Analysis of factors related to needle-stick and sharps injuries at a dental specialty university hospital and possible prevention methods.

Needle-stick and sharps injuries (NSIs) can happen even when dental health care workers (DHCWs) act in compliance with standard precautions to prevent transmitting blood-borne infections. The objective of this study was to investigate causes of NSIs that had occurred at a dental specialty university hospital during the past 12 academic years. A total of 215 NSIs were reported during the investigation period, and NSIs ascribed to female DHCWs (n = 148; 68.8%) were significantly (P < 0.05) more common than those ascribed to male DHCWs (n = 67; 31.2%). One hundred twenty-six NSIs (58.6%) were caused by DHCWs with little experience (P < 0.05), and 37 of those (17.2% of the total) were ascribed to undergraduate students during clinical training (P < 0.05). The NSIs occurred both during treatment (n = 119; 55.3%) and during cleaning up used devices (n = 89; 41.4%). The NSIs at the dental hospital occurred with a probability of 0.004% of total therapeutic opportunities. Prevention of NSIs should be the responsibility of dental students and DHCWs, and should be a part of education about infection control to prevent the nosocomial transmission of blood-borne pathogens.

Irrigation effectiveness of continuous ultrasonic irrigation system: An ex vivo study.

This study evaluated the irrigation extrusion and smear layer (SL) removal of continuous ultrasonic irrigation (CUI). Forty root canals were shaped, embedded in warm sterile saline agar colored with 1% acid red, and distributed into four groups; three groups had the final irrigation sequence of sodium hypochlorite and EDTA solutions performed by using syringe irrigation (SI), EndoVac (EV), and CUI, respectively. Group 4 had the canals ultrasonically irrigated with saline (CUIS). Irrigant extrusion was verified by a color change of the agar. The SL was evaluated under scanning electron microscope. Irrigant extrusion was observed in two and four teeth in the CUI and SI, respectively, whilst none had irrigant extruded in the EV. In term of SL, the CUI was comparable to EV at 1 and 3 mm but different to SI at 1 mm. Although CUI might clean the root canal system effectively, it was unable to avoid irrigant extrusion.

Effect of nickel titanium file design on the root surface strain and apical microcracks.

The aim of this study was to determine the effect of nickel titanium file design on the root surface strain generated and apical microcracks caused during canal shaping. Thirty-three mandibular incisors were distributed into LightSpeed X, FlexMaster and a control group. A strain gauge was fixed apically on the proximal root surface to determine the maximum strain during canal shaping. Except for the control group, all root canals were enlarged to size 50. Images were taken after removing the apical 1 and 2 mm of the root end. Mean maximum strain values and presence of microcracks were statistically compared using the t-test and chi-square test, respectively. During canal shaping, the strain increased cumulatively with mean maximum strains of 808.2 ± 228.8 and 525.1 ± 168.9 microstrain in LightSpeed X and FlexMaster, respectively (P = 0.004). Both systems caused comparable microcracks. Although LightSpeed X produced higher maximum strain, no difference in microcrack development was found between both systems.

Shaping ability of ProTaper NEXT, ProTaper Universal and iRace files in simulated S-shaped canals.

The aim of this study was to determine the shaping ability of three nickel-titanium endodontic file systems by comparing three parameters: canal deviation, apical foramen position and instrumentation time. A glide path was established in 30 simulated S-shaped canal blocks that were randomly assigned into three groups (n = 10): ProTaper Universal, ProTaper NEXT and iRace. Each group was instrumented per its manufacturer's directions. Pre- and postoperative images were superimposed to determine any canal deviation or change in apical foramen position. The instrumentation times were recorded. The iRace system resulted in the least mean canal deviation. The apical foramen position was least shifted by the iRace system. The iRace system also required the least instrumentation time. The iRace system demonstrated the most favourable shaping ability in all three parameters.

Performance of NiTi endodontic instrument under different temperatures.

The purpose of this study was to test nickel titanium (NiTi) instrument performance under different surrounding temperatures. Twenty-four superelastic NiTi instruments with a conical shape comprising a 0.30-mm-diameter tip and 0.06 taper were equally divided into 3 groups according to the temperature employed. Using a specially designed cyclic fatigue testing apparatus, each instrument was deflected to give a curvature 10 mm in radius and a 30° angle. This position was kept as the instrument was immersed in a continuous flow of water under a temperature of 10, 37, or 50 °C for 20 s to calculate the deflecting load (DL). In the same position, the instrument was then allowed to rotate at 300 rpm to fracture, and the working time was converted to the number of cycles to fracture (NCF). The statistical significance was set at p = 0.05. The mean DL (in N) and NCF (in cycles) of the groups at 10, 37, and 50 °C were 10.16 ± 1.36 and 135.50 ± 31.48, 13.50 ± 0.92 and 89.20 ± 16.44, and 14.70 ± 1.21 and 65.50 ± 15.90, respectively. The group at 10 °C had significantly the lowest DL that favorably resulted in the highest NCF. Within the limitations of this study, the surrounding temperature influences the cyclic fatigue resistance and DL of the superelastic NiTi instruments. Lower temperatures are found to favorably decrease the DL and extend the lifetime of the superelastic NiTi instrument. Further NiTi instrument failure studies should be performed under simulated body temperature.

A comparison between two negative pressure irrigation techniques in simulated immature tooth: an ex vivo study.

OBJECTIVES: This ex vivo study evaluated the irrigation efficacy of a new apical negative pressure system (ANP) in canals with simulated immature teeth, by comparing it to EndoVac (EV) system in terms of smear layer (SL) removal and irrigation extrusion. MATERIALS AND METHODS: Three millimetres of the root end of 40 single canalled lower incisors were resected and decoronated to standardize root canal length. After instrumentation, the specimens were embedded in warm normal saline agar coloured with 1 % acid red and randomly divided into four groups; one control group and three experimental groups. Except in the control group where distilled water was used as irrigant using positive pressure irrigation needle, the canals were irrigated with 6 % NaOCl and 17 % EDTA using the intracanal negative pressure needle (iNP) system, the EV system or 27G open-ended needle under positive pressure (PP). NaOCl extrusion was determined by observing a discolouration of the agar surrounding the root. The SL was evaluated by observing scanning electron microscope images based on a four-level scoring system. RESULTS: Two specimens with irrigant extrusion were observed in the iNP group, which was significantly different (logistic regression, p < 0.05) to EV and PP. There were no significant differences (Kruskall-Wallis test, p > 0.05) among the experimental groups in terms of SL removal, but all were significantly different to the control group. CONCLUSIONS: Irrigation with the iNP could be a viable alternative to EV as an apical negative pressure irrigation technique especially while treating immature teeth. CLINICAL RELEVANCE: ANP in canal cleanliness is recommended to be utilized in treating immature teeth where periapical tissues should be saved and stimulated. The iNP system might have the potential to avoid irrigant extrusion while cleaning the canal till the apical end.

Sympathetic Regulation of Tertiary Dentinogenesis via Beta-2 Adrenergic Receptor on Rat Odontoblasts.

INTRODUCTION: Beta-2 adrenergic receptor has been found within the osteoblast membrane meditating bone remodeling. Propranolol is a sympatholytic beta antagonist commonly used as long-term medication for the management of many common diseases such as hypertension. This study was performed to verify the presence of this receptor on odontoblasts in rats and, if present, to investigate its possible association with tertiary dentinogenesis. METHODS: Twenty male Sprague-Dawley rats (9 weeks old) were randomly assigned to 4 groups: CP0.8 group, cavity preparation + propranolol treatment (0.8 mg/day, n = 5); CP4 group, cavity preparation + propranolol treatment (4.0 mg/day, n = 7); CON group, cavity preparation + saline treatment (0.2 mL/day, n = 5); and NT group, no treatment (n = 3). Cavity preparation was performed on the mesial aspect of the maxillary first molars bilaterally. After 2 weeks, the tertiary dentinogenesis (CP0.8, CP4, and CON) was examined by hematoxylin-eosin staining, and the localization of beta-2 adrenergic receptor (NT) was examined by immunohistochemistry. RESULTS: The beta-2 adrenergic receptor immunoreactivity was observed in the odontoblastic layer in normal rat molar dental pulp. The tertiary dentinogenesis beneath the prepared cavity was significantly higher in the rats receiving 2-week systemic administration of propranolol than in those without the propranolol treatment. The higher-dose treatment of propranolol (P < .001) presented more effective up-regulation of tertiary dentinogenesis than the lower-dose treatment (P < .01). CONCLUSIONS: These results indicate that the sympathetic nervous system decreases tertiary dentin formation via beta-2 adrenergic receptors located on rat odontoblasts. It suggests that adrenergic beta antagonist is expected to use in the treatment of inducing tertiary dentin formation to protect dental pulp.

Periostin is a negative regulator of mineralization in the dental pulp tissue.

The dental pulp tissue is encased in hard tissue and surrounded by hard tissue-forming cells, but remains in a non-mineralized state itself, suggesting the presence of regulatory mechanisms precluding pulp mineralization. This study aimed to reveal the regulatory function of periostin (Postn), which is essential for osteoblast differentiation, for odontoblast differentiation/mineralization. We evaluated the effects of Postn overexpression and RNAi-mediated suppression in mouse dental papilla cells (MDPs) on the expression of odontoblastic markers and Notch signaling molecules, and on the formation of mineralized nodules. Localization of Postn in the dental pulp tissue of normal and cavity-prepared molars was observed immunohistologically. Enforced overexpression of Postn in MDPs induced down-regulation of odontoblastic markers and in vitro mineralization. Conversely, silencing of Postn mRNA in MDPs induced up-regulation of odontoblastic markers and ALP activity. Up- and down-regulation of Postn caused increased and decreased expression, respectively, of Notch signaling molecules. Postn expression was minimal in normal dental pulp, but was rapidly and globally increased in the whole pulp tissue of molar teeth at 1 day after cavity preparation, decreasing thereafter. These results indicate that Postn may be a negative regulator of odontoblast differentiation/mineralization, and that may exert its actions via Notch signals.

Detection of a second mesiobuccal canal in maxillary molars by swept-source optical coherence tomography.

INTRODUCTION: The purpose of this study was to investigate the ability of swept-source optical coherence tomographic (SS-OCT) imaging to detect a second mesiobuccal canal (MB2) in maxillary molars compared with visual inspection (VI) and dental operating microscopy (DOM). METHODS: Forty extracted human maxillary molars were examined. After the removal of half the crown and access cavity preparation, the existence of MB2 canals was confirmed based on cross-sectional images of each tooth obtained by micro-computed tomographic scanning as the reference standard. Then, the pulp chamber floor was scanned by SS-OCT imaging. Three dentists independently evaluated the SS-OCT images and the pulp chamber floor under VI and DOM for the presence of MB2 canals. The detection rate of MB2 canals for SS-OCT imaging, VI, and DOM was calculated, and statistical analysis was performed. RESULTS: MB2 canals existed in 19 of 40 teeth (47.5%) using micro-CT imaging. Sensitivity of DOM (0.947) was significantly higher than that of SS-OCT imaging (0.632). Specificity of SS-OCT imaging (0.714) was significantly higher than that of DOM (0.333). No statistically significant differences were found for accuracy among the 3 methods. Kappa values of SS-OCT, VI, and DOM were 0.526, 0.417, and 0.326, respectively. CONCLUSIONS: SS-OCT imaging is noninvasive, involves no ionizing radiation, and is accurate for the detection of MB2 canals.

Correlation between cyclic fatigue and the bending properties of nickel titanium endodontic instruments.

The effects of cyclic fatigue on bending properties of NiTi endodontic instruments were investigated. Sixteen Profiles(®) were divided into two groups (A, and B). The sequence of cantilever bending test and cyclic fatigue test was alternated repeatedly until file separation occurred. In the cyclic fatigue test, the instrument curvature was 19° in group A and 38° in group B. Fractographic examination was performed to determine fracture patterns. In group A, there were significant differences between the bending load values measured before the cyclic fatigue test and the last cantilever bending test before instrument fracture at each deflection (p<0.05). Fractographic examination showed the specific patterns of cyclic fatigue fracture. The stress required to induce martensitic transformation might be reduced due to the softening behavior caused by the cyclic fatigue under the relaxation condition of the superelasticity range (group A). The SEM images were able to display specific patterns indicating cyclic fatigue fracture.

Down-regulation of inflammatory mediator synthesis and infiltration of inflammatory cells by MMP-3 in experimentally induced rat pulpitis.

INTRODUCTION: Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. METHODS: Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. RESULTS: NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. CONCLUSIONS: These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.

Application of optical coherence tomography to identify pulp exposure during access cavity preparation using an Er:YAG laser.

OBJECTIVE: The study aimed to evaluate the ability of optical coherence tomography (OCT) to guide and identify pulp exposure using an erbium: yttrium-aluminum-garnet (Er:YAG) laser. BACKGROUND DATA: The Er:YAG laser has been proven to be effective in ablating dental hard tissue and offers advantages, as there is none of the vibration and noise you get with conventional methods, but it has limitations in relation to the tactile feedback that would aid in identification of entry into the pulp chamber. Based on depth-resolved optical reflectivity, OCT technology has been developed to provide high-resolution, cross-sectional images of the internal structure of biological tissues. MATERIALS AND METHODS: The pulp chambers of 20 human mandibular incisors were examined, and the average thickness of hard tissue covering the pulp chamber was assessed using micro-computed tomography (micro-CT) images. An Er:YAG laser was used to gradually penetrate the hard tissue over the pulp chamber under microscopic guidance. The preparation was constantly imaged using a swept-source OCT at 10 sec intervals until a pulp chamber exposure was identified using the technology. The pulp exposure was re-examined under the microscope and compared with micro-CT images for verification. RESULTS: The pulp exposures of 20 incisors were all verified microscopically and with micro-CT images. The thickness of hard tissue penetrated by the laser ranged from 0.44 to 1.69 mm. CONCLUSIONS: Swept-source OCT is a useful tool for identifying pulp exposure during access opening with the Er: YAG laser.

Endodontic instruments after torsional failure: nanoindentation test.

This study aimed to evaluate effects of torsional loading on the mechanical properties of endodontic instruments using the nanoindentation technique. ProFile (PF; size 30, taper 04; Dentsply Maillefer, Switzerland) and stainless steel (SS; size 30, taper 02; Mani, Japan) instruments were subjected to torsional test. Nanoindentation was then performed adjacent to the edge of fracture (edge) and at the cutting part beside the shank (shank). Hardness and elastic modulus were measured under 100-mN force on 100 locations at each region, and compared to those obtained from the same regions on new instruments. It showed that PF and SS instruments failed at 559 ± 67 and 596 ± 73 rotation degrees and mean maximum torque of 0.90 ± 0.07 and 0.99 ± 0.05 N-cm, respectively. Hardness and elastic modulus ranged 4.8-6.7 and 118-339 GPa in SS, and 2.7-3.2 and 52-81 GPa in PF. Significant differences between torsion-fractured and new instruments in hardness and elastic modulus were detected in the SS system used. While in PF system, the edge region after torsional fracture had significantly lower hardness and elastic modulus compared to new instruments. The local hardness and modulus of elasticity of endodontic instruments adjacent to the fracture edge are significantly reduced by torsional loading.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

OBJECTIVE: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. METHODS: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. RESULTS: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. CONCLUSION: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.

The influence of heat treatment on the mechanical properties of Ni-Ti file materials.

The purpose of this study was to investigate the influence of heat treatment on the mechanical properties of Ni-Ti file materials. Ni-Ti wire (1.00 mm ø) was processed into a conical shape with 0.30-mm diameter tip and 0.06 taper. Specimens were heated for 30 min at 300, 400, 450, 500 or 600°C. Non-heated specimens were used as controls. DSC, a cantilever-bending test and cyclic fatigue test were performed. Ms and Af for groups 400 and 450 were higher than those for others (p<0.05). The load/deflection ratios of groups 400, 450 and 500 were lower than that of group 600 (p<0.05). The bending load values at 2.0-mm deflection of groups 400, 450 and 500 were lower than those of group 300 and the control group (p<0.05). The NCFs of groups 400, 450 and 500 exceeded that of group 600(p<0.05). Changes in flexibility with heat treatment could improve the cyclic fatigue properties of Ni-Ti instruments.

Joint haemorrhage partly accelerated immobilization-induced synovial adhesions and capsular shortening in rats.

PURPOSE: To elucidate the effects of intra-articular haemorrhage on the joint capsule of immobilized knees in rats. METHODS: The unilateral knee joints were immobilized using a plastic plate and screws. Sham operated rats had only screws inserted. A single injection of fresh autologous blood was given postoperatively into the knee joints of the immobilized blood injection (Im-B) and the Sham blood injection (Sm-B) groups. Normal saline was administered for the immobilized-saline injection (Im-S) group. Sagittal sections were prepared from the medial midcondylar region of the knee and assessed with histological, histomorphometric, and immunohistochemical methods. The range of motion (ROM) was measured, and the mechanical property of the capsule was assessed by scanning acoustic microscope. RESULTS: Absorption of the injected blood was delayed and made severe adhesions in the Im-B group. The length of the synovial membrane in the Im-B group was significantly shorter than that of the other groups. The ROM was significantly restricted in the Im-B group compared with the other groups. The elasticity of the posterior capsule in the Im-B group was significantly lower than that in the Sm-B group. Iron deposition was observed in the Im-B and Sm-B groups. Strong immunoreactivities of CD68, TGF-ß1, and α-SMA were observed in the adhesion area of the Im-B group. Joint immobilization with blood injection caused severe capsular adhesion and limited range of motion. Immunostaining related to fibrosis increased with joint haemorrhage. CONCLUSION: Intra-articular haemorrhage with joint immobilization might be an accelerated risk factor for joint contracture. It is likely that leaving a haematoma inside an immobilized joint should be avoided.

Kinetics of Th17-related cytokine expression in experimentally induced rat periapical lesions.

Th17-related cytokines are essential factors in various pathological states, including inflammatory bone destruction. This study investigated the contribution of Th17-related cytokines to the progress of experimentally induced rat periapical lesions. Periapical pathoses were induced by unsealed exposure of the pulp chamber of the lower first molars. A variety of immunocompetent cells, including CD68(+) macrophages, Ia antigen(+) cells and TCRαß(+) T cells, were observed in the lesions. The expression levels of Th17-related cytokines, IL-17 and IL-23, and of pro-inflammatory cytokines, IL-1ß and IL-6, were significantly increased at 14 days (expansion stage) compared with normal periapical tissues. The expression levels of Foxp3, a regulatory T cell (Treg)-related gene, and of IL-10, an anti-inflammatory cytokine, were higher at 28 days (chronic stage) than at 14 days. These findings suggest that Th17-related cytokines may be primary contributors to the initiation of periapical bone destruction, and that lesion expansion may be regulated by anti-inflammatory mediators.

Wnt11 expression in rat dental pulp and promotional effects of Wnt signaling on odontoblast differentiation.

Congenital anomalies of wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) are frequently accompanied with tooth and dentin abnormality. The aim of this study was to investigate the effects of Wnt signaling on odontoblast differentiation of mouse dental papilla cells (MDPs). Mouse dental papilla cells were cultured in α-modified minimum essential medium containing 10% fetal bovine serum and antibiotics. Odontoblast differentiation was induced by bone morphogenic protein 2 (BMP2), and the expression of odontoblast-specific markers and Wnt-related signaling molecules was analyzed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Odontoblast differentiation was evaluated by dentin sialophosphoprotein (Dspp) and dentin matrix protein (DMP) 1 expression. Localization of ß-catenin in MDPs was detected by immunocytochemistry using an anti-ß-catenin antibody. Dspp expression in MDPs was upregulated in the presence of BMP2. Wnt5a, Wnt11, Lef1 and Tcf4 expression was upregulated in BMP2-treated MDPs. Wnt11 expression was detected in rat dental pulp in vivo, and particularly strong expression of Wnt11 was detected in odontoblasts. Enhanced Dspp and DMP1 expression and alkaline phosphatase activity induced by BMP2 were completely negated by the Wnt antagonist: IWR-1-endo treatment. Nuclear translocation of ß-catenin observed in BMP2-treated MDPs was also negated by IWR-1-endo treatment. These results indicate that Wnt signaling upregulates odontoblast marker expression in MDPs, suggesting a promoting effect of Wnt signaling on odontoblast differentiation.

Comparison of the time required to create secondary fracture of separated file fragments by using ultrasonic vibration under various canal conditions.

INTRODUCTION: Nickel-titanium files often separate because of mechanical fatigue. The purpose of this study was to determine safe preparation techniques for separated file removal by using ultrasonics. METHODS: Fifty nickel-titanium file fragments were divided into 5 groups. An ultrasonic tip was activated on a file fragment positioned between dentin blocks simulating several canal conditions: Group 1 consisted of the fragment protruding from a pair of straight dentin blocks. For group 2, the fragment was also positioned between 2 straight dentin blocks except one block was positioned 1 mm more apically than the other block, simulating a troughed area that is often created during file removal attempts. For groups 3-5, the fragment was positioned similarly as group 2 but between blocks with 30°, 45°, and 60° curvatures, respectively. The time it took for secondary fracture to occur was recorded, and the data were statistically analyzed. RESULTS: Fragments with dentin wall supporting on the opposite side of ultrasonic activation site resisted fracture significantly longer than those without it. Fragments in 30° and 45° curved blocks took significantly longer to fracture than the other groups (Fisher protected least significant difference, P < .05). CONCLUSIONS: Secondary fracture of separated files appeared to be reduced when the ultrasonic tip was applied to the inner curvature of the canal.

Determination of the functional space for fluid movement in the rat dentinal tubules using fluorescent microsphere.

OBJECTIVES: The size of the functional space available for hydrodynamic fluid movement between cellular components and the walls of dentinal tubules has not yet been investigated. We attempted to measure the space using small diameter fluorescent microspheres. METHODS: The coronal enamel of 144 rat molars was removed to expose the dentine, which was acid-etched. Fluorescent microspheres of different diameters (0.02-4.0µm) were applied to the exposed dentine for 60min before the rat jaws were cut into cryostat sections. The distribution and fluorescent intensities of the fluorescent microspheres were examined with confocal laser scanning microscope and analyzed using image analysis software. RESULTS: Microspheres with a diameter of 2.0-4.0µm were detected only on the surface of the cavities. A small number of microspheres with a diameter of 1.0µm accumulated primarily in the outer third of the dentine. Microspheres with a diameter of 0.2-0.5µm were found in the outer and middle thirds of the dentine. Microspheres with a diameter of 0.02-0.1µm accumulated in the middle and occasionally inner thirds of the dentine. Some of the microspheres measuring 0.02-0.04µm in diameter reached the dental pulp. CONCLUSIONS: The dentinal tubules in the inner third of the rat coronal dentine may have a space less than 0.1µm through which dentinal fluid can move, despite outward tapering of the dentinal tubules. Retrograde tapering may increase the pressure in the inner third of the dentine layer, and this elevated pressure may contribute to mechanical deformation of the content in the dentinal tubules.