bmal1 and Clock peaking during the light phase.^77,110 In an in vitro study, periodic opposite oscillations of Per2 and Bmal1 within 48 h can be detected in ALC cells after synchronization,¹¹¹ with BMAL1 and PER2 being reciprocally expressed in ameloblasts and transferred from the nucleus to cytoplasm every 12 h.¹⁹

Existing studies provide multiple lines of evidence that circadian rhythms influence ameloblast behaviour, including cell differentiation, cell morphological and functional transitions, energy metabolism, pH regulation and intercellular connections.

The differentiation of ameloblasts is known to be perturbed by circadian rhythm disruption. Studies have shown that abnormal circadian rhythms in pregnant mice can lead to delayed tooth germ development in their offspring. Histological observations have shown that upon circadian clock disruption, ameloblasts could not form a normal tall columnar structure but retained a short columnar structure, with the polarity of the corresponding odontoblasts being delayed as well.¹¹²

Evidence suggests that the maturation phase of amelogenesis has a rhythmic pattern, and this pattern may be related to circadian rhythms, as implied by its incremental features. AMELX is regulated by the core clock genes (Bmal1, Clock, Per1, and Per2), which oscillate at both the RNA and protein levels in a 24 h cycle.¹⁰⁹Amelx expression in 2-day postnatal mouse molars was measured every 4 h throughout a total duration of 48 h, with oscillations lasting approximately 24 h. The acrophase of AMELX level occurs during the light period, while the bathyphase occurs during the dark period,⁷⁸ which exhibits reciprocal oscillations comparable to the expression of clock genes. Besides Amelx, Ambn and Amtn have also been detected to be rhythmically expressed during enamel development.⁷⁷ During the dark period, however, the expression of maturation-stage specific genes involved in bicarbonate production (Car2), bicarbonate transport (Slc4a4) and enamel matrix endocytosis (Lamp1) are upregulated.⁷⁸

The transcription factors runt-related transcription factor 2 (RUNX2) and distal-less homeobox 3 (DLX3), regulate the rhythmic expression of several ameloblast-specific genes throughout a diurnal period of 24 h, via interaction with the core clock genes.¹¹³ Being regulated by the circadian rhythm, periodic changes in RUNX2 expression in ameloblasts play a key role in inhibiting secretory phase gene expression (e.g. AMELX and ENAM) while upregulating expression of maturation stage genes, such as KLK4.¹¹³ In contrast, DLX3 is upregulated during the secretory stage, which is critical in promoting the expression of AMELX and ENAM, while being downregulated during the maturation stage.¹¹³

Synchronizing signals from the central clock may also affect amelogenesis. Melatonin likely regulates enamel development because the melatonin 1a receptor (Mel1aR) is expressed by secretory ameloblasts as well as other enamel-forming cells such as the cells of the stratum intermedium and stellate reticulum.²⁵ As previously mentioned, SAs are primarily fueled by glycolysis while RAs are primarily fueled by OXPHOS for their energy metabolism. It has been proposed that the redox system exhibits an autonomous oscillation that interacts with the rhythmic oscillation of gene transcription within cells.³ This mechanism likely exists in ameloblasts, but more research is required to confirm this.

Clock also regulates cell adhesion in ameloblasts. Per2 knockdown was reported to alter the subcellular localization of key markers of cell adhesion like E-cadherin in ameloblast-lineage cell (ALC) line, which would imply that Per2 knockdown affected ameloblast organization and secretion.¹¹¹

Cell differentiation, cell morphological and functional transitions, energy metabolism, pH regulation and intercellular connections are all rhythmically regulated during enamel development. Effective coordination between these oscillators may be required to ensure normal ameloblast function and the formation of well-developed tooth enamel. A summary of section 3.2 is presented in Fig. 5.

Enamel hypoplasia (EH) is a quantitative dental enamel defect defined as a region of reduced enamel thickness with a smooth outline caused by disruptions in enamel formation during tooth development, which can be caused by inherited disorders or environmental insults.^114,115 EH, caused by genetic factors is termed amelogenesis imperfecta (AI),¹¹⁵ and if the developmental defect is caused by systemic origins affecting one or more molars, it is termed as molar-incisor hypo-mineralization (MIH).^116,117 Various oral healthcare problems are associated with EH such as poor oral hygiene, unsatisfactory aesthetic appearances, impaired masticatory function, dental sensitivity, increased dental care costs and requirement for lifelong extensive restorative care, in addition to detrimental effects on social interactions, which could severely impact the life quality of patients.^118,119 A previous review summarized various EH risk factors, such as maternal and infant nutrition, premature birth, infantile convulsions and infection.¹¹⁴ EH attributable to inherited genetic disorders is quite rare, whereas EH caused by environmental or epigenetic factors is more common. It has also been suggested that compared to genetic factors, environmental risk factors may also exert a greater influence on the occurrence of EH in primary maxillary incisor teeth.¹²⁰

Animal experiments have confirmed that circadian rhythm disruption leads to enamel hypoplasia in vivo. Because tooth development occurs during both the embryonic and postnatal periods, disruptions in either the maternal or postnatal circadian rhythms may disrupt enamel development. Maternal circadian rhythm disruption may affect their offspring. Studies have shown that neonatal mice exposed to all-dark or all-light environments have delayed enamel development, indicating that circadian rhythm disruption caused by photoperiod perturbations can impact amelogenesis.¹¹² This evidence thus validated that circadian rhythm disruption of either the postnatal or prenatal phase can exert negative effects on amelogenesis.

It has been validated that environmental circadian disruption leads to the dysregulation of circadian gene expression in vivo. Abnormal circadian rhythms have been reported to disrupt the formation of mandibular first molar germs in mice, characterized by reduced protein expression levels of BMAL1 and PER2, as well as decreased immunohistochemical staining of BMAL1 and PER2 in ameloblasts.¹¹¹ Neonatal mice exposed to an all-dark or all-light environment also exhibited significant changes in their expression level of melatonin receptors (MTs). Some studies have shown that the injection of MT2 selective inhibitor 4P-PDOT into E16 maternal mice results in abnormal teeth mineralization in their newborns but has no significant effect on the morphology of the tooth germ.¹¹² Electron microscopy has shown that there are abnormalities in multiple organelles, including the disappearance of mitochondrial cristae and vacuolar degeneration of cells, but the underlying mechanisms need to be elucidated. Additionally, the light-dark photoperiod also affects the expression of melatonin receptors. Neonatal mice exposed to full dark or full light display significant changes in melatonin receptor (MTs) expression.¹¹²

To date, several studies have revealed the mechanisms of circadian rhythm disruption associated with EH at the histological and molecular levels. Electron microscopy imaging revealed some obvious voids among ameloblasts and increased necrosis of ameloblasts.¹¹² Molecular dysregulation in ameloblasts was also observed, which is referred to as a downregulated expression of EMPs (i.e. AMELX), which in turn leads to reduced enamel matrix deposition.¹¹¹ Apart from EMPs, other proteins that are essential for enamel cell activities such as cytokeratin 14 (CK14) and filamentous actin (F-actin) were also observed to be downregulated.¹¹¹

It has also been demonstrated that targeting clock genes affects amelogenesis. Per2 knockdown inhibits ameloblast-lineage cell (ALC) differentiation and also affects ALC cell adhesion by altering the subcellular localization of E-cadherin.¹¹¹Nr1d1 over-expression resulted in the up-regulation of Amelx and the down-regulation of Mmp20 and Klk4.¹¹³ Meanwhile, overexpression of Bmal1 can lead to up-regulation of RNA expression levels of Amelx and Klk4, while overexpression of cry1 had the opposite effect.¹¹³

Animal studies have validated that Mmp20 or Klk4 mutation is associated with non-syndromic AI, which is manifested as teeth enamel being not fully mineralized.¹²¹Mmp20-null mice exhibit this phenotype with hypoplastic enamel, and the hypoplastic enamel would delaminate from the dentin soon after tooth eruption.¹²²Klk4-null mice exhibited an enamel hypomaturation phenotype and the enamel would abrade or fracture above the DEJ soon after tooth eruption.¹²³ Changes in the expression levels of Klk4 are closely associated with DLX3 over-expression or knockdown.¹²⁴ As mentioned previously, the transcription factor DLX3 is regulated by core clock genes, thus giving rise to the hypothesis that circadian rhythm disruption might lead to the dysregulation of enamel proteinases like KLK4 via changes in DLX3 expression.¹¹³

In vivo, photoperiod perturbations might also affect the peripheral circadian clock via SCN and its hormonal output pathway. Pregnant mice treated with melatonin receptor antagonists exhibit enamel tissue deficiency in their offspring that is associated with downregulated expression of MTs and AMELX.¹¹² Nevertheless, incremental lines can still be observed in mice dentin, even though SCN is partly or completely damaged. This evidence thus indicates that the peripheral clock and tissue-specific circadian rhythm in teeth might be more independent than previously assumed.¹²⁵ Hence, it is reasonable to postulate that the same condition might also exist in enamel development.

As previously stated, circadian rhythms may integrate multiple independent oscillators during enamel development, a disruption or loss of rhythm may cause these oscillators to lose their synchronization. Although their respective functions may not be significantly affected, the coordination and synchrony with each other might go out of balance. As a result, antagonistic biological processes occur simultaneously, or the functional protein is not in the optimal environment to function properly, which may be the reason why enamel can still form but developmental abnormalities occur. Much further research is still required to rigorously characterize the potential negative effects of circadian rhythm disruption on enamel development. A summary of section 4.1 is presented in Fig. 6.

Although a number of etiological hypotheses for EH have been postulated and the mechanisms have been revealed, the treatment for EH is still symptomatic, which means that the abnormal enamel can only be restored but not recovered. The treatment of enamel hypoplasia is a lifelong management, which requires the participation of multidisciplinary specialists in pediatrics, endodontics, periodontology, prosthodontics and orthodontics. This lifelong management requires a step-wise treatment plan, starting with the most conservative treatment regimen. The main treatment goals for EH patients are to prevent pain and early tooth loss, restore the appearance, function and aesthetics of dentition, protect tooth tissue and improve the quality of life.¹²⁶

The main treatment modalities include oral hygiene instruction, dietary guidance, tooth desensitization, tooth bleaching and restorative treatment. Among these, the treatment modality that is closest to the etiological treatment might be applying Casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) paste.^127,128 Although this treatment could aid enamel remineralization, it still cannot exert any effect on new enamel formation. Rehabilitative treatment can improve the function and aesthetic appearance of the tooth, which includes the use of direct or indirect composite resin, veneer, crown, fixed partial denture, removable partial denture, and implant.¹¹⁶ The treatment of the severe symptoms is quite complicated, and due to the abnormal properties of enamel, the process of restoration treatment also faces many challenges. For example, EH patients’ teeth are brittle so that sufficient resistance and retention cannot be easily approached and it is difficult to obtain a good bonding effect. EH patients are more susceptible to caries, leading to more challenges in restoration or maintenance. Some patients have insufficient intermaxillary distance and clinical crown height due to severe wear, which requires crown lengthening surgery.¹²⁹

Most evidence on AI treatment is from case reports and lacks randomized clinical trials, which means the quality of the evidence is unsatisfactory.^116,129 Hence, treating EH by using an evidence-based approach is difficult. Long-term oral treatment also imposes financial and care burdens on EH patients and their families.

Therefore, we conducted this review to provide novel insights into the understanding of tooth enamel development. The role of circadian rhythms in enamel development could be a possible future research direction, which could offer new perspectives on novel treatment and preventive strategies for EH.