Ubiquitination and Deubiquitination in Oral Disease.

Oral health is an integral part of the general health and well-being of individuals. The presence of oral disease is potentially indicative of a number of systemic diseases and may contribute to their early diagnosis and treatment. The ubiquitin (Ub) system has been shown to play a role in cellular immune response, cellular development, and programmed cell death. Ubiquitination is a post-translational modification that occurs in eukaryotes. Its mechanism involves a number of factors, including Ub-activating enzymes, Ub-conjugating enzymes, and Ub protein ligases. Deubiquitinating enzymes, which are proteases that reversely modify proteins by removing Ub or Ub-like molecules or remodeling Ub chains on target proteins, have recently been regarded as crucial regulators of ubiquitination-mediated degradation and are known to significantly affect cellular pathways, a number of biological processes, DNA damage response, and DNA repair pathways. Research has increasingly shown evidence of the relationship between ubiquitination, deubiquitination, and oral disease. This review investigates recent progress in discoveries in diseased oral sites and discusses the roles of ubiquitination and deubiquitination in oral disease.

Comparing the Effectiveness of Different Dentinal Desensitizing Agents: In Vitro Study.

OBJECTIVES: To evaluate the in vitro effectiveness of desensitizing agents in reducing dentine permeability. METHODS: The efficacy of desensitizing agents in reducing dentine permeability by occluding dentine tubules was evaluated using a fluid filtration device that conducts at 100 cmH2O (1.4 psi) pressure, and SEM/EDX analyses were evaluated and compared. Forty-two dentine discs (n = 42) of 1 ± 0.2 mm width were obtained from caries-free permanent human molars. Thirty dentine discs (n = 30) were randomly divided into 3 groups (n = 10): Group 1: 2.7% wt. monopotassium-monohydrogen oxalate (Mp-Mh oxalate), Group 2: RMGI XT VAR, and Group 3: LIQ SiO2. Dentine permeability was measured following treatment application after 10 minutes, storage in artificial saliva after 10 minutes and 7 days, and citric acid challenge for 3 minutes. Data were analysed with a repeated measures ANOVA test. Dentine discs (n = 12) were used for SEM/EDX analyses to acquire data on morphological changes on dentine surface and its mineral content after different stages of treatment. RESULTS: Desensitizing agents' application on the demineralized dentine discs exhibited significant reduction of permeability compared to its maximum acid permeability values. Mp-Mh oxalate showed a significant reduction in dentine permeability (p < 0.05) when compared to RMGI XT VAR and LIQ SiO2. On SEM/EDX analysis, all the agents formed mineral precipitates that occluded the dentine tubules. CONCLUSIONS: 2.7% wt. monopotassium-monohydrogen oxalate was significantly effective in reducing dentine permeability compared to RMGI XT VAR and LIQ SiO2.

Dentin tubule obturation of a bioglass-based dentin desensitizer under repeated exposure to lactid acid and brushing.

BACKGROUND: Dentin hypersensitivity is a frequent finding especially in periodontitis patients. Conventional treatment aims for obstruction of dentin tubules by disabling liquid and osmotic fluctuation to and from the pulpal chamber. A novel bioglass-based desensitizer was shown to obstruct tubules and to resist periodic exposure to lactic acid. Whether this obstruction is resistant to brushing had not been tested so far. Accordingly, the present study aimed to assess dentin tubule obstruction after repeated acid exposure and brushing. METHODS: Sixty dentin discs were cleaned with 17% EDTA, mounted into a pulp fluid simulator and randomly divided into 3 groups: No surface treatment in Group A, Seal&Protect® in group B and DentinoCer in group C. Discs were exposed to 0.1 M non-saturated lactic acid thrice and standardized brushing twice a day for 12 days. At baseline and after 2, 4 and 12 d samples were removed from the setting and prepared for top-view SEM analysis to assess tubule obstruction using the Olley score. Discs were then vertically cut and the section surface morphologically assessed using backscatter imaging. For both vertical and sectional surfaces EDX analysis was used to characterize the surface composition in the tubular and inter-tubular area. RESULTS: Group A showed clean tubular lumina at all time points. From day 2 onwards dentin showed exposed collagen fibers. Group 2 initially showed a complete surface coverage that flattened out during treatment without ever exposing tubules. At baseline, samples of Group C displayed a complete homogeneous coverage. From day 2 on tubules entrances with obstructed lumen became visible. While on day 4 and 12 the dentin surface exposed collagen fibers the lumina remained closed. EDX analysis of the vertical and horizontal views showed that P and Ca were predominant elements in both the inter- and tubular dentin while Si peaks were found in the tubule plugs. CONCLUSION: While group B displayed a packed layer on the surface during the whole investigation time group C samples lost their superficial layer within 48 h. Tubule plugs containing considerable Si proportions indicated previous presence of DentinoCer, while high Ca and P proportions suggest obturation by dentin-like material.

The Role of Transient Receptor Potential (TRP) Channels in the Transduction of Dental Pain.

Dental pain is a common health problem that negatively impacts the activities of daily living. Dentine hypersensitivity and pulpitis-associated pain are among the most common types of dental pain. Patients with these conditions feel pain upon exposure of the affected tooth to various external stimuli. However, the molecular mechanisms underlying dental pain, especially the transduction of external stimuli to electrical signals in the nerve, remain unclear. Numerous ion channels and receptors localized in the dental primary afferent neurons (DPAs) and odontoblasts have been implicated in the transduction of dental pain, and functional expression of various polymodal transient receptor potential (TRP) channels has been detected in DPAs and odontoblasts. External stimuli-induced dentinal tubular fluid movement can activate TRP channels on DPAs and odontoblasts. The odontoblasts can in turn activate the DPAs by paracrine signaling through ATP and glutamate release. In pulpitis, inflammatory mediators may sensitize the DPAs. They could also induce post-translational modifications of TRP channels, increase trafficking of these channels to nerve terminals, and increase the sensitivity of these channels to stimuli. Additionally, in caries-induced pulpitis, bacterial products can directly activate TRP channels on DPAs. In this review, we provide an overview of the TRP channels expressed in the various tooth structures, and we discuss their involvement in the development of dental pain.

Performance of a bioglass-based dentine desensitizer under lactid acid exposition: an in-vitro study.

BACKGROUND: Dentine hypersensitivity is especially frequent in patients with pronounced periodontal attachment loss. Aim of the treatment is an obstruction of the dentine tubules in order to inhibit liquid or osmotic motion, which is considered as trigger for pain sensations. Novel approaches aim for obstruction by calcium phosphate compounds in order to rely on biocompatible compounds. It was the aim of the study to optically investigate the morphology and to assess the fluid permeability of treated dentine surfaces. METHODS: Dentine discs were pretreated in an ultrasonic bath with 17% EDTA to clean the lumina of the dentine tubules. Samples of group A remained untreated while Seal&Protect® as a conventional desensitizer was applied for group B and DentinoCer in group C. Discs were mounted into a pulp fluid simulator (PFS) with a methylene blue solution in order to create a flow pressure of 0.5 bar. Over 12 d, discs were exposed three times per day to 0.1 M nonsaturated lactic acid. At baseline and after 2, 8 and 12 d samples were removed from PFS and prepared for SEM analysis. Tubule obstruction was assessed quantitatively using Olley scores and by qualitative description of the surface. Absorption spectrometry was used to assess the concentration of leaked methylene blue outside the samples in order to estimate dentine permeability. RESULTS: Untreated discs showed clean lumina of all tubules at all time points and magnifications. From day 2 onwards dentine showed exposed collagene fibers due to acid exposition. Seal&Protect® initially showed homogenous dentine surface coverage that got a more granulomatous aspect in the course of treatment time. Few samples showed sporadic tubules with open lumen at day 8 and 12. Group C showed samples with a homogeneous, even surface. Narrow slits in the superficial layer are visible from day 4 on, but the dentine surface remained invisible and dentine tubules were closed till the end of the investigation period. CONCLUSION: Over 12 d of lactid acid exposure, samples showed complete coverage of the dentine tubules in the chosen in-vitro-model when treated with Seal&Protect® or DentinoCer.

Improved reactive nanoparticles to treat dentin hypersensitivity.

The aim of this study was to evaluate the effectiveness of different nanoparticles-based solutions for dentin permeability reduction and to determine the viscoelastic performance of cervical dentin after their application. Four experimental nanoparticle solutions based on zinc, calcium or doxycycline-loaded polymeric nanoparticles (NPs) were applied on citric acid etched dentin, to facilitate the occlusion and the reduction of the fluid flow at the dentinal tubules. After 24 h and 7 d of storage, cervical dentin was evaluated for fluid filtration. Field emission scanning electron microscopy, energy dispersive analysis, AFM and Nano-DMA analysis were also performed. Complex, storage, loss modulus and tan delta (δ) were assessed. Doxycycline-loaded NPs impaired tubule occlusion and fluid flow reduction trough dentin. Tubules were 100% occluded in dentin treated with calcium-loaded NPs or zinc-loaded NPs, analyzed at 7 d. Dentin treated with both zinc-NPs and calcium-NPs attained the highest reduction of dentinal fluid flow. Moreover, when treating dentin with zinc-NPs, complex modulus values attained at intertubular and peritubular dentin were higher than those obtained after applying calcium-NPs. Zinc-NPs are then supposed to fasten active dentin remodeling, with increased maturity and high mechanical properties. Zinc-based nanoparticles are then proposed for effective dentin remineralization and tubular occlusion. Further research to finally prove for clinical benefits in patients with dentin hypersensitivity using Zn-doped nanoparticles is encouraged. STATEMENT OF SIGNIFICANCE: Erosion from acids provokes dentin hypersensitivity (DH) which presents with intense pain of short duration. Open dentinal tubules and demineralization favor DH. Nanogels based on Ca-nanoparticles and Zn-nanoparticles produced an efficient reduction of fluid flow. Dentinal tubules were filled by precipitation of induced calcium-phosphate deposits. When treating dentin with Zn-nanoparticles, complex modulus values attained at intertubular and peritubular dentin were higher than those obtained after applying Ca-nanoparticles. Zn-nanoparticles are then supposed to fasten active dentin remodeling, with increased maturity and high mechanical properties. Zinc-based nanogels are, therefore, proposed for effective dentin remineralization and tubular occlusion. Further research to finally prove for clinical benefits in patients with dentin hypersensitivity using Zn-doped nanogels is encouraged.

Effects of a Dicalcium and Tetracalcium Phosphate-Based Desensitizer on In Vitro Dentin Permeability.

The present study evaluated the effectiveness of a dicalcium and tetracalcium phosphate-based desensitizer in reducing dentin permeability in vitro. Dentin fluid flow was measured before and after treatment of dentin with patent dentinal tubules using 1 or 3 applications of the dicalcium and tetracalcium phosphate containing agent TeethmateTM (TM) and comparing the results with two sodium fluoride varnishes VellaTM (VLA) and VanishTM (VAN), after storage in artificial saliva for 24 h, 48 h and 7 days. Significant differences were observed among the 4 methods employed for reducing dentin permeability (p < 0.001) and the 3 post-treatment times (p < 0.001). VLA and VAN never achieved 50% permeability reductions consistently in any of the 3 time periods. Only the calcium phosphate-based desensitizer applied for 3 times consistently reduced dentin permeability by 50% after 24 h. When applied once, the permeability reduction of TM increased progressively over the 3 time periods. After 7 days, only one and three applications of the calcium phosphate-based desensitizer consistently reduced dentin permeability by more than 50%. Permeability reductions corresponded well with scanning electron microscopy examination of dentinal tubule orifice occlusion in dentin specimens treated with the agents. Overall, the dicalcium and tetracalcium phosphate-based desensitizer is effective in reducing dentin permeability via a tubule occlusion mechanism. The ability of the agent to reduce dentin permeability renders it to be potentially useful as a clinical dentin desensitizing agent, which has to be confirmed in future clinical studies. By contrast, the two sodium fluoride varnishes are not effective in dentin permeability reduction and should be considered as topical fluoride delivering agents rather than tubular orifice-blocking agents.

Expression of Pannexin3 in human odontoblast-like cells and its hemichannel function in mediating ATP release.

OBJECTIVE: The aim of this study is to investigate the expression of pannexin3 (Panx3) in human odontoblast-like cells (hOBs) and its hemichannel function in mediating ATP release. METHODS: RT-PCR and immunofluorescence analysis were used to detect the expression of pannexins (Panxs) in human dental pulp tissue and cultured cells. To determine the role of Panx3 in ATP release, hOBs were infected with Panx3-overexpression lentivirus, Panx3-shRNA lentivirus or control lentivirus and then stimulated with cold buffer. Intracellular ATP was monitored using quinacrine, and then semi-quantitatively analyzed. In the meantime, the ATP release was quantitatively analyzed using the bioluminescence method when the cells were exposed to cold stimulus. RESULTS: Panx3 mRNA and protein were found in dental pulp tissue and cultured cells. Upon cold stimulus, intracellular ATP was released into the extracellular space. Overexpression of Panx3 accelerated ATP release, whereas inhibition of Panx3 suppressed this process. CONCLUSION: Panx3 hemichannel is expressed in human odontoblast-like cells and mediates ATP release into the extracellular space.

Expression of ecto-ATPase NTPDase2 in human dental pulp.

Dental pulpal nerve fibers express ionotropic adenosine triphosphate (ATP) receptors, suggesting that ATP signaling participates in the process of dental nociception. In this study, we investigated if the principal enzymes responsible for extracellular ATP hydrolysis, namely, nucleoside triphosphate diphosphohydrolases (NTPDases), are present in human dental pulp. Immunohistochemical and immunofluorescence experiments showed that NTPDase2 was predominantly expressed in pulpal nerve bundles, Raschkow's nerve plexus, and in the odontoblast layer. NTPDase2 was expressed in pulpal Schwann cells, with processes accompanying the nerve fibers and projecting into the odontoblast layer. Odontoblasts expressed the gap junction protein, connexin43, which can form transmembrane hemichannels for ATP release. NTPDase2 was localized close to connexin43 within the odontoblast layer. These findings provide evidence for the existence of an apparatus for ATP release and degradation in human dental pulp, consistent with the involvement of ATP signaling in the process of dentin sensitivity and dental pain.

Lipopolysaccharide-induced pulpitis up-regulates TRPV1 in trigeminal ganglia.

Tooth pain often accompanies pulpitis. Accumulation of lipopolysaccharides (LPS), a product of Gram-negative bacteria, is associated with painful clinical symptoms. However, the mechanisms underlying LPS-induced tooth pain are not clearly understood. TRPV1 is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and hyperalgesia under inflammation or injury. Although TRPV1 is expressed in pulpal afferents, it is not known whether the application of LPS to teeth modulates TRPV1 in trigeminal nociceptors. By assessing the levels of protein and transcript of TRPV1 in mouse trigeminal ganglia, we demonstrate that dentinal application of LPS increases the expression of TRPV1. Our results suggest that the up-regulation of TRPV1 in trigeminal nociceptors following bacterial infection could contribute to hyperalgesia under pulpitis conditions.

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity.

Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo-sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo-sensitive TRP channels in cultured odontoblast-like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast-like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP-channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.

Adult rat odontoblasts lack noxious thermal sensitivity.

Dentin hypersensitivity is a common symptom treated in the dental clinic, yet the underlying cellular and molecular mechanisms are not clear. We hypothesized that odontoblasts detect noxious thermal stimuli by expressing temperature-sensing molecules, and investigated whether temperature-activated TRP channels (thermo-TRP channels), which are known to initiate temperature signaling, mediate temperature sensing in odontoblasts. mRNA expression of dentin sialophosphoprotein and collagenase type 1, odontoblast-specific proteins, was shown in acutely isolated adult rat odontoblasts by single-cell RT-PCR, while TRPV1, TRPV2, TRPM8, and TRPA1 were not detected. Application of noxious temperatures of 42 degrees C and 12 degrees C, as well as capsaicin, menthol, and icilin, agonists of thermo-TRP channels, failed to increase intracellular calcium concentration. Immunohistochemical study also revealed no expression of TRPV1. Thus, it is unlikely that odontoblasts serve as thermal sensors in teeth via thermo-TRP channels.

The effects of outward forced convective flow on inward diffusion of potassium across human dentin.

PURPOSE: To measure the inward flux of potassium across human dentin disks, in vitro, in the absence and then in the presence of a simulated pulpal pressure (PP), before and after brushing with two desensitizing dentifrices. MATERIALS AND METHODS: Dentin discs were made from extracted unerupted third molars and etched with 6% citric acid to remove the smear layers. The hydraulic conductance (Lp or outward fluid movement) of the discs was measured before and after brushing for 2 minutes with two desensitizing dentifrices, Colgate Sensitive Maximum Strength or Sensodyne Fresh Mint, both dentifrices containing 5% potassium nitrate. The potassium flux was measured at a pressure of 0 cm H2O, and a simulated pulpal pressure of 20 cm H2O. RESULTS: The results showed the Colgate-brushed specimens had lower Lp (P < 0.05) than specimens brushed with Sensodyne or with saline. The Colgate-brushed specimens had a corresponding lower K+ flux, at a PP = 0 cm H2O, than specimens brushed with saline, but were not significantly different than specimens brushed with Sensodyne dentifrice. However, when the pressure was increased from 0 to 20 cm H2O, to simulate outward dentin fluid pressure, there were significant decreases in K+ flux across all three treatment groups, but the K+ flux of the Colgate-brushed specimens were affected the least, resulting in the Colgate-brushed specimens having significantly (P < 0.05) greater K+ fluxes than the saline or Sensodyne dentifrice-brushed groups. The magnitude of reduction in K+ flux in going from 0 cm H2O to 20 cm H2O was demonstrated to be linearly related to the magnitude of the Lp of the brushed specimens. Hence, a small Lp corresponded to a small reduction in K+ flux, and a correspondingly large K+ flux at 20 cm H2O. An explanation of this phenomenon is that a decrease in the Lp of dentin, produced by brushing, corresponds to a decreased outward dentin fluid flow. This lowers the outward K+ convective flow and also lowers the inward K+ diffusion. However, since the decrease of the outward K+ convective flow is greater than the decrease of the inward K+ diffusion, and the net flux is the algebraic sum of these two terms, the result is greater net inward K+ flux through the dentin. Therefore, the Colgate-brushed specimens, which exhibited the lowest Lp, provided the greatest inward K+ flux at simulated dentin fluid pressure.

Penetration of varnishes into demineralized root dentine in vitro.

In this paper the penetration of three different varnishes employed in caries prevention (Duraphat, Fluor Protector and Cervitec) into demineralized dentine is quantified using confocal laser scanning microscopy. The results show that the varnish penetration into lesions about 85 microns in depth if for Cervitec about 35 microns and considerably less for Duraphat and Fluor Protector. The penetration is into the dentinal tubules and is influenced by dentinal tubule direction. The drying procedure--pretreatment of the dentine--influences the penetration, though sizeably only for Cervitec applications. This paper shows that varnish penetration into the tissue and presumably 'sealing' tubules completely or partly is valuable with respect to root caries prevention and hypersensitivity.

Dentin permeability and dentin sensitivity.

The hydrodynamic theory of dentin sensitivity is based on the premise that sensitive dentin is permeable throughout the length of the tubules. Such a condition may permit the diffusion of bacterial products across dentin to the pulp where they may cause irritation of pulpal soft tissues. However, the slow outward movement of dentinal fluid tends to flush the tubules free of exogenous substances. This balance between the inward diffusion of exogenous substances, whether bacterial or the active ingredients in desensitizing medicaments, and the cleansing action of dentinal fluid flow needs to be examined experimentally.

Dentine hypersensitivity. II. Effects produced by the uptake in vitro of toothpastes onto dentine.

Toothpastes arguably are the most common preparations used in the treatment of dentine hypersensitivity. Evidence for efficacy largely comes from clinical trials, and supports the role of the contained so-called "active" ingredient. How such compounds produce therapeutic benefit has received limited attention. This study measured the availability and uptake onto dentine of fluoride and metal ions contained in commercial and test toothpastes. The effects of exposure of dentine sections to these toothpastes, and abrasive only pastes in particular, was investigated by scanning electron microscopy and X-ray microanalysis. Aqueous extracts of the different toothpastes contained measurable concentrations of the incorporated metal and or fluoride, but usually considerably below that incorporated by the manufacturer. Extraction of dentine sections exposed to toothpaste slurries revealed levels of fluoride and metal ions, although post-treatment washing in water reduced these levels. Surface changes at 1 and 10 h were not consistent with the uptake of fluoride or metals. These changes were almost certainly produced by the contained abrasives, including calcium carbonate, dicalcium phosphate, alumina and silica. In particular fume silica progressively accumulated on the dentine surface to narrow and occlude dentinal tubules. Unlike other abrasives fume silica was resistant to removal by washing. It would appear that insufficient attention has been given to the therapeutic potential of toothpaste ingredients, particularly abrasives, to produce benefit by occlusion of dentinal tubules.

Dentine hypersensitivity. I. Effects produced by the uptake in vitro of metal ions, fluoride and formaldehyde onto dentine.

Dentine has been shown to possess affinity for a large number of varied compounds, many of which have been shown effective in clinical trials, for the treatment of dentine hypersensitivity. The mode of action of these compounds is unclear. The aim of this study was to investigate the uptake of metal ions, fluoride and formaldehyde in solution onto dentine in vitro and determine whether therapeutic effects could be mediated through occlusion of dentinal tubules. Etched dentine sections were exposed for 1 h to 1000 ppm solutions of fluoride and metal salts. Levels of fluoride and respective metals could be extracted and measured from the specimens. Saliva pretreatment had a variable but small effect on uptake of each ion, but post treatment washings reduced all levels of retained fluoride and metals. X-ray microanalysis indicated surface or immediate subsurface deposits of metals. However, surface changes were only consistently produced by zinc and more particularly tin salts. Both zinc and tin salts produced covering or obturation of tubules. The surface effects of zinc could largely be reversed by washing, but not those of tin. Formaldehyde alone or in the presence of saliva produced no effects. A 4-week study involving twice daily exposure of specimens to saliva and fluoride, metals or formaldehyde yielded essentially the same results. It is unlikely that, except for tin, the compounds tested achieve their apparent clinical effects mediated by direct occlusion of dentinal tubules.

Dentine hypersensitivity: effect of interactions between metal salts, fluoride and chlorhexidine on the uptake by dentine.

A large number of compounds may be applied to the teeth for preventive or therapeutic purposes, most notably in dentine hypersensitivity. The uptake of any one compound by tooth substances may clearly be affected by another to produce synergistic, additive or antagonistic effects. This study determined whether uptake interactions occurred between fluoride, chlorhexidine, strontium, tin and zinc. All compounds, in particular tin, showed considerable affinity for dentine. Fluoride reduced the uptake of zinc but not tin, strontium or chlorhexidine. Chlorhexidine increased the uptake of zinc, but not tin, strontium or fluoride. Zinc and tin decreased fluoride uptake but strontium had no such effect. Finally, chlorhexidine uptake was increased by zinc and tin but not by fluoride or strontium. Based on the theories proposed for the reaction of these various compounds with calcified tissues, explanations for the interactions can be made. The results appear relevant to some reported clinical effects of combinations of these compounds and indicate that such interactions may have advantageous or disadvantageous consequences depending on the basis for treatment.

Dentin-predentin complex and its permeability: physiologic overview.

The major channels for solute diffusion across dentin are the dentinal tubules. Since dentin permeation is proportional to the product of tubule number and diameter, both of which increase as the tubules converge on the pulp, we find that dentin permeability increases rapidly as the pulp chamber is approached. The presence of a smear layer of cutting debris on top of cut dentin decreases dentin permeability, especially when permeability is measured by fluid filtration. Further, intratubular material--such as mineral deposits, collagen fibrils, proteoglycan linings, bacteria, etc.--can greatly reduce dentin permeability. Although the presence of irregular or irritation dentin has been thought to greatly reduce dentin permeability, recent in vivo experiments in dogs indicate that the dentin permeability of freshly cut cavities prepared in sound dentin falls very rapidly (i.e., 50-60% in the first six hours) before any histologic changes can be detected, either in the pulp or the dentin. When dogs were depleted of their plasma fibrinogen, this rapid decline in dentin permeability following cavity preparation failed to take place. The results implicate leakage of plasma proteins from the underlying pulpal vessels. The proteins subsequently permeate the tubules, where they are either adsorbed to the tubule walls or physically trapped in such a way as to reduce dentin permeability.