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Matrix Gla Protein Inhibition of Tooth Mineralization
N.R. Kaipatur1,
M. Murshed1,2 and
M.D. McKee1,3,*
1 Faculty of Dentistry, McGill University, 3640 University Street, Montreal, QC, Canada H3A 2B2;
2 Department of Medicine, McGill University, Montreal, QC, Canada; and
3 Department of Anatomy and Cell Biology, McGill University, QC, Montreal
Correspondence: * corresponding author, marc.mckee{at}mcgill.ca
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ABSTRACT
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Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice, using an osteoblast/odontoblast-specific 2.3-kb Col1a1 promoter. Mandibles were analyzed by radiography, micro-computed tomography, light microscopy, histomorphometry, and transmission electron microscopy. While bone and tooth ECMs were established in the Col1a1-Mgp mice, extensive hypomineralization was observed, with values of unmineralized ECM from four- to eight-fold higher in dentin and alveolar bone when compared with that in wild-type tissues. Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors.
Key Words: matrix Gla protein • biomineralization • bone • dentin • cementum
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INTRODUCTION
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Mineralization of bone and tooth extracellular matrix (ECM) is a physiologic process regulated by mineral ion availability, proteins, and other molecular determinants (Boskey, 1981; Glimcher, 1998). In contrast, soft-tissue mineralization, also known as ectopic calcification, is a pathologic condition, commonly observed in many diseases, that impedes normal tissue function (Parfitt, 1969; Renie et al., 1984; Witteman et al., 1986; Hamerman, 1989; Giachelli, 2001).
Mineralization of skeletal and dental ECMs typically depends on homeostasis of calcium and inorganic phosphate mineral ions, the presence of a suitable protein scaffold, and the absence (or removal) of mineralization inhibitors (Boskey, 1981; Glimcher, 1998; Murshed et al., 2005). In healthy individuals, soft tissues do not normally mineralize, partly because they lack an abundant scaffolding ECM and consist mostly of cells (e.g., glands), or, where ECM is present, because potent mineralization-inhibiting molecules may be present in the tissue (e.g., skin, blood vessels) (Schinke et al., 1999).
In recent years, molecular determinants regulating mineralization have been identified. In addition to the SIBLING family members (Small Integrin-binding Ligand and N-linked Glycoproteins) (Fisher and Fedarko, 2003), the ubiquitous small-molecule inhibitor pyrophosphate and matrix Gla protein (MGP) are potent inhibitors of mineralization (Fleisch and Bisaz, 1962; Price, 1989; Luo et al., 1997; Ho et al., 2000; Addison et al., 2007). Gene-targeting experiments ablating Mgp in mice and pharmacologic treatments of rats to inhibit post-translational  -carboxylation of MGP, thus rendering it nonfunctional, have identified MGP as the essential (non-redundant) inhibitor of mineralization residing in the ECM of arteries and cartilage (Luo et al., 1995, 1997; Price et al., 1998; Howe and Webster, 2000).
MGP is a 14-kDa ECM protein which, in mice, contains 4 -carboxylated glutamic acid (Gla) residues (Price et al., 1983; Luo et al., 1995). Although originally isolated from bone, Mgp is not significantly expressed by osteoblasts in vivo, and the purification of MGP from skeletal tissues likely resulted from cartilage and blood vessels contained therein (Luo et al., 1995). Consistent with the high expression of Mgp in vascular smooth-muscle cells and in chondrocytes, Mgp-deficient mice show two major phenotypic abnormalities: extensive mineralization of the ECM in arteries, and premature mineralization of cartilage (Luo et al., 1997). These phenotypes are fully penetrant, and establish unambiguously that MGP functions as a potent mineralization inhibitor in blood vessels and cartilage. In humans, inactivating MGP mutations cause Keutel syndrome, where persons show chondrodysplasia and ectopic cartilage mineralization (Munroe et al., 1999).
To continue understanding how proteins regulate mineralization in bones and teeth, we generated transgenic mice using a 2.3-kb proximal Col1a1 promoter fragment (Rossert et al., 1995) to express Mgp (Col1a1-Mgp) specifically in osteoblasts, odontoblasts, and cementoblasts (Murshed et al., 2004). We reported previously that these mice show an inhibition of mineralization in bone (Murshed et al., 2004). Here we present an analysis of how targeted ectopic expression of Mgp affects tooth and alveolar bone mineralization.
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MATERIALS & METHODS
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Animals and Tissue Processing
Generation and screening of Col1a1-Mgp founders have been described previously (Murshed et al., 2004), and were approved by the Animal Use Committee of Baylor College of Medicine. Transgenic mice, and wild-type littermates, were killed at 1–12 wks of age. Mandibles were immersion-fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer. For immunohistochemistry, samples were fixed in 4% paraformaldehyde and decalcified with 8% EDTA.
Whole-mount Skeletal Preparation Staining
To visualize 2.3-kb Col1a1 promoter activity, we used Col1a1-lacZ mice as described previously (Rossert et al., 1995). Transgenic skeletons were fixed in cold 4% paraformaldehyde in PBS, and stained with X-gal (1 mg/mL) in buffer (100 mM HEPES, 5 mM DTT, 1 mM MgSO4 and 2% Triton) containing 4 mM potassium ferrocyanide and 5 mM potassium ferricyanide.
Immunohistochemistry
Paraffin sections of the mandibles were processed for immunohistochemistry with mouse anti-human MGP mono-clonal 3–15 (VitaK Inc., Maastricht, the Netherlands), followed by secondary antibody and a colorimetric detection kit (Vector Laboratories Inc., Burlingame, CA, USA). Controls consisted of omission of primary antibody, and incubations with unrelated immunoglobulins.
Radiography
High-resolution radiography images were taken under identical conditions by means of a Faxitron Model MX-20 (Faxitron X-ray Corporation, Wheeling, IL, USA). Digital images were recorded at 26 kV and 0.3 mA over a five-second exposure.
Micro-computed Tomography (microCT)
To analyze and quantify bone and tooth mineralization, we used an x-ray microtomograph (SkyScan1072, Kontich, Belgium) to scan 3 hemimandibles of each genotype for 30 min at 1.9 sec per scan (100 kV, 98 µA). Analyses were standardized at a lower grey threshold setting of 65, and an upper grey threshold setting of 255, established first for the wild-type hemimandibles and then applied to all samples. The regions quantified were equivalent between samples, such that 1000 x-ray slices, with increments of 15 µm, covered the entire hemimandible. Percentage mineralized tissue volume was determined for each hemimandible, and three-dimensional reconstructions were produced with the manufacturer’s software (3D-Creator).
Light Microscopy and von Kossa Staining
For histology, mandibles were dehydrated and embedded in LR White resin (Mecalab, Montreal, QC, Canada). Sections were stained with 3% silver nitrate (von Kossa reagent) coupled with exposure to light for mineral localization; counterstaining was with toluidine blue. Micrographs were taken with an optical microscope (model Leitz DMRBE, Leica, Wetzlar, Germany), a CCD camera, and Northern Eclipse software (v6.0, Empix Imaging, Mississauga, ON, Canada).
Histomorphometry
For histomorphometry, images from von Kossa- and toluidine-blue-stained sections were obtained digitally as above for each animal age (N = 3), and for bone, analyzed for area of osteoid ECM and area of mineralized bone ECM in both wild-type and Col1a1-Mgp mice by means of the imaging software. Briefly, bone ECM was manually outlined, and threshold levels were set to capture mineralized tissue regions; osteoid area was also measured. For teeth, percentage mineralized dentin in the lower incisor was similarly determined, with data being subdivided for root analogue dentin and crown analogue dentin.
Electron Microscopy
Ultrastructural observations were made by transmission electron microscopy (TEM) after grid-mounted sections were stained with tannic acid and uranyl acetate. Electron micrographs were recorded by means of a JEOL JEM-2000FX TEM operating at 80 kV and equipped with a Gatan 792 Bioscan Multiscan CCD camera (JEOL, Peabody, MA, USA).
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RESULTS
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Transgene Expression, Radiography, and microCT
X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) staining of skeletal preparations demonstrated the activity of the 2.3 kb Col1a1 promoter driving bacterial β-galactosidase in mandibular tissues (Fig. 1A ). Immunohistochemistry for MGP in dentin (Fig. 1B) and alveolar bone (Fig. 1C) confirmed the presence of the protein in Col1a1-Mgp mandibles, whereas MGP was not localized in the dentin (Fig. 1D) or bone (data not shown) of wild-type mice. Radiological and microCT comparisons of three-week-old wild-type and Col1a1-Mgp hemimandibles indicated a marked reduction of mineralized tissues—both teeth and bone—in the transgenic samples (Figs. 1E–1J). MicroCT images of the Col1a1-Mgp hemimandibles revealed a complete lack of both molar root and incisor root analogue mineralization (Figs. 1F,1I,1J), and decreased alveolar bone mineralization (Fig. 1F). Quantitative microCT of mineralized tissue volume (Fig. 2A) and relative mineralized tissue volume (Fig. 2B) showed decreased mineralization in the transgenic samples.
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Figure 1. Transgene promoter expression, MGP localization, and radiography and micro-computed tomography (microCT) of transgenic and wild-type mice. (A) X-gal staining showing the activity of the 2.3-kb Col1a1 promoter driving bacterial β-galactosidase (lacZ) expression in the mandible (oval outline) of a four-day-old Col1a1-LacZ mouse. (B–D) Immunohistochemistry showing MGP localization after transgene expression in three-week-old Col1a1-Mgp incisor root analogue dentin (asterisk, B) and surrounding alveolar bone (asterisk, C); no MGP is found in wild-type dentin (D) or alveolar bone. (E,F) Radiographs (insets) of three-week-old hemimandibles from wild-type and Col1a1-Mgp mice demonstrate overall increased radiolucency (hypomineralization) in the tooth root area and in the mandibular bone of the transgenic mice. MicroCT reconstructions along the mid-sagittal plane of the incisor in wild-type and transgenic hemimandibles confirm the complete lack of mineralized molar roots (white arrow) and incisor root analogue (black arrow), and show an increase in bone porosity in the alveolar bone, from decreased mineralization in the Col1a1-Mgp samples. (G–J) Reconstructed and single-slice cross-sectional microCT profiles at the level of the first molar similarly demonstrate a lack of mineralization of the tooth roots (molar, white arrows; incisor, black arrows) and surrounding alveolar bone in the Col1a1-Mgp mice. M1, first molar; M2, second molar; R, roots of molars; RA, root analogue of incisor; CA, crown analogue of incisor; I, incisor. Magnification bars equal 50 µm. (This Fig. is available in color online.)
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Figure 2. Total (A) and relative (B) mineralized tissue volumes obtained by quantitative microCT from hemimandibles of wild-type and Col1a1-Mgp mice ranging in age from 1–12 wks. Significant decreases in Col1a1-Mgp vs. wild-type mineralized tissue volumes are observed at most of the ages examined. MTV, mineralized tissue volume; TV, total tissue volume. N = 3 for each genotype. Data are shown as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.
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Light Microscopy and Histomorphometry
To confirm the x-ray-based analyses showing decreased mineralization in the Col1a1-Mgp hemimandibles, we examined undecalcified tissue samples by light microscopy after von Kossa staining for mineral. Unlike the fully mineralized teeth and bone in wild-type hemimandibles (Figs. 3A–3C), the Col1a1-Mgp tissues showed profound hypomineralization, apparent as increased osteoid (osteoidosis) in the alveolar bone, and unmineralized molar root dentin and unmineralized incisor root analogue dentin (Figs. 3D–3F). Crown dentin was less affected, and mineralized enamel formed only when there was mineralized crown dentin; where present, enamel structure was significantly altered (data not shown). Unmineralized osteodentin was frequently observed in the pulp chambers of molar roots (Fig. 3F), and abundant cellular cementum that was not mineralized accumulated at the apical tips of molar roots (see below). Histomorphometric analyses of incisor crown and root analogue dentin, and alveolar bone, in cross-sections of the hemimandibles at the level of the first molar (ages as indicated) all showed significantly decreased mineralization in the transgenic hemimandibles compared with age-matched littermates (Figs. 3G–3I).
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Figure 3. Light micrographs of von Kossa-stained (black areas, mineral) sections from undecalcified hemimandibles, and histomorphometry, of wild-type and Col1a1-Mgp mice. Wild-type incisors and bone at 1 wk (A) and 3 wks (B) of age, and wild-type first molar at 3 wks of age (C), showing well-mineralized tooth and bone extracellular matrices. Col1a1-Mgp incisors and bone at 1 wk (D) and 3 wks (E) of age, and Col1a1-Mgp first molar at 3 wks of age (F), showing well-developed tooth and bone extracellular matrices, but with an absence of, or substantial decreases in, mineralization. Molar root dentin, incisor root analogue dentin, and alveolar bone were all particularly hypomineralized, whereas molar crown dentin and incisor crown analogue dentin typically were less affected and showed breakthrough dentin mineralization, which interfaced with mineralized enamel. Osteodentin was also sometimes present in the pulp chambers of the Col1a1-Mgp molars. Histomorphometry of incisor root analogue (G) and crown analogue (H) dentin from wild-type and Col1a1-Mgp mice of different ages showing significantly higher amounts of unmineralized dentin in the transgenic mice. (I) Similarly, unmineralized osteoid in Col1a1-Mgp was substantially increased compared with that in wild-type littermates. En, enamel; Den, dentin; Os-Den, osteodentin. Magnification bars equal 100 µm. (G–I) N = 3 for each genotype. Data are shown as the mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001. (This Fig. is available in color online.)
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Tooth and Bone Ultrastructure
Ultrastructural morphology by TEM of the dentin in Col1a1-Mgp incisor teeth (undecalcified) revealed otherwise normal dentin structure, despite the absence of mineralization (Figs. 4A,4B). Most striking in this region was a complete absence of acellular cementum against the mantle dentin, and thus a well-developed attachment involving the periodontal ligament was lacking at this interface. Prominent within the root dentin ECM and visible at only higher magnifications were abundant, small mineralization foci (Fig. 4B, inset), but mineralization was restricted to these sites. In the abundant acellular cementum at the root tips (Figs. 4C,4D) of the Col1a1-Mgp molars, tissue structure seemed otherwise normal, but again, small mineralization foci were present throughout the ECM (Fig. 4D, inset). In Col1a1-Mgp alveolar bone, extensive areas of unmineralized osteoid were observed, within which were occasional regions of mineralized bone (Figs. 4E,4F). By TEM, abundant mineralization foci were likewise observed in the otherwise structurally normal osteoid (Fig. 4F, inset), but most striking was a layer of mineral at the mineralization front that was unique in density and texture relative to the adjacent mineralized bone (Fig. 4F).
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Figure 4. Light micrographs after von Kossa staining for mineral (black areas), and electron micrographs, of undecalcified three-week-old Col1a1-Mgp mandibular teeth and alveolar bone. Light micrographs in the left panels show regions selected for electron microscopy (box frame) as presented in the right panels. (A,B) Incisor dentin extracellular matrix and associated periodontal tissues show typical histology in terms of cellular and matrix distribution and organization, although acellular cementum was lacking at the root surface (asterisks). However, although there was a generalized absence of mineralization in the dentin, small mineralization foci were frequently observed by TEM throughout the matrix (inset). (C,D) In molars, as for dentin, the extracellular matrix of cellular cementum was generally unmineralized, and showed similar small mineralization foci dispersed throughout the matrix (inset). Osteodentin, which, like alveolar bone, showed variable levels of mineralization (but in this sample is not mineralized), was occasionally observed within the pulp. (E,F) Alveolar bone, while having profound osteoidosis, also had many areas of extensive, but incomplete, mineralization. Shown here are large unmineralized areas, and thickened osteoid seams. At the mineralization front adjacent to the thickened osteoid, an unusual band of mineral texture was observed (arrows) adjacent to the mineralized matrix proper. Again, small mineralization foci were distributed throughout the otherwise unmineralized osteoid (inset). Den, dentin; PDL, periodontal ligament; Od, odontoblast; Os-Den, osteodentin; C-Cem, cellular cementum; Ob, osteoblast. Magnification bars equal 50 µm (A,C,E), 5 µm (B,D,F), and 50 nm (insets).
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DISCUSSION
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Recent developments in molecular genetics and the use of genetically modified mouse models recapitulating human disease provide valuable research tools for the understanding of mineralized tissue biology. For many human genetic disorders associated with skeletal and dental abnormalities, which typically include either soft (hypomineralized) and/or brittle mineralized tissues—such as the dentin dysplasias, dentinogenesis imperfectas, and amelogenesis imperfectas—transgenic mouse models are now available (Gibson et al., 2001; Sreenath et al., 2003; Lopez Franco et al., 2005; Seedorf et al., 2007). These animal models have identified key genes involved in biologic mineralization, and their experimental use has provided important new insight into mechanisms underlying human disease.
Physiologic levels of MGP secreted by vascular smooth cells and chondrocytes inhibit mineralization of blood vessel and cartilage ECM (Luo et al., 1997). Given this mineral-inhibiting function, we generated a transgenic mouse line ectopically expressing the native Mgp gene in both bones and teeth (Murshed et al., 2004). The transgene was driven by a 2.3-kb proximal Col1a1 promoter known to be highly expressed in differentiated osteoblasts, odontoblasts, and cementoblasts (Rossert et al., 1995). Mgp transgene expression and MGP protein secretion in endochondral and intramembranous (cranial) bones resulted in decreased mineralization and osteoidosis. During the later course of this study, we noted that mandibular and maxillary bone and teeth were highly affected in these transgenic mice, and thus we report here on the characterization of these tissues.
In the present study, our analysis of Col1a1-Mgp mice by the various imaging methods all revealed almost complete abrogation of mineralization in tooth root ECM, and significant inhibition of alveolar bone matrix mineralization. In both cases, the ECMs of dentin, cellular cementum, and bone all seemingly assembled into a well-ordered and structured matrix, but failed to mineralize in the presence of abundant MGP. While mineralization is widely thought to be regulated by the phophorylated SIBLING proteins (Fisher and Fedarko, 2003), there are no cases where mutations or deletions of these proteins have led to a complete blockade in skeletal and/or dental mineralization. Our findings most closely resemble those of Beertsen et al. and Takano et al. (Beertsen et al., 1985; Takano et al., 1998), who, after injections of the mineral-inhibiting bisphosphonate EtidronateTM (1-hydroxyethyl-idene-1,1-bisphos-phonate, HEBP), likewise observed these mineralization defects. Despite the overall blockade in mineralization, there nevertheless is evidence that the mineralization process commences at certain sites within the ECM, appearing as small mineralization foci (containing many apatitic crystallites) among the collagen fibrils in the various bone and tooth ECMs. This observation indicates that initial mineral nucleation events remain possible even in the presence of very high levels of inhibitory MGP; however, once nucleated, mineralization does not propagate beyond these small foci. Eventually, however, significant mineralization does indeed occur in some locations, as seen by the limited regions of bone and tooth ECM that are extensively mineralized in the Col1a1-Mgp mice. Where this "breakthrough" mineralization has occurred, the unusually textured layer of mineral found at the mineralization front at the edge of the osteoid in the bone likely reflects the atypical events required to overcome this inhibition by MGP.
Surprisingly, as reported here for MGP and in the studies by Beertsen et al. and Takano et al. (Beertsen et al., 1985; Takano et al., 1998) using HEBP bisphosphonate, molar crown dentin and incisor root-analogue dentin showed delayed, but breakthrough, mineralization, with mineralized enamel interfacing with the mineralized dentin at these sites. This site-specific difference in dentin mineralization (i.e., crown vs. root dentin) is consistent with findings of differences in mineral-regulating, non-collagenous phosphoprotein distribution, abundance, and secretion rates at these two sites (Beertsen and Niehof, 1986; Takagi et al., 1988). Alternatively, these differences in mineralization may be attributable to differential Mgp gene expression and protein secretion levels in the crown odontoblasts vs. the root odontoblasts. Also suggested by these findings is that enamel mineralization may require induction by mineralization of dentin, a concept discussed many years ago (Arsenault and Robinson, 1989) and supported by our present study and the previous bisphosphonate inhibition work.
The absence of mineralized acellular cementum in Col1a1-Mgp mice teeth is also noteworthy, and indicates that a mineralized dentin substratum is required for ECM accumulation and mineralization characteristic of acellular cementogenesis. Both acellular cementum matrix and mineral were absent when root dentin was not mineralized, an observation likewise reported for teeth in HEBP-treated rats and in Akp2–/– (tissue-non-specific alkaline-phosphatase-deficient) mice (Beertsen et al., 1985, 1999; Waymire et al., 1995). Since apical cellular cementum assembled seemingly appropriately as an ECM with occluded cementocytes (but without mineralization) in molars of Col1a1-Mgp mice, a lack of acellular cementum more occlusally implies that mineralization within root mantle dentin is required for at least the initiation of acellular cementogenesis. These observations also suggest that mineralization occurring at the surface of mineralized root mantle dentin may precede the assembly of cementum proteins into an ECM.
Our present work has validated the use of the Col1a1-Mgp mouse as an animal model for the study of protein-based regulators of tooth (and bone) mineralization in vivo. The ability to inhibit mineralization of tooth and bone ECM by ectopic expression of a protein normally inhibiting soft-tissue (blood vessels and cartilage) mineralization underscores the likelihood that common mechanisms exist in the regulation of both physiologic mineralization and pathologic calcification—mechanisms that might also involve modulation of the mineral-regulating activities of one protein (including enzymes) by another. Importantly, our results from using this mouse model further strengthen the view that physiologic mineralization of bone and tooth ECMs is critically regulated by potent mineralization inhibitors.
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ACKNOWLEDGMENTS
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The authors thank G. Karsenty, T. Schinke, and S.-H. Lin for guidance and help with this work, and L. Malynowsky, J.-S. Binnette, and F. Al-Halabi for their skillful assistance. This work is funded by CIHR and NIH.
Received for publication November 16, 2007.
Revision received May 20, 2008.
Accepted for publication May 30, 2008.
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Journal of Dental Research, Vol. 87, No. 9,
839-844 (2008)
DOI: 10.1177/154405910808700907
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ABSTRACT
INTRODUCTION