Amelogenin X impacts age-dependent increase of frequency and number in labial incisor grooves in C57BL/6.

Labial grooves in maxillary incisors have been reported in several wild-type rodent species. Previous studies have reported age-dependent labial grooves occur in moderate prevalence in C57BL/6 mice; however, very little is known about the occurrence of such grooves. In the present study, we observed age-dependent groove formation in C57BL/6 mice up to 26 months after birth and found that not only the frequency of the appearance of incisor grooves but also the number of grooves increased in an age-dependent manner. We examined the molecular mechanisms of age-dependent groove formation by performing DNA microarray analysis of the incisors of 12-month-old (12M) and 24-month-old (24M) mice. Amelx, encoding the major enamel matrix protein AMELOGENIN, was identified as a 12M-specific gene. Comparing with wild-type mice, the maxillary incisors of Amelx-/- mutants indicated the increase of the frequency and number of labial grooves. These findings suggested that the Amelx gene impacts the age-dependent appearance of the labial incisor groove in C57BL/6 mice.

Weaker dental enamel explains dental decay.

Dental caries continues to be the most prevalent bacteria-mediated non-contagious disease of humankind. Dental professionals assert the disease can be explained by poor oral hygiene and a diet rich in sugars but this does not account for caries free individuals exposed to the same risk factors. In order to test the hypothesis that amount of amelogenin during enamel development can influence caries susceptibility, we generated multiple strains of mice with varying levels of available amelogenin during dental development. Mechanical tests showed that dental enamel developed with less amelogenin is "weaker" while the dental enamel of animals over-expressing amelogenin appears to be more resistant to acid dissolution.

Tracking endogenous amelogenin and ameloblastin in vivo.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

The use of mouse models to investigate shear bond strength in amelogenesis imperfecta.

Patients with amelogenesis imperfecta (AI) have defective enamel; therefore, bonded restorations of patients with AI have variable success rates. To distinguish which cases of AI may have good clinical outcomes with bonded materials, we evaluated etching characteristics and bond strength of enamel in mouse models, comparing wild-type (WT) with those having mutations in amelogenin (Amelx) and matrix metalloproteinase-20 (Mmp20), which mimic 2 forms of human AI. Etched enamel surfaces were compared for roughness by scanning electron microscopy (SEM) images. Bonding was compared through shear bond strength (SBS) studies with 2 different systems (etch-and-rinse and self-etch). Etched enamel surfaces of incisors from Amelx knock-out (AmelxKO) mice appeared randomly organized and non-uniform compared with WT. Etching of Mmp20KO surfaces left little enamel, and the etching pattern was indistinguishable from unetched surfaces. SBS results were significantly different when AmelxKO and Mmp20KO enamel surfaces were compared. A significant increase in SBS was measured for all samples when the self-etch system was compared with the etch-and-rinse system. We have developed a novel system for testing shear bond strength of mouse incisors with AI variants, and analysis of these data may have important clinical implications for the treatment of patients with AI.

The leucine-rich amelogenin peptide alters the amelogenin null enamel phenotype.

INTRODUCTION: The amelogenin proteins secreted by ameloblasts during dental enamel development are required for normal enamel structure. Amelx null (KO) mice have hypoplastic, disorganized enamel similar to that of human patients with mutations in the AMELX gene, and provide a model system for studies of the enamel defect amelogenesis imperfecta. Because many amelogenin proteins are present in developing enamel due to RNA alternative splicing and proteolytic processing, understanding the function of individual amelogenins has been challenging. PURPOSE: Our objective was to better understand the role of LRAP, a 59 amino acid leucine-rich amelogenin peptide, in the development of enamel. APPROACH: Teeth from transgenic mice that express LRAP under control of the Amelx regulatory regions were analyzed for mechanical properties, and transgenic males were mated with female KO mice. Male offspring with a null background that were transgene positive or transgene negative were compared to determine phenotypic differences using microcomputed tomography (microCT) and scanning electron microscopy (SEM). RESULTS: Nanoindentation revealed no differences between LRAP transgenic and wild-type murine enamel. Using microCT, LRAPKO enamel volume and density measurements were similar to those from KO mice. However, in etched samples examined by SEM, the organization of the enamel rod pattern was altered by the presence of the LRAP transgene. CONCLUSIONS: The presence of LRAP leads to changes in enamel appearance compared to enamel from KO mice. Expression of a combination of amelogenin transgenes in KO mice may lead to rescue of the individual characteristics of normal enamel.

Amelogenin- and enamelysin (Mmp-20)-deficient mice display altered birefringence in the secretory-stage enamel organic extracellular matrix.

Dental enamel is the most mineralized tissue of vertebrate organisms. Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. The formation of an ordered enamel organic extracellular matrix (ECM) seems be a crucial step for the proper formation of mineral phase. Polarizing microscopy demonstrates that the ordered supramolecular structure of the secretory-stage enamel organic ECM is strongly birefringent. In the present work we analyzed the birefringence of secretory-stage enamel organic ECM in amelogenin (Amelx)- and enamelysin (Mmp20)-deficient mice. Female Amelx+/- animals showed significant reduction in optical retardation values when compared with the Amelx+/+ subgroup (p=0.0029). The secretory-stage enamel organic ECM of the Amelx-/- subgroup did not exhibit birefringence. The secretory-stage enamel organic ECM of Mmp20-/- mice showed a significant decrease in optical retardation as compared with Mmp20+/+ and Mmp20+/- mice (p=0.0000). Mmp20+/- and Mmp20+/+ mice exhibited similar birefringence (p=1.0000). The results presented here support growing evidence for the idea that the birefringence of secretory-stage enamel organic ECM is influenced by the ordered supramolecular organization of its components.