Mechanical properties of mineralized collagen fibrils as influenced by demineralization.

Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.

Effects of polar solvents on the fracture resistance of dentin: role of water hydration.

Although healthy dentin is invariably hydrated in vivo, from a perspective of examining the mechanisms of fracture in dentin, it is interesting to consider the role of water hydration. Furthermore, it is feasible that exposure to certain polar solvents, e.g., those found in clinical adhesives, can induce dehydration. In the present study, in vitro deformation and fracture experiments, the latter involving a resistance-curve (R-curve) approach (i.e., toughness evolution with crack extension), were conducted in order to assess changes in the constitutive and fracture behavior induced by three common solvents-acetone, ethanol and methanol. In addition, nanoindentation-based experiments were performed to evaluate the deformation behavior at the level of individual collagen fibers and ultraviolet Raman spectroscopy to evaluate changes in bonding. The results indicate a reversible effect of chemical dehydration, with increased fracture resistance, strength, and stiffness associated with lower hydrogen bonding ability of the solvent. These results are analyzed both in terms of intrinsic and extrinsic toughening phenomena to further understand the micromechanisms of fracture in dentin and the specific role of water hydration.

Three-dimensional morphometry of the L6 vertebra in the ovariectomized rat model of osteoporosis: biomechanical implications.

This article summarizes the results of a three-dimensional study of changes in the morphology of the L6 rat vertebra at 120 days after ovariectomy (OVX), with estrogen replacement therapy used as a positive control. Synchrotron radiation microtomography was used to quantify the structural parameters defining trabecular bone architecture, while finite-element methods were used to explore the relationships between these parameters and the compressive elastic behavior of the vertebrae. There was a 22% decrease in trabecular bone volume (TBV) and a 19% decline in mean trabecular thickness (Tb.Th) with OVX. This was accompanied by a 150% increase in trabecular connectivity, a result of the perforation of trabecular plates. Finite-element analysis of the trabecular bone removed from the cortical shell showed a 37% decline in the Young's modulus in compression after OVX with no appreciable change in the estrogen-treated group. The intact vertebrae (containing its trabecular bone) exhibited a 15% decrease in modulus with OVX, but this decline lacked statistical significance. OVX-induced changes in the trabecular architecture were different from those that have been observed in the proximal tibia. This difference was a consequence of the much more platelike structure of the trabecular bone in the vertebra.

Mineral distribution and dimensional changes in human dentin during demineralization.

Many bonding agents require the dentin surface to be acid-etched prior to being bonded. Understanding the stability and morphology of the etched dentin surface is important for improving bond strength and reliability in these systems. In this study, the atomic force microscope was used to quantify dimensional changes that occur to fully hydrated dentin during demineralization with a pH 4.0 lactic acid gel. A high-resolution microtomography instrument, the x-ray tomographic microscope, was also used to quantify the mineral density distribution in the dentin as a function of etching time. The intertubular dentin surface shrank by less than 0.5 microns during etching, while the peritubular dentin receded at an initially rapid linear rate. The dentin surface retained its initial morphology, although it was more porous with the removal of the peritubular dentin. Beneath the etched surface, there were three major zones characterized by mineral density differences. The first zone was a fully demineralized collagen layer, subjacent to which was a partially demineralized zone of roughly constant mineral density. Immediately following the partially mineralized layer was normal dentin. The presence of the partially mineralized layer could be explained in terms of different transport rates in the peritubular and intertubular dentin.

Nanomechanical properties of hydrated carious human dentin.

Most restorative materials are bonded to caries-affected dentin that has altered structure. We tested the hypothesis that hydrated dentin of the transparent zone did not have increased hardness or elastic modulus. Nanoindentation by modified AFM was used to determine site-specific elastic modulus and hardness for components of hydrated dentin from 8 carious and non-carious human teeth. Indentations in intertubular dentin were made at intervals from pulp through the affected layers (subtransparent, transparent, and discolored zones). The values of intertubular dentin increased slightly from near the pulp into the transparent zone, then remained constant or decreased slightly through transparent dentin (E, 18.3 GPa; H, 0.8 GPa; confirming the hypothesis), and decreased markedly through the discolored region. Peritubular dentin values were unaltered in transparent dentin, and intratubular mineral had values between those of normal peritubular and intertubular dentin. Superficial areas contained distorted tubules without peritubular dentin or intratubular mineral.

Amelogenin-guided crystal growth on fluoroapatite glass-ceramics.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.

The threshold effects of Nd and Ho: YAG laser-induced surface modification on demineralization of dentin surfaces.

Laser irradiation alters the structure of dentin and produces surface layers that give the appearance of being more enamel-like. The laser-modified surface may be more resistant to demineralization; hence, many investigators are proposing continued development of the laser as a possible preventive treatment for caries. The purpose of this study was to explore the morphological changes that occur in dentin when treated at threshold illuminance with two clinically interesting laser wavelengths, and to evaluate the effectiveness of the laser-treated surface at resisting demineralization in an acid-gel solution. The Nd: YAG laser (wavelength 1060 nm) produced significant recrystallization and grain growth of the apatite, without the formation of second phases such as beta-tricalcium phosphate. This recrystallized surface layer showed resistance to demineralization; however, the layer did not provide protection of the underlying dentin from demineralization because of cracks and macroscopic voids that allowed for penetration of the demineralizing gel. The Ho: YAG laser-treated surface (wavelength 2100 nm) did not show significant evidence of recrystallization and grain growth, and only a trace amount of an acid-resistant layer was observed with demineralization. It is speculated that the Ho:YAG laser is coupling with absorbed water, and that the heat transfer from the water to the mineral phase is inefficient. For the purposes of creating a demineralization-resistant layer, threshold illuminance with both Nd: YAG and Ho: YAG was ineffective.

Ultrastructure and nanomechanical properties of cementum dentin junction.

The attachment between cementum and dentin has been given several definitions and nomenclature, including: interzonal layer, intermediate cementum, collagen hiatus, Hopewell-Smith's hyaline layer, and more commonly, cementum-dentin junction (CDJ). Understanding the attachment of two structurally dissimilar hard tissues such as cementum and dentin defined by a junction may provide information necessary to engineer functionally graded materials that can be used for efficient tooth restorations in clinical dentistry and other bioengineering applications. Hence, in this study, as a first step toward understanding the CDJ using a biomechanical approach, it was hypothesized that the CDJ between cementum and dentin is a wide zone with mechanical properties significantly lower than the neighboring tissues. The structure of the CDJ was studied using an atomic force microscope (AFM), and site-specific mechanical response of the three regions; cementum, CDJ, and dentin were determined using an AFM-nanoindenter under dry and wet conditions. The AFM results of the CDJ demonstrated a valley under dry conditions and a peak under wet conditions. The magnitude of the depth of the valley was approximately the same as the height of the peak of the CDJ, ranging from 10 to 40 microm. The nanomechanical properties under dry conditions indicated no significant difference (p > 0.05) in elastic modulus and hardness of the CDJ (Er = 17.5 +/- 2.7 GPa, H = 0.6 +/- 0.1 GPa) and cementum (Er = 18.7 +/- 2.5 GPa, H = 0.6 +/- 0.1 GPa). The mechanical properties of the CDJ were significantly lower (p << 0.05) than dentin (Er = 19.9 +/- 2.9 GPa, H = 0.6 +/- 0.1 GPa) under dry conditions. However, under more relevant hydrated conditions, the mechanical properties of CDJ (Er 3.0 +/- 0.7 GPa, H = 0.1 +/- 0.0 GPa) were significantly lower (p << 0.05) than those of cementum (Er 6.8 +/- 1.9 GPa, H = 0.2 +/- 0.1 GPa) and dentin (Er 9.4 +/- 2.3 GPa, H = 0.3 +/- 0.1 GPa). Based on the results from this study, it can be concluded that the CDJ can be regarded as a wide zone containing large quantities of proteins including collagen that contribute to hydration and significantly reduce mechanical properties, compared with the adjacent hard tissues, cementum, and dentin. The lower mechanical properties of the CDJ may make it possible for it to redistribute occlusal loads to the alveolar bone.

Micro-Raman spectroscopic investigation of dental calcified tissues.

The purpose of this study was to determine if dental calcified junctions (DEJs/CDJs) in human teeth contain different compositional phases compared to the adjacent dental calcified tissues. Peak positions and intensities were determined from micro-Raman spectra for PO(3-) (4) and the C--H modes and compared among the mineralized tissues and their junctions. Values of width were determined from the intersections of intensity regression lines through the junctions and in the adjacent tissues. The peaks were measured in 1-microm steps along a l00-microm line across the junction. High-resolution analysis revealed that PO(3-) (4) band peaks for dentin, the DEJ, enamel, the CDJ, and cementum were at the same position (959 cm(-1)), while for the C--H stretching mode a significant shift of 4.6 cm(-1) was found between enamel, the DEJ, and dentin. The mean width of the DEJ was 7.6 (+/- 2.8) microm using the PO(3-) (4) band and 8.6 (+/- 3.6) microm using the C--H stretching mode. Across the DEJ, the mineral content monotonically decreased from enamel to dentin while the organic component monotonically increased. The DEJ width was in agreement with prior nanoindentation studies. No width estimate was possible for the CDJ because the compositional differences between cementum and dentin were small.

Optical spectroscopy and imaging of the dentin-enamel junction in human third molars.

A 351-nm laser excitation source was used to perform autofluorescence microscopy of dentin, enamel, and the dentin-enamel junction (DEJ) to obtain information regarding their morphology and spectral characteristics. The emission spectra of these calcified dental tissues were different from one another, and this enabled the DEJ to be imaged and dimensionalized. The DEJ displayed sharp and clearly delineated borders at both its enamel and dentin margins. The dentinal tubules and the enamel prisms appeared to terminate abruptly at the DEJ. The median DEJ width was 10 microm, ranging from 7 to 15 microm, and it did not appear to depend on intratooth position.

Evaluation of a new modulus mapping technique to investigate microstructural features of human teeth.

Teeth contain several calcified tissues with junctions that provide interfaces between dissimilar tissues. These junctions have been difficult to characterize because of their small size. In this work a new technique using a combination of atomic force microscopy (AFM) and a force-displacement transducer was used to simultaneously study the surface topography and map mechanical properties of the junctions and adjacent hard tissues. Prepared specimens from human third molars were scanned by an AFM piezo-tube in contact mode. To measure the dynamic viscoelastic properties of the material a small sinusoidal force was superimposed on the contact force and the resulting displacement amplitude and the phase shift between the force and amplitude were measured. This force modulation technique was used to map the local variation of nanomechanical properties of intertubular dentin, peritubular dentin, enamel, dentin-enamel junction (DEJ) and peritubular-intertubular dentin junction (PIJ). This new technique allowed us to measure the widths of these junctions in addition to local variation in dentin and enamel without causing plastic deformation to the material and with 2 orders of magnitude increase in spatial resolution compared with previous studies that used discrete nanoindentation techniques. Due to the ability to analyze the sample line-by-line, the distribution functions associated with the width of the DEJ and PIJ were conveniently obtained for specific intratooth locations. The data suggested, for three third molar specimens, a DEJ width of 2-3 microm with full-width half-maximum (FWHM) of 0.7 microm and PIJ width of 0.5-1.0 microm with 0.3 microm FWHM. The intertubular dentin storage modulus variation was between 17 and 23 GPa with a mean value of 21 GPa. The range of storage modulus for enamel near the DEJ was between 51 and 74 GPa with a mean value of 63 GPa.

Tip-radius-induced artifacts in AFM images of protamine-complexed DNA fibers.

Isolated DNA fibers complexed with protamine (the chromosomal protein that packages DNA in mammalian sperm) have been produced by partially decondensing the highly compacted mouse sperm chromatin particle on a glass coverslip. These DNA fibers were then scanned with the atomic force microscope (AFM). While the smallest of the fibers appear in AFM images as ribbon-like structures 250-350 A wide and 10-25 A high, experiments indicate that these images are the result of a convolution of the imaging-tip's shape with the object's actual shape. In such convolutions the height of the object is affected only by the compressibility of the object, while the width is affected in addition by the sharpness of the tip. Images of polyamidoamine particles also appear to show this artifact. We have also deduced the tip's radius of curvature from images of sharp steps and attempt to demonstrate the artifacts associated with a relatively large imaging tip.

Atomic-force microscopic study of dimensional changes in human dentine during drying.

Six 1-mm thick sections of human dentine, three parallel to the occlusal surface and three perpendicular to the buccal surface, were prepared from non-carious third molars. The enamel was ground off, and the sections were polished with alumina powder to remove the smear layer. Each section was imaged by atomic-force microscopy with 20 nm horizontal and 0.1 nm vertical resolutions, initially while the samples were immersed in deionized water and then periodically during drying at room temperature. No dimensional changes over microscopic fields of view (scanned areas smaller than 50 x 50 microns) could be detected within the precision of the measurements (< 0.5%). Across the entire sample, however, vertical displacements of 10-20 microns were measured. Elasticity (Bernoulli beam) theory was used to calculate the engineering strain required to produce these displacements. The magnitude of the strain was 0.04% (SD = 0.01) in the buccal sections in the direction of the tubule axis and 0.09% (SD = 0.02) in the direction normal to the tubule axis. Also, the strain alternated between tension and compression across the samples. It was concluded that, as determined by using microscopic techniques, drying-induced strain is too small to require corrections for tubule size and tubule density.

Mechanical properties of human dental enamel on the nanometre scale.

Atomic force microscopy (AFM) combined with a nano-indentation technique was used to reveal the structure and to perform site-specific mechanical testing of the enamel of third molars. Nano-indentations (size<500 nm) were made in the cusp area to measure the mechanical properties of single enamel rods at different orientations. The influence of etching on the physical properties was studied and etching conditions that did not significantly alter the plastic-elastic response of enamel were defined. Elasticity and hardness were found to be a function of the microstructural texture. Mean Young's moduli of 87.5 (+/-2.2) and 72.2 (+/-4.5) GPa and mean hardness of 3.9+/-0.3 and 3.3+/-0.3 GPa were measured in directions parallel and perpendicular to the enamel rods, respectively. Analysis of variance showed that the differences were significant. The observed anisotropy of enamel is related to the alignment of fibre-like apatite crystals and the composite nature of enamel rods. Mechanical properties were also studied at different locations on single enamel rods. Compared to those in the head area of the rods, Young's moduli and hardness were lower in the tail area and in the inter-rod enamel, which can be attributed to changes in crystal orientation and the higher content of soft organic tissue in these areas.

A micromechanics model of the elastic properties of human dentine.

A generalized, self-consistent model of cylindrical inclusions in a homogeneous and isotropic matrix phase was used to study the effects of tubule orientation on the elastic properties of dentine. Closed-form expressions for the five independent elastic constants of dentine were derived in terms of tubule concentration, and the Young's moduli and Poisson ratios of peri- and intertubular dentine. An atomic-force microscope indentation technique determined the Young's moduli of the peri- and intertubular dentine as approx. 30 and 15 GPa, respectively. Over the natural variation in tubule density found in dentine, there was only a slight variation in the axial and transverse shear moduli with position in the tooth, and there was no measurable effect of tubule orientation. It was concluded that tubule orientation has no appreciable effect on the elastic behaviour of normal dentine, and that the elastic properties of healthy dentine can be modelled as an isotropic continuum with a Young's modulus of approx. 16 GPa and a shear modulus of 6.2 GPa.

Hardness and Young's modulus of human peritubular and intertubular dentine.

A specially modified atomic-force microscope was used to measure the hardness of fully hydrated peritubular and intertubular dentine at two locations within unerupted human third molars: within 1 mm of the dentine enamel junction and within 1 mm of the pulp. The hardness of fully hydrated peritubular dentine was independent of location, and ranged from 2.23 to 2.54 GPa. The hardness of fully hydrated intertubular dentine did depend upon location, and was significantly greater near the dentine enamel junction (values ranged from 0.49 to 0.52 GPa) than near the pulp (0.12-0.18 GPa). A Nanoindenter was used to estimate the Young's modulus of dehydrated peritubular and intertubular dentine from the unloading portion of the load displacement curve. The modulus values averaged 29.8 GPa for the peritubular dentine (considered to be a lower limit), and ranged from 17.7 to 21.1 GPa for the intertubular dentine, with the lower values obtained for dentine near the pulp.

Atomic force microscopy of acid effects on dentin.

Atomic force microscopy (AFM) was used in the examination of the early stages of acid treatment of dentin. Disks of highly polished dentin were initially examined under deionized water and following exposure to 0.025 M nitric acid for 20 s intervals from 0-100 s. Peritubular depth changes were linear (0.005 microns/s). The intertubular dentin surface initially moved at approximately 1/2 the peritubular rate and then reached a plateau as the demineralized collagen scaffold collapsed. There was no apparent difference in the tubule center-to-center distance during the treatment. Differences in the movement and morphology of the zones are of importance in dentin bonding applications relying on penetration of the demineralized dentin by adhesive monomers. The changes are probably related to the partial collapse of the collagen matrix. Alternatively, access to the apatite crystals and solubility may be higher in the peritubular zone. AFM appears to hold exceptional promise for the study of conditioning and priming agents for dentin bonding.

Dentin demineralization: effects of dentin depth, pH and different acids.

OBJECTIVES: This investigation sought to determine: 1) if dentin demineralization rates are proportional to acid concentration for demineralization in phosphoric acid (10% or 1.76 M, 0.025 M, 0.0001 M, with pH = 0.95, 2.0, 4.0 respectively); 2) if the etching characteristics are independent of dentin depth; and 3) if the etching characteristics for phosphoric acid were comparable to those for citric acid over a similar pH range. METHODS: Highly polished dentin disks from freshly extracted, non-carious, third molars were prepared with a reference layer. Samples were prepared from either superficial or deep coronal dentin. The samples were etched for periods of up to 30 min using phosphoric acid solutions (pH = 0.95, 2.0, 4.0) in a wet cell of an atomic force microscope (AFM). Depth changes with respect to the reference layer were determined for the intertubular and peritubular dentin to quantify structural changes. The results were compared with similar studies using citric acid (pH = 1.0, 2.15 or 3.4). Etching characteristics were statistically compared using 2-way repeated measures ANOVA at p < 0.05 and the Tukey's multiple comparison test. RESULTS: The relation between time and recession for peritubular dentin was initially linear. The intertubular dentin recession started rapidly but then reached a plateau within a very short interval for etching solutions at pH = 2.0 and 4.0. At the highest concentration, the recession decreased with time, but a clear plateau was not established. There was no statistical difference between peritubular etching rates of superficial and deep dentin surfaces with phosphoric acid at any concentration. There was also no difference in the intertubular dentin recession at the location of the plateau that depended on dentin depth. Etching rates increased dramatically with decreased pH for both phosphoric and citric acids, but were higher for citric acid than for phosphoric acid. SIGNIFICANCE: The AFM allowed quantification of changes during etching of wet dentin. Peritubular dentin etching rates increased with decreasing pH, as expected, but changes were not linear and were different for the two acids studied over a similar pH range. Intertubular dentin surface recession was small and plateaued for low concentrations. The peritubular etching rate and intertubular dentin recession did not depend on dentin depth.

The dentin substrate: structure and properties related to bonding.

OBJECTIVES: Dentin is a vital, hydrated composite material with structural components and properties that vary with location. These variations are reviewed along with alterations by physiological and pathological changes that allow classification into various forms of dentin. Structural characteristics and mechanical properties are reviewed and the limitations of our understanding of structure-property relationships for normal and modified forms of dentin are discussed with respect to their impact on dentin bonding. Recent progress in methods available to study dentin and its demineralization are emphasized with their promise to increase our understanding of dentin properties and structure. DATA SOURCES: Recent microstructural studies, focusing on scanning electron microscopy, atomic force microscopy and X-ray tomographic microscopy are included. A review of fundamental studies with emphasis on microstructurally sensitive methods, and prior reviews of basic mechanical properties are included with discussion of their correlation to composition and structure. STUDY SELECTION AND CONCLUSIONS: Emphasis in this work was placed on the major structural components of the tissue, including the collagen based organic matrix and its mineral reinforcement, the distribution of these components and their microstructural organization as related to mechanical properties and response to demineralization. Little information is included on biochemical and developmental studies or on non-collagenous proteins and other organic components for which limited understanding is available with respect to their role in structure-property relations and influence on bonding. In spite of the fact that the complexity of dentin precluded a comprehensive review, it is clear that local structural variations influence properties and impact nearly all preventive and restorative dental treatments. Much more work is needed in order to understand differences between vital and non-vital dentin, and dentin from extracted teeth. Although our knowledge is rudimentary in certain areas, increasingly sophisticated methods of studying dentin should provide the necessary information to model structure-property relations, optimize dentin bonding, and improve many aspects of preventive and restorative dentistry.

Coupled Nanomechanical and Raman Microspectroscopic Investigation of Human Third Molar DEJ.

The dentino-enamel junction (DEJ) connects enamel, that covers the outer surface of a tooth, to a thicker underlying dentin. The DEJ is a critical interface that permits joining these materials that have widely dissimilar mechanical properties. AFM-based nanoindentation and Raman microspectroscopy were used to define the width and composition of human molar DEJ. Indentation elastic modulus and hardness of enamel, dentin, and DEJ were determined along lines of indents made at 2 µm intervals across the DEJ. Indents made at maximum loads at each end of the indent lines were used to make visible markers allowing Raman microspectroscopy at 1 µm intervals across the DEJ, while using the nanoindent markers for orientation and location. Functional DEJ width estimates were made based on results from nanoindentation and Raman microspectroscopy. DEJ width estimates ranged from 4.7 (±1.2) µm to 6.1 (±1.9) µm based on hardness and 4.9 (±1.1) µm to 6.9 (±1.9) µm based on modulus. DEJ width based on Raman peak intensity variations were 8.0 (±3.2) µm to 8.5 (±3.1) µm based on the phosphate peak, and 7.6 (±3.2) µm to 8.0 (±2.6) µm for C-H stretching mode. These estimates are in the range of DEJ width estimates reported using nanoindentation.