Aspartic acid racemization of root dentin used for dental age estimation in a Polish population sample.

Precise age determination of unidentified bodies and human remains is one of the essential tasks of forensic science. The aim of this study was to assess the applicability of using the enantiomeric composition of aspartic acid racemization in root and crown dentin for dental age estimation using a Polish population sample. Coronal and root dentin from four teeth groups from the mandible were studied using gas chromatography with mass spectrometry. The results demonstrated a very high correlation between the chronological age and enantiomeric composition in both of the dentin samples. Individual linear equations of root dentin with correlation coefficients between 0.96 and 0.98 and a standard estimation error of ±2.95-4.84 years validated the application of aspartic acid racemization as a significant practical contribution to everyday forensic medical practice. Discrepancies in methodological aspects and modifications that simplify the protocol are presented.

Use of XRD and SEM/EDX to predict age and sex from fire-affected dental remains.

In fire scenarios, the application and accuracy of traditional odontological methods are often limited. Crystalline studies and elemental profiling have been evaluated for their applicability in determining biological profiles (age and sex) from human dentition, particularly fire- and heat-affected dental remains. Thirty-seven teeth were paired according to tooth type and donor age/sex for the analysis of crown and root surfaces pre- and post-incineration using X-ray diffraction (XRD) and scanning electron microscopy (SEM/EDX). In unburned crowns, carbon (C) content showed a positive correlation with age, whereas phosphorus (P) and calcium (Ca) contents showed a negative correlation with age. In unburned roots, C, P and Ca contents also showed significant changes that were opposite of those observed in the crowns. In relation to sex, females exhibited a higher C ratio than males, whereas males showed significantly higher levels of oxygen (O), P and Ca in unburned roots. Incineration resulted in an increase in the crystallite size that correlated with increasing temperature. No differences in hydroxyapatite (HA) crystallite size were found between age groups; however, unburned teeth from females exhibited a larger crystallite size than did those from males. The challenges of using XRD with a 3D sample were overcome to allow analysis of whole teeth in a nondestructive manner. Further studies may be useful in helping predict the temperature of a fire.

CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro.

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.

Analysis of the erosive effect of different dietary substances and medications.

Excessive consumption of acidic drinks and foods contributes to tooth erosion. The aims of the present in vitro study were twofold: (1) to assess the erosive potential of different dietary substances and medications; (2) to determine the chemical properties with an impact on the erosive potential. We selected sixty agents: soft drinks, an energy drink, sports drinks, alcoholic drinks, juice, fruit, mineral water, yogurt, tea, coffee, salad dressing and medications. The erosive potential of the tested agents was quantified as the changes in surface hardness (ΔSH) of enamel specimens within the first 2 min (ΔSH2-0 = SH2 min - SHbaseline) and the second 2 min exposure (ΔSH4-2 = SH4 min - SH2 min). To characterise these agents, various chemical properties, e.g. pH, concentrations of Ca, Pi and F, titratable acidity to pH 7·0 and buffering capacity at the original pH value (ß), as well as degree of saturation (pK - pI) with respect to hydroxyapatite (HAP) and fluorapatite (FAP), were determined. Erosive challenge caused a statistically significant reduction in SH for all agents except for coffee, some medications and alcoholic drinks, and non-flavoured mineral waters, teas and yogurts (P < 0·01). By multiple linear regression analysis, 52 % of the variation in ΔSH after 2 min and 61 % after 4 min immersion were explained by pH, ß and concentrations of F and Ca (P < 0·05). pH was the variable with the highest impact in multiple regression and bivariate correlation analyses. Furthermore, a high bivariate correlation was also obtained between (pK - pI)HAP, (pK - pI)FAP and ΔSH.

Age estimation in forensic sciences: application of combined aspartic acid racemization and radiocarbon analysis.

Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster because the age at death, birth date, and year of death as well as gender can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization, has shown reproducible and more precise results. In this study, we analyzed teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that aboveground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ((14)C), which has been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel, and 10 of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R(2) = 0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 1.0 +/- 0.6 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 +/- 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

Effect of acid etching time on demineralization of primary and permanent coronal dentin.

PURPOSE: To determine the effect of acid etching time on dentin calcium solubilization and to compare the solubilization rate of primary and permanent dentin when etched for the same period of time. METHODS: Flat coronal dentin surfaces were produced on primary (n=8) and permanent (n=8) sound teeth. Three 2 mm-diameter areas were delimited on each surface and assigned to 5, 10 or 15 seconds of acid etching. Five microliters of 35% H3PO4 were applied on each area during the preset times, and 4 microL were retrieved for analysis. The amount of calcium was determined colorimetrically using Arsenazo III reagent and expressed as microg Ca/mm2 of dentin. Data were subjected to two-way ANOVA, Tukey's test and linear regression (alpha=5%). RESULTS: For both primary and permanent dentin, a statistically significant correlation was observed between acid etching time and calcium concentration (primary dentin R2 = 0.79; permanent dentin R2 = 0.73). Mean (sd) values of dissolved primary dentin after 5, 10 and 15 seconds were 1.00 (0.25), 1.74 (0.11) and 2.30 (0.42), whereas for permanent dentin the values were 0.47 (0.13), 0.89 (0.36) and 1.38 (0.20) microg Ca/mm2, respectively. Greater calcium solubilization (P<0.05) was detected for primary dentin than for permanent dentin after all acid etching times.

Distribution of enamel crystallite orientation through an entire tooth crown studied using synchrotron X-ray diffraction.

The biomineralization of human dental enamel has resulted in a highly anisotropic and heterogeneous distribution of hydroxyapatite crystallites, which in combination with its high mineral content has resulted in one of the most durable and hardest tissues in the human body. In this study, we used position-sensitive synchrotron X-ray diffraction to quantify the spatial variation in the direction and magnitude of the preferred orientation of enamel crystallites across a whole tooth crown. Two-dimensional synchrotron X-ray diffraction images were collected with 300 µm spatial resolution over a series of six sequential tooth sections obtained from a single maxillary first premolar and were analyzed using Rietveld refinement. Both the magnitude and the direction of the crystallite orientation were found to have a high spatial heterogeneity. Areas of high crystallite alignment were directed perpendicular to the biting surfaces, which is thought to meet the functional requirements of mastication. The results may assist in our understanding of the structure-function relationship and of the evolutionary development of enamel.

Recovery and identification of mature enamel proteins in ancient teeth.

Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science.

Bond integrity and microleakage of dentin-bonded crowns cemented with bioactive cement in comparison to resin cements: in vitro study.

OBJECTIVE: The aim was to compare restorative marginal integrity of ceramic crowns luted with bioactive and resin cements using micro-computed tomography (micro-CT) microleakage evaluations and bond strength assessment. METHODS: Thirty molar teeth were prepared by sectioning and polishing for dentin exposure for resin cement build-ups. Teeth were randomly divided among three groups of cements: (1) bioactive (ACTIVA); (2) glass ionomer cement (GIC; Ketac Cem); and (3) resin luting agent (Nexus 3). Bonding regime and build-ups (4 mm × 2 mm) were performed using the recommended protocol. For microleakage assessment, 30 premolar teeth were prepared for dentin-bonded crowns using lithium disilicate ceramic and the computer-aided design and computer-aided manufacturing technique. Crowns were cemented with standard load, cement amount, and duration using three cements (Group A: bioactive; Group B: GIC; Group C: resin) and photopolymerized. Cemented crowns were evaluated for volumetric infiltration using micro-CT (Skyscan, Bruker 1173- at 86 kV, 93 µA, 620 ms) after immersion in 50% solution of silver nitrate (AgNO3) (24 hours). Shear bond strength (SBS) was assessed by fracture of cement build-ups at a cross-head speed of 0.5 mm/min in a universal testing machine. RESULTS: Mean SBS among bioactive (21.54 ± 3.834 MPa) specimens was significantly higher than that for GIC (14.08 ± 3.25 MPa) specimens (p < 0.01), but they were comparable to resin samples (p > 0.05) (24.73 ± 4.32 MPa). Microleakage was significantly lower in crowns luted with bioactive (0.381 ± 0.134) cement compared to GIC (1.057 ± 0.399 mm3) (p < 0.01) and resin (0.734 ± 0.166 mm3) (p = 0.014) cemented crowns. The type of luting agent had a significant influence on the microleakage of crowns and bond strength to dentin (p < 0.05). CONCLUSION: Bioactive cement exhibited less microleakage and comparable SBS to resin luting agents in in vitro conditions.

Human identification through DNA analysis of restored postmortem teeth.

The identification of human remains using DNA analysis can be extremely challenging and its success is certainly influenced by the time elapsed since death. In that context, intact teeth have been shown to be highly successful in DNA analysis. However, restored teeth are usually available and, surprisingly, these specimens have been poorly studied. In fact, there are no reports regarding forensic DNA analysis of those types of samples in real cases. Therefore, the aim of this study was to perform DNA typing on healthy and restored teeth from exhumed human remains, which had been buried for 46 years. A powder-free DNA extraction protocol specifically designed for teeth was followed and human DNA quantitation and degradation assessment was performed using an in-house qPCR assay. Samples were amplified with commercial human identification kits for autosomal and Y chromosome markers. The obtained DNA profiles were compared to those of a previously processed femur sample as well as a buccal swab from a putative son. One healthy and one restored tooth yielded complete, concordant and compatible DNA profiles with previously typed samples from the femur and the putative son. Biostatistical calculations supported the paternity relationship with a likelihood ratio greater than 11 million. The present study highlights the use of restored teeth in a real exhumation case and the powder-free approach specifically designed for the extraction of DNA from teeth is discussed.

Novel methodology for measuring intraoral wear in enamel and dental restorative materials.

OBJECTIVES: To test the hypotheses that (a) the chairside/handheld dental scanner combined with a metrology software will measure clinical wear in vivo in agreement with measurements from X-ray computed microtomography and; (b) polished monolithic zirconia does not cause accelerated wear of opposing enamel. MATERIALS AND METHODS: Thirty single crowns were randomized to receive a monolithic zirconia or metal-ceramic crown. Two non-restored opposing teeth in the same quadrants were identified to serve as enamel controls. After cementation, quadrants were scanned using an intraoral dental scanner. Patients were recalled at 6-months and 1-year for re-scanning. Scanned images were compared using a metrology software to determine maximum vertical wear of teeth. The accuracy of the scanning measurements from this new method was compared with X-ray computed microtomography (micro-CT) measurements. Statistical analysis was performed using Mann-Whitney U test to determine significant differences between wear of enamel against zirconia, metal-ceramic or enamel. Linear regression analysis determined agreement between measurements obtained using intraoral scanning and micro-CT. RESULTS: Regression analysis demonstrated that there is a quantitative agreement between depth and volume measurements produced using intraoral scanning and the micro-CT methodologies. There was no significant difference between the wear of enamel against polished monolithic zirconia crowns and enamel against enamel. CONCLUSIONS: Intraoral scanning combined with a matching software can accurately quantify clinical wear to verify that monolithic zirconia exhibited comparable wear of enamel compared with metal-ceramic crowns and control enamel. Agreement between the intraoral scanner and the micro-CT was 99.8%. Clinical Trials.gov NCT02289781.

Temporal and spatial expression of TGF-beta2 in tooth crown development in mouse first lower molar.

Transforming Growth Factor beta2 (TGF-beta2) is involved in the regulation of many important cellular processes during tooth development. In this study we systematically characterized the expression pattern of TGF-beta2 in vivo and further analyzed its possible roles during different developmental stages of mouse first lower molar using immunofluorescence histochemical method with confocal microscopy. TGF-beta2 signaling was detected in different developing stages in both dental epithelium and surrounding dental mesenchyme. For the first time, we found that the basement membrane and epithelial cells in the basal layer showed no immunostaining from embryonic day 11 to 13; the primary enamel knot and secondary enamel knot exhibited pronounced immunostaining with different expression patterns at embryonic day 14 and 16. In addition, the mature ameloblast lost immunoreactivity, but the secretory ameloblast still exhibited positive immunoreaction at day 2 of postnatal development. Collectively, the temporospatial distribution patterns of TGF- beta2, especially in the basement membrane, epithelial cells in the basal layer, enamel knot, mature odontoblast and ameloblast, suggested a close association between TGF-beta2 signaling and tooth crown development, and indicated that TGF-beta2 might participate in tooth initiation, epithelial morphogenesis, formation of dentine matrix, and ameloblast differentiation.

Analysis of collagen distribution in human crown dentin by confocal laser scanning microscopy.

The authors used confocal laser scanning microscope to analyze human crown dentin. Specimens from 10 teeth were divided in two groups, one of which was decalcified and stained with hematoxylin and eosin. In the second group an undecalcified section was analyzed. Both groups were scanned by confocal microscope to generate optically sectioned images. All of the analyzed samples presented an intense autofluorescent that was ascribed to collagens. The degree of autofluorescence intensity was variable and might be due to collagen expression. The results indicate that a confocal microscope may be of help in analyzing and defining the nature and extent of collagen fibrils in human dentin.

Crystallographic texture and mineral concentration quantification of developing and mature human incisal enamel.

For human dental enamel, what is the precise mineralization progression spatially and the precise timing of mineralization? This is an important question in the fundamental understanding of matrix-mediated biomineralization events, but in particular because we can use our understanding of this natural tissue growth in humans to develop biomimetic approaches to repair and replace lost enamel tissue. It is important to understand human tissues in particular since different species have quite distinct spatial and temporal progression of mineralization. In this study, five human central incisors at different stages of enamel maturation/mineralization were spatially mapped using synchrotron X-ray diffraction and X-ray microtomography techniques. From the earliest developmental stage, two crystallite-orientation populations coexist with angular separations between the crystallite populations of approximately 40° varying as a function of position within the tooth crown. In general, one population had significantly lower texture magnitude and contributed a higher percentage to the overall crystalline structure, compared to the other population which contributed only 20-30% but had significantly higher texture magnitude. This quantitative analysis allows us to understand the complex and co-operative structure-function relationship between two populations of crystallites within human enamel. There was an increase in the mineral concentration from the enamel-dentin junction peripherally and from the incisal tip cervically as a function of maturation time. Quantitative backscattered-electron analyses showed that mineralization of prism cores precedes that of prism boundaries. These results provide new insights into the precise understanding of the natural growth of human enamel.

Perceptibility and acceptability of CIELAB color differences in computer-simulated teeth.

OBJECTIVES: To determine the perceptibility and acceptability of tooth color differences using computer-generated pairs of teeth with simulated gingival displayed on a calibrated color monitor using appropriate signal detection theory methodology (SDT). METHODS: Twelve dental professionals (four from each of the following groups: dentists, dental auxiliaries, and fixed prosthodontic technicians) and four dental patients served as subjects. Responses to tooth color differences (DeltaE) were measured on each of the three principal axes of CIELAB color space (L*, a*, and b*). As a control, responses to DeltaE=0 (the false alarm rate) were also measured in the same experimental session. RESULTS: No group differences among subjects were found. All gave 50% match or acceptance points that averaged about 1.0 DeltaE units in the L* and a* directions, and 2.6 units in the b* direction. False alarm rates across all subjects averaged 27% (4-55%) and 28% (0.4-61%), respectively, for perceptibility and acceptability. A reanalysis of the data based on SDT, which takes subjects' false alarm rates into account, gave somewhat larger color difference thresholds. CONCLUSIONS: Color difference thresholds for our simulated teeth are generally in line with and extend results obtained with studies using "real" dental materials. No differences between thresholds for acceptability versus perceptibility were found. Furthermore, subjects often reported color differences when none existed, and this behavior needs to be factored into any determination of quality control standards for the fabrication of dental prostheses.

Variation in elemental composition of human teeth and its application for feasible species identification.

Identifying human remains is a primary task in forensic science. In this study, we propose a possible new technique, handheld X-ray fluorescence (HHXRF), for determining whether a suspected tooth is an authentic human tooth. A total of 444 teeth obtained from 111 human skulls (male=62, female=49) aged between 30-67 years (51.81±8.37 years) were used as subjects. The teeth were scanned by HHXRF to acquire their elemental profile. Differences in elemental composition were analyzed for different tooth positions (numbers 1-32), between crown and root, and between sexes (male and female); also, the proportion of elements in relation to different human ages was examined. Teeth from 20 different animal species, serving as non-human teeth samples, were used to distinguish between human and non-human teeth through a stepwise discriminant analysis. Our results revealed that different tooth positions, different regions (crown and root) of a tooth, and different sexes demonstrated disparities in the proportion of several elements. The accuracy rate of predicting sex based on the elemental profile of human teeth was 65.5%. Likewise, a dissimilar distribution of elements between human and non-human teeth was observed, leading to a high degree of correctness of 83.2% for distinguishing them. In conclusion, elemental analysis by HHXRF could serve as a promising candidate tool for identifying human teeth in forensic science, but is ineffective for sex determination.

Analysis of artificial radiocarbon in different skeletal and dental tissue types to evaluate date of death.

Radiocarbon dating, with special reference to the modern bomb-curve, can provide useful information to elucidate the date of death of skeletonized human remains. Interpretation can be enhanced with analysis of different types of tissues within a single skeleton because of the known variability of formation times and remodeling rates. Analysis of radiocarbon content of teeth, especially the enamel in tooth crowns, provides information about the date of formation in the childhood years and in consideration of the known timing of tooth formation can be used to estimate the birth date after 1950 ad. Radiocarbon analysis of modern cortical and trabecular bone samples from the same skeleton may allow proper placement on the pre-1963 or post-1963 sides of the bomb-curve as most trabecular bone generally undergoes more rapid remodeling than does most cortical bone. Pre-1963 bone formation would produce higher radiocarbon values for most trabecular bone than for most cortical bone. This relationship is reversed for formation after 1963. Radiocarbon analysis was conducted in this study on dental, cortical, and trabecular bone samples from two adult individuals of known birth (1925 and 1926) and death dates (1995 and 1959). As expected, the dental results correspond to prebomb-curve values reflecting conditions during the childhoods of the individuals. The radiocarbon content of most bone samples reflected the higher modern bomb-curve values. Within the bone sample analyses, the values of the trabecular bone were higher than those of cortical bone and supported the known placement on the pre-1963 side of the bomb-curve.

Effect of the Crystallization Process on the Marginal and Internal Gaps of Lithium Disilicate CAD/CAM Crowns.

The aim of this study is to quantify the effect of the crystallization process on lithium disilicate ceramic crowns fabricated using a computer-aided design/computer-aided manufacturing (CAD/CAM) system and to determine whether the effect of crystallization is clinically acceptable by comparing values of fit before and after the crystallization process. The mandibular right first molar was selected as the abutment for the experiments. Fifteen working models were prepared. Lithium disilicate crowns appropriate for each abutment were prepared using a commercial CAD/CAM system. Gaps in the marginal area and 4 internal areas of each crown were measured twice-before and after crystallization-using the silicone replica technique. The mean values of fit before and after crystallization were analyzed using a paired t-test to examine whether the conversion that occurred during crystallization affected marginal and internal gaps (α = 0.05). Gaps increased in the marginal area and decreased in the internal areas after crystallization. There were statistically significant differences in all of the investigated areas (P < 0.05). None of the values for marginal and internal fit of lithium disilicate CAD/CAM crowns after crystallization exceeded 120 µm, which is the clinically acceptable threshold.

Importance of tubule density to the fracture toughness of dentin.

OBJECTIVE: The fracture toughness of dentin is critical to the prevention of tooth fracture. Within the tooth crown, the mechanical properties of dentin are influenced by spatial variations in the density and diameter of the dentin tubules with distance from the pulp. There are also relevant changes to the microstructure of dentin with age. In this investigation the importance of tubule density to the fracture toughness of dentin was evaluated in "young" and "old" age groups. METHODS: The variations in microstructure (density and diameter of tubules) from young and old donor teeth were studied by means of optical microscopy. RESULTS: A reduction in the density and diameter of tubules was identified to occur with aging. An approach previously proposed to study the mechanical behavior of porous materials was used to model the fracture toughness of coronal dentin in terms of the tubule characteristics. Results were then compared with published results from previous studies. CONCLUSIONS: The model predictions were consistent with experimental results for the fracture toughness of dentin from young donor teeth, but overestimated the values that have been reported for "old" dentin.

Collagen cross-linking and ultimate tensile strength in dentin.

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.