Dental Developmental Defects: A Pilot Study to Examine the Prevalence and Etiology in a Population of Children between 2 and 15 Years of Age.

Dental development defects (DDDs) are quantitative and/or qualitative alterations produced during odontogenesis that affect both primary and permanent dentition. The etiology remains unknown, being associated with prenatal, perinatal, and postnatal factors. The aims were to identify the possible etiological factors, as well as the prevalence of DDDs in the primary and permanent dentition in a pediatric population. Two hundred twenty-one children between 2 and 15 years of age, patients of the master's degree in Pediatric Dentistry of the Complutense University of Madrid, were reviewed. DDDs were observed in 60 children. Next, a cross-sectional, case-control study was carried out (60 children in the control group and 60 children in the case group). The parents or guardians completed a questionnaire aimed at identifying associated etiological factors. The prevalence of DDDs in patients attending our master's program in both dentitions was 27.15%. Otitis, tonsillitis, high fevers, and medication intake stood out as the most relevant postnatal factors among cases and controls. The permanent maxillary right permanent central incisor and the primary mandibular right second molar were the most affected; there were no differences in relation to gender. One out of three children who presented DDDs in the primary dentition also presented DDDs in the permanent dentition. Prenatal and postnatal etiological factors showed a significant relationship with DDD alterations, considered risk factors for DDDs in both dentitions.

In vivo real-time assessment of developmental defects in enamel of anti-Act1 mice using optical coherence tomography.

The purpose of this study was to explore the feasibility of using optical coherence tomography (OCT) for real-time and quantitative monitoring of enamel development in gene-edited enamel defect mice. NF-κB activator 1, known as Act1, is associated with many inflammatory diseases. The antisense oligonucleotide of Act1 was inserted after the CD68 gene promoter, which would cover the start region of the Act1 gene and inhibit its transcription. Anti-Act1 mice, gene-edited mice, were successfully constructed and demonstrated amelogenesis imperfecta by scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectroscopy. Wild-type (WT) mice were used as the control group in this study. WT mice and anti-Act1 mice at 3 weeks old were examined by OCT every week and killed at eight weeks old. Their mandibular bones were dissected and examined by OCT, micro-computed tomography (micro-CT), and SEM. OCT images showed that the outer layer of enamel of anti-Act1 mice was obviously thinner than that of WT mice but no difference in total thickness. When assessing enamel thickness, there was a significant normal linear correlation between these methods. OCT could scan the imperfect developed enamel noninvasively and quickly, providing images of the enamel layers of mouse incisors.

Reconstructing tooth crown heights and enamel caps: A comparative test of three existing methods with recommendations for their use.

Studies of enamel growth and thickness, whether in paleoanthropology, bioarchaeology, or primatology, require measurements of crown height (CH), cuspal enamel thickness (CET), average (AET), and/or regional enamel thickness (RegAET) on complete, unworn crowns. Yet because fully unworn crowns are uncommon, three methods to bolster sample sizes by reconstructing slightly worn teeth have been developed: Profile, Polynomial, and Pen Tool. Although these methods have been tested for accuracy, no study has yet directly compared the three methods to assess their performance across CH, CET, AET, and RegAET measurements. Moreover, it is currently unclear how accurate the methods are when reconstructing crowns with varying degrees of wear. The present study addresses this gap in our understanding of how these methods perform on four key dental measurements, evaluates the degree of wear for which accurate crown reconstructions can be completed, and offers recommendations for applying these methods. Here, the methods are compared on Paranthropus robustus mandibular molars, a sample chosen because it exhibits variable morphology, presenting a challenge for reconstruction methods. For minimally worn teeth, Profile, Polynomial, and Pen Tool methods can be employed (in that order) for all measurements except CET, which cannot be reliably measured on reconstructions. For teeth with wear that obliterates the nadir of the occlusal basin or dentin horns, CH and AET can be measured using Profile and Polynomial reconstructions; however, no other measurements or methods were reliable. Recommendations provided here will make it possible to increase sample sizes and replicability, enhancing studies of enamel thickness and growth.

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ.

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2-/- (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.

Enamel thickness and growth rates in modern human permanent first molars over a 2000 year period in Britain.

OBJECTIVES: This study explores variation and trends in first molar enamel thickness and daily enamel secretion rates over a 2000 year period in Britain. METHODS: Permanent first molars (n = 89) from the Roman, Anglo-Saxon, and Medieval periods, as well as modern-day Britain, were analyzed using standard histological methods. Relative enamel thickness (RET) and linear measurements of cuspal and lateral thickness were calculated for mesial cusps. Daily secretion rates (DSRs) were calculated for inner, mid, and outer enamel regions in both cuspal and lateral enamel. Significant differences and trends were identified between samples using nonparametric statistical tests. RESULTS: Enamel thickness differed between some populations, but no temporal trends were identified. Early Anglo-Saxon molars had significantly thinner RET than both Late Anglo-Saxon (p < .00) and Medieval (p < .00) molars. Lateral enamel from the Roman molars was significantly thinner than the modern-day sample (p = .04). In contrast, a significant slowing trend in DSRs was observed across the more ancient to modern-day samples in every measured region except the mid-lateral enamel region. DISCUSSION: This study presents the first evidence for a gradual slowing in the daily rate that enamel is secreted in molars over the past 2000 years in Britain. However, this trend was not matched by consistent or significant positive or negative shifts in enamel thickness. These findings suggest that modern human molars of similar enamel thickness, from different modern and ancient populations, formed at different rates.

DNA sequencing reveals AMELX, ODAM and MMP20 variations in dental fluorosis.

OBJECTIVE: Dental fluorosis (DF) is a dental development disorder caused by chronic fluoride overconsumption. There are differences in the susceptibility to and severity of DF in studied populations. The objective of the present study was to determine if single-nucleotide variations (SNVs) in the genes Amelogenin (AMELX), Odontogenic Ameloblast Associated (ODAM) and Matrix Metalloproteinase 20 (MMP20) are associated with DF by evaluating the relationship between variations in these genes and the degree of DF severity. SUBJECTS AND METHODS: Schoolchildren from two regions of Durango State and Mexico City, Mexico, were studied. The DF phenotype was determined using the Thylstrup and Fejerskov (TF) index. DNA was obtained from the buccal mucosa of each participant, and the presence of the variations rs946252 in AMELX, rs1514392 in ODAM and rs1784418 in MMP20 was determined by bidirectional DNA sequencing. RESULTS: A total of 180 DNA samples from 30 schoolchildren from 2 areas of Durango State were sequenced and analyzed. Differences in the severity of DF were found between the study areas (p = 0.006). SNVs in theMMP20 gene were present in 76.9 % of the participants in the high fluoride concentration and lower DF severity area. CONCLUSION: AMELX and ODAM variations was not different between the two populations with respect to DF severity; however, the presence of rs1784418 differed between phenotypes with regard to susceptibility to DF. Therefore, MMP20 might be related to the various phenotypes of DF and may serve as a protective marker.

Enamel formation and growth in non-mammalian cynodonts.

The early evolution of mammals is associated with the linked evolutionary origin of diphyodont tooth replacement, rapid juvenile growth and determinate adult growth. However, specific relationships among these characters during non-mammalian cynodont evolution require further exploration. Here, polarized light microscopy revealed incremental lines, resembling daily laminations of extant mammals, in histological sections of enamel in eight non-mammalian cynodont species. In the more basal non-probainognathian group, enamel extends extremely rapidly from cusp to cervix. By contrast, the enamel of mammaliamorphs is gradually accreted, with slow rates of crown extension, more typical of the majority of non-hypsodont crown mammals. These results are consistent with the reduction in dental replacement rate across the non-mammalian cynodont lineage, with greater rates of crown extension required in most non-probainognathians, and slower crown extension rates permitted in mammaliamorphs, which have reduced patterns of dental replacement in comparison with many non-probainognathians. The evolution of mammal-like growth patterns, with faster juvenile growth and more abruptly terminating adult growth, is linked with this reduction in dental replacement rates and may provide an additional explanation for the observed pattern in enamel growth rates. It is possible that the reduction in enamel extension rates in mammaliamorphs reflects an underlying reduction in skeletal growth rates at the time of postcanine formation, due to a more abruptly terminating pattern of adult growth in these more mammal-like, crownward species.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model.

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry. KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth. Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

Combination of Runx2 and Cbfß upregulates Amelotin gene expression in ameloblasts by directly interacting with cis­enhancers during amelogenesis.

Amelotin (Amtn) is a recently identified enamel protein secreted by ameloblasts at late stage of enamel development. Runt­related transcription factor 2 (Runx2) in combination with the coactivator core­binding factor ß (Cbfß) regulates the early stages of tooth development. The aim of the present study was to investigate the role of Runx2 in the regulation of Amtn gene expression in ameloblasts. Immunohistochemistry was performed and the results revealed that Runx2 protein was predominantly expressed in the nuclei of ameloblasts during the transition stage and the maturation stage of enamel development, whereas Cbfß was expressed in ameloblasts from the secretory stage to the maturation stage. Reverse transcription­quantitative polymerase chain reaction results demonstrated that Runx2 knockdown decreased Amtn expression in ameloblast­lineage cells and co­expression of Runx2 and Cbfß in ameloblast lineage cells induced an upregulation in Amtn gene expression. Two putative Runx2­binding sites within the Amtn promoter were identified using bioinformatics analysis. Results of an electrophoretic mobility shift assay and chromatin immunoprecipitation indicated that Runx2/Cbfß bound to specific DNA sequences. Site­directed mutagenesis of the Runx2 binding sites within the Amtn promoter resulted in decreased basal promoter activity and did not affect the overexpressed Runx2/Cbfß. The results of the present study suggest that Runx2 upregulates Amtn gene expression via binding directly to Runx2 sites within the Amtn promoter during amelogenesis.

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo.

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Mineralization-defects are comparable in fluorotic impacted human teeth and fluorotic mouse incisors.

OBJECTIVE: Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. DESIGN: Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. RESULT: Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. CONCLUSION: The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that also in developing human teeth fluoride-stimulated local acidification of enamel could be a mechanism for developing fluorotic enamel.

[Expression and function of microRNAs in enamel development].

microRNAs (miRNAs) are endogenous short, noncoding RNAs that can negatively regulate gene expression post-transcriptionally. miRNAs are involved in multiple developmental events in various tissues and organs, including dental enamel development. Any disruption during enamel development may result in inherited enamel malformations. This article reviews the expression and function of miRNAs in enamel development.

Is resin infiltration an effective esthetic treatment for enamel development defects and white spot lesions? A systematic review.

OBJECTIVES: To determine if resin infiltration is an effective treatment for improving the esthetic appearance of tooth discoloration resulting from development defects of enamel (EDD) and white spot lesions (WSL) by means of a systematic review. DATA SOURCES: A comprehensive search was performed in PubMed, Scopus, Web of Science, LILACS, BBO Library, Cochrane Library, and SIGLE, as well as in the abstracts of IADR conference, and in the clinical trials registry. STUDY SELECTION: Clinical studies in patients with whitish tooth discoloration, in which the resin infiltration technique was applied, were included. Color masking was the primary outcome. The methodological quality and risk of biases of included papers was assessed using MINORS criteria for non-randomized (NRS) comparative studies and Cochrane Collaboration for randomized clinical trials (RCT). RESULTS: From a total of 2930 articles, 17 were assessed for eligibility and 11 remained in the qualitative synthesis. Four NRS and seven RCT studies were selected, the latter consisting of four full-text studies and three conference abstracts. Two studies were excluded from the quality assessment, due to overlapping results. The number of participants (treated teeth) ranged from 18 to 21 (38-74) in the NRS, and 20-83 (20-231) in the RCT studies. Post-orthodontic WSL were the most frequent treated lesions. Initial condition was used as control in the NR studies. In the RCT, resin infiltration was compared to non treatment, remineralization, or bleaching. Overall, partial or complete color masking of affected teeth was reported immediately after resin infiltration. Only two studies followed original outcomes up to one year and reported maintenance of original color masking. Two NR studies were assessed as "moderate" and one as "high" quality. Two RCT were classified as "low" risk of bias in the chosen key domains. The remaining four studies were considered "unclear" or "high" risk of bias. CONCLUSION: Although the partial or total masking effect of enamel whitish discoloration has been shown with resin infiltration, there is no strong evidence to support this technique based on the present clinical studies. CLINICAL SIGNIFICANCE: Enamel whitish discolorations in esthetically compromised areas are clinically undesirable. Minimally invasive approaches used as attempts to minimize the discoloration include the resin infiltration technique. The evidence for clinical recommendation of this technique is not strong, thus, further RCT studies with long-term follow-ups should be conducted.

State of the art of bulk-fill resin-based composites: a review / Revisión del estado actual de resinas compuestas bulk-fill

ABSTRACT. Introduction: ameloblasts are cells responsible for the production and mineralization of the organic matrix of enamel through several stages: pre-secretory, secretory, transition, and maturation. The organic matrix components are produced in the secretory phase. In the maturation phase, the organic component is removed and the mineralization process starts. This process requires the involvement of matrix metalloproteinase 20 (MMP-20), also called enamelysin. Several studies have shown the presence of MMP-20 in tooth development and its relationship to alterations in enamel formation. The objective was: to classify the different studies and laboratory techniques used to demonstrate the involvement of enamelysin in tooth development and its relation to pathologies during enamel formation. Methods: a systematic review was conducted with the following bibliographic databases: PubMed, Science-Direct, Hinari, and SciELO, in order to classify the different studies related to the involvement of MMP-20 in tooth development and the methods to detect its expression, between the years of 2009 and 2014. Results and conclusions: 11 in vitro models show that MMP-20 has specific cleavage sites for enamel matrix proteins. This process is altered by chemical composition, ions, and the presence of hydroxyapatite. Enamel morphology is altered in the knockout models. In human studies, MMP-20 has been associated with increased susceptibility to dental caries, enamel thickness, and dental agenesis.

RESUMEN. Introducción: el ameloblasto es la célula encargada de la producción y mineralización de la matriz orgánica del esmalte. Atraviesa varias etapas: la fase pre-secretora, secretora, de transición y maduración. En la fase secretora se producen los componentes de la matriz orgánica. En la fase de maduración se elimina el componente orgánico y se inicia el proceso de mineralización. Este proceso requiere de la participación de la metaloproteinasa de matriz 20 (MMP-20) o también llamada enamelisina. Diversos estudios demuestran la presencia de MMP-20 en el desarrollo dentario y su relación con alteraciones en la formación del esmalte. El objeto fue clasificar los diferentes estudios y técnicas de laboratorio empleadas que demuestren la participación de enamelisina en el desarrollo dentario y su relación con patologías en la formación del esmalte. Métodos: se realizó una revisión sistemática de la literatura con las siguientes bases bibliográficas: PubMed, Science-Direct, Hinari y SciELO, con el fin de clasificar los diferentes estudios relacionados con la participación de MMP-20 en el desarrollo dental y los métodos utilizados para detectar su expresión, entre los años de 2009 a 2014. Resultados y conclusiones: los modelos in vitro evidencian que MMP-20 tiene sitios específicos de escisión para las proteínas de matriz de esmalte. Este proceso se ve alterado por la composición química, iones, y la presencia de hidroxiapatita. En los modelos knockout la morfología del esmalte está alterada. En los estudios en humanos, se ha relacionado la MMP-20 con una mayor susceptibilidad de caries dental, el grosor completo de esmalte y agenesias dentales.

The role of enamelysin (MMP-20) in tooth development: systematic review / El papel de la enamelisina (MMP-20) en el desarrollo dentario: revisión sistemática

ABSTRACT. Introduction: ameloblasts are cells responsible for the production and mineralization of the organic matrix of enamel through several stages: pre-secretory, secretory, transition, and maturation. The organic matrix components are produced in the secretory phase. In the maturation phase, the organic component is removed and the mineralization process starts. This process requires the involvement of matrix metalloproteinase 20 (MMP-20), also called enamelysin. Several studies have shown the presence of MMP-20 in tooth development and its relationship to alterations in enamel formation. The objective was: to classify the different studies and laboratory techniques used to demonstrate the involvement of enamelysin in tooth development and its relation to pathologies during enamel formation. Methods: a systematic review was conducted with the following bibliographic databases: PubMed, Science-Direct, Hinari, and SciELO, in order to classify the different studies related to the involvement of MMP-20 in tooth development and the methods to detect its expression, between the years of 2009 and 2014. Results and conclusions: 11 in vitro models show that MMP-20 has specific cleavage sites for enamel matrix proteins. This process is altered by chemical composition, ions, and the presence of hydroxyapatite. Enamel morphology is altered in the knockout models. In human studies, MMP-20 has been associated with increased susceptibility to dental caries, enamel thickness, and dental agenesis.

RESUMEN. Introducción: el ameloblasto es la célula encargada de la producción y mineralización de la matriz orgánica del esmalte. Atraviesa varias etapas: la fase pre-secretora, secretora, de transición y maduración. En la fase secretora se producen los componentes de la matriz orgánica. En la fase de maduración se elimina el componente orgánico y se inicia el proceso de mineralización. Este proceso requiere de la participación de la metaloproteinasa de matriz 20 (MMP-20) o también llamada enamelisina. Diversos estudios demuestran la presencia de MMP-20 en el desarrollo dentario y su relación con alteraciones en la formación del esmalte. El objeto fue clasificar los diferentes estudios y técnicas de laboratorio empleadas que demuestren la participación de enamelisina en el desarrollo dentario y su relación con patologías en la formación del esmalte. Métodos: se realizó una revisión sistemática de la literatura con las siguientes bases bibliográficas: PubMed, Science-Direct, Hinari y SciELO, con el fin de clasificar los diferentes estudios relacionados con la participación de MMP-20 en el desarrollo dental y los métodos utilizados para detectar su expresión, entre los años de 2009 a 2014. Resultados y conclusiones: los modelos in vitro evidencian que MMP-20 tiene sitios específicos de escisión para las proteínas de matriz de esmalte. Este proceso se ve alterado por la composición química, iones, y la presencia de hidroxiapatita. En los modelos knockout la morfología del esmalte está alterada. En los estudios en humanos, se ha relacionado la MMP-20 con una mayor susceptibilidad de caries dental, el grosor completo de esmalte y agenesias dentales.

Analysis of enamel development using murine model systems: approaches and limitations.

A primary goal of enamel research is to understand and potentially treat or prevent enamel defects related to amelogenesis imperfecta (AI). Rodents are ideal models to assist our understanding of how enamel is formed because they are easily genetically modified, and their continuously erupting incisors display all stages of enamel development and mineralization. While numerous methods have been developed to generate and analyze genetically modified rodent enamel, it is crucial to understand the limitations and challenges associated with these methods in order to draw appropriate conclusions that can be applied translationally, to AI patient care. We have highlighted methods involved in generating and analyzing rodent enamel and potential approaches to overcoming limitations of these methods: (1) generating transgenic, knockout, and knockin mouse models, and (2) analyzing rodent enamel mineral density and functional properties (structure and mechanics) of mature enamel. There is a need for a standardized workflow to analyze enamel phenotypes in rodent models so that investigators can compare data from different studies. These methods include analyses of gene and protein expression, developing enamel histology, enamel pigment, degree of mineralization, enamel structure, and mechanical properties. Standardization of these methods with regard to stage of enamel development and sample preparation is crucial, and ideally investigators can use correlative and complementary techniques with the understanding that developing mouse enamel is dynamic and complex.

Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice.

Enamel development occurs in stages. During the secretory stage, a soft protein rich enamel layer is produced that expands to reach its final thickness. During the maturation stage, proteins are removed and the enamel matures into the hardest substance in the body. KLK4 is expressed during the transition from secretory to the maturation stage and its expression continues throughout maturation. KLK4 is a glycosylated chymotrypsin-like serine protease that cleaves enamel matrix proteins prior to their export out of the hardening enamel layer. Mutations in KLK4 can cause autosomal recessive, non-syndromic enamel malformations in humans and mice. Klk4 ablated mice initially have normal-looking teeth with enamel of full thickness. However, the enamel is soft and protein-rich. Three findings are notable from Klk4 ablated mice: first, enamel rods fall from the interrod enamel leaving behind empty holes where the enamel fractures near the underlying dentin surface. Second, the ~10,000 crystallites that normally fuse to form a solid enamel rod fail to grow together in the ablated mice and can fall out of the rods. Third, and most striking, the crystallites grow substantially in width and thickness (a- and b-axis) in the ablated mice until they almost interlock. The crystallites grow in defined enamel rods, but interlocking is prevented presumably because too much protein remains. Conventional thought holds that enamel proteins bind specifically to the sides of enamel crystals to inhibit growth in width and thickness so that the thin, ribbon-like enamel crystallites grow predominantly in length. Results from Klk4 ablated mice demonstrate that this convention requires updating. An alternative mechanism is proposed whereby enamel proteins serve to form a mold or support structure that shapes and orients the mineral ribbons as they grow in length. The remnants of this support structure must be removed by KLK4 so that the crystallites can interlock to form fully hardened enamel.