Mater. Res. Soc. Symp. Proc. Vol. 874 2005 Materials Research Society L5.15.

Elastic Modulus and Mineral Density of Dentine and Enamel in Natural Caries Lesions

Amanpreet K. Bembey1, Michelle L. Oyen2, Ching-Chang Ko2, Andrew J. Bushby1 and Alan
Boyde3.
1
Department of Materials, Queen Mary, University of London, London E1 4NS, UK
2
University of Minnesota, Minneapolis, MN 55455
3
Dental Institute, Queen Mary, University of London, London E1 1BB, UK

ABSTRACT

Dental tissues have been reported to show a considerable decrease in both their mineral
content and mechanical properties in carious lesions. The changed properties of dentine and
enamel have been shown to be dependent on crystal size and not only mineral content [1],
although the connectivity between the mineral crystals has been overlooked. Teeth with carious
lesions were sectioned, embedded in polymethylmethacrylate (PMMA) and diamond polished.
Nanoindentation and quantitative backscattered electron imaging were used to determine
relationships between the elastic modulus and mineral density of sound and carious regions
within dentine and enamel. The changes in elastic modulus with decreased mineralization for
dentine and enamel could not be explained by simple composite mechanics expressions relating
elastic modulus and mineral volume fraction. Finite element modeling of dentine and enamel as
a two-phase composite material at the ultrastructure level were used to demonstrate how changes
in the mineral phase connectivity can produce changes in the elastic modulus. Tissue models for
enamel, in which the mineral phase is both the major component of the structure (~ 85% by
volume) and highly interconnected, were consistent with the modulus of sound enamel. The
drastic change in enamel modulus with a relatively small change in mineral volume fraction
could be modeled as a decrease in mineral phase connectivity at nearly constant volume fraction.
The more gradual trend in the dentine data was also consistent with a structure that is initially
highly connected in the mineral phase, consistent with the known structure of dentine, and for
which the change in modulus is more directly related to changes in mineral content than mineral
connectivity.

INTRODUCTION

Mineralized tissues are composite materials with a mineral carbonated hydroxyapatite

(HA) phase and an organic hydrated phase, largely collagen in the calcified connective tissues
like bone and dentine, but there is no collagen in enamel. For composite materials with large
modulus mismatch between the component phases, a wide range of modulus values can be
achieved at constant composition (i.e. mineral volume fraction) by altering the geometrical
structure and arrangement of the component phases. The precise arrangement of the component
phases at the ultrastructural level is unclear for mineralized tissues such as teeth, and there is
associated uncertainty with the changes in tissue composition and structure due to disease
processes such as dental caries. Although there is certainly a loss of mineralization in caries, the
way that this affects the structure of the component phases - and in turn the modulus of the tissue
- is unclear, but of great interest in understanding the mechanical functionality of mineralized
tissues.

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15
 L5.15.2

It has been shown that elastic modulus values of hard tissues are difficult to predict based
only on mineral volume fraction [2]. Examination of the elastic modulus-mineral content
relationships for mineralized tissue composites are frequently undertaken by comparisons with
the upper and lower composite bounds, which correspond to the stiff phase or the compliant
phase being continuous in the structure. Bone demonstrates a wide range of modulus values at
nearly constant mineral volume fraction, occupying the entire region between the upper and
lower modulus bounds [2]. Dentine is similar in composition to bone, having close to 50%
mineral phase by volume but seems to have modulus values in the upper range of those seen for
bone [3]. The mineral phase in bone is at least partially continuous [4,5]. Enamel is highly
mineralized, with around 90% mineral. Analysis of enamel modulus indicates values that seem
to fall along a lower elastic modulus bound, indicating closely spaced but discrete mineral
particles [2], in agreement with developmental mechanisms. In the current study, dentin and
enamel, both sound and carious, are examined within a composite materials vision.

EXPERIMENTAL DETAILS

An upper second premolar extracted due to caries was washed in distilled water and
stored in 70% ethanol. The tooth was then sectioned mesiodistally to expose the center of the
approximal carious lesion. The cut surface was polished on successive abrasives down to 0.1m
diamond. The sectioned tooth was dehydrated in methanol, impregnated with MMA monomer
and polymerized in situ to fully embed the tissue at the molecular level. The embedded block
was trimmed and re-polished to re-expose the sectioned plane in the tooth at the surface of the
block, which was then coated with carbon [6].
The sample was imaged using quantitative backscattered electron imaging (qBSE, Zeiss
DSM962 with Kontron external control computer, 20kV, 17mm WD, 510-10Amps beam current
at sample). The qBSE calibration standards used were monobrominated, monoiodinated and
tetrabrominated dimethacrylates [7, 8]
Nanoindentation testing was performed using UMIS 2000 (CSIRO, Sydney, Australia)
using a spherical indenter tip (R = 5m) using the multiple partial loading technique [9, 10].
Each indentation test consisted of 40 increments, unloading to 75% of each load between
increments to a maximum load of 10mN. Elastic Modulus (E) was calculated as a function of
contact depth [11] for each load-partial unload data pair, and a mean value for E was derived for
each indentation site from the 10 deepest increments and used to represent the modulus at that
indentation site. This procedure has been shown to minimize artifacts from surface layers and
surface roughness [11]. Three arrays of indentations were carried out (1) across the sound
dentino-enamel junction (DEJ), (2) in partially demineralised carious dentine and, (3) in partially
demineralised carious enamel. Each array consisted of 527 indents (15m spacing) with deep
marker indents (50mN) to relocate the indent arrays for subsequent qBSE imaging at high
resolution (150: 20482048 pixels), as described by Ferguson et al. (2003) [12]. Mineral
density (mean gray level) within each defined indentation patch was derived from the qBSE data
using SigmaScan Pro 5 software (SPSS Inc., Chicago, IL, USA).

MODELING AND ANALYSIS

For examination of the experimental data in a composite materials framework,

mineralized tissue samples were considered as a two-phase organic-inorganic composite.
Experimental qBSE values of the tissue density (the composite density, c) were converted to
approximate volume fractions of mineral assuming an organic matrix phase density of 1 g/cm3,

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15
 L5.15.3

an inorganic filler phase density of 3.1 g/cm3, and using a rule-of-mixtures assumption. The
indentation data for elastic modulus were re-examined and compared with the Hashin-Shtrikman
bounds for a mineral phase with an elastic modulus (E) of 150 GPa and an organic phase with an
elastic modulus of 1.0 GPa (embedded matrix, earlier studies have shown that embedded tissues
behave as though the collagen is dehydrated [11,13]). Both phases were assumed to have a
Poisson's ratio of 0.3.
A series of 2D finite element (FE) models were developed in FlexPDE (PDE Solutions,
Antioch, CA) to represent a two-phase mineralized tissue composite with different
configurations of mineral (stiff) and compliant (organic) phase. The elastic modulus of the
compliant (gelatin/collagen) phase was assumed to be100 MPa and that of the stiff (mineral)
phase was set to 100 GPa. Poisson's ratio was assumed to be 0.3 for both phases.
Models were constructed to represent (1) a compliant matrix phase with stiff
particles; (2) a stiff matrix phase with compliant particles, (3) intermediate structures with
a compliant matrix phase were generated by making stiff bridging connecters between a
varying fraction of stiff particles in the direction of model loading. The intermediate
connectivity models (3) were designated by the fraction (Cx) of total possible bridging
connections, such that Cx = 0 corresponded to no bridges (similar to model 1) and Cx = 1
corresponded to all bridges (similar to model 2). Interparticle spacing was varied at constant
structure for each model to effectively change the mineral volume fraction at fixed structure, and
structural changes were examined at different interparticle spacings to examine structural
changes at approximately fixed mineral fraction. In models 1 and 2 the effect of anisotropic
(high aspect ratio) particles was also examined. Effective elastic modulus was computed for
each model structure by comparisons between composite structures and homogeneous stiff
structures with the same external geometry [5].

RESULTS

Experimental values of the indentation modulus are shown as a function of qBSE number
in Figure 1. Overall, with caries the mean modulus of dentine decreased from 24 to 16 GPa,
while the mean modulus of enamel decreased from 77 to 51 GPa. Images of sound (Figure 2a)
and carious (Figures 2b, 2c) tissue demonstrate morphological differences as well as changes in
grey scale associated with mineral density (especially in enamel, Fig. 2b).
Overall, a larger decrease in both mechanical and compositional properties was observed
in dentine than enamel. The elastic modulus for dentine decreased by 28% with only a 19%
decrease in mineral content. For enamel, the mean elastic modulus decreased by 24% and only
2% for mineral density, indicating that removal of a small amount of mineral has a greater effect
on mechanical properties of enamel.
Finite element model results are shown in Figures 3 for models (1)-(3). In all models,
discrete mineral reinforcing phase corresponds to modulus values similar to the lower Hashin-
Shtrikman bound, while fully-connected mineral phase structures corresponds to modulus values
similar to the upper Hashin-Shtrikman bound.
With changes in the mineral phase structure, both particle aspect ratio (Fig. 3a) or
interconnectivity between the particles by bridging elements (Fig. 3b), modulus values changed
dramatically at constant or nearly constant mineral phase volume fraction. The results for
partially connected structures (model 3, Fig 3b) with increasing connectivity are consistent with
previously reported data for rigorous modeling of interpenetrating phase composites near the
percolation threshold, in which a jump from the lower bound to the upper bound with small

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15
 L5.15.4

changes in volume fraction is associated with a transition in the stiff phase from discrete to
continuous [14].
Conversely, for changing scale of the structures (changing mineral volume fraction) with
fixed mineral phase structure for different base structures with varying connectivity (Fig. 3c)
modulus changes are gradual with mineral volume fraction and run approximately parallel to the
upper Hashin-Shtrikman composite bound.
120
Carious Dentine
Carious Enamel
100
Indentation Modulus E' / GPa

Sound Dentine
Sound Enamel
80

60

40

20

0
0 50 100 150 200 250
qBSE Gray Level
Figure 1: The relationship between mineral density and indentation modulus for dentine and
enamel in sound and carious condition. The demineralization process due to caries affects the
two tissues differently. qBSE Gray Levels of 0 and 255 are approximately equivalent to density
values of 1.7g/cm3 and 3g/cm3, respectively.

(a) (b) (c)

Figure 2: qBSE images (field width 585m) with indentation locations overlaid as black spots,
(a) Sound dentino-enamel junction with the upper divide showing enamel and the lower divide
dentine, (b) carious enamel, (c) carious dentine. Gray levels of 0 and 255 are approximately
equivalent to density values of 1.7g/cm3 and 3g/cm3, respectively.

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15
 L5.15.5

Effective Modulus, EEFF (GPa)

A B C
100 100 100

10 10 10

1 1 1

0.1 0.1 0.1

0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0
Volume Fraction Mineral, Vf

Figure 3: (A) Finite element results for varying particle aspect ratio in models (1) and (2). A
wide range of modulus values can be seen at constant mineral volume fraction depending on the
particle geometry. The controlling factor for modulus of high aspect ratio composites is the
interparticle spacing [5] (B) Finite element results for partially connected mineral networks
(model 3): changes in structural connectivity (changes in number of bridging elements) with
little change in mineral volume fraction; and (C) changes in mineral volume fraction with no
change in structural connectivity. Dashed lines in all figures are the Hashin-Shtrikman
composite bounds.

DISCUSSION

In re-examination of the experimental data for sound and carious mineralized tooth
structures, different patterns emerge for dentine and enamel tissues (Figure 4). Dentine data
demonstrate gradual changes in modulus with mineral volume fraction, and data for both normal
and carious dentine lie approximately parallel to an upper modulus bound. Thus the dentine data
are consistent with the results in Figure 3(c), and demineralization is associated in pure changes
in mineral content with no change in relative mineral organization and structure. In contrast,
enamel data demonstrate large decreases in modulus with small changes in mineral volume
fraction, demonstrating that changes in the relationships between the individual mineral particles
are dominating the behavior, associated with the modeling results in Figures 3a and 3b. The FE
model demonstrates that, to achieve the measured modulus values, the mineral must be the load
bearing phase and that the composite modulus is determined by the volume fraction and the
degree of connectivity of the mineral crystallites. In the indented regions of the caries lesion, the
demineralization had not progressed to the point where the percolation threshold was exceeded
in reverse, such that the soft tissue would never dominate the elastic response.
It is difficult to ascertain from modulus data alone whether the mineral phase is
continuous or discontinuous in the highly mineralized enamel. The steep changes in modulus at
fixed volume fraction here could be associated with one of two mechanisms: (i) physical
connectivity of the mineral network, as in Figure 3b, the high aspect ratio of the crystallites
contributing to the variation in modulus with prism orientation; or (ii) removal of the magnesium
and carbonate enriched centers of the enamel crystallites, which is known to occur in caries [15],

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15
 L5.15.6

140

120
Enamel
100

Modulus, E (GPa)
80

60

40
Dentine
20

0
0.0 0.2 0.4 0.6 0.8 1.0
Mineral Volume Fraction, Vf

Figure 4: Experimental data from Figure 1 after conversion of qBSE values to mineral
volume fractions. The dashed lines are Hashin-Shtrikman composite bounds.

Figure 3a. In either case, the high mineral content in enamel means that even a relatively minor
demineralization has a great effect on modulus.
In dentine, on the other hand, the mineral concentration is moderate. The observed
variability in sound dentine is small, both in modulus and in mineral content, and in carious
dentine a decrease in modulus results from a comparable percentage decrease in mineral content.

REFERENCES

1. L. Angker, C. Nocklands, M.V. Swain and N. Kilpatrick, Arch. Oral. Bio. 49, 369-378
(2004).
2. J.L. Katz. J Biomech. 4,455 (1971).
3. J.H. Kinney, M. Balooch, S.J. Marshall, G.W. Marshall, T.P. Weihs. Arch Oral Biol 41, 9
(1996).
4. C. Hellmich and F-J. Ulm, J Biomech 35, 1199 (2002).
5. M.L. Oyen and C-C. Ko, MRS Proceedings, 844, Y8.7.1 (2005).
6. A. Banerjee and A. Boyde, Caries Research. 32, 219 (1998).
7. A. Banerjee and A. Boyde, Scanning 19, 151 (1997).
8. V.L. Ferguson, A.J. Bushby, and A. Boyde, MRS Proceedings, 841, 3-8, (2005).
9. J.S. Field and M.V. Swain, J. Mater. Res. 8, 297 (1993).
10. A.J. Bushby, Nondestruct. Test. Eval. 17, 213 (2001).
11. A.J. Bushby, V.L. Ferguson and A. Boyde J. Mater. Res. 19, 249 (2004).
12. V.L. Ferguson, A.J. Bushby and A. Boyde J. Anat. 203, 191 (2003).
13. A.K. Bembey, V. Koonjul, A.J. Bushby, V.L. Ferguson and A. Boyde, MRS Proceedings,
841, 9-14, 2005.
14. L.D. Wegner and L.J. Gibson, Intl J of Mechanical Sciences 42, 925 (2000).
15. A. Boyde, J Dent Res. 58 special issue B:981 (1979).

Downloaded from https:/www.cambridge.org/core. UNSW Library, on 21 Mar 2017 at 03:24:56, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.
https://doi.org/10.1557/PROC-874-L5.15