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Volume 52 (4): 437-445, 2004
Copyright ©The Histochemical Society, Inc.
Immunolocalization of Enamelysin (Matrix Metalloproteinase-20) in the Forming Rat Incisor
Matrices Extracellulaires et Biominéralisation, EA 2496, Faculté de Chirurgie Dentaire, Université René Descartes Paris V, Montrouge, France (KB–B,DS,MG); Department of Clinical Dental Sciences, University of Liverpool, Liverpool, United Kingdom (RH); and U 532 INSERM, Hôpital Saint-Louis, Paris, France (SM)
Correspondence to: Michel Goldberg, Faculté de Chirurgie Dentaire, Université Paris V, 1, rue Maurice Arnoux, 92120 Montrouge, France. E-mail: mgoldod{at}aol.com
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In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino–enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.
(J Histochem Cytochem 52:437–445, 2004)
Key Words: matrix metalloproteinase-20 • TIMP-2 • stratum intermedium • ameloblast • odontoblast • enamel • amelogenin • dentin
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Introduction |
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AMELOGENESIS is the culmination of a cascade of events that begins with the secretion of an amelogenin-rich product appearing as a translucent gel in the forming part of the rat incisor and ends with mineralization in the maturing and pigmented enamel, where the organic matrix is strongly reduced. In this context, the organic matrix accounts for about 20% at the early stage of enamel formation and is reduced to 0.4% at the end of enamel maturation (Robinson et al. 1995
). This implies that specific enzymes degrade most of the transient matrix and that the residues are reabsorbed by post-secretory ameloblasts bearing a brush border similar in some respects to that of osteoclasts (Leblond and Warshawsky 1979). Studies of the proteinases present in the developing enamel suggested that metalloproteinases (MMPs) may be more important during initial enamel formation, whereas serine proteases are implicated mostly at the later stages during enamel maturation (Overall and Limeback 1988; Fukae et al. 1996; Tanabe et al. 1996; Robinson et al. 1998).
Amelogenin is the most abundant protein in the forming enamel and is hydrolyzed within hours after its secretion to the matrix (Smith et al. 1989). The cleavage of amelogenin is highly regulated during development and is believed to produce biologically active fragments that may have different functions from those of the intact molecule. The degradation of amelogenin at early stages of enamel formation implies that amelogenin-degrading enzymes are particularly important in the initial development of enamel. Enamelysin, or MMP-20, is an MMP with a specificity for amelogenin and is considered a predominant enzyme for the processing of enamel matrix. It was shown to have an Mr of about 54.1 kD in humans (Llano et al. 1997) and 51.9 kD in swine (Bartlett et al. 1996), whereas the lower molecular weight active species (43, 38, 33, 25, and 21 kD) described by Li et al. (1999) and Moradian–Oldak et al. (2001) probably represent autoproteolytic products. MMP-20 has the same domain structure as most other MMPs including a signal peptide, a propeptide involved in maintaining enzyme latency, a catalytic domain with a Zn-binding site, and a hinge region that links the catalytic domain to the C-terminal hemopexin-like domain (Bartlett and Simmer 1999). It has recently been shown that MMP-20 can be activated by MT1–MMP in human odontoblasts and pulp cells (Palosaari et al. 2002). MT1–MMP is considered the physiological activator of the gelatinase MMP-2 on the cell membrane in a mechanism that requires a complex of TIMP-2 with the pro-MMP-2. The implication of TIMP-2 in pro-MMP-20 activation remains to be clarified.
Cloned from porcine enamel organ by Bartlett et al. (1996), MMP-20 was believed to be exclusively expressed in teeth, but its presence in human tongue carcinoma cells (Vaananen et al. 2001) and in cultured granulosa cells in follicles of porcine ovaries (Kimura et al. 2001) has recently been described. In the developing teeth, MMP-20 is expressed primarily during the secretory to late transition stages of amelogenesis but not in enamel maturation. In situ hybridization (ISH) and immunohistochemical (IHC) investigations revealed the presence of MMP-20 in both ameloblasts and in odontoblasts and in pulp cells (Begue–Kirn et al. 1998; Fukae et al. 1998; Caterina et al. 2000).
In this study we used the continuously growing rat incisor to study the expression pattern of MMP-20 and its presumed substrate, amelogenin, during different stages in the formation of mineralized rat dental tissues.
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Antibodies
The anti-MMP-20 polyclonal antibody (PAb), developed in rabbit and directed against a synthetic peptide corresponding to the hinge region of the MP, was from Sigma (St Louis, MO) (M9183). Anti TIMP-1 monoclonal antibody (MAb) was from Oncogene Research Products (Beverly, MA) and TIMP-2 monoclonal antibody (CA-101) was a gift from Dr Raphael Fridman (Wayne State University; Detroit, MI) (Hoyhtya et al. 1994
). Amelogenin PAb was a gift from I. Slaby (Biora; Malmoe, Sweden).
Zymography and Immunoblotting Analyses
Segments of the forming part of the rat mandibular incisor, as defined by Leblond and Warshawsky (1979), were homogenized in an ice-cold buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl2, 0.9% NaCl, and 0.2% Triton X-100 with or without a cocktail of protease inhibitors (Set V, EDTA-free; Calbiochem, La Jolla, CA) using a potter homogenizer (two incisors in 500 µl buffer). The resulting homogenate was then briefly sonicated on ice (three times for 5 sec), cleared by centrifugation at 10,000 x g for 15 min at 4C, and analyzed for the presence of MMP-20 by zymography and immunoblotting. MMP-20 was activated by incubating lysates with 1 mM p-aminophenylmercuric acetate (APMA) for 30 min at 37C.
Casein zymography was performed using 10% SDS-polyacrylamide gels co-polymerized with 1 mg/ml casein, Extracts (5 µg) were mixed with Laemmli sample buffer without reducing agents and without heating and subjected to SDS-PAGE. After electrophoresis, gels were incubated (30 min at 22C) in renaturing solution (2.5% Triton X-100), rinsed briefly in distilled water, and incubated (24 hr at 37C) in developing buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl2 and 0.02% NaN3). The gels were then stained with 0.1% Coomassie Blue R-250 in a solution of 20% isopropanol and 10% acetic acid and destained in 20% isopropanol and 10% acetic acid to reveal gelatinolytic activity.
For immunoblotting analysis, 20 µg of protein lysate was subjected to Laemmli 12% SDS-PAGE under reducing conditions, followed by transfer to a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk in TBS (100 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 (T-TBS) and was then incubated for 2 hr at room temperature (RT) with either MMP-20 antibody at 1:1000 dilution, anti-TIMP-1 at 1:2000 dilution, or TIMP-2 at 1:2000 dilution in T-TBS. The membrane was washed and incubated with a 1:1000 dilution of an IgG–peroxidase conjugate for 2 ht at RT. Immunodetection of the antigen was performed using the Renaissance Western Blot Chemiluminescence Reagent Plus Kit from NEN Life Science (Boston, MA) according to the manufacturer's instructions.
Light and Immunoelectron Microscopy
The study design was approved by the Ethics Committee of the Faculty. Five Sprague–Dawley male rats (100 g body weight) were anesthetized with an IP injection of chloral hydrate (400 mg/ kg), then perfused intracardially with a solution containing 4% paraformaldehyde, buffered with 0.1 M sodium cacodylate, pH 7.2–7.4, for 24 hr at 4C. After several rinses in cacodylate buffer, the teeth were dissected out from the mandibles, dehydrated in graded ethanol, and without demineralization embedded in Paraplast. Five- to 7-µm-thick sections were treated in methanol to inhibit endogenous peroxidases. Sections were saturated at 4C overnight with 1% PBS–BSA and incubated in the primary antibody at 1:100 dilution for 2 hr at RT. The optimal working dilutions in terms of detection and acceptable background labeling were determined after testing a series of antibody concentrations between 1:50 to 1:200. A swine anti-rabbit IgG conjugated with peroxidase (DAKO; Glostrup, Denmark) in 1% PBS–BSA was used as a secondary antibody at 1:100 dilution, and the conjugates were revealed by using 30 mg DAB:100 ml plus H2O2 (30 vol) in PBS (40 µl/100 ml). Controls were performed by omitting or substituting the primary antiserum with non-immune serum.
Electron Immunogold Labeling
Five Sprague–Dawley male rats (100 g body weight) were anesthetized with an injection of chloral hydrate, then perfused intracardially with a solution containing 1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2–7.4. After 10 min the two mandibular incisors were dissected out. The segments were prepared from the forming part of the incisor (the first 6 mm). The undemineralized mandibular incisors were sliced transversely with a razor blade into three equal segments about 2 mm thick. The segments were immersed in the fixative solution for 1 hr at 4C, then immersed overnight in 0.2 M cacodylate buffer. After dehydration in graded ethanols, the segments were embedded in Lowicryl K4M and polymerized at -20C with UV light.
Sections were incubated in PBS–5% BSA at RT for 90 min and then incubated with the primary antibodies (1:150 dilution) for 2 hr at RT. After three rinses with PBS–1% BSA, sections were incubated with a 1:100 dilution of the secondary antibody, a goat anti-rabbit IgG coupled to 15-nm colloidal gold (AuroProbe GAR IgG-G15; Amersham, Poole, UK) for 90 min at RT. Sections were rinsed in PBS and stained with aqueous uranyl acetate for 5 min and then with lead citrate for 1 min. They were examined with a JEOL 100B electron microscope operating at 80 kV. Controls were performed by omitting or substituting the primary antiserum with non-immune serum.
Quantitative Evaluation
The presence and distribution of MMP-20 and the intensity of its staining were evaluated by electron microscopy on two distinct presecretory and secretory areas as described (Goldberg et al. 2003). On electron micrographs enlarged to a final magnification of x54,000, gold–antibody conjugates were scored on 40 to 90 squares per area (2 cm x 2 cm) and were expressed as the number of gold particles per µm2 (grain density) followed by the standard deviation. The standard deviation mean was also calculated. Grain density was calculated in the stratum intermedium, in the basal mitochondrion-rich area, and in the distal supranuclear area of presecretory and secretory ameloblasts. In the presecretory area, calculation was further carried out in the non-mineralized mantle dentin and in polarizing odontoblasts. In the secretory area, immunogold complexes were scored again in the stratum intermedium and in secretory ameloblast cell bodies and Tomes' processes. Gold particles were scored also in the outer interrod and rod enamel growth sites. Calculations were carried out in the older inner enamel near the dentino-enamel junction (DEJ), mantle dentin, circumpulpal dentin, and metadentin (the 0.5–2.5-µm-thick border at which dentin mineralization is initiated) (Goldberg and Septier 1996), predentin, odontoblast cell bodies and processes, and finally in pulp cells. Background labeling was scored in the lumen of vessels and in lateral parts of the section where no biological material was present. Statistical comparison between two mean values was carried out using Student's t-test, and only p
0.05 was considered significant.
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Biochemical Detection of MMP-20 in Developing Rat Incisor
The forming part of the mandibular rat incisor (first 6 mm) was extracted in the presence of protease inhibitors and was analyzed by zymography and Western blotting for the presence of MMP-20, TIMP-1 and TIMP-2. The zymogram in Figure 1A shows that, in addition to the three expected MMP-20 forms at 54 kD for the latent and 46–41 kD doublet for the active forms (Bartlett and Simmer 1999
), two extra higher MW bands at 78 kD and 98 kD could be detected. When the extraction was performed in the absence of proteinase inhibitors, the main lysis band was observed at 21 kD, indicating degradation of the proteinase during extraction. Immunoblotting analysis on extracts treated by APMA shows the appearance of the 46-kD, active form but not the other 41-kD form shown in zymography (Figure 1B). This may reflect sample variation because we noted significant quantitative differences in band intensities when we compared different extracts. However, we cannot exclude the possibility that this molecular species may represent another casein-degrading enzyme. These variations among samples may be due to the different developmental stages present in the continuously growing rat incisor and would strongly depend on the precise incision point at sampling. APMA treatment caused the disappearance of the 98-kD complex but the 78-kD band was not greatly influenced either by the APMA treatment or by the absence of protease inhibitors during extraction. The 78-kD band was also stained with TIMP-2 antibody but not with TIMP-1 (Figure 1C). Because it also corresponds in size to the sum of the two and demonstrates a resistance to proteolytic degradation, it may represent an MMP-20/TIMP-2 complex. The higher molecular forms of TIMP-1 may represent complexes of this inhibitor with other MMPs (Kolkenbrock et al. 1995; Kossakowska et al. 1998).
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Light Microscopic Immunostaining
The pattern of immunostaining of MMP-20 and amelogenin in the forming part of rat incisor was studied by light microscopy on different segments of the rat incisor corresponding to specific developmental stages. In the presecretory stage, at the onset of matrix secretion (Figure 2, top panel), amelogenin staining was detected in ameloblasts and in the presecretory odontoblasts, but not in the more mature odontoblasts (Figure 2A). At this stage, only the apical poles of ameloblasts were stained for MMP-20 (Figure 2B). At the later secretory stage (Figures 2C and 2D), the strongest staining of both amelogenin and MMP-20 was observed in the secretory ameloblasts and in the outer part of the forming enamel. In addition, strong staining of MMP-20, although not of amelogenin, was noted in the stratum intermedium.
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Staining of MMP-20 was also seen in the odontoblasts, in some pulp cells, and in the stellate reticulum. Weak staining of MMP-20 was also observed in the inner forming enamel, which was mostly associated with the holes located near the DEJ. Some staining was seen in predentin but was absent in dentin.
Electron Immunogold Staining
Here we compared the distribution patterns and staining intensities of MMP-20 at two stages of enamel formation, an early stage characterized by a basement membrane separating the presecretory ameloblasts from the non-mineralized dentin and a later stage of development in which translucent enamel was partially formed, although not fully matured.
At the early stage, the most striking observation was the high labeling intensity in the stratum intermedium (Figure 3A). In the presecretory ameloblasts and odontoblasts, staining was mainly cytosolic and nuclear, although a few vesicles were also stained. No labeling was detected in the non-mineralized dentin, basement membrane, matrix vesicles, or with collagen fibrils (not shown).
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At the more advanced stage of tooth formation in which secretion of enamel occurs, labeling in the stratum intermedium was still intense (Figure 3B). Again, staining was mostly cytosolic in the cell body and in the processes of secretory ameloblasts and odontoblasts (Figures 3C, 3D, 4A, and 4B). A few immunogold complexes were located in the rough endoplasmic reticulum and in vesicles. Enamel interrods or rod growth sites were labeled along the fibrous ribbon-like structures (Figure 3D). The inner enamel near the DEJ was more faintly stained. No labeling was seen in the mantle dentin, dentin, metadentin, and predentin (Figure 4B). In the pulp, cells were labeled but the collagenous intercellular matrix was not.
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The grain densities for the gold–antibody complexes scored in cellular and extracellular compartments of the early presecretory and the secretory forming parts of the incisor were quantified and are presented in Figures 5A and 5B. Significant variations can be observed at both stages of development, with maximal values for SI, ameloblasts, and odontoblasts. Staining decreased gradually from the ameloblasts through the forming enamel, reaching background levels near the DEJ. Although a slight increase in score was then noted in the different compartments of the dentin, its value remained in the background level.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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In this study we used the continuously growing rat incisor to study the expression pattern of MMP-20 and its presumed substrate, amelogenin, during the formation of mineralized rat dental tissues. MMP-20 has unique structural and enzymatic properties, being capable of degrading amelogenin, the major component of the enamel matrix, and is therefore believed to play a major role during enamel development. Although its expression pattern is restricted to the developing tooth, it has been localized in both ameloblasts and odontoblasts (Bartlett et al. 1996
; Begue–Kirn et al. 1998; Caterina et al. 2000). This is rather unexpected when we consider that these cells are of different developmental origins, epithelial for ameloblasts and neural crest-derived cells for odontoblasts, and that they produce different matrices, enamel, and dentin. Although the function of MMP-20 expressed in ameloblasts is believed to be the slow processing of enamel proteins leading to enamel maturation, the target for MMP-20 expressed by odontoblasts or its function is yet to be determined. Our IHC and immunogold electron microscopic results confirm the presence of MMP-20 protein in both ameloblasts and odontoblasts of rat incisor but show, in addition, that MMP-20 protein is localized in the same sites as amelogenin, suggesting that, in odontoblasts also, amelogenin may be the target substrate, although the possibility that MMP-20 degrades other substrates cannot be ruled out. However, in both cell types, their temporal expression does not precisely coincide. The expression of amelogenin appears to precede that of MMP-20. Although the expression of amelogenin in ameloblasts persists in the different developmental stages, in odontoblasts its expression is transient and is seen essentially in the pre-polarized odontoblasts, disappearing with their maturation. MMP-20 expression lags behind, appearing only in the secretory odontoblasts. This delay in MMP-20 expression was also observed in ameloblasts, in which at the early stage when amelogenin is already present in pre-ameloblasts, MMP-20 is observed only in the more mature ameloblasts localized at the tip of the arch.
The significance of the localization of amelogenin or MMP-20 in odontoblasts is not clear. Although it was postulated that low molecular weight amelogenin degradation products diffuse through the basement membrane separating preameloblasts and preodontoblasts to become trapped in the forming dentin (Sawada and Nanci 1995), the localization of amelogenin and MMP-20 to the same sites argues for local synthesis of both enzyme and substrate. This would be in accord with the localization of MMP-20 RNA in the odontoblasts by in situ hybridization (Caterina et al. 2000). The local degradation of amelogenin by MMP-20 would produce low molecular mass peptides that have been suggested to serve as signaling molecules. Indeed, two gene splice products of 5–9-kD amelogenin [A+4] and [A-4], synthesized by odontoblasts, were shown to interact with immature mesenchymal cells to initiate a change in the cell phenotype and the maturation pathway (Nebgen et al. 1999; Veis et al. 2000). However, in view of our results showing the localization of both MMP-20 and amelogenin at the same sites, it appears likely that the amelogenin peptides extracted from dentin by Nebgen et al. may also represent degradation products. Indeed, the amelogenin present in the tooth was suggested to be a complex mixture of gene isoforms and degradation products (Simmer 1995).
When we examine the staining of MMP-20 within the cell, the decreased density of gold–antibody complexes observed from the cell bodies to the processes favors the possibility of active secretion. This may be the case for the forming enamel, in which staining was strong in the outer forming enamel and gradually diminished near the DEJ, but not for the matrix secreted by odontoblasts, because dentin and predentin were unstained. In addition, examination of ultrathin sections revealed that the gold-antibody complexes were located mostly in the cytosol and the nucleus, with a few gold particles seen in the secretory vesicles. This is not a unique phenomenon. We have previously shown that MMP-3 is mostly associated with cytoskeletal structures or/and small structures that may be storage sites for MPs in the cytosol (Hall et al. 1999).
A striking observation by both light and electron microscopy is the strong concentration of MMP-20 in the SI, particularly in the earlier stages of enamel development during which staining appears even stronger than in the presecretory ameloblasts. We have no explanation for this high SI labeling and why relatively weaker staining was observed by IHC. It is unlikely that the MMP-20 is synthesized locally only to be transferred to ameloblasts or to the mineralization front. It is possible, however, that MMP-20 at this location contributes to the polarized organization of the enamel organ by the cleavage of laterally secreted enamel proteins that might otherwise accumulate in intercellular spaces (Ruch et al. 1975; Matsuo et al. 1988,1990).
The molecular forms of MMP-20 in the rat incisor were also investigated by casein zymography and immunoblotting. Zymographic analysis of extracts prepared in the presence of protease inhibitors revealed the 46- and 41-kD bands corresponding to the calculated active form and the 57-kD band to the proform of MMP-20, as was previously shown for human and bovine MMP-20 (Bartlett et al. 1996; Llano et al. 1997). We noted, however, that when different extracts were compared, although these three bands were consistently present the relative intensity of the latent vs active bands varied quite significantly. This may be due to the fact that the forming part of the rat incisor represents multiple stages of tooth formation expressing different stages of MMP-20 activation. Interestingly, when extraction of the tooth protein was performed in the absence of proteinase inhibitors, the main lysis band was observed at 21 kD, indicating degradation of the proteinase during extraction. This may suggest that the smaller molecular weight species previously reported (Moradian–Oldak et al. 2001) are not physiological but are processed during the extraction procedure, although it is not excluded that they can be, under other circumstances, also produced naturally.
The higher molecular weight 78-kD band revealed by both immunoblotting and zymograms was also stained with the TIMP-2 antibody but not with TIMP-1, and its molecular weight, as well as its resistance to proteolysis, may indicate a complex proMMP-20/TIMP-2. Such MMP/TIMP complexes that do not dissociate under SDS-PAGE conditions have been previously described (Kolkenbrock et al. 1995; Kossakowska et al. 1998). It might resemble the specific interaction of the proMMP-2 with TIMP-2 via the C-terminal end of proMMP-2, an interaction that is a prerequisite for the activation of MMP-2 by MT1–MMP (Strongin et al. 1995). It is interesting, in this respect, that proMMP-20, like proMMP-2, is also activated by MT1–MMP (Palosaari et al. 2002). However, further work will be necessary to determine the exact nature of the interaction of TIMP-2 with MMP -20.
Although the significance of MMP-20 and amelogenin in odontoblasts, but not in dentin, remains to be determined, its transient expression during cell maturation, as well as its intracellular location not linked to the classical secretion pathway, may reinforce the additional role previously proposed for MMP-20 in signaling and therefore in the crucial event of epithelo–mesenchymal interaction.
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Received for publication July 1, 2003; accepted December 17, 2003
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  L. F. Massa, V. Bradaschia-Correa, and V. E. Arana-Chavez Immunocytochemical Study of Amelogenin Deposition during the Early Odontogenesis of Molars in Alendronate-treated Newborn Rats J. Histochem. Cytochem., June 1, 2006; 54(6): 713 - 725. [Abstract] [Full Text] [PDF]
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 C. Chaussain-Miller, F. Fioretti, M. Goldberg, and S. Menashi The Role of Matrix Metalloproteinases (MMPs) in Human Caries J. Dent. Res., January 1, 2006; 85(1): 22 - 32. [Abstract] [Full Text] [PDF]
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