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Periodontal Breakdown in the Dmp1 Null Mouse Model of Hypophosphatemic Rickets
L. Ye1,*,
S. Zhang1,
H. Ke2,
L. F. Bonewald1 and
JQ. Feng3,*
1 Oral Biology, School of Dentistry, University of Missouri-Kansas City, 650 E 25th Street, Kansas City, MO 64108, USA;
2 Amgen, Amgen Center Drive, Thousand Oaks, CA 91320, USA; and
3 Department of Biomedical Sciences, Baylor College of Dentistry, Dallas, TX 75246, USA
Correspondence: * corresponding author, yeling{at}umkc.edu or jfeng{at}bcd.tamhsc.edu
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ABSTRACT
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Dentin Matrix Protein 1 (DMP1) is highly expressed in alveolar bone and cementum, which are important components of the periodontium. Therefore, we hypothesized that Dmp1 is critical for the integrity of the periodontium, and that deletion may lead to increased susceptibility to disease. An early-onset periodontal defect was observed in the Dmp1 null mouse, a mouse model of hypophosphatemic rickets. The alveolar bone is porous, with increased proteoglycan expression. The cementum is also defective, as characterized by irregular, punctate fluorochrome labeling and elevated proteoglycan. The osteocyte and cementocyte lacuno-canalicular system of both alveolar bone and cementum is abnormal, with irregular lacunar walls and fewer canaliculi. As a consequence, there is significant interproximal alveolar bone loss, combined with detachment between the periodontal ligament (PDL) and cementum. We propose that defective alveolar bone and cementum may account for the periodontal breakdown and increased susceptibility to bacterial infection in Dmp1 null mice.
Key Words: DMP1 • PDL • cementum • hypophosphatemic rickets • alveolar bone
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INTRODUCTION
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Hypophosphatemic rickets is characterized by osteomalacia, resulting in limb deformities due to phosphate wasting. Recently, we have shown that mutation or deletion ofleads to hypophosphatemic rickets (Feng et al., 2006). Persons with hypophosphatemic rickets have enlarged pulp chambers and increased predentin width (Goodman et al., 1998; Murayama et al., 2000; Pereira et al., 2004), consistent with what we have described previously in the Dmp1 null mouse (Ye et al., 2004). Immature calcospherites have been described in the dentin of hypophosphatemic rickets, suggesting delayed mineralization (Boukpessi et al., 2006).
Dentin Matrix Protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, was first isolated from dentin (George et al., 1993), and later from bone, cartilage, and cementum (MacDougall et al., 1998; Butler et al., 2002; Feng et al., 2002; Toyosawa et al., 2004). Although DMP1 is expressed in all mineralized tissues, the highest expression appears in osteocytes (Toyosawa et al., 2004). Recently, broader but much lower DMP1 expression has been reported in non-mineralized tissue, such as brain, kidney, and pancreas (Terasawa et al., 2004). DMP1 may function to induce differentiation of mesenchymal cells to odontoblast-like cells, thereby enhancing mineralization (Narayanan et al., 2001), and by functioning as a transcription factor to target the nucleus and activate osteoblast-specific genes (Narayanan et al., 2003).
We originally generated Dmp1 null mice to study the function of this gene (Feng et al., 2003, 2006; Ye et al., 2004, 2005). The null mice are normal at birth, but post-natally develop a profound tooth phenotype, such as enlarged pulp chambers and increased width of the predentin zone with reduced dentin thickness (Ye et al., 2004). Since DMP1 is highly expressed in cementum and alveolar bone, we sought to determine if deletion of this protein in these tissues is responsible for any periodontal defects. Our hypothesis was that DMP1 is critical for the integrity of alveolar bone and cementum, and that deletion of DMP1 in these tissues would lead to increased susceptibility to disease.
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MATERIALS & METHODS
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Animals
Mice deficient in Dmp1 were generated as described previously (Feng et al., 2003). To obtain Dmp1 null mice, we interbred Dmp1 heterozygous mice to generate homozygotes in the C57BL/6 background. In this paper, wild-type mice are also designated as WT; Dmp1 null mice are designated as KO. The animal protocol was approved by Institutional Animal Care and Use Committees at the University of Missouri-Kansas City.
Radiography and Micro-computed Tomography (Micro-CT)
Mandibles were examined on a Faxitron MX-20 Radiography System (Faxitron X-ray Corp., Buffalo Grove, IL, USA). Three-dimensional images of 12-month mandibles from WT and Dmp1 null mice were scanned with Micro-CT. Images were reconstructed with EVS Beam software with a global threshold at 1400 Hounsfield Units.
Resin Infiltrating and Acid-etching
To study the canalicular system of osteocytes, we polished the surface of methylmethacrylate-infiltrated alveolar bone. The surface was then acid-etched with 37% phosphoric acid for 2–10 sec and 5% sodium hypochlorite for 5 min, then coated with gold and palladium, and examined by means of a FEI/Philips XL30 Field Emission Environmental Scanning Electron Microscope (SEM) (Hillsboro, OR, USA) (Martin et al., 1978; Feng et al., 2006).
Histology and Histomorphometric Analysis
Mandibles from WT and Dmp1 null mice were fixed in 4% paraformaldehyde, followed by decalcification in 10% EDTA solution for 3 wks. Sections were stained with hematoxylin & eosin (H&E). Histomorphometric analysis was carried out with the Image Analysis System (AnalySIS, Lakewood, CO, USA). Three sections, at least 24 µm apart, were examined from each animal (5 animals in each group). Alveolar bone area and bone width were measured as reported (Barnett and Rowe, 1986; Nociti et al., 2001). Specifically, the alveolar bone area is defined as the interproximal bone area above the straight line connecting the root apices of the first and second mandibular molars. The alveolar bone width is measured at the coronal, middle, and apical thirds of interproximal bone between the first and second molars, respectively, and the mean value is used as the actual bone width. All results were expressed as mean ± standard deviation (SD).
Fluorochrome Labeling of Cementum
To analyze cementum formation rate, we injected mice 5 days apart with calcein (5 mg/kg, Sigma-Aldrich, St. Louis, MO, USA; intraperitoneal injection, i.p.), alizarin red (20 mg/kg, Sigma-Aldrich, i.p.), and again with calcein. The animals were killed 2 days after the final injection.
Immunohistochemistry
Immunostaining was performed as described previously (Ye et al., 2004), with rabbit polyclonal antibodies for DMP1 (Larmas et al., 2008), biglycan and decorin (gifts from Dr. Larry Fisher, NIDCR, Bethesda, MD, USA). We obtained negative controls by replacing the primary antibody with rabbit serum (Appendix Fig. 1 ).
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Figure 1. Dmp1 null mice develop early periodontal breakdown. (A) Histological pictures of interproximal areas between the first and second lower molars at different stages (3 wks, 3 and 6 mos). At 3 wks of age, the periodontal attachment is intact in the Dmp1 null mice. At 3 mos, the alveolar bone defect becomes evident, accompanied by detachment between the PDL and cementum, compared with the healthy periodontal tissue in WT control mice. At 6 mos, the periodontal defect becomes more severe in the KO mice. (B) Quantitative analyses of alveolar bone width and alveolar bone area between the first and second molars from ages of 3 wks to 3 mos, showing decreases with age. +/+, WT; –/–, KO; n = 5; *P < 0.05; **P < 0.01, data represent mean ± SD.(AQ)
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Statistical Analysis
Data from the histomorphometric analysis were analyzed by Student’s t test. P < 0.05 was considered statistically significant.
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RESULTS
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Dmp1 Null Mice Develop Early-onset Periodontal Breakdown
Unlike humans, mice do not naturally develop periodontal disease until extreme old age, usually older than 1 yr, and mainly in the maxilla (Page and Schroeder, 1982). In contrast, Dmp1 null mice develop an early-onset periodontal defect (Fig. 1A ). At the age of 3 wks, the Dmp1 null alveolar bone appears grossly normal in shape, although the bone is woven and immature in nature. However, at ages 3 and 6 mos, there is a severe bone defect, combined with periodontal ligament (PDL)/cementum detachment in Dmp1 null mice (Fig. 1A, lower panels). Quantitative histomorphometric analysis shows significant decrease of bone in KO mice at 3 mos, but not at 3 wks (Fig. 1B). There is no difference in bone height between WT and Dmp1 null mice (data not shown), suggesting that the bone defect in Dmp1 null interproximal bone is mainly vertical.
Dmp1 Null Mice Display Porous, Hypomineralized, Immature Alveolar Bone
Although signs of dentin defects, as reflected by enlarged pulp and thin dentin, were obvious at the age of 3 wks, the bone between the first and second molars appeared comparable between the WT and Dmp1 null mice (Fig. 2A, left lower panel, black arrows). By the age of 3 mos, the Dmp1 null alveolar bone appeared porous compared with that of the age-matched control (Fig. 2A, middle lower panel, black arrow). At the age of 6 mos, the bone defect was more striking (Fig. 2A, right lower panel, white arrow), and by the age of 12 mos, the bone became extremely porous, as determined by Micro-CT. Also note the misalignment of teeth due to severe bone loss. Histological sections from 3-week and 3-month Dmp1 null alveolar bone reveal immature woven bone, with more osteoblasts and bone marrow spaces, suggesting that DMP1 is required for maturation of woven bone into lamellar bone (Appendix Fig. 2).
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Figure 2. Dmp1 null mice have defective alveolar bone. (A) Representative radiographs of mandibles from three-week-, three-month-, and six-month-old mice (WT in upper panels, and KO in lower panels). The porous alveolar bone in the null animals is evident by the ages of 3 and 6 mos (arrows). (B) Representative micro-CT reconstructions of mandibles from 12-month-old animals. Note that there is severe bone loss in KO mice.
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Increased Proteoglycans and Irregular Osteocyte Lacuno-canalicular System in Alveolar Bone
Biglycan and decorin are members of a small leucine-rich proteoglycan family. Elevated biglycan and decorin lead to mineralization defects in the dentin sialophosphoprotein null tooth (Sreenath et al., 2003), and elevated biglycan protein expression is also observed in the Dmp1 null predentin (Ye et al., 2004). To address whether there are any changes in biglycan or decorin expression in the Dmp1 null alveolar bone, we used polyclonal antibodies to mouse biglycan or decorin (kind gifts from Dr. Larry Fisher, NIDCR) for immunostaining of the interproximal bone in 3-week- and 3-month-old mice. Dramatic increases in biglycan and decorin immunostaining were observed in the Dmp1 null alveolar bone (Fig. 3A, lower panels, white asterisks), suggesting either increased accumulation of these extracellular matrix proteins or greater exposure due to hypomineralization.
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Figure 3. Elevated proteoglycan expression and defective lacuno-canalicular morphology in Dmp1 null alveolar bone. (A) Immunostaining for biglycan and decorin on sections of alveolar bones from three-week- and three-month-old animals (WT in the upper panels, and KO in the lower panels). The increased staining of biglycan and decorin is evident (white asterisks). (B) SEM images of resin-infiltrated, acid-etched alveolar bone from three-week-old WT (left panels) and KO mice (right panels). Osteocyte lacunae were filled with resin, and the mineral was removed by 37% phosphoric acid and 5% sodium hypochlorite. The smooth canalicular walls from a representative lacuna are indicated with red arrows (lower left panel), while rough canalicular walls from a lacuna in KO bone are indicated by arrowheads (lower right panel). Note that the WT osteocyte lacuno-canalicular system is evenly distributed, but the KO osteocyte lacuno-canalicular system is disorganized, and lacunae are clustered. There is also a large amount of osteoid present in the matrix.
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Previously, we had shown a retardation of maturation of Dmp1 null osteoblasts into osteocytes (Feng et al., 2006). For further study of the morphological changes of the osteocyte lacuno-canalicular system in the Dmp1 null alveolar bone, resin-infiltrated sections were acid-etched to reveal a three-dimensional relief of the osteocyte lacuno-canalicular system, by two-dimensional SEM. Images showed that the WT osteocyte lacunae were highly organized and spaced regularly, generally in linear arrays. The canaliculi were generally straight and ran perpendicular to the long axis of the osteocyte, with numerous canaliculi with smooth walls (Fig. 3B, left panels). In contrast, the Dmp1 null osteocyte lacunae appeared to have rough walls, and were larger in size, with fewer canaliculi. The distribution of the lacunae appeared less organized and more clustered, while their canaliculi were less straight and smooth (Fig. 3B, right panels). The abnormal morphology of lacunae in the Dmp1 null mice suggests that DMP1 plays a role in the formation of the lacuno-canalicular system.
Abnormal Cementum and PDL in Dmp1 Null Mice
When the cementum and PDL were compared between Dmp1 null and WT mice by H&E staining, it was noted that the PDL cells were irregularly shaped. In the WT mice, the cuboid-shaped cementoblasts (Fig. 4A, left panel, red arrows) were regularly aligned on the surface of cementum, and a clear acellular cementum layer was present (Fig. 4A, left panel). In contrast, the acellular cementum layer in Dmp1 null mice was much thinner and more difficult to recognize. Second, there were few Dmp1 null cementoblasts, and they were no longer cuboid, but spindle-shaped (Fig. 4A, right panel). Third, the WT PDL layer was composed of different cell types, whereas the KO PDL layer was composed of fewer and spindle-shaped fibroblasts.
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Figure 4. Cementum and PDL in the Dmp1 null mouse are malformed. (A) H&E staining of section containing cementum, cementoblasts, PDL. The cuboidal cementoblasts aligned along the surface of the cementum in the WT (left panel), as indicated by the red arrows, are absent in the KO (right panel). Note that the cells in the WT PDL are mixed, with few spindle-like shapes (left panel), whereas the majority of the KO PDL cells are spindle-shaped (right panel). (B) Immunostaining of a one-month-old mandible for DMP1. Brown signal for DMP1 immunostaining in the acellular layer is indicated by a red arrowhead, and in the cellular layer by a red arrow. (C) Immunostaining with polyclonal antibodies against biglycan and decorin in cellular cementum. The increased expression of biglycan and decorin in KO cellular cementum is indicated by white asterisks in both ages of 3 wks and 3 mos.
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Previously, using Dmp1-lacZ knock-in mice, we reported that DMP1 is expressed in cementum (Feng et al., 2003). Using immunostaining for DMP1, we showed that the protein was also expressed in both acellular (red arrowhead) and cellular cementum (red arrow), as well as in alveolar bone (Fig. 4B, brown). Similar to alveolar bone, increases in biglycan and decorin were observed in the cellular cementum (Fig. 4C, lower panels, white asterisks). These proteoglycans may potentially cause or be the result of the defective process.
Next, the cementocyte lacunar morphology was compared between the WT and KO, by resin-infiltrating and acid-etching techniques. Similar to the abnormal KO osteocyte lacunae, the Dmp1 null cementocyte lacuna is rough on the lacunar wall, with few dendrites (Appendix Fig. 3). To evaluate the change of cementum formation rate, we performed fluorochrome injections and showed distinct labeling lines in WT cementum. In contrast, the labeling lines in Dmp1 null mice were sparse and diffuse (Appendix Fig. 3, white arrow), suggesting defective calcospherite maturation in the KO mice.
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DISCUSSION
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We have reported that Dmp1 null mice post-natally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, and increased width of the predentin zone with a reduced dentin wall (Ye et al., 2004). In this study, we show that Dmp1 null mice develop severe alveolar bone and cementum defects, which may explain the early periodontal breakdown in these mice. DMP1 is highly expressed in both alveolar bone and cementum. Loss of Dmp1 results in alveolar bone that is porous and immature, with a large amount of osteoid similar to that shown in bone of the appendicular skeleton (Feng et al., 2006). Cementum, a similar bony tissue, shows abnormal mineralization, with immature calcospherites as shown in bone (Feng et al., 2006) and in the dentin of hypophosphatemic individuals (Boukpessi et al., 2006).
Elevated protein expression of both biglycan and decorin was observed in both alveolar bone and cementum in the Dmp1 null mice. Biglycan and decorin are small leucine-rich proteoglycans, produced by osteoblasts and odontoblasts, and lack of these proteoglycans has been associated with defective mineralization in dentin (Sreenath et al., 2003). Decorin is also abundant in unmineralized bone matrices in the embryonic rat mandible, but could not be detected in mineralized matrices, suggesting that decreased proteoglycan is associated with normal matrix mineralization (Kamiya et al., 2001). In vitro studies have shown that recombinant decorin and biglycan can inhibit hydroxyapatite-induced crystal growth (Boskey et al., 1997; Sugars et al., 2003). Elevated expression of these proteoglycans in the matrix from Dmp1 null mice may be partially responsible for defective mineralization in alveolar bone and cementum.
Although bacteria play an important role in the development of periodontitis in the clinical patient, this does not appear to be the case in the Dmp1 null mice. Significant vertical bone defect is evident in the Dmp1 null mice as early as 3 mos, without any clear evidence of bacterial infection or inflammatory response. With increasing age (up to 12 mos; Appendix Fig. 4), Dmp1 null mice are more prone to bacterial infection, most likely due to alveolar bone defect and detachment between the PDL and cementum. Therefore, bacteria may not play a major role in the formation of the periodontal defects in Dmp1 null mice. However, to confirm this hypothesis, future experiments using antibiotics or a bacteria-free environment are planned.
It is of note that PDL cells in the Dmp1 null mouse have an abnormal spindle shape. We do not know if this is caused by an intrinsic defect in the PDL cells and/or is secondary to the defects in bone and cementum. However, immunostaining of tissue from the null mice did not reveal detectable expression of DMP1 in PDL cells, whereas significant DMP1 expression was observed in alveolar bone and cementum. Second, the Dmp1 null mice showed comparable alveolar bone and cementum at 3 wks, suggesting that PDLs in the Dmp1 null mouse still have osteogenic or cementogenic capability; therefore, we propose that their abnormal spindle shape may be secondary to the bone and cementum defects. Further in vitro primary PDL cell culture experimentation is needed to answer this question.
DMP1 expression increases selectively after mechanical loading in the tooth movement model (Gluhak-Heinrich et al., 2003) and in the ulna of loaded mice (Yang et al., 2005). Loading of bone in the Dmp1 null mouse produces more strain than in wild-type or heterozygous littermates under identical mechanical loads (Rios et al., 2005). These observations support the concept that DMP1 may be an important factor in the regulation of the osteocyte response to load. Both osteocyte and cementocyte lacunae were abnormal in these mice, and the canaliculi were reduced in number, suggesting abnormal bone fluid flow through these systems. Therefore, ablation of DMP1 may lead to pathological response to physiological mechanical loading, such as mastication. Overloading of the alveolar bone may also generate excessive microdamage, leading to greater bone loss (Frost, 1992, 1994). This, combined with the lack of support by the defective alveolar bone and cementum, may exacerbate bony defects.
In summary, we report severe alveolar bone and cementum defects in Dmp1 null mice most likely responsible for periodontal breakdown, especially in the interproximal area between molars, which worsens with age. Although these defects are distinct from clinical periodontitis, analysis of our data suggests that, without proper phosphate treatment and dental care, hypophosphatemic individuals may be predisposed to bacterial infection.
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ACKNOWLEDGMENTS
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This research is supported by NIDCR grants DE016977 and DE07294, and by NIAMS grants AR51587 and AR46798.
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/7/624/DC1.
Received for publication January 10, 2008.
Revision received April 20, 2008.
Accepted for publication May 7, 2008.
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Journal of Dental Research, Vol. 87, No. 7,
624-629 (2008)
DOI: 10.1177/154405910808700708
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ABSTRACT
INTRODUCTION