A Fourier transform infrared spectroscopy analysis of carious dentin from transparent zone to normal zone.

It is well known that caries invasion leads to the differentiation of dentin into zones with altered composition, collagen integrity and mineral identity. However, understanding of these changes from the fundamental perspective of molecular structure has been lacking so far. In light of this, the present work aims to utilize Fourier transform infrared spectroscopy (FTIR) to directly extract molecular information regarding collagen's and hydroxyapatite's structural changes as dentin transitions from the transparent zone (TZ) into the normal zone (NZ). Unembedded ultrathin dentin films were sectioned from carious teeth, and an FTIR imaging system was used to obtain spatially resolved FTIR spectra. According to the mineral-to-matrix ratio image generated from large-area low-spectral-resolution scan, the TZ, the NZ and the intermediate subtransparent zone (STZ) were identified. High-spectral-resolution spectra were taken from each zone and subsequently examined with regard to mineral content, carbonate distribution, collagen denaturation and carbonate substitution patterns. The integrity of collagen's triple helical structure was also evaluated based on spectra collected from demineralized dentin films of selected teeth. The results support the argument that STZ is the real sclerotic layer, and they corroborate the established knowledge that collagen in TZ is hardly altered and therefore should be reserved for reparative purposes. Moreover, the close resemblance between the STZ and the NZ in terms of carbonate content, and that between the STZ and the TZ in terms of being A-type carbonate-rich, suggest that the mineral that initially occludes dentin tubules is hydroxyapatite newly generated from odontoblastic activities, which is then transformed into whitlockite in the demineralization/remineralization process as caries progresses.

Chemical characterization of etched dentin in non-carious cervical lesions.

PURPOSE: Bonding to non-carious cervical lesion (NCCL) sclerotic dentin that involves acid etching continues to be a challenging problem due to its altered chemical structure. In the present study, the objective was to investigate the chemical response of NCCL sclerotic dentin to the different acid etching times. MATERIALS AND METHODS: Extracted human premolars affected with NCCLs were selected, and a cavity matching the natural lesion with respect to size and location was prepared on the lingual surface of each tooth to serve as the control. The dentin surfaces were treated for 15 s and 30 s using 37% phosphoric acid and then analyzed by Raman microspectroscopic mapping/imaging. RESULTS: NCCL dentin substrates had dramatic effects on the chemical profile of dentin demineralization. The spectral comparison showed that the demineralized layer generated by the acid treatment was highly irregular in terms of depth and mineral component retained, especially when NCCL sclerotic dentin was etched for 15 s. When the etching time was increased to 30 s, the demineralization of NCCL sclerotic dentin was more effective and comparable to the nonsclerotic control that was treated for 15 s. Different etching times affected the depth, degree, and profile of the dentin demineralization. CONCLUSION: The shorter etching time (ie, 15 s) might not be adequate for NCCL sclerotic dentin. However, the longer etching time (ie, 30 s) would induce much deeper demineralized dentin for nonsclerotic substrates. Thus, although extended etching times can be used to remove the hypermineralized layer, further studies are required to analyze the impact this might have on the dentin bonding.

Reduced antigenicity of type I collagen and proteoglycans in sclerotic dentin.

Antigenic alterations to the dentin organic matrix may be detected by an immunohistochemical approach. We hypothesized that alterations in the antigenicity of type I collagen and proteoglycans occur in sclerotic dentin under caries lesions. Transverse sections were prepared from carious teeth in the sclerotic zone and normal hard dentin. A double-immunolabeling technique was performed on these sections, with anti-type I collagen and anti-chondroitin 4/6 sulfate monoclonal primary antibodies. We used gold-conjugated secondary antibodies to visualize the distribution of intact collagen fibrils and proteoglycans by high-resolution SEM. For sclerotic dentin, labeling densities were 19.57 +/- 3.01/microm2 for collagen and 9.84 +/- 2.62/microm2 for proteoglycans. For normal hard dentin, values were 35.20 +/- 2.73/microm2 and 17.03 +/- 1.98/microm2, respectively. Distribution of intact collagen fibrils and proteoglycans in sclerotic dentin was significantly lower than in normal hard dentin. Reductions in antigenicity from the organic matrix of sclerotic dentin under caries lesions raise concern about the potential of intrafibrillar remineralization.

SEM evaluation of neodentinal bridging after direct pulp protection with mineral trioxide aggregate.

The purpose of this study was to observe the basic morphology and determine the chemical composition of neodentinal bridges adjacent white mineral trioxide aggregate (WMTA) when used as a direct pulp capping material. The experimental procedures were performed on six intact dogs' teeth. The pulps were exposed and cavities were filled with WMTA. After 2 weeks, neodentinal bridge formation was evaluated by scanning electron microscope (SEM) of cross-sections of the specimens and electron probe microanalysis (EPMA) of the pulpal surfaces. Results of SEM observation showed that the most characteristic reaction of pulp cells was the intimate connection of cell processes and secreted extracellular fibres with the crystals of the pulp capping material. Results of EPMA indicated that the mineralisation of neodentinal bridge formation occurred progressively from the periphery to the central area. Based on these results, it appears that WMTA has the potential to be used as a direct pulp capping material during vital pulp therapy.

Synthetic octacalcium phosphate-enhanced reparative dentine formation via induction of odontoblast differentiation.

Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP.

Regional strengths of bonding agents to cervical sclerotic root dentin.

The regional bond strengths of three current-generation bonding systems (All Bond 2, Scotchbond Multi-Purpose, and Clearfil Liner Bond 2) were measured in natural wedge-shaped defects in the cervical area of extracted human teeth. A microtensile testing method was used to compare the strengths of resin bonds made to occlusal margins with those made to gingival margins. Controls consisted of normal teeth which had artificial wedge-shaped defects, of the same depth and dimension, created with a high-speed bur. The results indicated that there were no regional differences in bond strength, although bonds made to natural lesions were from 20 to 45% lower than those made to normal dentin in artificially created wedge-shaped defects, depending on the bonding agent. Scanning electron microscopy revealed that Clearfil Liner Bond 2 created the thinnest hybrid layers, which were difficult to measure in the natural lesions. The natural lesions contained sclerotic dentin, whereas the artificial lesions were composed of normal dentin. Although the bond strengths to sclerotic dentin were lower than those to normal dentin, the absolute values (ca. 16 to 17 MPa) were high relative to previous-generation bonding agents.

In vitro bond strengths and SEM evaluation of dentin bonding systems to different dentin substrates.

In comparison to enamel, bonding to normal dentin is a greater challenge because of its organic constituents, fluid-filed tubules, and variations in intrinsic composition. Bonding to sclerotic dentin is even more difficult. To evaluate the shear bond strengths of four adhesive systems to dentin substrates with different levels of mineralization, 120 extracted human teeth were randomly assigned to three groups (n = 40). After mid-coronal dentin was exposed, groups of specimens were artificially hypermineralized by immersion in a remineralizing solution, demineralized by means of an acetic acid demineralizing solution, or stored in distilled water to model sclerotic, carious, and normal dentin, respectively. Resin composite was bonded to dentin by use of commercial adhesive systems. After the specimens were thermocycled, shear bond strengths were determined in an Instron universal testing machine. Dentin substrates and resin/dentin interfaces were examined by SEM. For each adhesive system, the mean shear bond strength to normal dentin was significantly higher than that to either of the other substrates. Shear bond strengths to hypermineralized dentin were significantly higher than those to demineralized dentin with all adhesives except Prisma Universal Bond 3.

Acid profiles and pH of carious dentin in active and arrested lesions.

Organic acids in caries lesions play important roles in initiation and progress of dental caries. We investigated relationships between clinical types of dentin caries and acid profile or pH in the lesions. Caries lesions in dentin from 76 permanent teeth were classified into active, arrested, situated beneath a restoration, and unclassified types. The pH of carious dentin was distinctly lower than that of sound dentin (p < 0.001). Carious dentin with a high percentage of lactate had a lower pH than that with a high percentage of acetate and propionate (p < 0.001). Dentin from active lesions showed a mean pH of 4.9, and the dominant acid was lactate (mean percentage, 88.2). In contrast, carious dentin from arrested lesions showed a higher pH, 5.7, with acetate and propionate as the dominant acids (mean percentages of acetate and propionate, 64.0 and 18.2, respectively). The acid profile (mean percentages of acetate and propionate, 54.0 and 27.7, respectively) and pH (mean 5.8) of carious dentin sampled from lesions beneath a restoration were similar to those of dentin from arrested lesions. This study showed a clear relationship between clinical classification of dentin caries and acid profile and pH, suggesting that both factors are important in dentin caries etiology.

Histological and scanning electron microscopy assessment of various vital pulp-therapy materials.

Pulp capping and pulpotomy procedures were performed on 15 male mongrel dogs. Three materials were used: calcium hydroxide, acid-etched dentin bonding, and mineral trioxide aggregate. Six of the animals were killed at 50 days and nine were killed at 150 days. Samples from 11 dogs were used for histological evaluation, and the remaining dog samples were used for scanning electron microscopy evaluation. Each slide was graded histologically according to previously published criteria. Scanning electron microscopy analysis was performed, and the weight percentage of elements found in the dentin of a nontreated tooth versus the bridge formed in the exposed specimen was established. By evaluating pulp inflammation in vital pulp-therapy treatments, it was found that mineral trioxide aggregate was not significantly different from the untreated control group, both in pulp-capping procedures at 50 days (p = 0.357) or 150 days (p = 0.198) and pulpotomy procedures at 50 days (p = 0.357) or 150 days (p = 0.198). Moreover, histologically mineral trioxide aggregate was a considerably better material than calcium hydroxide or acid-etched dentin bonding in maintaining the integrity of the pulp.

Quantitative assessment of dentin bridge formation following pulp-capping in miniature swine.

A quantitative assessment of dentin bridges was conducted on sections prepared from teeth capped with four pulp-capping agents (Bioglass, Life, Demineralized Dentin Matrix, and Teflon) in a micro-swine model. The degree of mineralization of the dentin bridges relative to the adjacent primary dentin was measured using a computer-based image analysis of microradiographs prepared from the sections. The rate of formation of the dentin bridge was measured from fluorescent bands formed in the same sections by a Demeclocycline dentin marker. There were no statistically significant differences in the relative mineral densities of the dentin bridges and the rate of dentin bridge formation under the four pulp-capping agents. These findings support the suggestion that components of the extracellular matrix rather than pulp-capping agents may be important in the formation and mineralization of dentin bridges in repairing dental pulps.

Calcium salts deposition in rat connective tissue after the implantation of calcium hydroxide-containing sealers.

This study was conducted to observe the rat subcutaneous connective tissue reaction to implanted dentin tubes that were filled with mineral trioxide aggregate, Sealapex, Calciobiotic Root Canal Sealer (CRCS), Sealer 26, and the experimental material, Sealer Plus. The animals were sacrificed after 7 and 30 days, and the specimens were prepared for histological analysis after serial sections with a hard-tissue microtome. The undecalcified sections were examined with polarized light after staining according to the Von Kossa technique for calcium. At the tube openings, there were Von Kossa-positive granules that were birefringent to polarized light. Next to these granulations, there was irregular tissue, like a bridge, that was Von Kossa-positive. The dentin walls of the tubes exhibited a structure highly birefringent to polarized light, usually like a layer, in the tubules. These results were observed with all the studied materials, except the CRCS, which didn't exhibit any kind of mineralized structure. The results suggest that among the materials studied, the CRCS could have the least possibility of encouraging hard tissue deposition.

Resin bonding to cervical sclerotic dentin: a review.

Several reports have indicated that resin bond strengths to noncarious sclerotic cervical dentine are lower than bonds made to normal dentine. This is thought to be due to tubule occlusion by mineral salts, preventing resin tag formation. The purpose of this review was to critically examine what is known about the structure of this type of dentine. Recent transmission electron microscopy revealed that in addition to occlusion of the tubules by mineral crystals, many parts of wedge-shaped cervical lesions contain a hypermineralised surface that resists the etching action of both self-etching primers and phosphoric acid. This layer prevents hybridisation of the underlying sclerotic dentine. In addition, bacteria are often detected on top of the hypermineralised layer. Sometimes the bacteria were embedded in a partially mineralised matrix. Acidic conditioners and resins penetrate variable distances into these multilayered structures. Examination of both sides of the failed bonds revealed a wide variation in fracture patterns that involved all of these structures. Microtensile bond strengths to the occlusal, gingival and deepest portions of these wedge-shaped lesions were significantly lower than similar areas artificially prepared in normal teeth. When resin bonds to sclerotic dentine are extended to include peripheral sound dentine, their bond strengths are probably high enough to permit retention of class V restorations by adhesion, without additional retention.

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.

Fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation in rat molars.

OBJECTIVE: The purpose of this study was to examine changes in distribution of fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation. STUDY DESIGN: Class V cavity preparations were prepared on maxillary first molars of 12 rats. The dentin and pulps were observed histologically and immunohistochemically for fibrinogen and fibronectin at 6 hours and 1, 2, and 3 days after the preparation. RESULTS: At 6 hours and 1 day after cavity preparation, positive staining for fibrinogen was noted in the exudative lesion and in the dentinal tubules under the cavity preparation. Fibronectin staining in the exudate showed a pattern with close similarity to the fibrinogen staining. At 3 days after cavity preparation, the irregularly shaped predentin under the cavity preparation showed strong positive staining for fibronectin. CONCLUSIONS: Fibrinogen/fibrin and fibronectin are present during the healing process of the dentin-pulp complex after cavity preparation.

Thickness and morphology of resin-infiltrated dentin layer in young, old, and sclerotic dentin.

The purpose of the present study was to evaluate the morphology of the resin tags and the resin-infiltrated dentin layer (RIDL) of several bonding systems in superficial vs deep young, old, and sclerotic human dentin. Dentin was obtained after the removal of occlusal enamel from extracted molars. Phosphoric acid gels (35-37%) were used to etch dentin before the application of bonding systems (OptiBond FL; Prime & Bond 2.0; Scotchbond Multi-Purpose Plus and Scotchbond 1; One Step). Each bonded specimen was then sectioned into two halves. One half was polished using a standard procedure to evaluate RIDL thickness and morphology by SEM. The other half was demineralized and deproteinized to evaluate the presence and the morphology of resin tags. RIDL was thinner in superficial dentin than in deeper dentin for all the materials tested regardless of the type of dentin. Sclerotic and old dentin showed thinner RIDL, with short resin tags, and fewer lateral branches than normal dentin.

Dentin bonding: effect of degree of mineralization and acid etching time.

This in vitro study verifies whether there are differences between bonding to hypermineralized dentin and normal dentin and if longer acid etching can improve the bond strength to this modified substrate without damaging the bond to normal dentin. Forty-two extracted human molars with chronic occlusal caries were transversally cut with a diamond saw under refrigeration. The occlusal surfaces were ground until the carious lesion was removed, exposing the sclerotic dentin in the center and polished to 600/grid. A 35% phosphoric acid (3M) was applied for 15 seconds in 15 specimens. SingleBond (3M) adhesive system was applied and a hybrid resin composite (Filtek Z250, 3M) was inserted in four 1-mm increments and light-cured. The remaining 15 molars were prepared in the same manner, but with an acid etching time of 30 seconds. After 24 hours in water, the specimens were cut in two perpendicular directions to obtain a cross section of approximately 0.7 mm2 (n=25). A visual examination was conducted to select sticks between the two groups: sclerotic dentin (G15S or G30S) and normal dentin (G15N or G30N). Sticks without 100% sclerotic dentin (translucent area) or those with normal areas were not tested. Two-way ANOVA computed the mu-TBS data taking into consideration dentin type and acid etching time. The dentin Knoop hardness number (KHN) of the sticks was verified. A t-test compared the KHN data between sclerotic and normal dentin. Twelve additional molars (n=6) were prepared to observe the interface under a SEM. The mean (+/- SD) microtensile bond strengths (mu-TBS) were: G15S=56.4(+/- 14.9), G15N=69.7(+/- 17.2), G30S=63.2(+/- 15.6) and G30N=67.7(+/- 13.3). Two-way ANOVA showed higher mu-TBS to normal dentin than sclerotic dentin. Duncan's Post Hoc showed G15N had higher mean mu-TBS than G15S. Other comparisons were not significantly different. The t-test showed statistically higher microhardness in sclerotic dentin than in normal dentin (p<0.0001). The hybrid layer (HL) formation was observed in all specimens without gap formation in any region. In sclerotic dentin (G15S), the HL was very thin, with minimal resin tags in the dentinal tubules and, when present, they were shorter. Doubling the etching time (G30S) resulted in more resin tags with an HL formation on peritubular dentin. The HL on normal dentin was thicker when it was acid etched for 30 seconds (G30N). Numerous resin tags were present with both etching times. The results suggest that the higher mineral amount in sclerotic dentin makes it difficult to bond to this substrate, resulting in a lower mu-TBS. However, doubling the etching time resulted in mu-TBS similar to normal dentin.

Variability of clinical dentin substrates.

Clinical trials remain the only conclusive research of dentin adhesive performance, but variables unique to the oral environment may influence the results. Dentin variability is one factor that may account for extreme variations in clinical evaluations. Despite the encouraging results of laboratory studies, clinical trials with adhesive systems have been disappointing. Current adhesives are reported to react with dentin surfaces by chemical and/or micro-mechanical mechanisms. Dentin surfaces, such as cervical abrasions, are subject to intraoral changes and dentin tubules commonly become obturated by the growth of peritubular dentin or by the precipitation of calcific deposits within the tubules. The resulting sclerotic dentin may be less receptive to current dentin adhesives. An examination of clinically aged dentin surfaces has revealed an inordinate variability in tubular morphology. The more sclerotic dentin present, the less effective was dentin conditioning and resin composite adaptation. Early correlations with current clinical trials appear to substantiate that the greatest failure of restorations occurred in sclerotic lesions.

Surface morphology and chemical characterization of abrasion/erosion lesions.

PURPOSE: To describe the dentin surface morphology of abrasion/erosion lesions and to chemically characterize in vivo samples of sclerotic dentin. MATERIALS AND METHODS: Baseline polyvinylsiloxane impressions of eight in vivo caries-free lesions were taken. Dentin was collected from retention grooves for FTIR photoacoustic spectroscopic analysis. The cavity preparation was etched for 30 seconds with 37% phosphoric acid, rinsed, and dried. Impressions were taken of the etched surfaces. Epoxy resin dies were made of baseline and etched impressions, sputter-coated and examined at x1000 with the SEM. RESULTS: Lateral dentin tubule orientation was observed at gingival margins and on occlusal walls. Open cross-sectional tubules were seen at the depth of the groove. The mineral/protein ratio in the FTIR/PAS spectra of in vivo unetched sclerotic dentin samples suggested an increased mineral content.

Resin bonding to sclerotic, noncarious, cervical lesions.

Noncarious, cervical, wedge-shaped, sclerotic lesions are commonly encountered in clinical practice. In such lesions, dentin has been pathologically altered, often resulting in partial or complete obliteration of the dentinal tubules. These lesions are known to respond to etching and bonding differently from normal dentin, leading to complications during clinical treatment. A search of the literature was performed to obtain background information on the most commonly cited etiologic factors, clinical diagnoses, and morphologic and chemical characterizations along with an extensive review of all potential obstacles to bonding the most recent adhesives to such a dentinal substrate. Recent progress in adaptive strategies to render dentin more receptive to resin bonding is emphasized in this article, and the major drawbacks of these strategies are discussed.

Tenascin and fibronectin expression after pulp capping with different hemostatic agents: a preliminary study.

This study investigated the expression of extracellular matrix glycoproteins tenascin (TN) and fibronectin (FN) in pulp repair after capping with calcium hydroxide (CH), following different hemostasis protocols. Class I cavities with a pulp exposure were prepared in 42 human third molars scheduled for extraction. Different hemostatic agents (0.9% saline solution, 5.25% sodium hypochlorite and 2% chlorhexidine digluconate) were used and pulps were capped with CH cement. After 7, 30 or 90 days, teeth were extracted, formalin-fixed, and prepared for immunohistochemical technique. Hemostatic agents did not influence the expression of TN and FN. Both glycoproteins were found in the entire the pulp tissue and around collagen fibers, but were absent in the mineralized tissues. In the predentin, TN showed positive immunostaining and FN had a variable expression. Within 7 days post-treatment, a slightly more pronounced immunostaining on the pulp exposure site was observed. Within 30 days, TN and FN demonstrated a positive expression around the dentin barrier and at 90 days, a thin and linear expression of TN and FN was delimitating the reparative dentin. In conclusion, hemostatic agents did not influence TN and FN expression. Immunostaining for TN and FN was seen in different regions and periods, demonstrating their role in pulp repair.