Intertubular Dentin Research Articles

Effect of Various Concentrations of Sodium Hypochlorite on Primary Dentin: An in vitro Scanning Electron Microscopic Study

Sodium hypochlorite solutions have been evaluated for their effects in bonding procedures as they are found to deplete or remove the organic portion of the dentin, particularly the collagen fibrils. The aim of this in vitro study was to assess and compare the efficacies of 1%, 2.5%, 5% and 10% NaOCl at 30, 60 and 120s on etched primary dentin. 84 primary anterior teeth were ground to expose a flat dentin area on the buccal surface. The specimens were divided into fourteen groups of six each based on the dentin surface treatment (35% phosphoric acid etching for 7 seconds-AE and/or NaOCl application), NaOCl solution concentrations (1%, 2.5%, 5% and 10%) and time of application (0, 30, 60 and 120s). Specimens were prepared for SEM and photomicrographs were taken of the surface and were scored against a five point scale, based on the smear layer and amount of collagen removed. The scores were submitted to Kruskal-Wallis and Mann Whitney tests. This study showed the presence of smear layer in the control group. The group treated with Acid Etchant showed a demineralized pattern of dentin with exposure of dentin tubules and collagen fibrils network on the intertubular and peritubular dentin which was not significantly different from the groups treated with 1% and 2.5% NaOCl. Groups treated with 5% NaOCI were not statistically different from each other the surface was corroded but collagen fibrils were not completely depleted. Groups treated with 10% NaOCl were not statistically different from each other and showed complete removal of collagen fibrils with wider tubular apertures and several secondary tubules on peritubular and intertubular dentin. Higher concentrations of NaOCl solutions (5% and 10%) produced significant changes in the etched primary dentin. The higher the concentration of the NaOCI solution, the lower can be the time for the application of the solution for the complete removal of collagen fibrils.

In situ study of the gelatinase activity in demineralized dentin from rat molar teeth

Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris–CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin–enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.

Ultra-structural observation of human enamel and dentin by ultra-high-voltage electron tomography and the focus ion beam technique

Human enamel and dentin are hard and brittle mineralized tissues. It is difficult to prepare thin specimens (under 200nm) of these tissues for transmission electron microscope observation without demineralizing them. We present a novel method of creating three-dimensional ultra-structural images of human enamel and dentin, using the focus ion beam (FIB) method and ultra-high-voltage electron microscope tomography. Thin specimens (less than 2μm) of enamel and dentin were prepared using the FIB method. This method is appropriate for nano-fabrication of thin specimens for brittle materials such as enamel and dentin. It allows penetration of an ultra-high-voltage electron beam of a 3000kV maximal acceleration voltage into a specimen, enabling projections of the specimen to be taken. It facilitates tomography of the enamel rods, sheaths, dentinal tubules and collagen fibrils with a high resolution of 2nm. The component percentages in ultra-structures of dentin can be expressed numerically by using three-dimensional reconstruction images of tomograms. The matrix of peritubular dentin differed from that of intertubular dentin by having relatively fewer collagen fibrils. The major advantage of this method is its ability to visualize ultra-structural tomograms of highly calcified specimens without demineralization.

Multiphoton imaging of the dentine‐enamel junction

Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin-enamel junction (DEJ) based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpendicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low intensity line on the 2PEF images and this was never previously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine-enamel junction in the first 12 μm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo-proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions.

Thermal effects and morphological aspects of human dentin surface irradiated with different frequencies of Er:YAG laser

This study reports the effects on micromorphology and temperature rise in human dentin using different frequencies of Er:YAG laser. Sixty human dentin fragments were randomly assigned into two groups (n = 30): carious or sound dentin. Both groups were divided into three subgroups (n = 10), according to the Er:YAG laser frequency used: 4, 6, or 10 Hz (energy: 200 mJ; irradiation distance: 12 mm; and irradiation time: 20 s). A thermocouple adapted to the tooth fragment recorded the initial temperature value (°C); then, the temperature was measured after the end of the irradiation (20 s). Morphological analysis was performed using images obtained with scanning electron microscope. There was no difference between the temperatures obtained with 4 and 6 Hz; the highest temperatures were achieved with 10 Hz. No difference was observed between carious and sound dentin. Morphological analyses revealed that all frequencies promoted irregular surface in sound dentin, being observed more selectively ablation especially in intertubular dentin with tubule protrusion. The caries dentin presented flat surface for all frequencies used. Both substrates revealed absence of any signs of thermal damage. It may be concluded that the parameters used in this study are capable to remove caries lesion, having acceptable limits of temperature rise and no significant morphological alterations on dentin surface.

Short-term PTH administration increases dentine apposition and microhardness in mice

ObjectiveThe purpose of this study is to investigate the effects of intermittent parathyroid hormone (PTH) administration on the apposition rate and structural features of dentine from mouse incisors. MethodsYoung male A/J Unib mice were treated daily for 6 and 10 days with 40μg/kg of hPTH 1-34 or a vehicle. Dentine apposition rates measured by fluorescent labels (tetracycline and calcein) and alkaline phosphatase (ALP) plasma levels were evaluated after 6 days of treatment. Knoop microhardness testing and element content measurements in at.% of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentine were performed by Energy Dispersive X-ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) after 10 days of treatment. ResultsHistometric analysis revealed an increase of 5% in the apposition rate of dentine and 25% in the ALP plasma levels in the PTH treated group. In addition, knoop microhardness testing revealed that the animals treated with PTH had a greater microhardness (11%). EDX microanalysis showed that PTH treatment led to increases in P (23%) and Ca (53%) at.% content, as well as the Ca/P ratio (24%) in peritubular dentine. The chemical composition of intertubular dentine did not vary between the groups. ConclusionsThese findings indicate that intermittent administration of hPTH (1-34) increases apposition and mineralization of the dentine during young mice incisor formation.

Linking Microstructure and Nanochemistry in Human Dental Tissues

Mineralized dental tissues and dental pulp were characterized using advanced analytical transmission electron microscopy (TEM) methods. Quantitative X-ray energy dispersive spectroscopy was employed to determine the Ca/P and Mg/P concentration ratios. Significantly lower Ca/P concentration ratios were measured in peritubular dentine compared to intertubular dentine, which is accompanied by higher and variable Mg/P concentration ratios. There is strong evidence that magnesium is partially substituting calcium in the hydroxyapatite structure. Electron energy-loss near-edge structures (ELNES) of C-K and O-K from enamel and dentine are noticeably different. We observe a strong influence of beam damage on mineralized dental tissues and dental pulp, causing changes of the composition and consequently also differences in the ELNES. In this article, the importance of TEM sample preparation and specimen damage through electron irradiation is demonstrated.

Mechanical heterogeneity of dentin at different length scales as determined by AFM phase contrast

In this study we sought to gain insights of the structural and mechanical heterogeneity of dentin at different length scales. We compared four distinct demineralization protocols with respect to their ability to expose the periodic pattern of dentin collagen. Additionally, we analyzed the phase contrast resulting from AFM images obtained in tapping mode to interrogate the viscoelastic behavior and surface adhesion properties of peritubular and intertubular dentin, and partially demineralized dentin collagen fibrils, particularly with respect to their gap and overlap regions. Results demonstrated that all demineralization protocols exposed the gap and overlap zones of dentin collagen fibrils. Phase contrast analyses suggested that the intertubular dentin, where the organic matrix is concentrated, generated a higher phase contrast due a higher contribution of energy dissipation (damping) than the highly mineralized peritubular region. At increasing amplitudes, viscoelasticity appeared to play a more significant contribution to the phase contrast of the images of collagen fibrils. The overlap region yielded a greater phase contrast than the more elastic gap zones. In summary, our results contribute to the perspective that, at different length scales, dentin is constituted of structural features that retain heterogeneous mechanical properties contributing to overall mechanical performance of the tissue. Furthermore, the interpretation of phase contrast from images generated with AFM tapping mode appears to be an effective tool to gain an improved understanding of the structure and property relationship of biological tissues and biomaterials at the micro- and nano-scale.

Effects of heating on the mechanical and chemical properties of human dentin

ObjectivesWe had previously discovered that the flexural and tensile strengths of human dentin were 2–2.4 times greater after being heated to 140°C, and deduced that the generation of higher-density structures and therefore dehydration probably promoted the increased strength. Our test hypotheses were that intertubular dentin, which constitutes a major part of organic components, was selectively affected by heating, and such changes could happen without critical damages to the basic structure of dentin type I collagen. MethodsMicro-mechanical changes of human dentin by heating at 140°C were investigated by nano-indentation. Chemical changes in dentin collagen after heating were also investigated by X-ray diffraction study, a microscopic Fourier transform infrared (micro-FTIR) and a laser Raman spectroscopic analyses, and a cross-linking analysis by high-performance liquid chromatography. ResultsThe results of nano-indentation showed that the micro-hardness of intertubular dentin increased after heating at 140°C to 1.8 times more than unheated dentin; on the other hand, peritubular dentin was unchanged. Results of X-ray diffraction showed that the lateral packing of collagen molecules shrank from 13.6±0.3 to 10.6±0.1Å after heating, but the shrinkage reversed to the original after rehydration for seven days. After heating, no substantial chemical changes in the collagen molecules were detected in tests by micro-FTIR or Raman analyses, or by cross-linking analysis. SignificanceThese results suggest that intertubular dentin, which contains most of the type I collagen, was selectively affected by heating at 140°C without critical damage to its collagen.

Etch and rinse versus self-etching adhesives systems: Tridimensional micromechanical analysis of dentin/adhesive interface

The purpose of this study was to evaluate stress distribution in the hybrid layer produced by two adhesive systems using three-dimensional finite element analysis (FEA). Four FEA models (M) were developed: Mc, a representation of a dentin specimen (41×41×82μm) restored with composite resin, exhibiting the adhesive layer, hybrid layer (HL), resin tags, peritubular dentin, and intertubular dentin to simulate the etch-and-rinse adhesive system; Mr, similar to Mc, with lateral branches of the adhesive; Ma, similar to Mc, however without resin tags and obliterated tubule orifice, to simulate the environment for the self-etching adhesive system; Mat, similar to Ma, with tags. A numerical simulation was performed to obtain the maximum principal stress (σmax). The highest σmax in the HL was observed for the etch-and-rinse adhesive system. The lateral branches increased the σmax in the HL. The resin tags had a little influence on stress distribution with the self-etching system.

Comparison of Shear Bonding Strength for Two Different Etching Systems in Canine and Human Dentin

Canine dentin has increased size and number of dentinal tubules compared to human dentin. With less intertubular surface area for bonding to composite material in dog teeth, bonding shear strength may be affected negatively. This study was designed to compare the shear bonding strength of canine and human dentin. It also compared bond strength of two different acid etch systems when used with the same composite resin. The shear bond strength of composite cylinders to dentin was measured in 30 extracted canine cuspid teeth and a similar number of human molar teeth. Shear bond strength was 40% greater for human compared with canine dentin. This significantly different (P < 0.05) result validates the hypothesis that dentin bond strength is positively correlated with the area of intertubular dentin. Results also indicated that shear bond strength was greater in the self-etching bond system compared with the etch and rinse system, although this difference was not significant.

Effects of gamma radiation on hard dental tissues of albino rats using scanning electron microscope – Part 1

In the present study, 40 adult male albino rats were used to study the effect of gamma radiation on the hard dental tissues (enamel surface, dentinal tubules and the cementum surface). The rats were irradiated at 0.2, 0.5, 1.0, 2.0, 4.0 and 6.0 Gy gamma doses. The effects of irradiated hard dental tissues samples were investigated using a scanning electron microscope. For doses up to 0.5 Gy, there was no evidence of the existence of cracks on the enamel surface. With 1 Gy irradiation dose, cracks were clearly observed with localized erosive areas. At 2 Gy irradiation dose, the enamel showed morphological alterations as disturbed prismatic and interprismatic areas. An increase in dentinal tubules diameter and a contemporary inter-tubular dentine volume decrease were observed with higher irradiation dose. Concerning cementum, low doses,<0.5 Gy, showed surface irregularities and with increase in the irradiation dose to≥1 Gy, noticeable surface irregularities and erosive areas with decrease in Sharpey's fiber sites were observed. These observations could shed light on the hazardous effects of irradiation fields to the functioning of the human teeth.

Chemical composition and structure of peritubular and intertubular human dentine revisited

ObjectiveCurrently there is still a debate about whether peritubular dentine (PTD) is non-collageneous or collageneous tissue. The chemical composition and structure of human PTD and intertubular dentine (ITD) was re-visited in this study. DesignThe dentine tubular region including ITD and PTD prepared from human third molars was in situ detected by means of micro-Raman spectroscopy (μRs) and atomic force microscopy (AFM). ResultsFrom the μRs study, it was found that the mineral/matrix ratios (phosphate vs. CH2) in PTD were ∼3 times of those in ITD. For the mineral, the differences between PTD and ITD were small, but still detectable. For the organic matrix, the intensity ratios of amide III to CH2 in ITD were ∼1.5 times of those in PTD, indicating the structural differences. In addition, there was a higher proline/hydroxyproline content in ITD than that in PTD. However, the overall Raman peak contour in the amide regions (I & III) was similar, indicating collagen might still exist in both the ITD and PTD. An in situ AFM observation of the dentinal tubular region during EDTA etching confirmed that dentine collagen ran across from the ITD into the PTD. ConclusionA phenomenon similar to that observed in the dentine–enamel junction is proposed to explain the above results. It is demonstrated that the μRs–AFM approach can be used to provide an insight into the structure of small dental tissues at the micron or sub-micron scale.

Nanomechanical Properties of Endodontically Treated Teeth

Introduction Although it is apparent that teeth become more susceptible to fracture after root canal treatment, the contributing factors for this are not completely established. The purpose of this study was to determine whether there are changes in nanomechanical properties of dentin in root canal–treated teeth compared with non–root canal–treated control teeth. Methods Atomic force microscopy–based nanoindentation testing was performed on root canal–treated teeth and age- and type-matched control teeth. Radicular intertubular dentin was indented in 6 locations, and triplicate measurements were averaged. Paired t tests were used to compare root canal–treated teeth with control teeth. Results The moduli of elasticity were 17.8 ± 2.9 GPa and 18.9 ± 2.9 GPa for root canal–treated teeth and controls, respectively; the hardness values for the 2 groups were 0.84 ± 0.25 GPa and 0.84 ± 0.18 GPa, respectively. Neither the modulus of elasticity nor the hardness differed between groups ( P > .05). Conclusions It appears that root canal treatment does not result in nanomechanical changes to radicular intertubular dentin.

Nanoscopic dynamic mechanical properties of intertubular and peritubular dentin

An experimental evaluation of intertubular and peritubular dentin was performed using nanoindentation and Dynamic Mechanical Analysis (DMA). The objective of the investigation was to evaluate the differences in dynamic mechanical behavior of these two constituents and to assess whether their response is frequency dependent. Specimens of hydrated coronal dentin were evaluated by DMA using single indents over a range of parametric conditions and using scanning probe microscopy. The complex (E∗), storage (E′) and loss moduli (E″) of the intertubular and peritubular dentin were evaluated as a function of the dynamic loading frequency and static load in the fully hydrated condition. The mean complex E∗ (19.6 GPa) and storage E′ (19.2 GPa) moduli of the intertubular dentin were significantly lower than those for peritubular dentin (E∗=31.1 GPa, p<0.05; E′=30.3 GPa, p<0.05). There was no significant influence of dynamic loading frequency on these measures. Although there was no significant difference in the loss modulus (E″) between the two materials (p>0.05), both constituents exhibited a significant increase in E″ with dynamic load frequency and reduction in the quasi-static component of indentation load. The largest difference in dynamic behavior of the two tissues was noted at small quasi-static indentation loads and the highest frequency.

Insights into the structure and composition of the peritubular dentin organic matrix and the lamina limitans

Dentin is a mineralized dental tissue underlying the outer enamel that has a peculiar micro morphology. It is composed of micrometer sized tubules that are surrounded by a highly mineralized structure, called peritubular dentin (PTD), and embedded in a collagen-rich matrix, named intertubular dentin. The PTD has been thought to be composed of a highly mineralized collagen-free organic matrix with unknown composition. Here we tested the hypothesis that proteoglycans and glycosaminoglycans, two important organic structural features found in dentin, are key participants in the microstructure and composition of the PTD. To test this hypothesis dentin blocks were demineralized with 10 vol% citric acid for 2 min and either digested with 1 mg/ml TPCK-treated trypsin with 0.2 ammonium bicarbonate at pH 7.9 (TRY) or 0.1 U/mL C-ABC with 50 mM Tris, 60 mM sodium acetate and 0.02% bovine serum albumin at pH 8.0 (C-ABC). TRY is known to cleave the protein core of dentin proteoglycans, whereas C-ABC is expected to selectively remove glycosaminoglycans. All specimens were digested for 48 h in 37 °C, dehydrated in ascending grades of acetone, immersed in HMDS, platinum coated and imaged using an FE-SEM. Images of demineralized dentin revealed a meshwork of noncollagenous fibrils protruding towards the tubule lumen following removal of the peritubular mineral and confirmed the lack of collagen in the peritubular matrix. Further, images revealed that the peritubular organic network originates from a sheet-like membrane covering the entire visible length of tubule, called lamina limitans. Confirming our initial hypothesis, after the digestion with C-ABC the organic network appeared to vanish, while the lamina limitans was preserved. This suggests that glycosaminoglycans are the main component of the PTD organic network. Following digestion with TRY, both the organic network and the lamina limitans disappeared, thus suggesting that the lamina limitans may be primarily composed of proteoglycan protein cores. In summary, our results provide novel evidence that (1) PTD lacks collagen fibrils, (2) PTD contains an organic scaffold embedded with mineral and (3) the PTD organic matrix is manly composed of glycosaminoglycans, whereas the lamina limitans is primarily made of proteoglycans protein cores.

Quantitative Analysis of the Effect of Irrigant Solution Sequences on Dentin Erosion

Objective The purpose of the study was to examine the level of erosion on root canal wall dentin caused by immersion in different irrigant solutions in alternative sequences. Methods Dentin specimens from teeth with one root canal were instrumented and randomly divided into five groups. Each group was subjected to 17% EDTA, 17% ethyleneglycoltetraacetic acid (EGTA), or 10% citric acid (CA) and 5.25% sodium hypochlorite (NaOCl) varying the time of irrigant exposure and the order of the irrigants. The specimens were examined by scanning electron microscopy (SEM), and randomized digital images of the dentin surface were taken. The area of tubule openings was measured by a semiautomatic method. Results No erosion was detected when demineralizing agents were used as a final rinse after NaOCl. However, the erosion of peritubular and intertubular dentin was detected when EDTA, EGTA, or CA were used first followed by 5.25% NaOCl ( P < .05), and an increase over 100% in the area of dentin tubule openings was measured ( P < .01). Conclusions NaOCl used as a final irrigant solution after demineralization agents causes marked erosion of root canal dentin.

Near tubule and intertubular bovine dentin mapped at the 250 nm level

In this study, simultaneous diffraction and fluorescence mapping with a (250nm)(2), 10.1keV synchrotron X-ray beam investigated the spatial distribution of carbonated apatite (cAp) mineral and elemental Ca (and other cations including Zn) around dentin tubules. In 1μm thick sections of near-pulp root dentin, where peritubular dentin (PTD) is newly forming, high concentrations of Zn, relative to those in intertubular dentin (ITD), were observed adjacent to and surrounding the tubule lumens. Some but not all tubules exhibited hypercalcified collars (high Ca signal relative to the surrounding ITD), and, when present, the zone of high Ca did not extend around the tubule. Diffraction rings from cAp 00.2 and 11.2+21.1+30.0 reflections were observed, and cAp was the only crystal phase detected. Profiles of Ca, Zn and cAp diffracted intensities showed the same transitions from solid to tubule lumen, indicating the same cAp content and organization in ITD far from the tubules and adjacent to them. Further, the matching Ca and diffraction profiles demonstrated that all of the Ca is in cAp or that any noncrystalline Ca was uniformly distributed throughout the dentin. Variation of 00.2 and 11.2+21.1+30.0 diffracted intensity was consistent with the expected biaxial crystallographic texture. Extension of X-ray mapping from near 1μm resolution to the 250nm level, performed here for dentin and its tubules, will provide new understanding of other mineralized tissues.

Effect of dentin age and acid etching time on dentin bonding.

To compare the bond strengths of young and old dentin using a single-bottle etch-and-rinse adhesive system with two acid-etching times (15 s and 30 s), and to examine the correlation between dentin hardness and bond strength. Twenty-four molars from 18- to 22- or 55- to 60-year-old patients were prepared to expose the occlusal dentin, cut into two equal parts, and assigned to four groups (G), varying the etching time (15 s and 30 s) and dentin age (young [Y]or old [O]): G15Y, G30Y, G15O and G30O. After etching, AdperSingleBond (3M ESPE) adhesive was applied and the tooth was built up with a composite (Filtek Z250). The specimens were prepared for the microtensile bond strength test (μTBS) at a crosshead speed of 0.5 mm/min and the interfaces were observed under SEM. Also, the Knoop hardness (KHN) of young vs old dentin was measured. The mean (± SD) μTBS (MPa) were: G15Y = 45.9 (± 10.7)a, G15O = 34.1 (± 9.4)b, G30Y = 48.6 (± 14.3)a and G30O = 47.7 (± 11.0)a. Two-way ANOVA showed no difference in μTBS between young and old dentin. Old dentin acid etched for 15 s (G15O) had a lower μTBS than when acid etched for 30 s (G30O). Dentin hardness was higher for old dentin (67.0 ± 4.8KHN) than young dentin (63.7 ± 2.9KHN) (p < 0.004). No correlation between μTBS and dentin hardness was observed. Resin tags were larger and more numerous for young dentin. The hybrid layer formed in intertubular old dentin (G15O) was very thin. Bonding to old dentin with 30 s of etching time resulted in higher bond strength and more homogeneous hybrid layer formation than dentin acid etched for 15 s.

Microstructure of dental hard tissues in fossil and recent xenarthrans (Mammalia: Folivora and Cingulata)

A striking difference between xenarthrans and other mammals is the complete loss of tooth enamel in all members but the earliest armadillos. However, sloth and armadillo teeth show structured wear facets, which in all other mammals are formed by tooth enamel. How is that possible? Here, I report about an analysis of fossil and recent xenarthran dental hard tissue microstructure. It shows that osteodentine is not exclusive to fossil Cingulata, but also occurs in some recent taxa. Furthermore, I found profound modifications of orthodentine architecture in comparison to other mammals. Remarkable features are (a) a larger proportion of the highly mineralized, collagen-free peritubular dentine, and (b) a modified architecture of the odontoblastic process with frequent interconnections between the extensions and unusually intensive branching of the extensions forming a complex meshwork, penetrating the intertubular dentine matrix. The orthodentine microstructural build-up is unique in Folivora and Cingulata.