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J Dent Res 83(10):807-810, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Collagen Cross-linking and Ultimate Tensile Strength in Dentin

P.A. Miguez1,2, P.N.R. Pereira1,2, P. Atsawasuwan2, and M. Yamauchi2,*

1 Department of Operative Dentistry, CB# 7455, and 2 Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7455, USA;

* corresponding author: mitsuo_yamauchi{at}dentistry.unc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have indicated differences in bond strength of dental materials to crown and root dentin. To investigate the potential differences in matrix properties between these locations, we analyzed upper root and crown dentin in human third molars for ultimate tensile strength and collagen biochemistry. In both locations, tensile strength tested perpendicular to the direction of dentinal tubules (undemineralized crown = 140.4 ± 48.6/root = 95.9 ± 26.1; demineralized crown = 16.6 ± 6.3/root = 29.0 ± 12.4) was greater than that tested parallel to the tubular direction (undemineralized crown = 73.1 ± 21.2/root = 63.2 ± 22.6; demineralized crown = 9.0 ± 3.9/root = 16.2 ± 8.0). The demineralized specimens showed significantly greater tensile strength in root than in crown. Although the collagen content was comparable in both locations, two major collagen cross-links, dehydrodihydroxylysinonorleucine/its ketoamine and pyridinoline, were significantly higher in the root (by ~ 30 and ~ 55%, respectively) when compared with those in the crown. These results indicate that the profile of collagen cross-linking varies as a function of anatomical location in dentin and that the difference may partly explain the site-specific tensile strength.

KEY WORDS: collagen matrix • ultimate tensile strength • dentin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The process of conventional dentin caries treatment involves the removal of caries by cavity preparation exposing sound dentin, partial demineralization of dentin with an acidic gel or solution, and subsequent restoration with adhesive materials. For successful adhesion, the formation of a hybrid layer, composed of collagen fibrils embedded with adhesive resin, is apparently important (Nakabayashi et al., 1982). In the past, several studies have shown differences in bond strength between crown and root dentin, although the results are somewhat conflicting (Nakajima, 1991; Berry and Powers, 1994; Burrow et al., 1996; Yoshiyama et al., 1996). The cause of this site-dependent bond strength is not well-understood. Since the integrity and stability of collagen fibrils are the structural basis for hybrid layer formation, we hypothesized that the site-dependent bond strength observed may be due to the varied biochemical and mechanical property of collagen in the respective sites of dentin. To investigate the site-dependent collagen quality, we analyzed and compared the tensile strength, collagen content, and collagen cross-linking in crown and upper root dentin of human third molars.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ultimate Tensile Testing and Scanning Electron Microscopy
Human third molars free of caries (from subjects 18–25 yrs old) were used with approval of the Institutional Review Board of the University of North Carolina. Thirty-four teeth were sliced parallel to their long axis, with a thickness of 0.5 ± 0.1 mm, by means of a low-speed diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA) under water cooling. The slices were divided into ‘crown’ and ‘root’ groups, and were further trimmed to an hourglass shape to produce a 0.25-mm2 area (Sano et al., 1994a). For the crown specimens, the trimmed area was at the center of the slice, 2 mm above the pulp chamber. For the root specimens, the trimmed area was equidistant between the pulp wall and cementum at 3 mm below the cemento-enamel junction. Of the total number of slabs (n = 120), 60 were trimmed so that the tubules would run parallel to the tensile force (parallel group), and the other half so that the tubules would run perpendicular (perpendicular group) to the tensile force. In addition, half of each group (n = 30) was demineralized with 0.5 M EDTA at a pH of 7.4 for 10 days (Fig. 1Go). Complete demineralization was confirmed by x-ray.



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  		Figure 1. Sample preparation for microtensile test. The dentin samples for biochemical analyses were collected from the same area where the microtensile test was performed. The tooth was cut parallel to its long axis (a), indicated by a thin dotted line, and the slices were further divided into crown and root, indicated by a thick dotted line. The microtensile test was performed either parallel (b) or perpendicular (c) to the direction of the dentinal tubules.

 
Specimens were attached to a Ciucchi Jig, a device which consists of two stainless-steel components which slide away from each other when the apparatus is subjected to tensile force, thus pulling the specimen apart (Paul et al., 1999). This device was mounted to an E-Z Test machine (Shimadzu, Kyoto, Japan), and tested in tension at a crosshead speed of 1 mm/min (Sano et al., 1994b). All data were subjected to two-way ANOVA and Fisher’s PLSD test (p < 0.05) (Statview® Software Version 5.0.1, SAS, Cary, NC, USA). After the ultimate tensile strength of undemineralized and demineralized dentin was tested, some specimens were randomly selected and fixed in 10% neutral buffered formalin for at least 8 hrs, rinsed with de-ionized water for 1 min, dehydrated in ascending grades of ethanol, immersed in hexamethyldisilazane (HMDS) for 10 min, and air-dried (Perdigão et al., 1999). Samples were placed on SEM stubs, gold-sputter-coated, and analyzed for morphology under a scanning electron microscope (JSM 6300, JEOL USA Inc., Peabody, MA, USA).

Collagen Analyses
Sixty teeth were sliced into 1.5-mm-thick slabs as described above, and dentin samples obtained from the same areas as used for the ultimate tensile strength testing were collected, pulverized in liquid N2 (Spex Freezer Mill, Metuchen, NJ, USA), washed with distilled water, and lyophilized. To obtain sufficient amounts for biochemical analyses, we pooled dentin samples as described below. To determine the collagen content, we pooled dentin samples from 18 teeth. Using a Varian/Waters HPLC system (Varian 9050 and 9012; Varian, Walnut Creek, CA, USA) fitted with a strong cation exchange column (AA911; Transgenomic, San Jose, CA, USA) (Yamauchi and Shiiba, 2002), we hydrolyzed an aliquot (~ 1 mg) with 6 N HCl and subjected it to amino acid analysis (n = 9) to determine the hydroxyproline content. For reducible and non-reducible fluorescent cross-link analyses, samples of dentin from 3 teeth were pooled from a total of 27 teeth, demineralized with 0.5 M EDTA, pH 7.4, for 1 wk at 4°C, washed with distilled water, and lyophilized. Approximately 2 mg of the demineralized collagen from each pool of sample (n = 9) was then reduced with standardized NaB3H4, hydrolyzed, and subjected to amino acid and cross-link analyses (n = 9) as described previously (Yamauchi and Katz, 1993). The reducible cross-links (dehydrodihydroxylysinonorleucine [deH-DHLNL], its ketoamine, and dehydrohydroxylysinonorleucine [deH-HLNL]) and precursor aldehydes (Hylald and Lysald) were analyzed as their reduced forms (DHLNL, HLNL, DHNL, and HNL, respectively). The non-reducible, fluorescent cross-links, pyridinoline and deoxypyridinoline, were measured at the same time with the use of an online fluorescence flow monitor.

Pyrrole, an Ehrlich’s reagent (p-dimethylaminobenzaldehyde)-positive non-reducible cross-link (Scott et al., 1981), was also analyzed. Samples were pooled from 27 teeth and demineralized as described above. A quantity of approximately 2 mg of demineralized samples was digested with trypsin (Kuboki et al., 1981; Yamauchi et al., 1981), and an aliquot of the digest was subjected to hydroxyproline analysis as described above, and the rest to pyrrole analysis by the slightly modified method of Scott et al.(1981). Briefly, the tryptic digest was dissolved in 250 µL of 5% sodium dodecyl sulphate and 250 µL of 10% p-dimethylaminobenzaldehyde in 4 M perchloric acid (all Fisher Scientific, Pittsburgh, PA, USA). After 10 min, the amount of pyrrole was measured by absorbance at 572 nm.

All cross-links and aldehydes were quantified as a mole per mole of collagen (Yamauchi and Katz, 1993), and each value was analyzed by one-way ANOVA and Fisher’s PLSD for the variable location (p < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
For undemineralized specimens, mean ultimate tensile strength values of root vs. crown, when analyzed within the same tubule direction, were not significantly different (parallel p = 0.1418, perpendicular p = 0.0566, respectively) between the two groups. Within each group, the ultimate tensile strength of dentin tested parallel to the tubules was significantly lower than the tensile strength of dentin tested perpendicular to the tubule direction (root p = 0.0008, crown p = 0.0025, respectively).

The demineralized specimens had significantly greater ultimate tensile strength values in the root than in the crown (parallel p = 0.0001, perpendicular p = 0.0091), when tested in the same tubule direction. Like the undemineralized specimens, when ultimate tensile strength was tested parallel to the tubules, the values were less than those tested perpendicular to the tubules (root p = 0.0109, crown p = 0.0066) (TableGo).


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  		Table. Ultimate Tensile Strength of Undemineralized and Demineralized Human Dentin in Crown vs. Root
 
The direction of the tubules in the specimens tested, either parallel or perpendicular, was confirmed by low-magnification scanning electron microscopy (data not shown).

Based on the hydroxyproline analysis, the collagen contents in both root and crown were comparable (131.99 ± 21.5 and 166.49 ± 49.4 µg of collagen/mg of dentin, respectively).

Regarding the results of the cross-link analyses, in all samples analyzed, 2 aldehydes (DHNL and HNL), 2 reducible cross-links (DHLNL and HLNL), and 3 non-reducible cross-links (pyridinoline, deoxypyridinoline, and pyrrole) were identified (Fig. 2Go). The major aldehyde, DHNL, the major reducible cross-link, DHLNL, and the major non-reducible cross-link, pyridinoline, were all significantly higher in the root when compared with those of the crown (p < 0.005, p < 0.001, and p < 0.005, respectively). The minor aldehyde (HNL) and cross-links (HLNL, deoxypyridinoline) were comparable in quantity between these two groups. A pyrrole cross-link was relatively minor in dentin, and no statistical difference was found for this cross-link between the two groups (0.12 ± 0.04 and 0.11 ± 0.02 mol/mol of collagen in root and crown, respectively).



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  		Figure 2. Collagen cross-link analysis in crown and root dentin (n = 9). (a) Cross-link precursor aldehydes, (b) reducible cross-links, and (c) pyridinoline cross-links. Asterisks indicate significant differences (p < 0.005, p < 0.001, and p < 0.005, respectively) between the two groups.

 
The ratios of both DHLNL to HLNL and pyridinoline to deoxypyridinoline were significantly higher in the root than in the crown, i.e., 5.17 vs. 4.19 and 15.67 vs. 14.29, respectively (p ≤ 0.0001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanical properties of dentin have been attributed mainly to the packing and density of mineral particles (Marshall et al., 1997; Kinney et al., 2001), the amount of intertubular dentin, and/or tubule density (Konishi et al., 2000). In this study, we demonstrated that the collagen matrix alone significantly contributes to the tensile strength of dentin. The levels of contribution were found to be different between the two different locations tested, i.e., 25 to 30% in the root vs. 11 to 12% in the crown. The results also showed that ultimate tensile strength is greater when tested perpendicular to the tubules for both groups. This is in agreement with a recent report by Inoue et al.(2003), but not with reports from other studies (Watanabe et al., 1996; Inoue et al., 2002). The inconsistency is possibly due to the difference in the substrates used (i.e., human vs. bovine teeth). SEM images of the specimens used for this study demonstrated that the majority of the fibrils run either perpendicular or oblique to the tubule direction (Fig. 3Go). The results therefore indicate that a tensile force applied to collagen fibrils longitudinally results in higher ultimate tensile strength values than when applied perpendicularly.



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  		Figure 3. Direction of the collagen fibrils (arrows) in relation to the dentinal tubules in an undemineralized crown specimen (SEM at 20,000X).

 
When demineralized, the root had significantly higher ultimate tensile strength than the crown, although there was no difference when the undemineralized dentin was tested. This may be due to the differences in the content and quality of mineral, and/or to the non-collagenous matrix molecules that are extractable with EDTA, between the two groups tested.

The major finding of this study was that although the collagen content was comparable, the collagen cross-linking was significantly different between root and crown dentin. In the root, the contents of the major precursor aldehyde (Hylald) and its major cross-linking products, both reducible (DHLNL) and non-reducible (pyridinoline) cross-links, were significantly higher than those in crown. In addition, the ratios of both DHLNL to HLNL and pyridinoline to deoxypyridinoline were significantly higher in the root than in the crown. The mechanisms that cause the quantitative and qualitative differences in collagen cross-linking between the two locations are not clear at this point. It is possible that, during dentinogenesis, the enzymes that are responsible for oxidative deamination of {epsilon}-amino groups of lysine and hydroxylysine (i.e., lysyl oxidase) and lysine hydroxylation (i.e., lysyl hydroxylases) of collagen are differentially expressed/activated by odontoblasts, depending on the location and stage of dentin formation (Uzawa et al., 1999). This may lead to the site-specific cross-linking pattern, leading to the different levels of ultimate tensile strength found in this study. Although the difference in collagen cross-linking may partly explain the variations in ultimate tensile strength found in the two locations, the potential contributions of non-collagenous proteins should also be investigated.

The finding of the site-specific quality (i.e., cross-linking/mechanical property) of collagen matrix in dentin may imply that the degree of demineralization with phosphoric acid/other acids, the stability and durability of the hybrid layer, and bond strength may also vary as a function of location. The potential relationship among numbers and types of cross-links to mineral loss, organic matrix degradation, and dentin mechanical properties must be further investigated.


   ACKNOWLEDGMENTS
 
This study was supported by NIH-NIDCR grant DE10489 and by NASA grant NAG2-1596. This paper is based on a thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Operative Dentistry, School of Dentistry. A preliminary report was presented at the IADR General Session in Göteborg, Sweden, June, 2003.

Received January 14, 2004; Last revision July 20, 2004; Accepted July 25, 2004


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Berry EA, Powers JM (1994). Bond strength of glass ionomers to coronal and radicular dentin. Oper Dent 19:122–126.[ISI][Medline]

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Inoue T, Takahashi H, Nishimura F (2002). Anisotropy of tensile strengths of bovine dentin regarding dentinal tubule orientation and location. Dent Mater J 21:32–43.[ISI][Medline]

Inoue S, Pereira PN, Kawamoto C, Nakajima M, Koshiro K, Tagami J, et al. (2003). Effect of depth and tubule direction on ultimate tensile strength of human coronal dentin. Dent Mater J 22:39–47.[ISI][Medline]

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