Artificial Caries Resistance in Enamel after Topical Fluoride Treatment and 445 nm Laser Irradiation.

OBJECTIVE: This in vitro study is aimed at investigating the caries preventive effectiveness of 445 nm diode laser in combination with topical fluoridation. MATERIALS AND METHODS: A total of 30 caries-free bovine teeth were used in this study. Eighteen teeth were covered with nail varnish except four windows on the labial surface. The windows were assigned to no treatment/control (C), laser (L) (0.3 W, 60 s, and 90 J/cm2), fluoride (F), and fluoride followed by laser (FL) treatment groups. Artificial caries lesions were created, and the teeth were sectioned and investigated under polarized light microscopy for quantitative measurement of the resulted lesion depth. Ten teeth were used for surface temperature measurement and two teeth for scanning electron microscopy (SEM). Extra twelve human molars were used for the intrapulpal temperature measurement. The absorbance of fluoride at 445 nm was measured. RESULTS: The means of lesion depth for the C, L, F, and FL groups were 123.48 (±21.93), 112.33 (±20.42), 99.58 (±30.68), and 89.03 (±30.38) µm, respectively. The pairwise differences of the L, F, and FL groups compared with the C group were significant (p < 0.05). The differences between groups were tested: FL versus L p=0.02, F versus L p=0.16, and FL versus F p=0.91, and the difference of the F versus FL was not significant (p=0.91). Temperature increment at the enamel surface and pulp roof were ∆T = 16.67 (±4.11) and 2.12 (±0.66)°C, respectively. The topical fluoride absorbance at 445 nm is five orders higher than that at 810 nm. SEM shows that after laser irradiation the enamel surface was intact and without thermal damage. CONCLUSIONS: The 445 nm laser irradiation may be useful for caries prevention, and its effectiveness is lower than those previously achieved using the argon ion laser.

In Vitro Evaluation of Microleakage and Microhardness of Ethanolic Extracts of Propolis in Different Proportions Added to Glass Ionomer Cement.

OBJECTIVE: To evaluate the effect of ethanolic extracts of propolis (EEP) addition in different proportions to glass ionomer cement (GIC) on microleakage and microhardness of GIC. STUDY DESIGN: The cement was divided into four groups: one using the original composition and three with 10%, 25%, and 50% EEP added to the liquid and then manipulated. For microleakage assessment, sixty primary molars were randomly divided into four groups (n=15). Standard Class II cavities were prepared and then filled with EEP in different proportions added to GICs. Microleakage test was performed using a dye penetration method. The data were analyzed using one-way ANOVA and Mann-Whitney U tests (α = 0.05). Disc shaped specimens were prepared from the tested GIC to determine Vickers hardness (VHN). The data were analyzed using one-way ANOVA and post hoc Tukey test (α = 0.05). RESULTS: There were no statistically significant differences between the groups in terms of microleakage (p > 0.05). There were statistically significant differences between the VHN values of groups (p < 0.05). Increasing addition of EEP to GIC statistically significantly increased VHN value of GIC (p < 0.05). CONCLUSIONS: The addition of EEP to GIC increased the microhardness of the GIC and did not adversely affect the microleakage. Thus, it might be used during routine dental practice due to its antibacterial properties.

Morphological aspects and physical properties of enamel and dentine of Sus domesticus: A tooth model in laboratory research.

This study aims to describe and analyze morphological and physical properties of deciduous teeth of Sus domesticus. Ultrastructural analysis, mineral composition and microhardness of enamel and dentine tissues were performed on 10 skulls of S. domesticus. External anatomic characteristics and the internal anatomy of the teeth were also described. Data regarding microhardness and ultrastructural analysis were subjected to statistical tests. For ultrastructural analysis, we used the analysis of variance (ANOVA) with Tukey's post hoc (p≤0.05) test. In the analysis of microhardness, the difference between the enamel and dentine tissues was analyzed by a Student's t test. Values were expressed as mean with standard error. The results of ultrastructural analysis showed the presence of an enamel prism pattern. A dentinal tubule pattern was also observed, with a larger diameter in the pulp chamber and the cervical third, in comparison to middle and apical thirds. We observed an average microhardness of 259.2kgf/mm(2) for enamel and 55.17kgf/mm(2) for dentine. In porcine enamel and dentine, the chemical elements Ca and P showed the highest concentration. The analysis of internal anatomy revealed the presence of a simple root canal system and the occurrence of main canals in the roots. The observed features are compatible with the functional demand of these animals, following a pattern very similar to that seen in other groups of mammals, which can encourage the development of research using dental elements from the pig as a substitute for human teeth in laboratory research.

The expression of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and HCN2 in the rat trigeminal ganglion, sensory root, and dental pulp.

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and 2 (HCN2) are abundantly expressed in primary sensory neurons and contribute to neuronal excitability and pathological pain. We studied the expression of HCN1 and HCN2 in the rat trigeminal ganglion (TG) neurons and axons in the dental pulp, and the changes in their expression following inflammation, using light- and electron-microscopic immunocytochemistry and quantitative analysis. HCN1 and HCN2 were expressed predominantly in large-sized, neurofilament 200-immunopositive (+) or parvalbumin+ soma in the TG whereas they were expressed mostly in unmyelinated and small myelinated axons in the sensory root. The expression was particularly strong along the plasma membrane in the soma. In the dental pulp, majority of HCN1+ and HCN2+ axons coexpressed calcitonin gene-related peptide. They were expressed mainly in the peripheral pulp and pulp horn where the axons branch extensively in the dental pulp. The expression of HCN1 and HCN2 in TG neurons increased significantly in rats with experimentally induced inflammation of the dental pulp. Our findings support the notion that HCN1 and HCN2 are expressed mainly by both the soma of mechanosensitive neurons in the TG and peripheral axons of nociceptive neurons in the sensory root, and may play a role in the mechanisms of inflammatory pain from the dental pulp.

The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars.

Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs). However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored. We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells' proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P < 0.05). However, this enhancement was inconsistent when the cells were cultured in 1% PL or in 10% PL; 10% PL significantly inhibited cell proliferation and was therefore excluded from further differentiation testing. Culture medium containing 5% PL also significantly promoted the mineralized differentiation of DPSCs, as indicated by the measurement of alkaline phosphatase activity and calcium deposition under mineral-conditioned media (P < 0.05). Scanning electron microscopy and modified Ponceau trichrome staining showed that the cells treated with 5% PL and mineralizing media were highly capable of integrating with the HA/TCP biomaterials and had fully covered the surface of the scaffold with an extensive sheet-like structure 14 d after seeding. In addition, 5% PL showed significantly positive effects on tissue regeneration in two in vivo transplantation models. We conclude that the appropriate concentration of PL enhances the proliferation and mineralized differentiation of human DPSCs both in vitro and in vivo, which supports the use of PL as an alternative to FBS or a nonzoonotic adjuvant for cell culture in future clinical trials. However, the elucidation of the molecular complexity of PL products and the identification of both the essential growth factors that determine the fate of a specific stem cell and the criteria to establish dosing require further investigation.

STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.

Effects of low-power red laser on dentine-pulp interface after cavity preparation. An ultrastructural study.

OBJECTIVE: Studies on the influence of low-power red laser on the repair of dental structures are very scarce. This study investigated the effects of the laser therapy on the ultrastructure of the dentine-pulp interface after conservative class I cavity preparation. DESIGN: Two female volunteers with 8 premolars indicated for extraction for orthodontic reasons were recruited. Class I cavities were prepared and the teeth were randomly divided into two groups. The first group received treatment with a GaA1As laser, lambda=660nm, power of 30mW and energy dose of 2J/cm(2), directly and perpendicularly into the cavity in a single visit. After the irradiation, the cavities were filled with composite resin. The second group received the same treatment, except by the laser therapy. RESULTS: Twenty-eight days post-preparation, the teeth were extracted and processed for transmission electron microscopy analysis. Two sound teeth, without cavity preparation, were also studied. The irradiated group presented odontoblast process in higher contact with the extracellular matrix and the collagen fibrils appeared more aggregated and organised than those of control group. These results were also observed in the healthy teeth. CONCLUSION: These findings suggest that laser irradiation accelerates the recovery of the dental structures involved in the cavity preparation at the predentine region.

Radiological and microscopic aspects of the denticles.

In this paper we have realized a study on 43 patients on which the retro-alveolar radiological exam has shown the existence of a calcareous structure within the pulp chamber having in view a better knowledge of this structure and its etiology, way of forming and pathological implications. For 16 of the patients the radiological exam was supplemented by a microscopic examination. Radiologically, the denticles were noticed more often in the pulp chamber of the molars. The examination of the histological samples has evidenced aspects such as the size, shape and structure of the denticles. The concentric disposition of the tissue elements shows that in the formation of a denticle there is a "center" around which a conjunctive substance avid of calcium salts is deposited. The microscopic aspects noticed on large denticles allowed us to sustain the hypothesis that in a large pulp chamber many denticles form simultaneously and grow up and merge generating a large-sized unique calcareous structure.

[Changes of dentin, dental pulp and periodontium tissue in tail-suspended rats].

OBJECTIVE: To investigate the metabolic changes of calcium and phosphorus in dentin, dental pulp and periodontium in tail-suspended rats, and the functions of TGF-beta 1, c-fos, collagen-I and collagen IV in dentin, dental pulp and periodontium. METHOD: Relative percentage contents of Ca, P in dentin, dental pulp and periodontium were measured with scanning electron microscope and energy spectrum analytical system in 3 groups of rats. The expression of TGF-beta 1, c-fos, collagen-I and collagen IV were also observed. RESULT: In the suspension group, the relative percentage content of Ca declined significantly, while P increased slightly. There were no significant differences of Ca, P in alveolar bone. The expressions of TGF-beta 1, c-fos and collagen-I declined, but the expression of collagen-IV in pulp vessel increased. There were no significant changes of expressions of TGF-beta 1, c-fos, collagen-I and collagen-IV in the vicinity of PDL. After adopting artificial countermeasures, the above expressions restored partly. CONCLUSION: Weightlessness might cause abnormal mineralization in dentin, and 1.5 G artificial countermeasures could eliminate the above changes of mineral metabolism. The poor mineralization of dentin might be associated with the reduced secretion of TGF-beta 1, c-fos and collagen-I in tail-suspended rats.

Ca-binding domains in the odontoblast layer of rat molars and incisors under normal and pathological conditions.

We recently reported the presence of high concentrations of a Ca-binding matrix in the circumpulpal dentin of rat incisors which had been prevented from mineralization by a systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), a type of bisphosphonates, thus suggesting the role of the putative Ca-binding matrix in the appositional mineralization of circumpulpal dentin (TAKANO et al., 1998, 2000; OHMA et al., 2000). In this study, we examined the distribution of Ca-binding domains in the pulp tissue of normal rat teeth and its changes under the influence of HEBP, in order to identify and clarify the role of the Ca-binding matrix in the physiological process of dentin mineralization. Observation of the normal rat tooth pulp showed occasional, tiny extracellular deposits of Ca-enriched material in the odontoblast layer, associated primarily with pericapillary regions. Such deposits were immunopositive for dentin sialoprotein (DSP), displayed high levels of X-ray peaks for calcium and phosphorus, and showed a drastic increase in amount by daily injections of HEBP. A brief vascular perfusion of high Ca-containing solution in normal animals caused the extensive deposition of Ca-P complexes along the basolateral membranes of odontoblasts but not in the other regions of the pulp tissue. These data suggest the existence of DSP-enriched extracellular Ca-binding domains in the odontoblast layer and also indicate a novel Ca-binding property of the basolateral membranes of odontoblasts. Since DSP is primarily synthesized as dentin sialophosphoprotein (DSPP) and later cleaved into dentin phosphophoryn (DPP) and DSP in odontoblasts, and since DSP has no notable affinity for Ca, the sites of DSP-immunopositive Ca-P deposits in the odontoblast layer may also contain DPP, a highly phosphorylated acidic protein having a strong binding property for calcium. Characteristic Ca-binding properties seen in the odontoblast layer appear to be related to the regulation of the appositional mineralization of circumpulpal dentin.

The influence of preparation technique and sodium hypochlorite on removal of pulp and predentine from root canals of posterior teeth.

AIM: To test the null hypothesis that removal of pulp tissue and predentine from root canals of multirooted teeth is not significantly different when performed by one of two mechanical preparation techniques in conjunction with either water or 3% sodium hypochlorite (NaOCl) irrigation. METHODOLOGY: Forty-six freshly extracted premolar or molar teeth, with pulps vital at the time of extraction, were assigned to four groups balanced by anatomy (group 1, step-back filing/NaOCl; group 2, step-back filing/water; group 3, automated rotary/NaOCl; group 4, automated rotary/water). Preparation was undertaken by a single operator and the volumes of irrigant recorded for each tooth. One root from each tooth was prepared for transverse histological assessment at eight levels, including one at and two apical to the working length. A validated index was used to measure the presence and location of residual pulp tissue and predentine. RESULTS: No statistically significant differences were found in the frequency with which residual pulp tissue or predentine were observed amongst the groups. Coronal parts were more frequently devoid of pulp tissue and predentine than the apical. Canals were frequently occluded with residual pulp tissue apical to the working length. There was a trend for better pulp and predentine removal in accessible parts of the canal as a function of rotary preparation and NaOCl irrigation. The isthmus had residual pulp tissue less frequently in the NaOCl groups. Accessory anatomy was unaffected regardless of technique or irrigant. CONCLUSIONS: Pulp tissue and predentine removal were not significantly different between a step-back filing and an automated rotary preparation technique in conjunction with water or NaOCl.

Efficacy of final irrigation--a scanning electron microscopic evaluation.

The purpose of this in vitro study was to determine the degree of removal of pulpal remnants and smear layer from root canals after final irrigation with three different solutions. During instrumentation the step-back preparation and 1% NaOCl were used. The final 4-min, 30-ml irrigation varied as follows: group I, 10 ml of 1% NaOCl + 10 ml of 10% citric acid + 10 ml of distilled water; group II, 15 ml of 0.5% NaOCl + 15 ml of EDTA-T; and group III, 10 ml of 5% NaOCl + 10 ml of 3% H2O2 + 10 ml of 5% NaOCl. Scanning electron microscopic photomicrographs were evaluated for the mean number of visible open dentinal tubules by three observers. The largest number of visible tubules in the three groups was in the cervical third, followed by the middle and apical thirds. There was no statistically significant difference between groups I and II when third was compared with third; however, groups I and II had significantly more visible dentinal tubules than group III.

Histopathologic effects of kinetic cavity preparation for the removal of enamel and dentin. An in vivo animal study.

Recent developments in technology, direct placement restorative materials, and cavity preparation design have renewed interest in kinetic cavity preparation, a term to describe the use of air-abrasion for removal of tooth structure. This study compared the pulpal response of 120 teeth in mixed-breed dogs treated with four kinetic cavity preparation combinations of pressure (80 psi and 160 psi) and aluminum oxide particle sizes (27 microns and 50 microns) to those treated with high-speed rotary burs. Class V buccal preparations were made and restored with an interim material. Teeth were collected 72 hours after surgery, decalcified, sectioned, stained with hematoxylin and eosin, and blindly evaluated by two examiners at the minimal dentin thickness. Samples were graded for extent of displacement, disruption, inflammation, and necrosis of pulpal structures. Differences between groups were analyzed with the use of Bonferroni-adjusted multiple Mann-Whitney-Wilcoxon tests with p < 0.05 being significant. Higher pressures and smaller particles yielded significantly fewer pulpal effects than the high-speed treated teeth whereas lower pressures and larger particles were not significantly different for most effects evaluated. No adverse soft tissue effects were noted when kinetic cavity preparation was directed at attached gingiva.

Mineral content of the dentine remaining after chemomechanical caries removal.

Although the dentine remaining after chemomechanical caries removal appears sound by normal clinical criteria, no definitive evidence has yet been obtained to confirm that the dentine surface is in fact mineralised. The aim of this study was to use backscattered electron (BSE) imaging and electron probe micro-analysis (EPMA) to ascertain the level of mineralisation of the dentine remaining in cavities prepared by this technique. Carious dentine was removed from carious lesions by means of N-monochloro-DL-2-aminobutyric acid (NMAB) or NMAB containing 2 mol/l urea. Sections of teeth in which caries removal was complete by normal clinical criteria were examined by EPMA and BSE. Dentine adjacent to the pulp was found to be less mineralised than the surrounding dentine. Although the superficial layer of dentine remaining on the cavity floors frequently appeared to have a slightly reduced mineral content, the results clearly indicated that there was no significant difference between this dentine and the underlying sound dentine.

Histological observations of hard tissue barrier formation in amputated dental pulp capped with alpha-tricalcium phosphate containing calcium hydroxide.

This study was performed to evaluate the pulpal response to alpha-tricalcium phosphate (alpha TCP) containing calcium hydroxide (Ca(OH)2). The dental pulps of monkeys were amputated and dressed with four agents: alpha TCP, alpha TCP containing 1% Ca(OH)2, alpha TCP containing 5% Ca(OH)2, and Ca(OH)2 being used as a control. The pulpal responses were histologically evaluated after 4 and 8 weeks. The pulp tissue treated with alpha TCP proliferated above the level of the original wound surface, and a thin layer of hard tissue barrier was formed directly against the capping agent. The barrier demonstrated atubular matrix lined with flattened or cuboidal cells, but occasionally appeared irregular in form. Ca(OH)2 dressing resulted in destruction of pulp tissue, with a thick hard tissue barrier being formed below the level of the exposure site. The barrier consisted coronally of osteodentin and pulpally of tubular dentin lined with odontoblast-like cells. By contrast, 1% Ca(OH)2 added to alpha TCP produced a slight proliferation of pulp tissue. An atubular matrix barrier, pulpally lined with cuboidal cells, formed above the exposure site. It was later followed by the formation of tubular matrix lined with columnar cells. Teeth treated with 5% Ca(OH)2 showed a thin necrotic layer and a thick barrier formation. The barrier was composed of tubular dentin-like tissue lined with odontoblast-like cells. It would appear that alpha TCP containing a small amount of Ca(OH)2 may be clinically useful as a capping agent, as it induced consistent hard tissue formation, without excessive destruction of underlying pulp tissue.

[Effects of CO2 laser irradiation on microstructure of dentin, dentin adhesion of resin composites and dental pulp].

This study was designed to investigate the effects of CO2 laser irradiation on the dentin microstructure, dentin adhesion of resin composites and dental pulp. An artificial caries lesion was produced on the bovine dentin, immersing in 0.4 mol/l acetic acid. Three kinds of dentin (sound dentin, outer or inner caries dentin) surfaces were pretreated by the laser irradiation, acid etching or no-treatment. The tensile adhesion-test of these pretreated dentin surfaces was performed. The changes in these dentin surfaces by the various treatments including the laser irradiation and the resin-dentin interfaces were examined by the SEM. Furthermore, histopathological study using monkey teeth was conducted to examine the effect of the laser irradiation on the dental pulp. Histopathological changes in the pulp were found at 3 and 90 days after the operation. The findings were as follows: 1. The outer highly and inner partially decalcified layers produced in the bovine dentin were each approximately 200 microns in thickness. 2. The bond strength of the resin composites to the laser pretreated sound dentin was approximately 40 kgf/cm2. Those of the laser pretreated outer and inner decalcified dentins were about 60 kgf/cm2. 3. At the initial stage, the laser irradiation (output power 3W : irradiation period 0.5 second) exhibited a slight pulpal response, producing irritation dentin formation in the latter stage without any severe histopathological change.

Matrix vesicles isolated from apical pulp of rat incisors: crystal formation in low Ca x Pi ion-product medium containing beta-glycerophosphate.

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.