Proteolytic activity and degradation of bovine versus human dentin matrices.

OBJECTIVE: Non-human teeth have been commonly used in research as replacements for human teeth, and potential dissimilarities between the dental tissues should be considered when interpreting the outcomes. To compare the proteolytic activity and degradation rate of bovine and human dentin matrices. METHODOLOGY: Dentin beam specimens were obtained from human molars (n=30) and bovine incisors (n=30). The beams were weighed hydrated and after complete dehydration to obtain the mineralized wet and dry masses. Then, the beams were demineralized in 10 wt% phosphoric acid. Next, 15 beams from each substrate were randomly selected and again dehydrated and weighed to obtain the initial demineralized dry mass (DM). Then, the beams were stored in saliva-like buffer solution (SLBS) for 7, 14 and 21 days. SLBS was used to evaluate hydroxyproline (HYP) release after each storage period. The remaining beams of each substrate (n=15) were tested for initial MMP activity using a colorimetric assay and then also stored in SLBS. DM and MMP activity were reassessed after 7, 14 and 21 days of incubation. The data were subjected to two-way ANOVA tests with repeated measures complemented by Bonferroni's tests. Unpaired two-tailed t-tests were also used (p<0.05). RESULTS: Similar water and inorganic fractions were found in human and bovine dentin, while human dentin had a higher protein content. The most intense proteolytic activity and matrix deterioration occurred short after dentin was demineralized. Both substrates exhibited a sharp reduction in MMP activity after seven days of incubation. Although human dentin had higher MMP activity levels, greater HYP release and DM loss after seven days than bovine dentin, after 14 and 21 days, the outcomes were not statistically different. CONCLUSION: Bovine dentin is a suitable substrate for long-term studies involving the degradation of dentin matrices.

Tissue response to white mineral aggregate-based cement containing barium sulfate as alternative radiopacifier: A randomized controlled animal study.

The purpose of this study was to investigate the tissue reaction stimulated by BaSO4 - and Bi2 O3 -containing White MTA Angelus, in comparison with Bi2 O3 -containing white Portland cement, and white ProRoot MTA. Thirty-six adult male Wistar rats (Rattus norvegicus), weighing between 250 and 300 g, were distributed into three groups (n = 12) in accordance with the period of sacrifice (15, 30, and 60 days). Four polyethylene tubes filled with the tested cements were implanted into the dorsum of each rat. Lateral wall of the tubes served as the negative control. After the experimental periods, the animals were euthanized by overdose of pentobarbital anesthetic solution, and the specimens were prepared for microscopic analysis under ×50, ×100, and ×400 magnifications. Inflammatory scores (0-3) were used to grade the tissue reaction. Data were analyzed by the Kruskal-Wallis test and Dunn's test for individual comparisons (p < .05). A mild to moderate inflammatory tissue reaction was observed at the 15-day period, which decreased over the course of the periods for all cements, except for Portland cement. There was no significant difference among the tissue responses for ProRoot MTA, BaSO4 - and Bi2 O3 -containing White MTA Angelus at the 60-day period (p > .05). The Portland group had moderate inflammatory reaction at the final period of analysis, which was statistically different when compared to the other groups (p < .05). The microscopic findings of this animal study suggest that the addition of BaSO4 to White MTA Angelus does not hampers the biocompatibility of the cement.

Proteolytic activity and degradation of bovine versus human dentin matrices

Abstract Non-human teeth have been commonly used in research as replacements for human teeth, and potential dissimilarities between the dental tissues should be considered when interpreting the outcomes. Objective: To compare the proteolytic activity and degradation rate of bovine and human dentin matrices. Methodology: Dentin beam specimens were obtained from human molars (n=30) and bovine incisors (n=30). The beams were weighed hydrated and after complete dehydration to obtain the mineralized wet and dry masses. Then, the beams were demineralized in 10 wt% phosphoric acid. Next, 15 beams from each substrate were randomly selected and again dehydrated and weighed to obtain the initial demineralized dry mass (DM). Then, the beams were stored in saliva-like buffer solution (SLBS) for 7, 14 and 21 days. SLBS was used to evaluate hydroxyproline (HYP) release after each storage period. The remaining beams of each substrate (n=15) were tested for initial MMP activity using a colorimetric assay and then also stored in SLBS. DM and MMP activity were reassessed after 7, 14 and 21 days of incubation. The data were subjected to two-way ANOVA tests with repeated measures complemented by Bonferroni's tests. Unpaired two-tailed t-tests were also used (p<0.05). Results: Similar water and inorganic fractions were found in human and bovine dentin, while human dentin had a higher protein content. The most intense proteolytic activity and matrix deterioration occurred short after dentin was demineralized. Both substrates exhibited a sharp reduction in MMP activity after seven days of incubation. Although human dentin had higher MMP activity levels, greater HYP release and DM loss after seven days than bovine dentin, after 14 and 21 days, the outcomes were not statistically different. Conclusion: Bovine dentin is a suitable substrate for long-term studies involving the degradation of dentin matrices.

In vivo and in vitro radiotherapy increased dentin enzymatic activity.

OBJECTIVES: The present study investigated the effects of in vitro and in-vivo radiotherapy on endogenous enzymatic activity in dentin using gelatin zymography and in-situ zymography. METHODS: Gelatin zymographic assays were performed on protein extracts obtained from dentin powder of sound non-irradiated (NRT), in vitro irradiated (VTRT) and in vivo irradiated (VIRT) human teeth. Their proteolytic activities were quantified using band densitometric evaluation. For in-situ zymography, dentin specimens from NRT, VIRT and VTRT were covered with fluorescein-conjugated gelatin and examined with confocal laser-scanning microscopy. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified and statistically analyzed. In-situ zymography data were statistically analyzed using Kruskal-Wallis ANOVA and Dunn's multiple comparison procedures (α = 0.05). RESULTS: No difference between in vitro and in vivo radiotherapy treatment was found. Both VTRT and VIRT groups showed increase in MMP-9 expression when compared to NRT group. Significant increases (p < 0.05) in gelatinolytic activity (26 % for VTRT; 55 % for VIRT) were observed when compared to the NRT group. CONCLUSION: Radiotherapy increase endogenous enzymatic activity in non-restored dentin.

Insights into cathepsin-B activity in mature dentin matrix.

OBJECTIVE: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. DESIGN: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates' cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. RESULTS: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. CONCLUSIONS: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.

Chlorhexidine preserves the hybrid layer in vitro after 10-years aging.

OBJECTIVE: The present study investigated the ability of a chlorhexidine (CHX)-containing primer (0.2% aqueous solution) to inhibit dentinal enzymes, preserve the hybrid layer (HL) and remain within the HL, after 10 years of aging in artificial saliva at 37°C. METHODS: Non-carious extracted molars were assigned to two groups, cut into slabs exposing middle/deep dentin, etched and bonded with Adper Scotchbond 1XT (SB1XT) with or without 0.2% CHX aqueous solution pretreatment. Composite build-ups were made, and the specimens were cut in 1-mm thick bonded sticks. In situ zymography was performed on freshly prepared specimens (T0) and specimens aged for 10 years (T10-yr) at 37°C in artificial saliva, to investigate endogenous gelatinolytic activity within the HL. At T10-yr, specimens were also decalcified and embedded in epoxy resin for TEM analysis. Micro-Raman spectroscopy was performed at T0 and T10-yr to evaluate the chemical profiles in intertubular dentin and the HL. RESULTS: In situ zymography showed less pronounced enzymatic activity in the CHX-pretreated group (p<0.05) regardless of aging, maintaining a similar level of fluorescence at T0 and T10-yr (p>0.05). TEM results showed that 98% of the HL had been degraded in the control group, while 95% of the HL was intact in the experimental group. Moreover, all the Raman spectra peaks assigned to CHX could be identified only in the CHX-pretreated group (T0 and T10-yr). SIGNIFICANCE: In vitro, CHX remains in the HL after 10 years with its inhibitory effect preserved. This may be the underlying factor for HL preservation after this long aging period.

Proteolytic activity, degradation, and dissolution of primary and permanent teeth.

BACKGROUND: Primary and permanent teeth composition may influence dissolution and degradation rates. AIM: To compare the dissolution and degradation of primary and permanent teeth. DESIGN: Enamel and dentin powders were obtained from primary molars and premolars and incubated within different pH buffers. Calcium and inorganic phosphate release was quantified in the buffers by atomic absorption and light spectrophotometry. A colorimetric assay was used to assess the MMP activity of primary dentin (PrD) and permanent dentin (PeD). Collagen degradation was assessed by dry mass loss, change in elastic modulus (E), and ICTP and CTX release. Data were submitted to ANOVA and Tukey's tests (α = 0.05). RESULTS: Similar dissolution was found between PrD and PeD after 256 hours. At pH 4.5, enamel released more minerals than dentin whereas at pH 5.5 the inverse result was observed. MMP activity was similar for both substrates. PrD showed higher dry mass loss after 1 week. In general, greater reduction in E was recorded for PrD. Higher quantities of ICTP and CTX were released from PrD after 1 week. CONCLUSIONS: Primary and permanent teeth presented similar demineralization rates. Collagen degradation, however, was faster and more substantial for PrD.

Ultra-high-speed videography of resin-dentin interface failure dynamics under tensile load.

OBJECTIVES: Ultra-high-speed (UHS) videography was used to visualize the fracture phenomena at the resin-dentin interface during micro-tensile bond strength (µTBS) test. We also investigated whether UHS videography is applicable for failure-mode analysis. METHODS: Ten human mid-coronal dentin surfaces were bonded using Clearfil SE Bond either in self-etching (SE) or etch-and-rinse (ER) mode. After 24-h water storage, the samples were cut into beams for µTBS test and tested at a cross-head speed of 1 mm/min. The fracture phenomena at the bonded interface were captured using a complementary metal-oxide-semiconductor digital UHS camera at 299,166 frames per second. The failure modes were classified using UHS videography, followed by scanning electron microscopy (SEM) analysis. The failure-mode distributions determined by UHS videography and SEM analysis were statistically analyzed using Fisher's exact test with Bonferroni correction. RESULTS: The crack-propagation speed exceeded 1,500 km/h. No significant difference was found between the SEM and UHS videography failure-mode distributions in the SE mode. A significant difference appeared between them in the ER mode. Significant differences in the incidence of cohesive failures within the adhesive and at the adhesive-composite interface between the SE and ER modes were identified by both SEM and UHS videography. SIGNIFICANCE: UHS videography enabled visualization of the fracture dynamics at the resin- dentin interfaces under tensile load. However, the resolution at such high frame rate was insufficient to classify the failure mode as precisely as that of SEM. Nevertheless, UHS videography can provide more detailed information about the fracture origin and propagation.

Polymer conjugation optimizes EDTA as a calcium-chelating agent that exclusively removes extrafibrillar minerals from mineralized collagen.

During development of mineralized collagenous tissues, intrafibrillar mineralization is achieved by preventing mineralization precursor inhibitors that are larger than 40 kDa from entering the collagen fibrils. Such a property is incorporated in the design of a calcium chelator for dentin bonding in the etch-and-rinse technique that selectively demineralizes extrafibrillar apatite while leaving the intrafibrillar minerals intact. This strategy prevents complete demineralization of collagen fibrils, avoids collapse of collagen that blocks resin infiltration after air-drying, and protects the completely demineralized fibrils from bacteria colonization and degradation by endogenous proteases after resin bonding. In the present study, a water-soluble glycol chitosan-EDTA (GCE) conditioner was synthesized by conjugation of EDTA, an effective calcium chelator, to high molecular weight glycol chitosan, which exhibits weak chelation property. The GCE conjugate was purified, characterized by FTIR, 1H NMR, isothermal titration calorimetry and ICP-AES, and subjected to size exclusion dialysis to recover molecules that are >40 kDa. The optimal concentration and application time for etching dentin were determined by bond strength testing to ensure that the dentin bonding results were comparable to phosphoric acid etching, and maintained equivalent bond strength after air-drying of the conditioned collagen matrix. Extrafibrillar demineralization was validated with transmission electron microscopy. Inhibition of endogenous dentin proteases was confirmed using in-situ zymography. The water-soluble GCE dentin conditioner was non-cytotoxic and possessed antibacterial activities against planktonic and single-species biofilms, supporting its ongoing development as a dentin conditioner with air-drying, anti-proteolytic and antibacterial properties to enhance the durability of bonds created using the etch-and-rinse bonding technique. STATEMENT OF SIGNIFICANCE: The current state-of-the-art techniques for filling decayed teeth with plastic tooth-colored materials require conditioning the mineralized, biofilm-covered, decayed dentin with acids or acid resin monomers to create a surface layer of completely- or partially-demineralized collagen matrix for the infiltration of adhesive resin monomers. Nevertheless, fillings prepared using these strategies are not as durable as consumers have anticipated. Conjugation of polymeric glycol chitosan with EDTA produces a new conditioner for dentin bonding that demineralizes only extrafibrillar dentin, reduces endogenous protease activities and kills biofilm bacteria. The high molecular weight glycol chitosan-EDTA is non-cytotoxic to the key regenerative players within the dentin-pulp complex. This advance permits dry bonding and the use of hydrophobic resins.

Carbodiimide inactivation of matrix metalloproteinases in radicular dentine.

OBJECTIVES: The present in vitro study evaluated the effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a cross-linking agent used as an additional therapeutic primer for luting fiber posts to radicular dentine to prevent hybrid layer degradation. METHODS: Root canal treatment was performed on 80 extracted single-rooted human teeth. A 10-mm post space was prepared and pecimens were randomly assigned to four groups (n=20) according to the bonding system: 1) All Bond 3 (Bisco); 2) All Bond 3 + 0.3M EDC; 3) Prime&Bond XP (Dentsply Sirona); 4) Prime&Bond XP + 0.3M EDC. In groups 2 and 4, EDC was applied on phosphoric acid-etched dentine for 1 min. Fiber posts (RelyX Fiber Post, 3M ESPE) were luted with a dual-cured resin cement (Core-X flow, Dentsply Sirona). Slices were prepared for micro push-out test and interfacial nanoleakage evaluation of the coronal and apical region of the canal space after 24 h and 1 year storage in artificial saliva. In-situ zymography was performed to investigate endogenous matrix metalloproteinase activities within the hybrid layer. Results were statistically analysed with three-way ANOVA test or Chi Square test. Statistical significance was set at α=0.05. RESULTS: No significant influence was identified between the two adhesives. The use of EDC significantly improved fiber post bond strength at 1 year but not at 24 h. Application of 0.3 M EDC prior to bonding significantly reduced gelatinolytic activities within the radicular hybrid layers. CONCLUSIONS: Carbodiimide was effective in preserving fibre post bond strength over time, through reducing the activities of intra-radicular endogenous proteases. CLINICAL SIGNIFICANCE: Inhibition of matrix metalloproteinases using EDC over radicular dentin could play an important role in bond strength preservation. However, the clinical relevance of these findings needs to be proven.

Biochemical and immunohistochemical identification of MMP-7 in human dentin.

OBJECTIVES: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. METHODS: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. RESULTS: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (∼20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. CONCLUSION: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. CLINICAL SIGNIFICANCE: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.

Optimizing resin-dentin bond stability using a bioactive adhesive with concomitant antibacterial properties and anti-proteolytic activities.

Secondary caries and hybrid layer degradation are two major challenges encountered in long-term resin-dentin bond stability. As a link between resin and dentin, adhesives that possess both antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. In the present study, a new quaternary ammonium methacryloxy silane (QAMS) prepared from sol-gel chemistry was incorporated into experimental adhesives to examine their antimicrobial effect and anti-proteolytic potential. This functional methacrylate resin monomer contains polymerizable methacryloxy functionalities as well as a positively-charged quaternary ammonium functionality with a long, lipophilic -C18H37 alkyl chain for puncturing the cell wall/membrane of surface-colonizing organisms. Antibacterial testing performed using agar diffusion test, live/dead bacterial staining and colony-forming unit counts all indicated that the QAMS-containing adhesives killed Streptococcus mutans and Actinomyces naeslundii in a dose-dependent manner via a predominant contact-killing mechanism. Gelatinolytic activity within the hybrid layers created by these adhesives was examined using in-situ zymography. Hybrid layers created with 0% QAMS-containing adhesive exhibited intense green fluorescence emitted by the hydrolyzed fluorescein-conjugated gelatin, with 4-fold increase in enzymatic activity compared with an experimental adhesive containing 5% QAMS. Taken together, incorporation of 5% QAMS in the experimental adhesive provides simultaneous antimicrobial and anti-proteolytic activities that are crucial for the maintenance of long-term resin-dentin bond integrity. STATEMENT OF SIGNIFICANCE: Durability of resin-dentin interfacial bond remains a clinically-significant challenge. Secondary caries caused by bacteria and the degradation of hybrid layers via endogenous dentin proteases are two important contributors to the poor resin-dentin bond durability. The present study developed a new 5% QAMS-containing adhesive that provides simultaneous antimicrobial and dentin protease inhibition functions to extend the longevity of resin-dentin bonds.

Water-associated attributes in the contemporary dentin bonding milieu.

OBJECTIVES: The water-associated attributes of resin-dentin interfaces created by contemporary adhesives are important determinants of bond integrity and stability. In the present work, these attributes were estimated from the perspectives of causality, to examine the behavior of the first and most-recently launched versions of universal adhesives when applied in either the etch-and-rinse mode or the self-etch mode. METHODS: The immediate cause of interfacial permeability and the time-dependent cause of water sorption were investigated in conjunction with the intermediate effect of interface degradation and the more long-term effect of loss of mechanical strength, before and after thermomechanical cycling. The results were compared with control etch-and-rinse and self-etch adhesives. RESULTS: Although the introduction of this new class of universal adhesives has brought forth significant changes to the dental adhesion arena, including more application options, reduced bonding armamentarium and increased user friendliness, the water-associated attributes that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large, when compared with benchmarks established by former classes of adhesives. CONCLUSION: It appears that the current trend of adhesive development has brought forth significant changes but lacks the vigor that demarcates progress and technological sublimity. CLINICAL SIGNIFICANCE: The advent of the user friendly universal adhesives has brought forth significant changes to the dental adhesion arena. However, the elements that are critical for making resin-dentin bonds more durable to environmental challenges and less susceptible to degradation have remained unchanged at large.

Inhibitory effect of curcuminoid pretreatments on endogenous dentin proteases.

The aim of this study was to evaluate the effect of curcuminoids on the dentin endogenous protease activity. Demineralized dentin were pretreated with 50 or 100 µM of three different curcuminoids for 60 s and incubated up to 3 months. Untreated beams served as controls. Dry dentin mass was measured after incubation. Aliquots were analyzed for the quantity of ICTP and CTX releases for MMP and cathepsin-K mediated degradation, respectively. The effect of curcuminoids on matrix-bound MMP and soluble rhMMP-9 were measured using an activity assay. Data were subjected to repeated-measures-ANOVA (α=0.05). Gelatinolytic activity was analyzed using zymography. ICTP and CTX release and dry mass loss of curcuminoid-treated groups were significantly lower than the control. Inhibition of rhMMP-9 varied from 29-49% among curcumonoid-treated groups, whereas no inhibition was observed at untreated control (p>0.05). Results were confirmed by zymography. The study showed that the pretreatment of dentin matrices by curcuminoids decreases endogenous protease activity-mediated degradation in dentin.

Zein based magnesium oxide nanoparticles: Assessment of antimicrobial activity for dental implications.

MgO nanoparticles have been recently discovered as an antibacterial, however, they limited by property degradation due to agglomeration. The addition of a coating agent, such as a zein polymer, is effective in preventing agglomeration without affecting nanosized properties. The aim of this study was to assess the antimicrobial property of MgO nanoparticles when coated with a zein polymer against several oral bacteria and fungi. This was done by utilizing various assessment techniques. The ultimate aim is to use these nanoparticles in dental preparations. The antimicrobial activity of zein-coated MgO nanoparticles at different concentrations of 0.5, 1 and 2% were tested against four different microorganisms: Staphylococcus aureus, Streptococcus mutans and Enterococcus faecalis (gram positive bacteria), and Candida albicans (as oral fungus). Two different techniques were utilized: the Kirby-Bauer test, and a modified direct contact test. The results indicated that the antibacterial effect of 1% or 2% zein-coated MgO nanowires were statistically significant (p<0.05) against the four organisms studied: S. mutans, S. aureus, E. faecalis and C. albicans. Zein-coated MgO nanoparticles are a new human friendly and potent antimicrobial agent that can be incorporated in the formulation of a variety of new dental materials and products that should provide improvements in dental care and oral health.

Removal of matrix-bound zoledronate prevents post-extraction osteonecrosis of the jaw by rescuing osteoclast function.

Unlike other antiresorptive medications, bisphosphonate molecules accumulate in the bone matrix. Previous studies of side-effects of anti-resorptive treatment focused mainly on systemic effects. We hypothesize that matrix-bound bisphosphonate molecules contribute to the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). In this study, we examined the effect of matrix-bound bisphosphonates on osteoclast differentiation in vitro using TRAP staining and resorption assay, with and without pretreatment with EDTA. We also tested the effect of zoledronate chelation on the healing of post-extraction defect in rats. Our results confirmed that bisphosphonates bind to, and can be chelated from, mineralized matrix in vitro in a dose-dependent manner. Matrix-bound bisphosphonates impaired the differentiation of osteoclasts, evidenced by TRAP activity and resorption assay. Zoledronate-treated rats that underwent bilateral dental extraction with unilateral EDTA treatment showed significant improvement in mucosal healing and micro-CT analysis on the chelated sides. The results suggest that matrix-bound bisphosphonates are accessible to osteoclasts and chelating agents and contribute to the pathogenesis of BRONJ. The use of topical chelating agents is a promising strategy for the prevention of BRONJ following dental procedures in bisphosphonate-treated patients.

Release of ICTP and CTX telopeptides from demineralized dentin matrices: Effect of time, mass and surface area.

OBJECTIVE: The present study evaluated the influence of time, mass and surface area of demineralized dentin collagen matrices on telopeptides release. The hypotheses tested were that the rates of ICTP and CTX release by matrix bound endogenous proteases are 1) not time-dependent, 2) unrelated to specimen mass, 3) unrelated to specimen surface area. METHODS: Non-carious human molars (N=24) were collected and randomly assigned to three groups. Dentin slabs with three different thicknesses: 0.37mm, 0.75mm, and 1.50mm were completely demineralized and stored in artificial saliva for one week. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptidases. Activity of MMPs in the aging medium was evaluated using fluorometric activity assay kit. RESULTS: A statistically significant (p<0.05) decrease in the release of both ICTP and CTX fragments over time was observed irrespective of the specimen thickness. When data were normalized by the specimen mass, no significant differences were observed. Releases of ICTP and CTX were significantly related to the aging time as a function of surface area for the first 12h. Total MMP activity, mainly related to MMP-2 and -9, decreased with time (p<0.05). SIGNIFICANCE: Because the release of collagen fragments was influenced by specimen storage time and surface area, it is likely that cleaved collagen fragments closer to the specimen surface diffuse into the incubation medium; those further away from the exposed surface are still entrapped within the demineralized dentin matrix. Bound MMPs can only degrade the substrate within the limited zone of their molecular mobility.

Experimental use of an acrolein-based primer as collagen cross-linker for dentine bonding.

OBJECTIVES: The objective of the present study was to investigate the long-term effect of 0.01% acrolein (ACR) aqueous solution, employed as an additional primer, on the mechanical durability and enzymatic activity of resin-dentine interfaces created with a simplified etch-and-rinse adhesive. METHODS: Dentine surfaces were etched with 35% phosphoric acid for 15s, rinsed and blot-dried. Specimens were then assigned to: Group 1: dentine pre-treated with 0.01% ACR aqueous solution for 1min and bonded with Adper Scotchbond 1 XT (SB1XT), a 2-step etch-and-rinse adhesive; Group 2: SB1XT was applied on untreated acid-etched dentine (control). Resin composite build-ups were made using Filtek Z250. Microtensile bond strength was tested by stressing sectioned specimens to failure immediately or after 1year of storage in artificial saliva at 37°C. Zymography and in-situ zymography assays were performed for examining dentine matrix metalloproteinase (MMP) activities. RESULTS: The use of 0.01% ACR as conditioning primer appeared to have contributed better to preservation of bond strength over time without affecting immediate bond strength. Zymography and in-situ zymography showed reduction in MMP activities after the application of ACR. CONCLUSION: Dentine collagen cross-linking produced by an ACR-based primer increases the longevity of resin-dentine bonds by reinforcement of the adhesive interface and reduction of dentine MMP activities. Further studies are required to evaluate the potential in vivo and in vivo cytotoxicity of ACR. CLINICAL SIGNIFICANCE: The acrolein-based primer is a potentially useful clinical bonding tool because it demonstrates good collagen cross-linking ability within a clinically-acceptable working time. Although a low ACR concentration was employed in the present study, the cytotoxicity of ACR should be tested prior to clinical use.

Cross-linking effect on dentin bond strength and MMPs activity.

OBJECTIVE: The objectives of the study were to evaluate the ability of a 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)-containing primer to improve immediate bond strength of either self-etch or etch-and-rinse adhesive systems and to stabilize the adhesive interfaces over time. A further objective was to investigate the effect of EDC on the dentinal MMPs activity using zymographic analysis. METHODS: Freshly extracted molars (n=80, 20 for each group) were selected to conduct microtensile bond strength tests. The following groups were tested, immediately or after 1-year aging in artificial saliva: G1: Clearfil SE (CSE) primer applied on unetched dentin, pretreated with 0.3M EDC water-solution for 1min and bonded with CSE Bond; G2: as G1 but without EDC pre-treatment; G3: acid-etched (35% phosphoric-acid for 15s) dentin pretreated with 0.3M EDC, then bonded with XP Bond (XPB); Group 4 (G4): as G3 without EDC pre-treatment. Further, gelatinase activity in dentin powder treated with CSE and XPB with and without EDC pre-treatment, was analyzed using gelatin zymography. RESULTS: The use of 0.3M EDC-containing conditioner did not affect the immediate bond strength of XPB or CSE adhesive systems (p>0.05), while it improved the bond strength after 1year of aging (p<0.05). Pre-treatment with EDC followed by the application of CSE resulted in an incomplete MMPs inactivation, while EDC pretreatment followed by the application of XPB resulted in an almost complete inactivation of dentinal gelatinases. SIGNIFICANCE: The µTBS and zymography results support the efficacy of EDC over time and reveal that changes within the dentin matrix promoted by EDC are not adhesive-system-dependent.

Mechanism of bioactive molecular extraction from mineralized dentin by calcium hydroxide and tricalcium silicate cement.

OBJECTIVES: The objective of the present study was to elucidate the mechanism of bioactive molecule extraction from mineralized dentin by calcium hydroxide (Ca(OH)2) and tricalcium silicate cements (TSC). METHODS AND RESULTS: Transmission electron microscopy was used to provide evidence for collagen degradation in dentin surfaces covered with Ca(OH)2 or a set, hydrated TSC for 1-3 months. A one micron thick collagen degradation zone was observed on the dentin surface. Fourier transform-infrared spectroscopy was used to identify increases in apatite/collagen ratio in dentin exposed to Ca(OH)2. Using three-point bending, dentin exposed to Ca(OH)2 exhibited significant reduction in flexural strength. Using size exclusion chromatography, it was found that the small size of the hydroxyl ions derived from Ca(OH)2 enabled those ions to infiltrate the intrafibrillar compartment of mineralized collagen and degrade the collagen fibrils without affecting the apatite minerals. Using ELISA, TGF-ß1 was found to be extracted from dentin covered with Ca(OH)2 for 3 months. Unlike acids that dissolve the mineral component of dentin to release bioactive molecules, alkaline materials such as Ca(OH)2 or TSC released growth factors such as TGF-ß1 via collagen degradation. SIGNIFICANCE: The bioactive molecule extraction capacities of Ca(OH)2 and TSC render these dental materials excellent for pulp capping and endodontic regeneration. These highly desirable properties, however, appear to be intertwined with the untoward effect of degradation of the collagen matrix within mineralized dentin, resulting in reduced flexural strength.