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RESEARCH REPORTS |
1 Department of Cytokine Biology and 2 Department of Biomineralization, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA; and
3 Laboratory for the Study of Calcified Tissues and Biomaterials, Faculté de médecine dentaire, Université de Montréal, Montreal, QC, Canada H3C 3J7;
* corresponding author, jbartlett{at}forsyth.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: enamelysin • amelogenin • amelogenesis • FTIR • microhardness
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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During tooth development (amelogenesis), enamel crystallites begin forming at the dentino-enamel junction on freshly mineralized dentin in a matrix comprised of tooth-specific proteins (Deutsch et al., 1991). Amelogenesis consists of at least 3 stages: the secretory, transition, and maturation stages of enamel development. During the secretory stage, very thin ribbons of carbonated apatite mineral form. These ribbons are only a few apatitic unit cells in thickness (about 10 nm), with a width of approximately 30 nm (Cuisinier et al., 1992; Daculsi and Kerebel, 1978), and may grow to extend through the entire depth of the enamel layer. During the transition and maturation stages of amelogenesis, a marked decrease in organic matter is observed (Robinson et al., 1988a), with a concomitant increase in mineral deposition. As a result, the ribbons thicken, thereby allowing rod patterns to become more visible. These general features of amelogenesis are remarkably constant among different species (Robinson et al., 1988b).
Two proteases are known to play a role in dental enamel formation. Matrix metalloproteinase 20 (MMP-20, enamelysin) is expressed during the secretory and transition stages, but not during most of the maturation stage (Bartlett et al., 1996, 1998; Hu et al., 2002). Conversely, kallikrein 4 (KLK-4, EMSP1) is expressed during the transition through maturation stages of enamel development (Simmer et al., 1998; Hu et al., 2000, 2002). So, it is thought that MMP-20 is involved in the regulation of axial growth of enamel crystals, while KLK-4 plays a role in their thickening. Previously, the MMP-20 gene was deleted from the mouse genome. The resulting dental enamel possessed an altered enamel matrix rod pattern, had hypoplastic enamel that delaminated from the dentin, and had a deteriorating enamel organ morphology as development progressed (Caterina et al., 2002). In this study, we hypothesized that the physical and chemical properties of MMP-20 null enamel would also be significantly diminished compared with those of controls. We assessed enamel composition and physical properties by performing microhardness testing, FTIR spectroscopy, and ashing experiments.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Fourier Transform Infrared Spectroscopy (FTIR)
Erupted portions of maxillary incisors from adult wild-type and MMP-20 null mice were dehydrated with ethanol and acetone and embedded in a hard-formulation epoxy embedding medium (EMbed 812, EMS, Hatfield, PA, USA). The samples were ground and polished to 0.25 µm with a diamond paste (Buehler, Lake Bluff, IL, USA). Enamel and dentin from the erupted portions of incisors were studied in ATR and reflectance modes with the use of a Multiscope FTIR microscope coupled with a Spectrum One FTIR spectrometer (PerkinElmer, Wellesley, MA, USA). The ATR spectra were collected (4000 cm–1–700 cm–1 range) at a resolution of 4 cm–1 with 32 accumulations per run. Reflectance spectra were collected from ~ 2500 µm2 areas of the samples at 4000 cm–1-550 cm–1 intervals with a resolution of 4 cm–1 and with 64 accumulations per run. Background spectra were collected from a gold mirror. We processed the reflectance spectra using the Kramers-Kronig transform routine in the Spectrum 5.0 program to obtain an absorbance-like spectrum (Kanton, 1998; Lippert et al., 1998; Tesch et al., 2001). Corrected maximum heights of protein Amide I absorption peaks in the 1600–1700 cm–1 region and 
3 PO4 absorption peaks in the 1000–1100 cm–1 region were measured in all spectra. We used the Amide I/3 PO4 ratio to assess the extent of mineralization in wild-type controls and MMP-20 null enamel.
Tissue Preparation, Micro-weighing, and Ashing
Adult C57BL6 mice were killed by CO2 asphyxiation, and hemimandibles and hemimaxillae were removed and immersed in liquid nitrogen. The tissues were freeze-dried at -55°C for 48 hrs (Labconco, Kansas City, MO, USA). The bone and enamel organs covering the incisors were removed, and the exposed enamel surfaces were wiped gently with dry Kimwipes. The enamel layer on each incisor was transected with a scalpel blade into a series of 0.5-mm-long strips, from apical toward incisal ends of the tooth. Each strip was removed, dried overnight at 50°C, weighed, placed in an Isotemp Muffle Furnace (Fisher Scientific, Pittsburgh, PA, USA), and heated at 575°C for 18–24 hrs. The samples were re-weighed (referred to as ‘ash weight’). This procedure vaporized organic material and any bound water present in the enamel strips (Hiller et al., 1975). The ‘ash weight’ therefore represents the amount of mineral present in a given enamel strip. For the purposes of this study, protein content of strips was defined as the difference between starting dry weight minus final ash weight. The percent mineral by weight in each strip was calculated by the equation (ash weight/dry weight) x 100. The raw data from strips were then pooled into 3 defined developmental stages (Table
) according to alignment procedures described elsewhere (Smith et al., 2004). Data processing and statistical analyses were performed with Version 6 of Statistica for Windows (Statsoft Inc., Tulsa, OK, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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3 PO4 absorption bonds is routinely used for the assessment of the mineral-to-matrix ratios of various mineralized tissues (Paschalis et al., 1996; Tesch et al., 2001; Aparicio et al., 2002). The Amide I/3 PO4 maximum height ratio was significantly higher in the MMP-20 null enamel samples (0.078) compared with the controls (0.016) for the spectra obtained in ATR mode (Fig. 1A). The differences were also significant in reflectance mode (Fig. 1B): MMP-20 null 0.04 ± 0.007 (n = 4) vs. wild-type control 0.007 ± 0.0004 (n = 4). Analysis of these data demonstrates that the MMP-20 null enamel contains more protein per unit volume than the wild-type control enamel (see also Fig. 2C).
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3 PO4 maximum height ratio in the spectra observed from MMP-20 null and wild-type dentin. No significant differences were observed between these groups for the dentin analyses, so the data were merged to generate a single averaged ratio. The ratio for dentin was 0.31 ± 0.025 (n = 3) and 0.16 ± 0.012 (n = 4) in ATR and reflectance modes, respectively. Assuming that fully mature wild-type enamel and dentin are 96% and 70% mineral by weight, respectively (Nanci, 2003), and assuming that a linear dependence exists between the Amide I/PO4 ratio and mineral content, we calculate that, in the regions sampled, the mature enamel mineral content of the MMP-20 null mouse was 88–90% by weight. This represents a reduced mineral content of 6–8% when compared with the 96% value attributed to the wild-type enamel.
To quantify mineral differences between the MMP-20 null and control mice in a separate assay system, we performed enamel ashing studies (Table
, Fig. 2). No differences in gross mineral (ash) weight or percent mineral were detected in comparison of mandibular with maxillary incisors (Table, non-parametric tests, line 1), and all subsequent analyses of effects by group (MMP-20 null vs. wild-type) and by stage (secretory, early mature, nearly mature) were performed with ‘Jaw’ excluded as a factor. The mean weight of mineral increased dramatically by stage for all teeth examined (Table, ash weight), but the weight of mineral present in MMP-20 null enamel was always less than the weight of mineral detected in wild-type controls at the corresponding stage (Table, +/+ vs. -/- per Stage; Fig. 2A). These differences were least noticeable for the secretory stage (not significant) and most dramatic for nearly mature enamel, where the gross weight of mineral present in the enamel of MMP-20 null animals was approximately one-half the amount detected in wild-type controls (Table, significant; Fig. 2A). Given the half-mineral content in enamel from null mice vs. controls, the protein content of the MMP-20 null enamel started at proportionally normal levels, but increased significantly thereafter and did not plateau as the enamel progressed through the early and nearly mature stages (Fig. 2B). Mineral:protein ratio analyses (Fig. 2C) confirmed that the null mouse enamel proteins were not properly re-absorbed as the enamel matured. Interestingly, the mineral did not appear to be as dramatically affected when data were adjusted so that equal weights (by weight) of enamel from experimental and control samples were assessed for mineral content (Table; Fig. 2D). No significant differences between treatment groups were detected for this parameter when data were analyzed en masse (Table, non-parametric tests, line 2) or by stage for the secretory and early maturation stages (Table, bottom; Fig. 2D). However, for the nearly mature enamel, a significant difference was observed. The percent mineral by weight was lower by about 12% in the MMP-20 null mice compared with that in wild-type controls (74.6 ± 7.6% vs. 86.9 ± 4.4% N=16; Table, last line, Fig. 2D). This result was unexpected, because MMP-20 is not expressed in nearly mature enamel. Although mature wild-type enamel could not be dissected, mature MMP-20 null enamel was dissectible and was assessed for mineral content. The average percent mineral content by weight of enamel extracted from mature MMP-20 null incisors was 80.1% ± 3.3% (N = 12). This was a reduction of approximately 16% when compared with the 96% value attributed to fully mature enamel.
Hardness is an essential mechanical property of enamel. To assess how the structural abnormalities and differences in organic content identified in this study affect hardness, we performed a series of Knoop microhardness tests on mature enamel from MMP-20 null and wild-type incisors (Catarina et al., 2002). Micro-indentations were created in each of 2 maxillary incisors from MMP-20 null and wild-type mice. The average hardness for wild-type enamel (N = 65 indentations) was 327 KHN10 (SD ± 70) and 207 KHN10 (SD ± 63) for MMP-20 null enamel (N = 30 indentations). The difference was highly significant (p = 0.005), and the results demonstrated that the mature null enamel was approximately 37% softer than normal enamel (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The most surprising result was that the biggest difference in percent mineral by weight between MMP-20 null animals and controls occurred in the nearly mature enamel (Fig. 2), when MMP-20 is normally no longer expressed and is not catalytically active (Smith et al., 1996). This suggests that the initial MMP-20 cleavages are important for the eventual removal of the protein matrix as the enamel rods grow in width and thickness. Of note is that MMP-20 tends to cleave amelogenin within the C-terminal area (Ryu et al., 1999), whereas the subsequent proteinase KLK-4 tends to cleave amelogenin within the N-terminal area (Ryu et al., 2002). So, perhaps the initial MMP-20-mediated amelogenin C-terminal cleavages during the secretory stage are necessary for the later maturation-stage removal of amelogenin from the enamel matrix. Alternatively, the absence of MMP-20 may indirectly affect the normal process of ameloblast protein re-absorption during the maturation stage. The demonstrated lack of an enamel prism pattern in the MMP-20 null enamel could adversely affect the efficiency of the re-absorption process (Caterina et al., 2002). Perhaps, as enamel crystals mature, the altered prism/rod geometry increasingly restrains proper protein removal.
The results from the FTIR analyses did differ somewhat from those from the ashing studies. FTIR analyses were performed on fully mature erupted enamel, and ashing studies were performed primarily on enamel that was not yet fully mature. Mature enamel was too hard and tightly bound to the dentin to be dissected from the wild-type animals. However, mature erupted enamel for the ashing experiments was dissectible from certain regions of the MMP-20 null incisors. Ashing studies identified the percent mineral by weight for the mature MMP-20 null enamel as approximately 16% less than the 96% norm, whereas FTIR analyses identified an approximate 6–8% reduction in this value. Although the values are somewhat different, both techniques demonstrated the same general trend.
Another interesting finding was that the mature enamel from the null mouse contained between 7 and 16% less weight percent of mineral than controls, but was approximately 37% softer. Previously, the MMP-20 null mouse enamel was demonstrated to lack a defined enamel prism pattern, and this lack of a prism pattern may account for the significantly decreased enamel hardness values (Caterina et al., 2002). It is also possible that the null mouse enamel is more porous than normal, and analyses of mineral on a per unit volume basis rather than a per unit weight basis could have revealed larger mineral content differences (Hiller et al., 1975; Robinson et al., 1988a). Regardless of the explanation, our findings are supported by recent results demonstrating that hydrated, hypomineralized, aprismatic human enamel can have small decreases (5%) in mineral content, with large decreases (86%) in hardness (Mahoney et al., 2004).
In conclusion, analysis of these data demonstrates that MMP-20 performs enamel matrix protein cleavages that are essential, either directly or indirectly, for the proper removal of proteins during the maturation stage of enamel development, when MMP-20 is no longer expressed. The analysis also suggests that a non-porous decussating enamel prism structure is essential for maintaining the extreme hardness of fully mature dental enamel.
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ACKNOWLEDGMENTS |
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Received April 4, 2004; Last revision September 29, 2004; Accepted September 29, 2004
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
NM), and more protein appears to be retained in the nearly mature enamel of the MMP-20 null mice (B, NM). Ratio (C) and percent mineral by weight (D) differences are significant only relative to nearly mature enamel (NM; see also Table