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RESEARCH REPORT |
1 Endocrinology Section, Department of Medicine, University of Wisconsin, H4/556 CSC (5148), 600 Highland Ave., Madison, WI 53792, USA;
3 Research Division, Hospital for Special Surgery, 535 East 70th St., New York, NY, 10021, USA;
4 Department of Materials Science & Engineering, 1509 University Ave., Madison, WI 53706, USA;
5 Geriatrics Research, Education, and Clinical Center, William S. Middleton VAMC, 2500 Overlook Terrace, Madison, WI 53705, USA; and
6 Osteoporosis Research and Clinical Center, 2870 Marshall Ct., Madison, WI 53705, USA
* corresponding author, rdb{at}medicine.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: collagen type I • dentinogenesis imperfecta • osteogenesis imperfecta • biomechanics • tooth calcification
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Classic features of dentinogenesis imperfecta include discoloration of teeth, increased tooth fragility, and obliteration of the pulp chambers. Histological and ultrastructural abnormalities reported in human dentinogenesis imperfecta include a decreased number of irregularly shaped dental tubules, remnants of capillary inclusions, obliterated pulps, morphological abnormalities of dental collagen fibers, and mineralization defects (Lukinmaa et al., 1987; Lygidakis et al., 1996; Waltimo et al., 1996; Hall et al., 2002). Not all dentinogenesis imperfecta is associated with osteogenesis imperfecta, however, since mutations in dentin sialophosphoprotein can also cause a syndrome with similar clinical manifestations (Xiao et al., 2001; Zhang et al., 2001).
The Col1a2oim (oim) mutation is a widely studied model of chain deficiency osteogenesis imperfecta. The mutation is a 1 base deletion in the gene encoding the 
2 chain of type I procollagen (Chipman et al., 1993), resulting in premature termination of the nascent peptide and nonsense-mediated degradation of the message. Homozygous mutant (oim/oim) animals display marked skeletal fragility, suffering spontaneous fractures with usual cage activity and husbandry-related handling (Chipman et al., 1993; Camacho et al., 1999). Heterozygous (oim/+) animals have a more subtle biomechanical impairment, appearing grossly normal but with diminished performance on three-point bend and torsion testing (Saban et al., 1996; Camacho et al., 1999). Moreover, homozygous oim/oim teeth are fragile, so that our standard husbandry conditions for these animals include the provision of powdered food.
Type 1 collagen is the major structural protein in dentin as well as bone, so we reasoned that mice harboring the oim mutation should exhibit a dental phenotype. We previously described the morphological and ultrastructural features of oim/oim and oim/+ teeth (Lopez Franco et al., 2005). In this study, we extend our previous work by performing nanoindentation of teeth from animals having the +/+, oim/+, and oim/oim Col1a2 genotypes, addressing the question of whether the mutation causes a measurable alteration of dentin material properties. We hypothesized, based on previous experimental evidence of an increased mineral:matrix ratio and decreased post-yield performance in oim/oim bone, that Young’s modulus would be increased in the mutant teeth in a gene-dosage-dependent fashion. Further, we expected that dentin near the dento-enamel junction would have greater Young’s modulus than that near the pulp chamber, reflecting the longer time since its synthesis and deposition.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Nanoindentation
We studied 3 mice per genotype and 1 tooth per mouse. Upper incisors were fractured at the level of the junctional epithelium, and nanoindentation was performed at 2 sites (Fig
.). The "far from pulp" region was chosen to be in proximity to the dento-enamel junction, while the "near the pulp" site was chosen to be in proximity to the pulp chamber, but beyond the border of the predentin. Following polishing, we performed at least 3 indents/site/tooth. Loading was applied with the Hysitron (Minneapolis, MN, USA) triboscope apparatus and a diamond Berkovitch tip to a maximum of 3000 µN, with a five-second hold before unloading. We analyzed the data according to the Oliver and Pharr method (Oliver and Pharr, 1992), as applied by the instrument software to the 95%-50% unloading limb of the load-deformation curves. We calibrated the instrument and determined machine compliance with a fused quartz standard. Young’s modulus (E) and Meyer hardness (H) were calculated from the raw data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Genotype was not significant, with F = 1.08. Post hoc analysis by Tukey’s test revealed that only the oim/+ genotype displayed a significant difference between locations (p = 0.0052). The same qualitative pattern held for the other genotypes, but fell short of statistical significance. Similarly, greater hardness in mutant teeth apparently did not reach significance.
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). There was no evidence of a significant interaction between genotype and location, with F = 3.41. Post hoc testing revealed a significant difference between locations only for the oim/+ genotype (p = 0.0096). Near the dento-enamel junction, the +/+ and oim/+ genotypes differed significantly (p = 0.016). Near the pulp chamber, Young’s modulus did not differ significantly among genotypes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Young’s modulus in a composite material (Ec) such as bone or dentin is estimated to lie between the sum of the component materials’ Young’s moduli (Ei),
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and the reciprocal of the sum of the reciprocals of the component materials’ Young’s moduli,
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sweighted according to the volume fraction (Vi) of each component in the composite.
It is instructive to consider the ranges of moduli that would be predicted in oim based on these simple assumptions. In previous work, we demonstrated a 20% decrease in oim/oim bone collagen content relative to +/+ and an intermediate collagen content in heterozygotes (Camacho et al., 1999), and regardless of genotype, mineral comprised approximately 65% to 70% of the dry mass. Young’s modulus of apatite is ~110 GPa (Walsh and Christiansen, 1995), while that of collagen in compression is about 2 orders of magnitude less (Zioupos et al., 1999), thus contributing negligibly to the Young’s modulus of the composite. The theoretical maximum Young’s modulus of dry dentin is therefore approximately 78 GPa, and the minimum Young’s modulus is approximately 0.3 GPa. The measured values in our study fell in the middle of this range. Both wetting and the presence of other minerals with Young’s moduli lower than that of apatite will lower measured Young’s modulus of the composite dentin. In particular, calcium carbonate’s Young’s modulus is ~35 to 70 GPa, and it makes up approximately 1 to 2% of bone mineral. In tibiae, we previously found that carbonate in oim/oim animals was ~23% less than in either oim/+ heterozygotes or +/+ mice (Camacho et al., 1999). Moreover, Phillips and colleagues, using neutron activation analysis, measured the concentrations of various minerals in femora and incisors of mice harboring the oim mutation (Phillips et al., 2000), finding a mutant allele dosage-dependent decrease in Mg in teeth and an increase in F in heterozygotes relative to both homozygotes. The CaCO3 and Mg concentrations were both low enough that their quantitative impact on Young’s modulus would be small, but the qualitative impact of substituting Mg for Ca or carbonate for phosphate in the apatite crystal is unknown. In our earlier description of the oim dental phenotype, we observed a reduction in the number of dentinal tubules in a mutant-allele-dependent manner (Lopez Franco et al., 2005). These contribute "empty space" to the dentin matrix, whose loss would be expected to increase Young’s modulus. Notably, empty space in the matrix is not reflected in quantitative analyses of the matrix’s composition. Reduced collagen and carbonate content are also possible contributing reasons for our observation that the oim mutation increased Young’s modulus. In reality, behavior is still more complex, since each mechanism of loading—i.e., compression, bending, tension, and torsion—will have an impact on the measured material properties of the composite.
Our results agree with those of Grabner et al.(2001), who found that Vickers hardness increased in embedded cortical bone in an oim dosage-dependent manner, ranging from ~0.6 GPa for +/+ to ~0.9 GPa for oim/oim. They also measured mineralization by backscattered electron microscopy, reporting that the mutation also increases mineralization, agreeing with our prior observation that the Fourier transform infrared (FTIR)-spectroscopy-determined mineral-to-matrix ratio was increased by the oim mutation (Camacho et al., 1996). Our mechanical results also agree with our previous observation of greater Young’s modulus in femoral cortical bone (Misof et al., 2005). This is at variance with observations at larger scales (e.g., by dual-energy x-ray absorptiometry or ash percentage) that reveal lower mineral density in osteogenesis imperfecta. This apparent discrepancy is most readily explained by the inclusion of non-mineralized tissue space in the larger-scale measurements, reflecting a decrease in mineralized tissue mass rather than a decrease in its degree of mineralization.
Our results also agree in part with those of Kinney and coworkers (Kinney et al., 2003). These investigators studied human dentin in normal teeth and teeth from patients with a clinical diagnosis of dentinogenesis imperfecta type 2 (no evidence of osteogenesis imperfecta). Unlike our data, they found that the dentinogenesis imperfecta dentin had a lower, not a higher, Young’s modulus than did normal dentin. The difference in the molecular lesions underlying dentinogenesis imperfecta in their samples and ours may account for this disparity. Their values for normal-dentin Young’s modulus were consistent with ours and were greater at the dento-enamel junction and smaller close to the pulp chamber. They demonstrated that this reflected the degree of dentin mineralization as determined by synchrotron radiation CT scanning. Our observations regarding hardness were generally consistent with those of Kinney et al., but went beyond them in considering the location dependence of hardness. The dentin at the dento-enamel junction has a greater tissue age than that adjacent to the pulp chamber, and therefore has had a longer period to undergo mineralization.
Our study has some important limitations that readers should keep in mind. First, we examined dentin only in compression, so material properties in other loading regimes were not addressed. Second, we examined only dry specimens in these experiments. Third, our sample sizes were small, limiting our ability to detect differences among genotypes and sites. This is particularly apparent with regard to hardness, where the observed differences among genotypes fell short of significance, as did the differences between locations in the homozygous genotypes. Fourth, we examined only a single age and a single osteogenesis imperfecta model, so the generality of our results to abnormal chain (in distinction to chain deficiency) forms of osteogenesis imperfecta cannot be assumed. The same limitation also holds for dentinogenesis imperfecta unassociated with osteogenesis imperfecta, and it is imperative that both osteogenesis imperfecta and dentinogenesis imperfecta be considered as clinical syndromes representing multiple disease states.
These limitations notwithstanding, we have demonstrated that both hardness and Young’s modulus increase as a function of distance from the pulp chamber in all genotypes, consistent with continued mineralization following initial synthesis and secretion. It is well-established that the volumes of intertubular and peritubular dentin vary as a function of location in the tooth, with dentin near the dento-enamel junction containing a greater fraction of intertubular dentin (Pashley, 1989). Furthermore, intertubular dentin is more heavily mineralized than peritubular dentin. In this regard, it is interesting to note that recent evidence shows that collagen is present only in the intertubular dentin (Gotliv et al., 2006), so that a change in the relative amounts of inter- and peri-tubular dentin as a result of the mutation could contribute to our observations. In animals harboring the oim mutation, Young’s modulus was increased, but without a simple mutant allele dosage relationship. Tooth fragility in mice harboring the oim mutation cannot be explained by deficiencies of hardness or Young’s modulus, and so defects in plastic behavior should be sought. Future progress in our understanding of the mechanical properties of dentin will benefit from the application of mechanical testing protocols that can address fatigue damage and the plastic regime.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received December 15, 2005; Last revision June 23, 2006; Accepted July 26, 2006
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REFERENCES |
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