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Micromechanical Property Recovery of Human Carious Dentin Achieved with Colloidal Nano-β-tricalcium Phosphate
1 Department of Oral Biomaterials and Technology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; and Correspondence: * corresponding author, yookun{at}dent.showa-u.ac.jp
Reconstitution of carious dentin has been recognized as difficult, because it progresses by loss of collagen polymerization and by demineralization under acidic conditions. Recently, colloidal alkaline nano-calcium phosphate, prepared by electrical discharge in a buffered physiological saline solution, has been shown to be effective in the formulation of a bone-like biocomposite by simply being mixed with acidic collagen solution. It was hypothesized that colloidal calcium phosphate was suitable for the reconstitution of carious dentin. Natural caries lesions in dentin from permanent teeth were exposed to colloidal hydroxyapatite and β-tricalcium phosphate for 10 days. The micromechanical properties of these tissues were evaluated by nano-indentation. The elastic modulus of human carious dentin improved after samples were immersed in colloidal β-tricalcium phosphate. The mineral density of carious dentin exposed to β-tricalcium phosphate increased more than that immersed in hydroxyapatite. However, since it was not directly proportional to micromechanical recovery, mineral density alone was not a sufficient indicator of mechanical behavior.
Key Words: Nanoindentation • dentin • calcium phosphate • collagen
Dental caries is one of the most widespread and costly infectious diseases remaining to be overcome (Mishra et al., 2006). Human teeth normally undergo continuous balanced demineralization and remineralization in the oral environment (Yamaguchi et al., 2006). If this balance is disrupted, particularly when caused by acidogenic bacteria, demineralization will progress, leading to a deterioration of the structure through a process known as dental caries. Traditional clinical practice has recommended complete removal of softened and discolored dentin, to eliminate infected tissue and create a hard foundation to support a proposed restoration such as composite resin or metal alloys. The routine usually followed has been to remove all demineralized carious dentin, by hand instrumentation or with a round bur, until sound normal dentin forms the entire pulpal floor. This is carried out particularly for non-adhering metal replacement restorative procedures. This technique is far from ideal, because excessive sound dentin is removed, as was advocated by Dr. G.V. Black. Reconstitution of carious dentin is a more desirable clinical approach than the traditional clinical practice. However, it has been thought of as being more difficult than that for enamel, because it involves two dissimilar phases: organic type-I collagen and inorganic apatite nanophases that have a specific spatial relationship with one another (He et al., 2003). The bio-composite structure of dentin is denatured by the demineralization and subsequent breakdown of collagen polymerization under acidic carious conditions (Tjäderhane et al., 1998). This implies that remineralization alone is insufficient for the total recovery of demineralized carious dentin. Calcium phosphate particles are promising candidates for remineralization of calcified tissue, because they are the main inorganic components of dentin and yield complex macromolecular assemblies (Perkin et al., 2005). Commercially available pure calcium phosphates are not suitable as experimental samples, because of their lower reactivity and penetration within the lesion due to their particle size (Kurashina et al, 1997; Zhao et al., 2002; Shibata et al., 2005). However, nanoscale calcium phosphates might increase the specific surface area available for conjugation of dentin organic matrix that remains in caries lesions. Preparation of calcium phosphate particles by electrical discharge in a buffered physiological saline solution has several advantages over traditional techniques (Shibata et al., 2004, 2005; Takashima et al., 2004). Specifically, it can generate various highly crystalline carbonated nanoscale (approx. 100-nm-diameter) calcium phosphate compounds, such as hydroxyapatite (HA) or β-tricalcium phosphate (β-TCP), without yielding additional impurities. The nanoscale calcium phosphates prepared by discharge are associated with improved performance in vivo (Yamamoto et al., 2006). The alkaline colloidal nano-calcium phosphates, especially colloidal β-TCP, are also attractive for the synthesis of human bone-like collagen composites, by simply being mixed with acidic type-I collagen solution. This occurs because of the catalysis of the polyphosphate chain produced during the mixing, as specifically found in the case of colloidal β-TCP/collagen composite (Shibata et al., 2005). Mineralized tissues, such as bone and dentin, are comprised of organic biomolecules integrated with oriented carbonated calcium phosphate nano-crystals (He et al., 2003). For carious dentin to be reconstituted, recovery of collagen polymerization and re-mineralization must be achieved at the same time. Thus, nano β-TCP prepared by electrical discharge might be expected to be useful in the reconstitution of carious dentin. An understanding of the mechanical properties of dentin is particularly important, especially the measurement of the mechanical properties of carious dentin, since this provides a direct means of assessing the effectiveness of its reconstitution (Kinney et al., 2003a). However, conventional mechanical tests, such as compressive, tensile, and bending tests, cannot be readily applied to human dentin, because of the local variations in structure of carious or sound dentin structures within a small area. Alternatively, nano-indentation allows for a more comprehensive assessment of the mechanical properties of dentin (He et al., 2006). Many tests can be conducted on a small selected region of the dentin, and accurate measurements can be made in different areas of the tooth. The present study was designed to investigate the reconstitution of carious dentin by the use of alkaline colloidal calcium phosphates. The mechanical properties of sound and carious dentin, with and without immersion in colloidal calcium phosphates, were measured with the use of an Ultra Micro-Indentation System (UMIS, CSIRO, Lindfield, Australia). Local mineralization adjacent to the respective micro-indentations was evaluated by energy-dispersive x-ray (EDX) spectroscopy under a scanning electron microscope (SEM) (XL-30, Philips, Eindhoven, The Netherlands). We hypothesized that carious dentin treated with nano-β-TCP would show better recovery of micromechanical properties than that treated with nano-HA.
We used 2 physiological buffered solutions—Hanks’ balanced salt solution (HBSS) without addition of organic molecules, and modified simulated body fluid (m·SBF)—to prepare colloidal HA and colloidal β-TCP, respectively (Shibata et al., 2005). Briefly, one piece of platinum foil (100 x 50 x 0.1 mm) was used as the power supply cathode, and another (10 x 30 x 0.1 mm) as the counter-electrode. Each piece was immersed in 100 mL of HBSS without organic molecules and m·SBF. Subsequently, discharge was maintained at 2.5 A and 100 V for 270 sec. Alkaline HA and β-TCP were prepared by removal of supernatants after processing (pH 11.3, particle diameter approx. 100 nm).
Human carious molar teeth extracted for periodontal or orthodontic reasons were used in this study under a protocol approved by the Ethics Committee of the University of Sydney, Australia (see APPENDIX). Sectioned samples (Fig. 1
The indentation experiments were performed with the use of a nano-based indentation system (Ultra Micro-Indentation System, UMIS-2000, CSIRO, Lindfield, Australia). A Berkovich indenter (Synton, Lyss, Switzerland) was used for this study. Fused silica acted as the standard calibration material for the determination of nano-indenter Berkovich tip and instrument compliance (Oliver and Pharr, 2004). Indentations (9 tests) were placed in both sound and carious dentin at a maximum force of 20 mN. Indentation tests were conducted at a loading/unloading rate of 1 mN/sec, along with a hold time at maximum load of 60 sec, to enable creep relaxation to occur. Typically, 20 datapoints on the loading and unloading portions of the curves were generated. The distance between indents was maintained at more than 50 µm to avoid any influence of residual stresses from adjacent indentations. After immersion in colloidal calcium phosphates, indentations were re-applied adjacent to the initial indented region before immersion. The hardness and elastic modulus were calculated from the force-displacement curves by means of the UMIS proprietary software. A Poisson’s ratio of 0.3 was used for this material, and the Oliver-Pharr method was used (Oliver and Pharr, 2004) for calculations (Fig. 2A  ).
The samples were subsequently coated with a very thin carbon film. An EDX equipped with SEM (XL-30, Philips, Eindhoven, The Netherlands) was used to produce back-scattered electron images and determine the elemental composition adjacent to each indentation. The aim of this analysis was the comparison of calcium/phosphate ratios between HA- and β-TCP-immersed samples. The authors expected that the mineral deposition of the β-TCP-immersed samples would be better than that of HA-immersed samples. Nine indentations (n = 9) performed in both sound and carious dentin, respectively, within the individual teeth before and after immersion in colloidal calcium phosphates were compared. The results were expressed as the mean ± SD, and analyzed statistically by analysis of variance (ANOVA) with follow-up by a modified Tukey t test. Significant differences were considered to exist when p < 0.01. These tests were repeated on all samples, and their reproducibility was confirmed. Representative sample data are shown below.
The indenter penetration depths at maximum loading before creep on sound dentin, carious dentin, HA-immersed sample, and β-TCP sample were 1.83 ± 0.08 µm, 2.45 ± 0.12 µm, 2.47 ± 0.06 µm, and 2.10 ± 0.05 µm, respectively. The force-displacement curves of sound dentin were unchanged after immersion in either solution. As well, no differences appeared between carious dentin with or without colloidal HA (data not shown). A comparison of the force-displacement curves of carious dentin treated with or without β-TCP showed that the initial loading component of carious dentin after immersion in colloidal β-TCP improved and was much closer to that of sound dentin. However, more creep was observed with the treated carious dentin at maximum load than was seen for untreated carious dentin (Fig. 2B  ).
The elastic modulus (E) and hardness (H) of carious dentin (E = 4.46 ± 0.76 GPa, H = 0.17 ± 0.03 GPa) were significantly lower (p < 0.01) than those of sound dentin (E = 9.93 ± 1.62 GPa, H = 0.30 ± 0.04 GPa) before immersion (Fig. 3
Representative EDX spectra of the samples are shown (Fig. 4 ). The calcium/phosphate atomic ratio of sound dentin was around 1.61 ± 0.01; that of carious dentin with colloidal β-TCP recovered to 1.54 ± 0.01, whereas that of the HA-exposed sample was 1.41 ± 0.01. The mineral content of carious dentin was significantly higher (p < 0.01) when exposed to colloidal β-TCP than that exposed to HA.
The loading component of the force-displacement curves showed that, initially, there was less displacement in carious dentin treated with colloidal β-TCP. Since mineral density in the treated samples had increased, we conclude that colloidal β-TCP treatment improved the resistance to indenter penetration. In the case of a metallic or elastic-plastic material, this increased resistance may be associated with an increased hardness or yield stress and elastic modulus. However, in the case of a porous material, such as demineralized carious dentin, the force-displacement curve and creep response is associated with fluid displacement and inelastic deformation of the remnant collagen/apatite structure. That is, the deformation is influenced by the permeability of the carious dentin. The higher stiffness of the unloading curve, however, appears to be responsible for the higher elastic modulus of the β-TCP-treated samples. Since more creep was observed, this suggests that there had been a reduction in permeability and consolidation or densification of the structure, thus leading to a lower hardness than in sound dentin. This implies that the β-TCP nanoparticles may have incompletely filled the porous residual carious dentin. However, even though the particle diameters and pH values of both colloidal calcium phosphates were the same (Shibata et al., 2005), the calcium/phosphate ratio of carious dentin treated with colloidal HA was significantly lower and showed no micromechanical property recovery. Thus, we assume that this recovery was caused not only by particle penetration, but also by some chemical reactions between the residual structure and β-TCP particles. This phenomenon can also be interpreted on the basis of the following recent studies. It has been proposed that apatite mineral occupies two sites within the collagen scaffold of dentin: intra-fibrillar (inside the periodically spaced gap zones in the collagen fibril) and extra-fibrillar (in the interstices between fibrils) (Kinney et al., 2003a). Extra-fibrillar mineral is more readily dissolved than intra-fibrillar in carious regions (Kinney et al., 2003b). This situation suggests that dentin remineralization may occur preferentially in extra-fibrillar rather than in intra-fibrillar sites (Suppa et al., 2006). However, extra-fibrillar mineralization demonstrates limited improvement of the micromechanical properties of the dentin (Kinney et al., 2003a). These authors also suggest that intra-fibrillar remineralization may play a crucial role in elastic stiffness of caries-affected dentin. Since effective mineralization is created between intra-fibrillar collagen molecules via the formation of specific cross-linking that yields a proper gap zone (Saito et al., 2006), intermolecular cross-linking recovery of denatured collagen may play a pivotal role in recovery of the micromechanical properties of carious dentin through intra-fibrillar remineralization. Previously, we demonstrated that bone-like collagen biocomposite can be formulated by simply mixing an acidic collagen solution and alkaline colloidal β-TCP, rather than colloidal HA (Shibata et al., 2005). In our earlier study, collagen molecules were polymerized together to form a highly cross-linked β-TCP/collagen composite (Shibata et al., 2005). The dispersed collagen molecules in β-TCP/collagen composite showed higher intermolecular assembly and covalent bonding between the Ca++ and RCOO– ions of the collagen fibers than that of HA/collagen, as determined by x-ray photoelectron spectroscopy. These reactions were greatly increased by the catalysis of polyphosphate generated during mixing. As predicted from previous work, the present study showed that the recovery of elastic properties was achieved in carious dentin only with colloidal β-TCP. The process of reconstitution of carious dentin can thus be explained as follows. Colloidal β-TCP particles were partially dissolved in the acidic carious regions. Subsequently, residual intermolecular collagen cross-linking was recovered in a manner similar to that proposed in our previous study. For colloidal β-TCP, the dissolved ions combined with appropriate intra-fibrillar sites, and crystallization occurred. The present study demonstrated that the elastic modulus of carious dentin treated with colloidal β-TCP was improved and approached that of sound dentin, despite the fact that mineral density was still lower than that of sound dentin. This also supports the concept that remineralization occurs at intra-fibrillar rather than extra-fibrillar sites. The authors hypothesize that, since the chemical reactions were incomplete, because the β-TCP nanoparticles were not well-dissolved under the carious conditions, intermolecular collagen cross-linking was not fully achieved. Therefore, deposited minerals were displaced during indentation holding time at maximum loading. Within the caries-affected area, the hypermineralized transparent zone becomes layered in a natural remineralization process, thought to be the result of a dentin-pulp reaction to mild stimulation in an attempt to block acid penetration (Kidd and Joyston-Bechal, 1997). However, results from a recent microscopic study suggest that intra-fibrillar remineralization is difficult, even in a biological setting, because the collagen fibrils are sparse in this region, appear collapsed and swollen, and exhibit extensive branching when compared with those in sound dentin (Suppa et al., 2006). Although micromechanical property recovery was limited, results of the present study suggested the possibility of intra-fibrillar remineralization in caries-affected dentin. Therefore, colloidal β-TCP can be considered a promising candidate for further remineralization studies, despite the fact that the micromechanical improvement was limited. Until recently, evidence of structural recovery of carious enamel and dentin has been largely based on results obtained by densitometry (Arends et al., 1997). However, recent studies suggest that mineral concentration alone may not be an appropriate endpoint for assessing the success or failure of these processes in dentin (Kinney et al., 2003a; Suppa et al., 2006). Since the mechanical properties of carious dentin treated with colloidal calcium phosphates were also not directly proportional to mineral density in the present study, the authors suggest that micromechanical studies are more appropriate and should be the method applied in future remineralization studies. In conclusion, when compared with colloidal HA, colloidal β-TCP was shown to achieve better recovery of micromechanical properties in carious dentin. Micromechanical evaluations should be included in future remineralization studies, since it was shown here that mineral density alone is not sufficient as an indicator of mechanical behavior.
This work was supported by MEXT, HAITEKU (2006), a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for the Encouragement of Young Scientists (B) from The Ministry of Education, Culture, Sports, Science and Technology of Japan.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/3/233/DC1. Received for publication April 4, 2007. Revision received October 29, 2007. Accepted for publication November 27, 2007.
Journal of Dental Research, Vol. 87, No. 3,
233-237 (2008)
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ABSTRACT
INTRODUCTION
A, where dP/dh is the slope of the unloading curve at maximum force. (B) A representative comparison of the force-displacement curves of carious dentin treated with or without β-TCP (n = 9). The initial loading component of carious dentin after immersion in colloidal β-TCP appears substantially improved and is much closer to that of sound dentin. However, as can be observed, more creep occurs with this sample at maximum load than for untreated carious dentin.
