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Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING protein, found in bone and dental tissues. The purpose of this study was to determine whether a 23-amino-acid peptide derived from MEPE (Dentonin or AC-100) could stimulate dental pulp stem cell (DPSC) proliferation and/or differentiation. DPSCs were isolated from erupted human molars, and the mitogenic potential of Dentonin in DPSCs was measured by BrdU immunoassay and cell-cycle gene SuperArray. Differentiation of DPSCs with Dentonin was characterized by Western blot and by osteogenesis gene SuperArray. Dentonin enhanced DPSC proliferation by down-regulating P16, accompanied by up-regulation of ubiquitin protein ligase E3A and human ubiquitin-related protein SUMO-1. Enhanced cell proliferation required intact RGD and SGDG motifs in the peptide. This study shows that Dentonin can promote DPSC proliferation, with a potential role in pulp repair. Further studies are required to determine the usefulness of this material in vivo.

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.

In this study, we characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of human dental pulp stem cells (DPSCs). DPSCs were capable of forming ectopic dentin and associated pulp tissue in vivo. Stromal-like cells were reestablished in culture from primary DPSC transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. The odontogenic potential of 12 individual single-colony-derived DPSC strains was determined. Two-thirds of the single-colony-derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one-third. These results indicate that single-colony-derived DPSC strains differ from each other with respect to their rate of odontogenesis. Taken together, these results demonstrate that DPSCs possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation.

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta'-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72(-/-)), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72(-/-) mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72(-/-) ameloblasts with retained proteins in the enamel matrix during maturation stage. H(+)/Cl(-) exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72(-/-) maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72(-/-) mice as compared to wildtype controls. Moreover, Wdr72(-/-) ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 − with Na+. Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 −/− mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR −/− and AE2a,b −/− mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Calcium (Ca2+) is a critical component of tooth enamel, dentin, and the surrounding extracellular matrix. Ca2+ also may regulate tooth formation, although the mechanisms for such action are poorly understood. The Ca2+-sensing receptor (CaR) that is expressed in the parathyroid gland, kidney, bone, and cartilage has provided a mechanism by which extracellular Ca2+ can regulate cell function. Because these tissues play an important role in maintaining mineral homeostasis and because Ca2+ is hypothesized to play a crucial role in tooth formation, we determined whether the CaR was present in teeth. In this study, using immunohistochemistry, CaR protein was detected in developing porcine molars localized in the predentin (pD), early secretory-stage ameloblasts, maturation-stage smooth-ended ameloblasts (SA), and certain cells in the stratum intermedium. CaR protein and messenger RNA (mRNA) were detected also in an immortalized ameloblast-like cell line (PABSo-E) using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and Northern analysis. Based on the observation that the CaR is expressed in cultured ameloblasts, we determined whether increments in medium Ca2+ concentration could activate the intracellular Ca2+ signal transduction pathway. In PABSo-E cells, increasing extracellular Ca2+ in the medium from 0 (baseline) to 2.5mM or 5.0 mM resulted in an increase in intracellular Ca2+ above baseline to 534 +/- 69 nM and 838 +/- 86 nM, respectively. Taken together, these results suggest that the CaR is expressed in developing teeth and may provide a mechanism by which these cells can respond to alterations in extracellular Ca2+ to regulate cell function and, ultimately, tooth formation.

Summary Purpose: The Erbium:YAG laser has been shown to be safe and effective for caries removal and cavity preparation in adults. In this study, we report a prospective parallel controlled randomized multicenter clinical trial of this laser for dental caries removal and cavity preparation in children. Methods: At two separate sites, a total of 124 patients from 4 to 18 years old, having at least one tooth with caries requiring restoration, were randomized for treatment in a 2:1 ratio, laser to conventional dental drill. Caries were removed, the teeth were restored and follow-up evaluations were completed after 3 months. Determination of safe and effective treatment included four criteria: 1) acceptable caries removal, 2) acceptable cavity preparation, 3) pulp vitality, and 4) intact and serviceable restoration. Results: All 42 drill procedures and 81 out of 82 laser procedures were found to be successful in terms of safety and effectiveness. No significant difference in pain reported was found between drill or laser treatments, and no complications or adverse events were reported after treatment or at any other time during the study. Subject satisfaction with treatment procedures as reported was equivalent in the laser and drill groups. The only significant difference found between treatment groups was in the greater use of anesthesia during drilling procedures. Conclusions: The Erbium:YAG laser is safe and effective for both caries removal and cavity preparation in children.

Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Highly mineralized tooth enamel develops from an extracellular matrix chiefly comprised of amelogenins formed by splicing of 7 (human) or 9 (rodent) exons secreted from specialized epithelial cells known as ameloblasts. Here we examined the role of the 59 amino acid alternatively spliced amelogenin known as leucine rich amelogenin peptide (LRAP) on enamel formation, using transgenic murine models in which LRAP overexpression is driven by an amelogenin promoter (TgLRAP). Beginning in the secretory stage of mouse amelogenesis, we found a reduced thickness of enamel matrix and a loss of Tomes' processes, followed by upregulated amelogenin mRNA expression, inhibited amelogenin secretion and loss of cell polarity. In the presecretory stage (P0) amelogenin m180 mRNA expression was increased 58 fold along with a 203 fold increase in MMP-20 expression and 3.5 and 3.2 fold increased in respectively enamelin and ameloblastin. When LRAP was overexpressed on an amelogenin knockout mouse model, the ameloblasts were not affected. Further, expression of the global chromatin organizer and transcription factor SATB1 was reduced in secretory stage TgLRAP ameloblasts. These findings identify a cellular role for LRAP in enamel formation that is not directly related to directing enamel crystal formation as is reported to be the primary function of full length amelogenins. The effect of LRAP overexpression in upregulating amelogenins, MMP-20 and SATB1, suggests a role in protein regulation critical to ameloblast secretion and matrix processing, to form a mineralized enamel matrix.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric poi

The aim of the study was to investigate the efficacy of the use of xylitol-containing tooth-wipes in preventing dental caries in young children. In a double-blinded randomized controlled clinical trial, 44 mothers with active caries and their 6- to 35-month-old children were randomized to xylitol-wipe or placebo-wipe groups. The children’s caries scores were recorded at baseline and 1 year. Salivary levels of mutans streptococci and lactobacilli were enumerated at baseline, 3, 6, and 12 months. Data were analyzed by intent-to-treat modeling with imputation for caries lesions and a linear mixed-effect model for bacterial levels. Significantly fewer children in the xylitol-wipe group had new caries lesions at 1 year compared with those in the placebo-wipe group (P < 0.05). No significant differences between the two groups were observed in levels of mutans streptococci and lactobacilli at all time-points. Daily xylitol-wipe application significantly reduced the caries incidence in young children as compared with wipes without xylitol, suggesting that the use of xylitol wipes may be a useful adjunct for caries control in infants ( Clinicaltrials.gov registration number CT01468727).

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair. © 2012 S. Karger AG, Basel.

Amelogenin gene organization varies from 6 exons (1,2,3,5,6,7) in amphibians and sauropsids to 10 in rodents. The additional exons are exons 4, 8, 9, and "4b", the latter being as yet unidentified in AMELX transcripts. To learn more about the evolutionary origin of these exons, we used an in silico approach to find them in 39 tetrapod genomes. AMEL organization with 6 exons was the ancestral condition. Exon 4 was created in an ancestral therian (marsupials + placentals), then exon 9 in an ancestral placental, and finally exons "4b" and 8 in rodents, after divergence of the squirrel lineage. These exons were either inactivated in some lineages or remained functional: Exon 4 is functional from artiodactyls onward; exon 9 is known, to date, only in rodents, but could be coding in various mammals; and exon "4b" was probably coding in some rodents. We performed PCR of cDNA isolated from mouse and human tooth buds to identify the presence of these transcripts. A sequence analogous to exon "4b", and to exon 9, could not be amplified from the respective tooth cDNA, indicating that even though sequences similar to these exons are present, they are not transcribed in these species. © 2012 International & American Associations for Dental Research.

Dental fluorosis occurs as a result of excess fluoride ingestion during tooth formation. Enamel fluorosis and primary dentin fluorosis can only occur when teeth are forming, and therefore fluoride exposure (as it relates to dental fluorosis) occurs during childhood. In the permanent dentition, this would begin with the lower incisors, which complete mineralization at approximately 2–3 years of age, and end after mineralization of the third molars. The white opaque appearance of fluorosed enamel is caused by a hypomineralized enamel subsurface; with more severe dental fluorosis, pitting and a loss of the enamel surface occurs, leading to secondary staining (appearing as a brown color). Many of the changes caused by fluoride are related to cell/matrix/mineral interactions as the teeth are forming. At the early maturation stage, the relative quantity of amelogenin protein is increased in fluorosed enamel in a dose-related manner. This appears to result from a delay in the removal of amelogenins as the enamel matures. In vitro, when fluoride is incorporated into the mineral, more protein binds to the forming mineral, and protein removal by proteinases is delayed. This suggests that altered protein/mineral interactions are in part responsible for retention of amelogenins and the resultant hypomineralization that occurs in fluorosed enamel. Fluoride also appears to enhance mineral precipitation in forming teeth, resulting in hypermineralized bands of enamel, which are then followed by hypomineralized bands. Enhanced mineral precipitation with local increases in matrix acidity may affect maturation stage ameloblast modulation, potentially explaining the doserelated decrease in cycles of ameloblast modulation from ruffleended to smooth-ended cells that occur with fluoride exposure in rodents. Specific cellular effects of fluoride have been implicated, but more research is needed to determine which of these changes are relevant to the formation of fluorosed teeth. As further studies are done, we will better understand the mechanisms responsible for dental fluorosis.

The response of ameloblasts to long-term (6 weeks) expo- sure to 100 ppm fluoride was examined in continuously erupting mandib- ular incisors of female Sprague-Dawley rats as compared to control rats receiving a similar diet (Teklad L-356) but no sodium fluoride in their drink- ing water. After treatment, animals from both groups were perfused intra- vascularly with glutaraldehyde, and the incisors were removed and pro- cessed for light microscope morphometric analyses directly from 1 μm thick Epon sections. Other animals were injected intravenously with cal- cein (green fluorescence) followed 4 hours later by xylenol orange (red fluorescence) in order to reveal smooth-ended ameloblast modulation bands and thereby allow quantification of parameters related to the cre- ation and movement of modulation waves within the maturation zone of these teeth. The results indicated that rat incisors expressed four major changes in normal amelogenesis which could be attributed to the chronic fluoride treatment. First, ameloblasts produced a thinner than normal enamel layer by the time they completed the secretory stage and entered the maturation stage of amelogenesis. Second, enamel organ cells within the maturation zone, especially those from the papillary layer, were shorter in height than normal. Third, ameloblasts related to maturing enamel in areas where it was partially soluble and/or fully soluble in EDTA modu- lated at a rate that was much slower than normal. In some locations amelo- blasts remained ruffle-ended for as much as 30% longer than normal per cycle. This upset the usual pattern such that fewer total modulation cycles were completed per unit time by these ameloblasts. Fourth, enamel pro- teins were lost from the maturing enamel layer at a rate that was about 40% slower than normal. The data suggested that ameloblasts detected the de- lay in the extracellular breakdown and/or loss of enamel proteins and they responded by remaining ruffle-ended for longer intervals than usual (pos- itive feedback). © 1993 Wiley-Liss, Inc.

Transforming growth factor β, TGF-β, is expressed during tooth formation and can induce pre-odontoblast differentiation and formation of functional odontoblast-like cells in vitro. In addition, exogenous TGF-β can increase reparative dentin formation, presumably by acting on odontoblasts. In this study, we examined the tooth phenotype of transgenic mice, in which TGF-β2 expression is directed by the osteocalcin promoter. Previous studies have shown that these mice have a bone phenotype that resembles that of human osteoporosis, including the existence of spontaneous fractures. Microhardness testing of the enamel and dentin showed no differences in the molars of these transgenic mice as compared with those of their wild-type littermates. Consistent with the increase in bone mineral apposition rate previously reported in these mice, the dentin apposition rate appeared to be increased in the TGF-β2-overexpressing mice. Thus, in teeth, as in bone, TGF-β2 appears to stimulate the synthesis and deposition of matrix. Further studies are needed to understand the effect of TGF-β2 on distinct mineralized tissues (bone, dentin, and cementum) and to determine whether exogenous TGF-β2 may be useful for tooth repair.

Objective: A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralised amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals. Design: Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analysed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM. Results: As compared to rh174 amelogenin, p.P70T mutant displayed significantly increased sizes of the assemblies, higher binding affinity to apatite, and decreased crystal height. Conclusion: Pro-70 plays an important structural role in the biologically relevant amelogenin self-assembly. The disturbed regularity of amelogenin nanospheres by this single mutation resulted in an increased binding to apatite and inhibited crystal growth.

Two low molecular weight amelogenin gene splice products (A+4 and A-4), used for pulp capping after surgical pulp exposure in the rat first maxillary molars, have been shown to induce the formation of a reparative dentinal bridge. A-4 can induce mineralization of the atubular orthodentinal type to totally fill the root pulp canal with a homogeneous mineralized structure. The two molecules recruit latent adult pulp progenitors to form actively dividing cells, which differentiated into cells bearing an osteoblast-odontoblast phenotype to produce an extracellular matrix implemented in reparative mineralization. We conclude that pulp capping with bioactive molecules will direct new approaches in biodentistry.

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin alpha2beta1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl- channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone. We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells. (C) 2009 Elsevier Inc. All rights reserved.

Amelogenins are extracellular matrix proteins expressed at the secretory and the maturation stages of enamel formation. To characterize amelogenin transcripts from these two stages, rat incisor enamel-organ mRNA was hybridized to a mouse amelogenin cDNA. Northern analysis of the mRNA showed four amelogenin transcripts at the secretory stage. Two of these were larger than previously described, and would code for high molecular-weight amelogenins (> 40 kDa). These transcripts were similar in size to at least three bovine amelogenin mRNAs. At the maturation stage, one transcript slightly smaller than the major amelogenin transcript from the secretory stage was apparent. These differences may indicate a selective splicing of amelogenin mRNA in the maturation stage as compared to the secretory stage of enamel development.

Enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation. Fluorosed enamel is characterized by a retention of amelogenins in the early-maturation stage, and by the formation of a more porous enamel with a subsurface hypomineralization. The mechanisms by which fluoride affects enamel development include specific effects on both the ameloblasts and on the developing enamel matrix. Maturation-stage ameloblast modulation is more rapid in fluorosed enamel as compared with control enamel, and proteolytic activity in fluorosed early-maturation enamel is reduced as compared with controls. Secretory enamel appears to be more susceptible to the effects of fluoride following acute fluoride exposure, such as may occur with the use of fluoride supplements. However, both human and animal studies show that the transition/early-maturation stage of enamel formation is most susceptible to the effects of chronic fluoride ingestion at above-optimal levels of fluoride in drinking water.

It has not been established whether transmission of mutans streptococci occurs between unrelated children older than 4 years of age. The aim of the study was to investigate the possible transmission of mutans streptococci genotypes from child to child in kindergarten. We studied 96 children (ages 5–6 yrs) in three San Francisco Bay Area public schools. Mutans streptococci colonies from each child were isolated from selective culture on Mitis Salivarius Sucrose Bacitracin agar. We used arbitrary primed polymerase chain reactions to determine the mutans streptococci genotypes. Two children (not siblings) in each of the three schools (6%) shared an identical amplitype of S. mutans, unique to each pair. The 19 S. sobrinus amplitypes were found in 12 children, and all were unique to each child. The presence of matching genotypes of S. mutans demonstrates horizontal transmission of this species between unrelated children aged 5–6 years.

Calcium is a key component of the mineralized enamel matrix, but may also have a role in ameloblast cell differentiation. In this study we used human ameloblast lineage cells to determine the effect of calcium on cell function. Primary human ameloblast lineage cells were isolated from human fetal tooth buds. Cells were treated with calcium ranging from 0.05 to -1.8 mM. Cell morphology was imaged by phase contrast microscopy, and amelogenin was immunolocalized. Proliferation of cells treated with calcium was measured by BrdU immunoassay. The effect of calcium on mRNA expression of amelogenin, Type 1 collagen, DSPP, amelotin, and KLK-4 was compared by PCR analysis. Von Kossa staining was used to detect mineral formation after cells were pretreated with calcium. Calcium induced cell organization and clustering at 0.1 and 0.3 mM concentrations. Increasing concentrations of calcium significantly reduced ameloblast lineage cell proliferation. The addition of 0.1 mM calcium to the cultures upregulated expression of amelogenin, Type I collagen, and amelotin. After pretreatment with 0.3 mM calcium, the cells could form a mineralized matrix. These studies, which utilized human ameloblast lineage cells grown in vitro, showed that the addition of calcium at 0.1 and 0.3 mM, induced cell differentiation and upregulation of amelogenin Type I collagen and amelotin.

Intake of excess amounts of fluoride during tooth development cause enamel fluorosis, a developmental disturbance that makes enamel more porous. In mild fluorosis, there are white opaque striations across the enamel surface, whereas in more severe cases, the porous regions increase in size, with enamel pitting, and secondary discoloration of the enamel surface. The effects of fluoride on enamel formation suggest that fluoride affects the enamel-forming cells, the ameloblasts. Studies investigating the effects of fluoride on ameloblasts and the mechanisms of fluorosis are based on in vitro cultures as well as animal models. The use of these model systems requires a biologically relevant fluoride dose, and must be carefully interpreted in relation to human tooth formation. Based on these studies, we propose that fluoride can directly affect the ameloblasts, particularly at high fluoride levels, while at lower fluoride levels, the ameloblasts may respond to local effects of fluoride on the mineralizing matrix. A new working model is presented, focused on the assumption that fluoride increases the rate of mineral formation, resulting in a greater release of protons into the forming enamel matrix.

Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro41 is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.

The repair of dental pulp by direct capping with calcium hydroxide or by implantation of bioactive extracellular matrix (ECM) molecules implies a cascade of four steps: a moderate inflammation, the commitment of adult reserve stem cells, their proliferation and terminal differentiation. The link between the initial inflammation and cell commitment is not yet well established but appears as a potential key factor in the reparative process. Either the release of cytokines due to inflammatory events activates resident stem (progenitor) cells, or inflammatory cells or pulp fibroblasts undergo a phenotypic conversion into osteoblast/odontoblast-like progenitors implicated in reparative dentin formation. Activation of antigen-presenting dendritic cells by mild inflammatory processes may also promote osteoblast/odontoblast-like differentiation and expression of ECM molecules implicated in mineralization. Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process.

Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by substrate competition assay, pull-down assay, and surface plasmon resonance (SPR). The results showed that the binding of the P41T mutant amelogenin for MMP20 was significantly lower than that of wild-type amelogenin. Our study supports a model in which the P41T mutation reduces the interactions between amelogenin and MMP20, leading to decreased degradation of amelogenin by MMP20, and resulting in AI.

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogenin-modulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells.

This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.

Fluorosed enamel is caused by exposure to fluoride during tooth formation. The objective of this study was to determine whether epithelial ameloblast-lineage cells, derived from the human enamel organ, are directly affected by micromolar concentrations of fluoride. Cells were cultured in the presence of fluoride, and proliferation was measured by BrdU incorporation. The effect of 0, 10, or 20 μM fluoride on apoptosis was determined by the flow cytometry apoptotic index. The effects of fluoride on gene expression were investigated by SuperArray microarray analysis and real-time PCR. Fluoride had a biphasic effect on cell proliferation, with enhanced proliferation at 16 μM, and reduced proliferation at greater than 1 mM F. Flow cytometry showed that both 10 μM and 20 μM NaF significantly increased the apoptotic index of ameloblast-lineage cells. There was no general effect of fluoride on gene expression. These results indicate multiple effects of micromolar fluoride on ameloblast-lineage cells.

Initiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. Objective In this study, we used a transgenic mouse model which drives overexpression of an activated form of TGF-β2 under control of the osteocalcin promoter, to investigate the role of TGF-β2 in the dental mesenchyme, on enamel formation. Design Dentin and enamel were imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Dentin mechanical properties were characterized for hardness and elasticity, following nanoindentation with a modified AFM. Pores found in enamel were quantified and compared using image analysis software (Scion Image™). Results The elastic modulus of dentin was significantly reduced in the male TGF-β2 overexpressor mice as compared to male wildtype mice, with no significant differences between female mice. Similarly, there were significantly more pores in enamel of the male transgenic mice as compared to male wildtype mice, with no significant differences between female mice. In situ hybridization of the continuously erupting incisor confirmed that osteocalcin expression was limited to the odontoblast cell layer at all stages of tooth formation. Conclusion TGF-β2 overexpression in the dentin matrix, results in sex-linked differences in dentin and enamel formation.

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 microM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 microM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 microM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.

Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.

Mice are commonly considered the model mammal for many biomedical studies. In this work, mouse and human dentin were compared to specify structural and mechanical differences to establish a baseline for comparison of dental tissues between these species. Atomic force microscopy revealed tubules of about 1.0 to 1.6 μm in diameter as the main structural feature in dentin of both species. Nanoindentation yielded the elastic modulus about 15% lower in murine intertubular dentin while the hardness was almost equal. Dynamic stiffness mapping confirmed the lower elastic properties and also revealed that the peritubular region of increased mineralization around tubules is drastically reduced or maybe absent in murine dentin of this age. This study suggests that structural and mechanical differences need to be considered when murine dentin is used as a model system.

Fluoride is associated with a decrease in the incidence of dental caries, but excess fluoride can lead to enamel fluorosis, a defect that occurs during tooth enamel formation. In fibroblasts, the Arhgap gene encodes a RhoGAP, which regulates the small G protein designated RhoA. Fluoride treatment of fibroblasts inactivates RhoGAP, thereby activating RhoA, which leads to elevation of filamentous actin (F-actin). Since RhoA is a molecular switch, our hypothesis is that in ameloblasts, fluoride may alter the cytoskeleton through interference with the Rho signaling pathway. Our objective was to measure the effects of sodium fluoride on F-actin using tooth organ culture and confocal microscopy. The results indicated that cellular responses to fluoride include elevation of F-actin in ameloblasts. It was concluded from immunohistochemistry, RT-PCR and confocal approaches that the components of the Rho pathway are present in ameloblasts, and that the response to fluoride involves the Rho/ROCK pathway.

Summary Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin. Objective: The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation. Design: DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot. Results: Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFβ-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated. Conclusion: DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.

Summary Tooth enamel is formed by ameloblasts, which are derived from the epithelial cells of the enamel organ. Objective: The purpose of this study was to grow human ameloblast-like epithelial cells in culture. Design: Human fetal tooth organs were isolated, and the cells were separated by digestion in collagenase/dispase. The cells were cultured in KGM-2 media with and without serum and at different calcium concentrations. The expression of enamel matrix proteins was analyzed by RT-PCR and cytokeratin 14 was detected by immunohistochemistry. The cells were further characterized by osteogenesis/odontogenesis-related DNA array. Results: Cells isolated from the tooth organs grown in KGM-2 media containing 2–10% serum, were mixture of cobblestone and spindle shaped cells. Culturing these cells in KGM-2 with 0.05 mM calcium was selective for cobblestone ameloblasts-like cells (CAB), which were immunopositive for cytokeratin 14. Amelogenin, ameloblastin, enamelin, MMP-20 and KLK-4 were detected in CAB cells by RT-PCR. Osteogenesis SuperArray analyses could not detect the presence of typical molecules related to mesenchymal odontoblast or osteoblast lineage cells in these cultures. Conclusions: These studies showed that cobblestone-shaped ameloblast-like cells are selected from the tooth organ cells, by culture in KGM-2 media with 0.05 mM calcium.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.

Dental caries in young children frequently appears first on the maxillary primary incisors, where the liquids ingested by the infant sucking on a bottle or breast remain pooled away from salivary flow. This so-called early childhood caries can rapidly progress to result in rampant destruction of the primary dentition. ECC can affect children from all socioeconomic classes but is most often found in children of new immigrants or those with lower socioeconomic status. The treatment of ECC is costly, and treatment relapses are frequent. Prevention and treatment of ECC focus on inhibition of the growth of oral bacteria. Strategies include reducing the frequency of exposure to bacterial substrates, as well as controlling the growth of oral bacteria. Topical antibacterials such as 10 percent povidone iodine show promise for future use in the inhibition of ECC in young children.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine for an adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N-terminal to the cleavage site for enamel matrix metalloproteinase-20 (MMP-20) in amelogenin. MMP-20 releases the tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate at which MMP-20 hydrolyses mutated amelogenin relative to unmutated amelogenin. A full-length recombinant human amelogenin and a mutated amelogenin with a substitution of proline by threonine were expressed and purified by ammonium sulphate precipitation and reverse phase HPLC. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the recombinant proteins, which resulted in fragments with a mass predicted for TRAP. The proteolytic site was also modelled as substrates by two synthetic peptides, SYGYEPMGGWLHHQ and SYGYETMGGWLHHQ, selected from residues 36 to 49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. These two peptides were labelled at their N- and C-termini respectively by using rhodamine and biotin. After digestion with MMP-20, the truncated peptides were separated by avidin-labelled magnetic Dynal beads and were identified by mass spectrometry. These results demonstrated that both oligopeptides were cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. Enzyme kinetics showed that the k(cat)/K(m) of rMMP-20 against the unmutated amelogenin peptide was 21 times greater than that against the mutated peptide. This study suggests that the reduced rate of TRAP formation by a single amino acid substitution alters enamel matrix hydrolysis by MMP-20, which may result in amelogenesis imperfecta.

Amelogenin proteins are assumed to control the calcification of dental enamel with a nanoscale precision that facilitates the formation of fibrous apatite crystals organized in a remarkable microstucture. In this study, recombinant full-length human amelogenin induced protein-guided mineralization and the formation of an enamel-like composite material at specific physical-chemical conditions as observed by atomic force microscopy (AFM). Amelogenin bound specifically to fluoroapatite crystals (FAP) of a glass-ceramic substrate at Ca2+ and PO43- concentrations similar to in-vivo conditions and at pH 8. Layers up to 400 nm high, containing elongated crystals, formed on the (001)-planes of FAP within 24h in supersaturated solutions. In contrast, (hk0)-faces grew by only 10-30 nm, but showed nanospheres aligned parallel to the c-axis of FAP. At pHs different from 8, proteins bound non-specifically to substrate and layers on FAP reached only 5-15 nm thickness. Micro-Raman spectroscopy and AFM revealed the formation of a composite material that resembled a structure and composition comparable to human enamel. These observations suggest that certain conditions are required to activate amelogenin to control and promote crystal growth of apatite along the c-axis and to synthesize an enamel-like material.

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.

Human deciduous and permanent teeth exhibit different developmental processes, morphologies, histological characteristics and life cycles. In addition, their pulp tissues react differently to external stimuli, such as the pulp sensitivity test, dental trauma and pulp therapy materials. These suggest differences in gene expression and regulation, and in this study we compared gene-expression profiles of the human dental pulp from deciduous and permanent teeth. Pulp tissues from permanent premolars and deciduous molars aged 11-14 years were extirpated and mRNA was isolated for cDNA microarray analysis, and quantitative real-time PCR (qPCR). Other teeth were used for immunohistochemical analysis (IHC). Microarray analysis identified 263 genes with a twofold or greater difference in expression level between the two types of pulp tissue, 43 and 220 of which were more abundant in deciduous and permanent pulp tissues, respectively. qPCR analysis was conducted for eight randomly selected genes, and the findings were consistent with the cDNA microarray results. IHC confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was broadly expressed in deciduous dental pulp tissue, but minimally expressed in permanent dental pulp tissue. Immunohistochemical analysis showed that calbindin 1 (CALB1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and gamma-aminobutyric acid A receptor beta 1 (GABRB1) were abundantly expressed in permanent predentin/odontoblasts, but only minimally expressed in deciduous dental pulp tissue. These results show that deciduous and permanent pulp tissues have different characteristics and gene expression, suggesting that they may have different functions and responses to therapies focused on pulp or dentin regeneration.