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Objective: This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience., Design: In-situ pellicles were collected from 41 children (aged 4-6 years) with different caries status; 17 of them were caries-free (dmf = 0), 12 had dental restorations but no current caries (dmf ≥ 2) and 12 had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30 min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling., Results: There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B., Conclusion: Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The objectives of this study were to investigate changes in the activity and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in permanent teeth with or without exposure to radiotherapy, and the role of proteinase inhibitors in their inactivation. In situ zymography and immunofluorescence assays were performed to evaluate the activity and expression of two key gelatinases (MMP-2 and MMP-9) in sections of permanent molars, assigned to irradiated and nonirradiated subgroups. Dental fragments were exposed to radiation at a dose of 2 Gy fractions for 5 consecutive days until a cumulative dose of 60 Gy was reached. To evaluate the effect of protease inhibitors on MMPs, teeth were immersed in 0.5 mL of 0.12% chlorhexidine digluconate (CHX), 0.05% sodium fluoride (NaF), 400 μM polyphenol epigallocatechin-3-gallate (EGCG), or distilled water (control) for 1 h. Fluorescence in the dentinoenamel junction (DEJ) was evaluated in 3 areas of the tooth: cervical, cuspal, and pit. These regions were photographed using a fluorescence microscope at 1.25× and 5× magnifications. Results were analyzed using the D'Agostino-Person normality test, and the Kruskal-Wallis, Dunn, and Wilcoxon tests for intergroup and paired comparisons (α = 0.05). The fluorescence intensity/mm2 in the DEJ at the three regions studied was higher in the irradiated teeth (p < 0.05) than in the nonirradiated teeth, revealing regions of expression of MMP-2 and MMP-9 by immunofluorescence. Postradiotherapy treatment with different solutions (CHX, NaF, and EGCG) led to lower fluorescence intensity/mm2 in irradiated teeth than in the control group (distilled water; p < 0.05), as a result of MMP inactivation. In conclusion, irradiation increased gelatinase activity in all regions of the DEJ. Treatment with 0.12% CHX, 0.05% NaF, and 400 μM polyphenol EGCG postradiotherapy inactivated enzyme activity., (© 2018 S. Karger AG, Basel.)

Vacuolar H + -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by μCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Objective: Recent studies suggested that head and neck radiotherapy increases active forms of matrix metalloproteinases (MMPs) in the dentin-enamel junction (DEJ), leading to enamel delamination and radiation-related caries. This study aimed to assess the expression and activity of the gelatinases MMP-2 and MMP-9 in the DEJ and dentin-pulp complex tissues of teeth irradiated in vivo., Study Design: Thirty-six teeth were studied, including 19 irradiated and 17 non-irradiated controls. In situ zymography was used to investigate the gelatinolytic activity in the micromorphologic components of enamel, DEJ, dentin-pulp complex, and caries. Immunohistochemical analysis was conducted on the demineralized samples to assess MMP-2 and MMP-9 expression levels in the DEJ, dentin-pulp complex components, and caries., Results: No statistically significant differences were detected between groups in gelatinolytic activity or in MMP-2 expression levels (P > .05). Odontoblast MMP-9 expression was reduced in the irradiated group (P = .02)., Conclusions: The study rejected the hypothesis that MMP-2 and MMP-9 would be overexpressed or more activated in the DEJ and dentin-pulp complex of irradiated teeth. Direct effects of radiation should not be regarded as an independent factor for explaining radiation-related caries onset and progression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.

Objectives: We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases., Materials and Methods: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0-6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses., Results: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentine and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22kDa and was immunolocalized predominantly to the morphological dentine enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70Gy, subsequent incubation at 37°C for 3-6 months with or without prior irradiation caused the proportion of Mr=24-22kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70Gy radiation also contained relatively more 24 and 22kDa MMP-20 than those of healthy age-related teeth., Conclusion: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentine-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The formation of an intraoral biofilm is primarily determined by initial bioadhesion processes, including molecular interactions. Therefore, this study aimed to establish fluorescent labelling protocols to enable the simultaneous visualization of different pellicle enzymes, extracellular glucans and adherent bacteria throughout the initial phase of biofilm formation., Design: In situ formed biofilm samples were collected on enamel and dentine slabs that were fixed on buccal sites of individual splints, being worn by 5 subjects. After an intraoral slab exposure from 30min to 8h, the following specially adapted fluorescent labelling assays were performed and analyzed by epifluorescent microscopy: pellicle-amylase, -lysozyme, -peroxidase and -glycosyltransferases B, C and D were marked with specific primary antibodies and then visualized by the aid of different fluorescently labelled secondary antibodies (Texas Red, DyLight 488, FITC). Afterwards the same samples were subjected to a combined DAPI-/Concanavalin A-staining to determine adherent bacteria and glucans., Results: All fluorescence labelling assays were successfully established to visualize pellicle enzymes, glucans and adherent bacteria at different times of biofilm formation. The combination of the labelling protocols showed a characteristic agglomeration of glucans and bacteria as well as an increased concentration of the pellicle enzymes in the initial phase of bioadhesion., Conclusion: Fluorescent labelling techniques are a valuable supplement of dental research as they provide an insight into the mutual interactions of different biofilm determinants in situ. Based hereon, information could also be deduced about the influence of oral therapeutics on individual caries susceptibility., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Intramembrane proteases are critically involved in signal transduction and membrane protein turnover. Signal-peptide-peptidase-like 2a (SPPL2A), a presenilin-homologue residing in lysosomes/late endosomes, cleaves type II-oriented transmembrane proteins. We recently identified SPPL2A as the enzyme controlling turnover and functions of the invariant chain (CD74) of the major histocompatibility complex II (MHCII) and demonstrated critical importance of this process for B cell development. Surprisingly, we found that SPPL2A is critical for formation of dental enamel. In Sppl2a knockout mice, enamel of the erupted incisors was chalky white and rapidly eroded after eruption. SPPL2A was found to be expressed in enamel epithelium during secretory and maturation stage amelogenesis. Mineral content of enamel in Sppl2a⁻/⁻ incisors was inhomogeneous and reduced by ∼20% compared to wild-type mice with the most pronounced reduction at the mesial side. Frequently, disruption of the enamel layer and localized detachment of the most superficial enamel layer was observed in the knockout incisors leading to an uneven enamel surface. In Sppl2a null mice, morphology and function of secretory stage ameloblasts were not noticeably different from that of wild-type mice. However, maturation stage ameloblasts showed reduced height and a characteristic undulation of the ameloblast layer with localized adherence of the cells to the outer enamel. This was reflected in a delayed and incomplete resorption of the proteinaceous enamel matrix. Thus, we conclude that intramembrane proteolysis by SPPL2A is essential for maintaining cellular homeostasis of ameloblasts. Because modulation of SPPL2A activity appears to be an attractive therapeutic target to deplete B cells and treat autoimmunity, interference with tooth enamel formation should be investigated as a possible adverse effect of pharmacological SPPL2A inhibitors in humans., (Copyright © 2013 American Society for Bone and Mineral Research.)

Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children's caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p < .001) and PE (194.8; p = .005). There was no significant difference in the salivary arginolytic activity among children with different caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children's age, caries status, and dentition (p < .001), with healthy plaque predicting higher ADS activity compared with diseased plaque. Plaque arginine metabolism varies greatly among children and tooth sites, which may affect their susceptibility to caries.

Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization.

Enamel formation involves highly orchestrated intracellular and extracellular events; following development, the tissue is unable to regenerate, making it a challenging target for tissue engineering. We previously demonstrated the ability to trigger enamel differentiation and regeneration in the embryonic mouse incisor using a self-assembling matrix that displayed the integrin-binding epitope RGDS (Arg-Gly-Asp-Ser). To further elucidate the intracellular signaling pathways responsible for this phenomenon, we explore here the coupling response of integrin receptors to the biomaterial and subsequent downstream gene expression profiles. We demonstrate that the artificial matrix activates focal adhesion kinase (FAK) to increase phosphorylation of both c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Jun (c-Jun). Inhibition of FAK blocked activation of the identified matrix-mediated pathways, while independent inhibition of JNK nearly abolished phosphorylated-c-Jun (p-c-Jun) and attenuated the pathways identified to promote enamel regeneration. Cognate binding sites in the amelogenin promoter were identified to be transcriptionally up-regulated in response to p-c-Jun. Furthermore, the artificial matrix induced gene expression as evidenced by an increased abundance of amelogenin, the main protein expressed during enamel formation, and the CCAAT enhancer binding protein alpha (C/EBPα), which is the known activator of amelogenin expression. Elucidating these cues not only provides guidelines for the design of synthetic regenerative strategies and opportunities to manipulate pathways to regulate enamel regeneration, but can provide insight into the molecular mechanisms involved in tissue formation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl(-/-), aka Akp2(-/-)) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl(-/-) mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl(-/-) mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP., (Copyright © 2012 American Society for Bone and Mineral Research.)

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ., (© 2011 Eur J Oral Sci.)

The crowns of matrix metalloproteinase 20 (Mmp20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and Mmp20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in Mmp20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the Mmp20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in Mmp20 null mice because of hypomineralization at the DEJ., (© 2011 Eur J Oral Sci.)

Dental enamel is a hypermineralized tissue, containing only trace amounts of organic components. During enamel formation, matrix metalloproteinase 20 (MMP20) processes proteins comprising enamel matrix and facilitates hypermineralization. In the human genome, 24 distinct MMP genes have been identified. Among these genes, MMP20 is clustered with eight other genes, including MMP13, and all these clustered genes show phylogenetically close relationships. In this study, we investigated MMP20 and closely related MMP genes in various tetrapods and in a teleost fish, fugu. In the genome of the chicken, a toothless tetrapod, we identified degraded exons of MMP20, which supports the previous proposition that MMP20 is important specifically for enamel formation. Nevertheless, for unknown reasons, we failed to identify MMP20 in the platypus genome. In the opossum, lizard, and frog genomes, MMP20 was found clustered with MMP13. Furthermore, in the fugu genome, we identified an MMP20-like gene located adjacent to MMP13, suggesting that MMP20 arose before the divergence of ray-finned fish and lobe-finned fish. The teleost tooth surface is covered with enameloid, a hypermineralized tissue different from enamel. Thus, we hypothesize that MMP20 could have been used in an ancient hypermineralized tissue, which evolved into enameloid in teleosts and into enamel in tetrapods., (© 2011 Eur J Oral Sci.)

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes., (© 2011 Eur J Oral Sci.)

Kallikrein-related peptidase 4 (KLK4) is critical for proper dental enamel formation. Klk4 null mice, and humans with two defective KLK4 alleles have obvious enamel defects, with no other apparent phenotype. KLK4 mRNA or protein is reported to be present in tissues besides teeth, including prostate, ovary, kidney, liver, and salivary gland. In this study we used the Klk4 knockout/NLS-lacZ knockin mouse to assay Klk4 expression using β-galactosidase histochemistry. Incubations for 5 h were used to detect KLK4 expression with minimal endogenous background, while overnight incubations susceptible to false positives were used to look for trace KLK4 expression. Developing maxillary molars at postnatal days 5, 6, 7, 8, and 14, developing mandibular incisors at postnatal day 14, and selected non-dental tissues from adult wild-type and Klk4(lacZ/lacZ) mice were examined by X-gal histochemistry. After 5 h of incubation, X-gal staining was observed specifically in the nuclei of maturation-stage ameloblasts in molars and incisors from Klk4(lacZ/lacZ) mice and was detected weakly in the nuclei of salivary gland ducts and in patches of prostate epithelia. We conclude that KLK4 is predominantly a tooth-specific protease with low expression in submandibular salivary gland and prostate, and with no detectable expression in liver, kidney, testis, ovary, oviduct, epididymis, and vas deferens., (© 2011 Eur J Oral Sci.)

Objectives: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play important roles in dentine formation, caries progression and hybrid layer degradation. This study tested the hypothesis that the distribution and concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 are different at different depths of human coronal dentine, including odontoblasts., Methods: Protein localization was performed using immunohistochemistry. Co-localization of the MMPs and their inhibitors was conducted using immunofluorescence double labelling. Protein concentrations were measured by ELISA and gelatinolytic potential was assessed with gelatine zymography., Results: MMP-2 was the main gelatinase in dentine and was concentrated in the odontoblasts, deep dentine and the dentinoenamel junction. TIMP-2 was co-localized with MMP-2 mainly in the odontoblasts but its concentration was low. Both MMP-9 and TIMP-1 showed a decreasing distribution from the deep to the superficial dentine layers; however, the concentration of TIMP-1 was much higher than that of MMP-9. The gelatinolytic potential of dentine protein extracts decreased gradually from deep to superficial dentine., Conclusions: The concentrations and distribution patterns of MMP-2, MMP-9, TIMP-1 and TIMP-2, and the gelatinolytic potential of dentine matrix are variable along different dentine depths. Thus, differential collagen degradation potentials may be expected depending upon the depth in which dentine is exposed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20 (+/-) mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F(-)) treatment ad libitum, the Mmp20 (+/-) mice had F(-) tissue levels similar to those of Mmp20 (+/+) mice. No significant difference in enamel hardness was observed between the F(-)-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20 (+/-) mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis.

Dental enamel formation occurs extracellularly and establishment of an ordered enamel organic extracellular matrix (ECM) seems to be crucial for proper construction of the enamel mineral phase. Polarizing microscopy shows that the ordered supramolecular structure of the secretory stage enamel organic ECM exhibits strong birefringence. We reported earlier that this birefringence is lost in unfixed specimens, probably due to extensive proteolytic cleavage of enamel proteins. Therefore, we investigated the association between enamel proteinase activities by analyzing the effects of metallo- and serine proteinase inhibitors in situ on the birefringence of the secretory stage enamel organic ECM. Male rats were used in the present study. After sacrifice, distal 10 mm fragments of upper incisors were removed and immersed for 15 h under continuous shaking at 37°C in one of the following solutions: 1) 10 mM Tris, pH 8.0; 150 mM NaCl (negative control, n = 8); 2) 2% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2 (positive control, n = 5); 3) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline (n = 9); 4) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM phenylmethyl-sulfonyl fluoride (PMSF) (n = 8); 5) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline; 2 mM PMSF (n = 9). Samples then were immersed in fixative solution for 24 h and processed to obtain 5 μm thick longitudinal sections of the secretory stage enamel organic ECM. The sections were immersed in 80% glycerin for 30 min and analyzed by transmitted polarizing light microscopy. 1,10-Phenanthroline (inhibitor of metalloproteinases) and 1,10-phenanthroline + PMSF (inhibitor of serine proteinases) clearly prevented a decrease in the optical retardation of birefringence brightness from the tissue. PMSF alone promoted a slight preservation of the birefringence exhibited by the secretory stage enamel organic ECM. Rapid loss of birefringence in secretory stage enamel organic ECM that is not fixed immediately is caused by enamel proteinases and the activity of metalloproteinases seems to lead to preliminary degradation of the enamel organic ECM, which in turn facilitates subsequent serine proteinase activity.

Fluorosed enamel can be porous, mottled, discolored, hypomineralized, and protein-rich if the enamel matrix is not completely removed. Proteolytic processing by matrix metalloproteinase-20 (MMP20) and kallikrein-4 (KLK4) is critical for enamel formation, and homozygous mutation of either protease results in hypomineralized, protein-rich enamel. Herein, we demonstrate that the lysosomal proteinase cathepsin K is expressed in the enamel organ in a developmentally defined manner that suggests a role for cathepsin K in degrading re-absorbed enamel matrix proteins. We therefore asked if fluoride directly inhibits the activity of MMP20, KLK4, dipeptidyl peptidase I (DPPI) (an in vitro activator of KLK4), or cathepsin K. Enzyme kinetics were studied with quenched fluorescent peptides with purified enzyme in the presence of 0-10 mM NaF, and data were fit to Michaelis-Menten curves. Increasing concentrations of known inhibitors showed decreases in enzyme activity. However, concentrations of up to 10 mM NaF had no effect on KLK4, MMP20, DPPI, or cathepsin K activity. Our results show that fluoride does not directly inhibit enamel proteolytic activity.

Background: It has been suggested that host matrix metalloproteinase-2 (MMP-2) present in dentin may be involved in caries progression, however, its response to caries is not known. Bone sialoprotein (BSP) has been implicated in dentin mineralization and MMP-2 modulation., Objective: To identify and compare the distribution of MMP-2 and BSP in healthy human coronal dentin and those with early caries., Methods: Freshly extracted 3rd molars and premolars with and without early caries were fixed, demineralized and subjected to immunohistochemistry using a monoclonal anti-MMP-2 antibody and monoclonal anti-BSP antibody with an avidin-biotin complex method. Immunoreactivity was visualized with 3,3'-diaminobenzidine substrate and observed under light microscopy., Results: Immunohistochemical analysis revealed that MMP-2 and BSP are not detected in the tubule lumens of healthy dentin. However, intense immunoreactivity for MMP-2 and BSP was detected in association with the full length of the caries-affected dentinal tubules. The MMP-2 and BSP at the dentino-enamel junction appeared unaltered., Conclusion: The results indicate that MMP-2 and BSP may be actively secreted by odontoblasts in response to carious insult. MMP-2 and BSP accumulation in the caries-affected dentinal tubules may indicate their potential involvement in the host defense mechanism which results in calcification of regions affected by the carious process., (Copyright © 2011 S. Karger AG, Basel.)

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

Objective: Calpain is a calcium ion-dependent cysteine protease, consisting of two primary isoforms (calpain1/calpain2) which mediate crucial cellular functions. The activity of the calpains is tightly regulated by the endogenous inhibitor calpastatin. Calpains have been detected in several studies during the embryonic and foetal stages. The aim of this study is to investigate the temporal transition of typical calpains and their inhibitor calpastatin during odontogenesis., Design: We used the first molar of foetal ICR mice from embryonic day (E) 14 to postnatal day (PN) 7. Using laser microdissection and semi-quantitative real-time PCR, we investigated calpain1, calpain2 and calpastatin expressions in each enamel epithelium, inner enamel epithelium, stellate reticulum and outer enamel epithelium., Results: We found calpain1 and calpain2 mRNA increased in the all enamel epithelia between E18 and PN1. In addition calpastatin mRNA expression increased in the ameloblasts from PN1 to PN7. The immunohistochemistry results demonstrated that calpain1/calpain2 was present in the distal side of ameloblasts from PN1 to PN7, and calpastatin was present in the extracellular enamel matrix from E16 to PN1. Furthermore calpain1/calpain2 was present in the dentin, and calpastatin was detected in dentin producing odontoblasts and predentin at PN7., Conclusions: In this study the temporal transition of calpain1, calpain2 and calpastatin mRNA and the immunolocalization are identified during tooth development. Our results indicate that the calcium-dependent proteases may play an important role in mouse molar development and extracellular calpain and calpastatin may be involved in molar mineralization.

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and from wild-type mouse was carried out using microarrays and validated using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. When comparing expression profiles at each time-point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR-alpha knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR-alpha knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR-alpha knockout had no significant effect on molar morphology, the results suggest that active PPAR-alpha signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR-alpha knockout tooth germ.

Kallikrein-4 (KLK4) is a serine protease expressed during enamel maturation, and proteolytic processing of the enamel matrix by KLK4 is critical for proper enamel formation. KLK4 is secreted as an inactive zymogen (pro-KLK4), and identification of its activator remains elusive. Dipeptidyl peptidase I (DPPI) is a cysteine aminopeptidase that can activate several serine proteases. In this study, we sought to examine DPPI expression in mouse enamel organ and determine if DPPI could activate KLK4. Real-time PCR showed DPPI expression throughout amelogenesis, with highest expression at maturation, and immunohistochemical staining of mouse incisors confirmed DPPI expression by ameloblasts. We demonstrate in vitro that DPPI activates pro-KLK4 to cleave a fluorogenic peptide containing a KLK4 cleavage site. Examination of mature enamel from DPPI null mice by FTIR showed no significant accumulation of protein; however, microhardness testing revealed that loss of DPPI expression significantly reduced enamel hardness.

It is well known that exposure to environmental cadmium causes itai-itai (ouch-ouch) disease. However, the exact mechanism underlying this bone disease remains unresolved. By focusing on the calcification mechanism, we examined developing tooth enamel in rats exposed to cadmium to test the hypothesis that cadmium exposure may cause defects in crystal formation. Electron microscopy revealed the presence of perforated crystals in developing tooth enamel, indicating that the process of crystal nucleation may have been interrupted by cadmium exposure. Furthermore, biochemical analyses revealed that the catalytic activity of carbonic anhydrase in the immature enamel matrix declined remarkably despite the fact that quantitative reduction of this enzyme was insignificant, suggesting that the decline of catalytic activity may have resulted from the replacement of zinc with cadmium ions. Therefore, we concluded that the poor catalytic activity of cadmium-binding carbonic anhydrase might hinder the nucleation process, leading to an impairment in mineralization that causes itai-itai disease.

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions., (Copyright 2008 S. Karger AG, Basel.)

Organic matrix degradation and crystal maturation are extracellular events that occur simultaneously during enamel biomineralization. We hypothesized that enamel proteases control amelogenin-mineral interaction, which, in turn can affect crystal nucleation, organization, and growth. We used a recombinant amelogenin (rP172), a homolog of its major cleavage product (rP148), and a native amelogenin lacking both N- and C-termini (13k). We compared apatite binding affinity between amelogenins and their digest products during proteolysis. We further compared binding affinity among the 3 amelogenins using a Langmuir model for protein adsorption. Amelogenin-apatite binding affinity was progressively reduced with the proteolysis at the C- and N- termini by recombinant pig MMP-20 (rpMMP20) and recombinant human kallikrein-4 (rhKLK4), respectively. The binding affinity of amelogenin to apatite was found to be in the descending order of rP172, rP148, and 13k. Analysis of our data suggests that, before its complete degradation during enamel maturation, stepwise processing of amelogenin by MMP-20 and then KLK4 reduces amelogenin-apatite interaction.

Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory-stage ameloblasts. Enamel protein-cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. KLK4 is secreted by transition- and maturation-stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.

Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20(-/-)mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4-5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.

During screening of a rat incisor enamel organ cDNA library by signal peptide trap technology, we identified a DNA fragment matching a predicted translation sequence for rat carbonic anhydrase 6 (CA6). This result was unexpected because CA6, to date, has been associated primarily with secretions from glandular tissues. To further characterize this observation, reverse transcription-polymerase chain reaction (RT-PCR) amplifications were carried out on total RNA extracted from freeze-dried secretory and maturation-stage rat incisor enamel organs. A cDNA fragment of the expected size was detected in control samples from rat salivary glands as well as within maturation-stage enamel organ samples. This CA6 RT-PCR fragment was further cloned and sequenced and found to match the nucleotide sequence 770-1079 from clone XM&#95;216584 of GenBank. Northern blot analyses with the rat CA6 cDNA fragment confirmed its expression relative to maturation-stage enamel organ samples. It is at present unclear whether the CA6 expressed by enamel organ cells is secreted into the enamel layer or into the intercellular spaces of the enamel organ itself to assist in neutralizing excess protons arising from the growth of apatite crystals during the maturation stage of amelogenesis.

The activities of two proteases--enamelysin (MMP-20) and kallikrein 4 (KLK4)--are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion.

Extracellular matrix proteins control the formation of the inorganic component of hard tissues including bone, dentin, and enamel. The structural proteins expressed primarily in the enamel matrix are amelogenin, ameloblastin, enamelin, and amelotin. Other proteins, like biglycan, are also present in the enamel matrix as well as in other mineralizing and nonmineralizing tissues of mammals. In addition, the presence of sulfated enamel proteins, and "tuft" proteins has been examined and discussed in relation to enamel formation. The structural proteins of the enamel matrix must have specific protein-protein interactions to produce a matrix capable of directing the highly ordered structure of the enamel crystallites. Protein-protein interactions are also likely to occur between the secreted enamel proteins and the plasma membrane of the enamel producing cells, the ameloblasts. Such protein-protein interactions are hypothesized to influence the secretion of enamel proteins, establish short-term order of the forming matrix, and to mediate feedback signals to the transcriptional machinery of these cells. Membrane-bound proteins identified in ameloblasts, and which interact with the structural enamel proteins, include Cd63 (cluster of differentiation 63 antigen), annexin A2 (Anxa2), and lysosomal-associated glycoprotein 1 (Lamp1). These and related data help explain the molecular and cellular mechanisms responsible for the removal of the organic enamel matrix during the events of enamel mineralization, and how the enamel matrix influences its own fate through signaling initiated at the cell surface. The knowledge gained from enamel developmental studies may lead to better dental and nondental materials, or materials inspired by Nature. These data will be critical to scientists, engineers, and dentists in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein-protein interactions involving the key dental proteins must be identified and understood. The scope of this review is to discuss the current understanding of protein-protein interactions of the developing enamel matrix, and relate this knowledge to enamel biomineralization.

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.

Matrix metalloproteinases (MMPs) have been expressed during root development and periodontal tissue formation, whereas it is not known if these MMP molecules are enzymatically active to degrade the extracellular matrices (ECMs). The present study was designed to investigate the gelatinolytic and collagenolytic activity in rat molar root and incisor development. Three-week old rat mandibles were frozen and cut without fixation or decalcification and processed for in situ zymography using substrates gelatin and collagen. The enzymatic activity was assessed according to the intensity of fluorescence due to the lysis of the substrates. Odontoblasts, predentin, cementum, bone and the enamel matrix showed the high activity. The present study demonstrated MMP activity in calcified tissues using in situ zymography for the first time and the possible involvement of the MMP activity in molar root and incisor development and periodontal tissue formation.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10(6) fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 microM fluoride was compared by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 microM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 microM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization., (Copyright 2002 Wiley-Liss, Inc.)

Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase. and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid., (Copyright 2002 Wiley-Liss, Inc.)

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.

Environmental contamination with heavy metals leads to their uptake by the body. Dental tissues are known to harbor metals in concentrations related to the exposure at the time of dentin or enamel formation. Several enzymes involved in the metabolism of extracellular matrix components have been shown to be inhibited by excess metal ions. Enamel matrix proteinases seem to play a central role in the maturation of dental enamel, and inhibition of these enzymes by metals could interfere with amelogenesis. We have investigated the effect of lead, cadmium and zinc on the activity of enamel matrix proteinases using a colorimetric assay, casein and gelatin zymography, and an assay of enamel protein degradation in vitro. All three metals inhibited the proteolysis, as shown by the three methods employed, in concentrations as low as 110 microM. This study showed that contaminant metals can inhibit proteinolysis when a crude enamel extract containing enamel proteases is tested in vitro. Therefore, the presence of heavy metals in enamel matrix could interfere with amelogenesis due to alteration in normal proteolysis.

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.