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J Dent Res 84(11):1021-1025, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Cementum and Dentin in Hypophosphatasia

T. van den Bos1, G. Handoko1, A. Niehof1, L.M. Ryan2, S.P. Coburn3, M.P. Whyte4, and W. Beertsen1,*

1 Department of Periodontology, Academic Center for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam, and Vrije Universiteit, Louwesweg 1, 1066 EA Amsterdam, The Netherlands;
2 Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA;
3 Department of Chemistry, Indiana University-Purdue University, Fort Wayne, IN, USA; and
4 Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, MO, USA;

* corresponding author, W.Beertsen{at}acta.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypophosphatasia (HPP) often leads to premature loss of deciduous teeth, due to disturbed cementum formation. We addressed the question to what extent cementum and dentin are similarly affected. To this end, we compared teeth from children with HPP with those from matched controls and analyzed them microscopically and chemically. It was observed that both acellular and cellular cementum formation was affected. For dentin, however, no differences in mineral content were recorded. To explain the dissimilar effects on cementum and dentin in HPP, we assessed pyrophosphate (an inhibitor of mineralization) and the expression/activity of enzymes related to pyrophosphate metabolism in both the periodontal ligament and the pulp of normal teeth. Expression of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) in pulp proved to be significantly lower than in the periodontal ligament. Also, the activity of NPP1 was less in pulp, as was the concentration of pyrophosphate. Our findings suggest that mineralization of dentin is less likely to be under the influence of the inhibitory action of pyrophosphate than mineralization of cementum.

KEY WORDS: alkaline phosphatase • hypophosphatasia • mineralization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypophosphatasia (HPP) is a rare, inherited bone disease characterized by reduced activity of the tissue non-specific (liver/bone/kidney) isoenzyme of alkaline phosphatase (TNSALP), due to deactivating mutations within the TNSALP gene (Whyte, 2001). A consistent feature of HPP is premature loss of deciduous teeth (Chapple, 1993), which is attributed to disturbed cementum formation (e.g., Beumer et al., 1973; Kjellman et al., 1973; Lundgren et al., 1991). With respect to the dentition, it is still unclear whether HPP becomes manifest specifically in the cemental tissues, or may also affect the dentin. Although several authors (e.g., Beumer et al., 1973; Hu et al., 2000) have reported that dentin has reduced thickness and mineral content, others (e.g., Baab et al., 1985; El-Labban et al., 1991) did not find any defects. It was the aim of the present study to determine whether cementum and dentin are similarly affected in HPP. We addressed this question in a group of well-defined HPP patients vs. matched healthy controls. Next to microscopic and chemical analyses of the dental hard tissues, we studied the expression/activity of enzymes in the periodontal ligament and pulp that are thought to be involved in the regulation of pyrophosphate (PPi), a potent natural inhibitor of hydroxyapatite formation [TNSALP, nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), progressive ankylosis protein (ANK); Ho et al., 2000; Terkeltaub, 2001; Nociti et al., 2002].


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Teeth
From seven HPP patients (five patients with the childhood type; two with odontohypophosphatasia; see Appendix Table 1), we obtained spontaneously shed deciduous teeth. These were immediately placed (by the parents) in 70% ethanol. In this way, 31 HPP teeth were collected: 19 incisors and 12 canines. Patient age ranged from 1.2 to 11.6 yrs at the time of tooth loss. For four patients, the periodontal condition was recorded (oral hygiene, bleeding on probing, clinical attachment level). All of these patients showed no severe inflammatory periodontal disease. Vital control teeth (n = 23; no pulp disease) with intact periodontium (no signs of loss of clinical attachment level), matched according to age and tooth type, were obtained from 20 healthy children and immediately frozen at –80°C.

The use of human subjects satisfied the requirements of the review board of the Research Institute of ACTA. Informed consent and assent were obtained from the parents or guardians of those < 18 yrs of age. Consent was obtained from those > 18 yrs old.

Mineral Content
Teeth were sectioned (~ 100 µm) parallel to their longitudinal axis by means of a diamond saw (Metals Research, Cambridge, UK) under constant water cooling (neutral pH) (Fig. 1AGo).



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  		Figure 1. Sampling of dentin. (A) ± 100-µm-thick longitudinal sections were used for LM, EM, and biochemical analyses. (B) Dentin was sampled at 4 zones: crown (C) and root (R1-3). Samples were taken by means of a dental bur (1 mm diameter) running at low speed to prevent overheating. For microradiography, 200-µm-thick transverse sections were prepared (dashed lines). (C) Mineral density was determined at sites 1–4. P = pulp; E = enamel.

 
Longitudinal mid-pulp sections were used for microscopy and microradiography; adjoining sections were used for dentin sampling (Fig. 1BGo). Samples were hydrolyzed in 6 N HCl and their phosphate, calcium, and hydroxyproline contents determined (Beertsen et al., 1999). Data were presented as calcium or phosphate per hydroxyproline.

Microscopy
Slices from 16 control teeth and 28 HPP teeth were decalcified and processed for LM and EM (Beertsen et al., 1999) (3 HPP and 7 control teeth could not be used due to poor section quality). Sections were scored for: presence or absence of acellular and cellular cementum; % root surface covered by bacterial plaque; and % root surface attached to periodontal ligament.

Microradiography
We used a diamond band-saw (Well Diamond Wire Saw, type 3242, Well, LeLocle, Switzerland) to prepare 200-µm-thick transverse sections from longitudinal sections of 5 randomly selected HPP (each from one patient) and 5 matched control teeth (each from one individual) (Fig. 1BGo). Samples were microradiographed by an x-ray generator (Philips, model PW1729, Philips, Eindhoven, NL). Mineral density was determined (Fig. 1CGo) by TMR1.25e software (Inspektor Research Systems, Amsterdam, NL).

Real-time PCR
Expression levels of genes coding for enzymes involved in PPi metabolism were assessed in the periodontal ligament and pulp of normal teeth ("fresh" HPP teeth were not available). Third molars free from caries and periodontal disease (n = 14; each from one individual, aged 18–30 yrs) were placed in RNAlater (Ambion, Huntingdon, UK) immediately upon extraction, and stored at –80°C. The periodontal ligament was collected from the middle part of the root of each tooth; pulp was sampled by means of a reamer. Tissues were placed in Trizol, and total RNA was isolated according to manufacturer’s guidelines. RNA was quantified by means of a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The synthesis of cDNA, the design of the primers (Appendix Table 2), and the performance of RT-PCR of duplicate samples in the ABI Prism 7000 (40 cycles) were as described previously (Kerkvliet et al., 2003). The PCR reactions of the different amplicons had equal efficiencies. ß2-microglobulin was used as the housekeeping gene. This gene was equally expressed in periodontal ligament and pulp. Samples were normalized for the expression of ß2-microglobulin by calculation of the cycle threshold ({triangleup}Ct) (Ctß2-microglobulin – Ctgene of interest), and the expression of the different genes is expressed as 2–({triangleup}Ct).

NPP1 Activity and PPi Concentration
To obtain sufficient tissue for the analyses, we used another set of third molars, free from caries and periodontal disease (n = 20; each from one individual, aged 18–30 yrs). Immediately after extraction, they were frozen at –20°C. Periodontal ligament and pulp were sampled (previous paragraph) and homogenized by sonification for 30 sec at 4°C in 0.05 M Tris, pH 7.8, 0.1% Triton, 1.6 mM MgCl2. After centrifugation for 15 min at 12,000 g, the supernatant was analyzed for DNA content (Picogreen method, Molecular Probes, Eugene, OR, USA), and NPP1 activity, with either 1 mM p-nitrophenyl phenyl-phosphonate (Hosoda et al., 1999) or 2 mM p-nitrophenylthymidine 5-monophosphate (Sigma) (Huang et al., 1994) as the substrate, and the activity of TNSALP was measured (van den Bos and Beertsen, 1999). PPi in the supernatant was assessed with a pyrophosphate reagent (from Sigma) according to the manufacturer’s guidelines. Enzyme activities and PPi concentrations were related to the amount of DNA.

Statistics
Data are presented as mean ± SD and were analyzed by the Wilcoxon signed-rank test and linear regression analysis, with GraphPad Software (San Diego, CA, USA). Differences were considered significant when p < 0.05 (two-tailed).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histology
Whereas in control teeth the roots were completely covered by periodontal ligament, in HPP teeth the percentage of periodontal ligament coverage was far less (21 ± 28%). Instead, the roots of the HPP teeth were largely covered by plaque (57 ± 27%), extending apically from the cemento-enamel junction. With regard to the acellular cementum, a continuous layer was observed in all control teeth. In the HPP teeth, however, no uninterrupted cementum layer could be distinguished (Figs. 2AGo, 2BGo). In only 3 of the 28 HPP specimens (patients #3 and #7) were small patches of acellular cementum (2–7 µm thick) observed. At many sites, dentinal tubules appeared to be exposed to the periodontal ligament, having led, in some places, to migration of periodontal ligament cells into the open-ended tubules (Fig. 2BGo). Electron microscopy revealed that, in the HPP specimens, the collagen fibrils of the periodontal ligament were separated from those of the dentin by a thin, electron-dense, non-fibrillar layer about 5 nm wide (Figs. 2CGo, 2DGo). In some specimens, individual collagen fibrils from the dentin bridged this gap and seemed to be continuous with fibrils in the periodontal ligament (Fig. 2DGo).



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  		Figure 2. Microscopic appearance of dental-periodontal interface in HPP and control specimens (A-E) and dentin mineral content (F). (A,B) Light microscopy of a control (A) and an HPP tooth (B). Note the absence of acellular cementum (c) in HPP. The arrow marks a periodontal ligament cell that has migrated into the orifice of a dentinal tubule. d = dentin; p = periodontal ligament. (C,D) EM showing interface between dentin (d) and periodontal ligament (p) of a HPP tooth. Acellular cementum is absent. Note the presence of a thin, electron-dense, amorphous layer covering the interface. Occasionally, individual collagen fibrils seem to cross the interface (arrow; shown at higher magnification in D). (E) Cellular cementum (cc) around the apex of an HPP tooth. (F) Relationship between HPP tooth age and mineral content in the 4 dentin compartments (C, R1-3; expressed as µg Ca/µg hydroxyproline).

 
With respect to the cellular cementum, a well-developed layer was observed in 7 out of 22 HPP teeth (patients #3, #6, and #7; Fig. 2EGo). When this layer was present, its thickness ranged from 18 to 345 µm. (Six HPP teeth could not be assessed because of severe root resorption in the apical region.)

Mineral Content
Mineral content of dentin revealed no statistically significant differences between the control and patient groups (Appendix Tables 3 and 4). No gender-related differences were detected, and no differences were found among tooth types (data not shown). However, among the 4 dentin compartments (in both HPP and control groups), significant variations were documented, with the highest mineral density being recorded coronally and the lowest apically. When the mineral content of HPP dentin was plotted as a function of tooth age, a significant increase was noted (Fig. 2FGo), both for calcium and for phosphate content. Similar maturation of dentin was seen in control teeth (data not shown).

Biochemical Analyses
TNSALP and ANK mRNA levels, relative to ß2-microglobulin, were comparable in pulp and periodontal ligament (Fig. 3AGo). NPP1 mRNA levels, however, showed marked differences between the 2 tissues, periodontal ligament being 6.6 times higher than pulp. This was also the case for NPP1 activity and PPi concentration. Differences between the 2 tissues in expression of collagen I-{alpha}1 and osteonectin, proteins not directly associated with phosphate metabolism, were less pronounced (Fig. 3AGo).



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  		Figure 3. Biochemical analyses of pulp and periodontal ligament specimens (A-C). (A) mRNA expression of NPP1, ANK, TNSALP, collagen I-{alpha}1, and osteonectin relative to ß2-microglobulin in periodontal ligament (open bars) and pulp (dashed bars) from normal teeth (mean ± SD; n = 14 for all datasets). (B) Activities of NPP1 (substrates: p-nitrophenyl phenylphosphonate and p-nitrophenylthymidine 5-monophosphate, respectively) and TNSALP (substrate: p-nitrophenylphosphate) in periodontal ligament (open bars) and pulp (dashed bars) of normal teeth (n = 20). Activities are given as mU/µg DNA. (C) Concentration of PPi relative to DNA content in periodontal ligament (open bar) and pulp (dashed bar) of normal teeth (n = 20). *p < 0.05; **p < 0.01; ***p < 0.001 according to the Wilcoxon signed-rank test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Histological examination of 28 deciduous teeth prematurely lost from seven children with HPP featuring TNSALP deficiency, in comparison with 16 matched control teeth, revealed that the periodontal attachment apparatus was severely compromised. Our observations clearly showed that cementum formation was almost completely abolished in HPP, not only for acellular cementum but also for cellular cementum. In only one out of seven patients did a deciduous tooth show cellular cementum, while acellular cementum was absent. Our observations in humans are at variance with those in TNSALP knock-out mice which showed blockage of acellular cementum formation but continuation of cellular cementum matrix formation (Beertsen et al., 1999).

Due to the cementum defect in HPP teeth, the collagenous fibrils of the periodontal ligament were not connected with the root via Sharpey’s fibers. In most places, the periodontal ligament was separated from the dentin by a 5-nm-wide, electron-dense layer containing non-fibrillar material. Occasionally, this layer seemed to be bridged by individual collagenous fibrils (Fig. 2DGo), not unlike what has been reported in mice following prolonged administration of the bisphosphonate HEBP, a synthetic analogue of PPi that blocks acellular cementum formation (Beertsen et al., 1985).

Although the periodontal condition could not be assessed in all HPP patients, we are confident that loss of deciduous teeth in HPP is not necessarily due to periodontal infection (see also Chapple, 1993). We postulate that the apical growth of the biofilm is the result rather than the cause (El-Labban et al., 1991) of loss of attachment in HPP.

Interestingly, in none of the HPP specimens were abnormalities seen in mineral density at any level of the dentin through the tooth. Also, maturation of the dentin occurred at a rate similar to that in the matched control teeth. The fact that hard tissue formation was especially compromised in the periodontal area (where the cementogenic capacity is present), and not in the pulp region, suggests that mineral homeostasis in the 2 tissues is regulated differently.

One of the major functions of TNSALP is the hydrolysis of PPi, a potent natural inhibitor of hydroxyapatite crystal growth (Terkeltaub, 2001). Thus, the difference between the 2 tissues could perhaps be due to differences in PPi regulation. The concentration of this molecule in normal teeth was indeed much higher in the periodontal ligament than in the pulp. These data concur with the expression and activity of the PPi-generating enzyme NPP1 in the periodontal ligament which was much higher compared with that in the pulp. This may suggest that different regulatory mechanisms operate for mineralization in these two tissues. Our findings may also suggest that this property of the pulp could be helpful in the context of its repair potential (Magloire et al., 2001).


   ACKNOWLEDGMENTS
 
We are grateful to Jaap Veerkamp and Rob Exterkate for their help. Financial support was provided by the Netherlands Institute for Dental Sciences (IOT) and the Shriners Hospitals for Children, USA.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received December 22, 2004; Last revision July 27, 2005; Accepted July 27, 2005


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Baab DA, Page RC, Morton T (1985). Studies of a family manifesting premature exfoliation of deciduous teeth. J Periodontol 56:403–409.[ISI][Medline]

Beertsen W, Niehof A, Everts V (1985). Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the formation of dentin and periodontal attachment apparatus in the mouse. Am J Anat 174:83–103.[ISI][Medline]

Beertsen W, vandenBos T, Everts V (1999). Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation. J Dent Res 78:1221–1229.[Abstract/Free Full Text]

Beumer J, Trowbridge HO, Silverman S, Eisenberg E (1973). Childhood hypophosphatasia and the premature loss of teeth. A clinical and laboratory study of seven cases. Oral Surg Oral Med Oral Pathol 35:631–640.[ISI][Medline]

Chapple I (1993). Hypophosphatasia: dental aspects and mode of inheritance. J Clin Periodontol 20:615–622.[ISI][Medline]

El-Labban NG, Lee KW, Rule D (1991). Permanent teeth in hypophosphatasia: light and electron microscopic study. J Oral Pathol Med 20:352–360.[ISI][Medline]

Ho AM, Johnson MD, Kingsley DM (2000). Role of mouse ank gene in control of tissue calcification and arthritis. Science 289:265–270.[Abstract/Free Full Text]

Hosoda N, Hoshino SI, Kanda Y, Katada T (1999). Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans. Eur J Biochem 265:763–770.[ISI][Medline]

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Huang R, Rosenbach M, Vaughn R, Prowedini D, Rebbe N, Hickman S, et al. (1994). Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells by transforming growth factor-beta. J Clin Invest 94:560–567.

Kerkvliet EH, Jansen IC, Schoenmaker T, Beertsen W, Everts V (2003). Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts. Matrix Biol 22:217–227.[ISI][Medline]

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Whyte MP (2001). Hypophosphatasia. In: The metabolic and molecular bases of inherited disease. 8th ed. Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, editors. New York: McGraw-Hill Book Company, pp. 5313–5329.




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