T. Maravic

Dentin bonding systems: From dentin collagen structure to bond preservation and clinical applications

OBJECTIVES Efforts towards achieving durable resin-dentin bonds have been made for decades, including the understanding of the mechanisms underlying hybrid layer (HL) degradation, manufacturing of improved adhesive systems, as well as developing strategies for the preservation of the HL. METHODS This study critically discusses the available peer-reviewed research concerning the formation and preservation of the HL, the mechanisms that lead to the degradation of the HL as well as the strategies to prevent it. RESULTS The degradation of the HL occurs through two main mechanisms: the enzymatic degradation of its collagen fibrils, and the leaching of the resin from the HL. They are enabled by residual unbound water between the denuded collagen fibrils, trapped at the bottom of the HL. Consequently, endogenous dentinal enzymes, such as the matrix metalloproteinases (MMPs) and cysteine cathepsins are activated and can degrade the denuded collagen matrix. Strategies for the preservation of the HL over time have been developed, and they entail the removal of the unbound water from the gaps between the collagen fibrils as well as different modes of silencing endogenous enzymatic activity. SIGNIFICANCE Although there are many more hurdles to be crossed in the field of adhesive dentistry, impressive progress has been achieved so far, and the vast amount of available research on the topic is an indicator of the importance of this matter and of the great efforts of researchers and dental material companies to reach a new level in the quality and longevity of resin-dentin bonds.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE (P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls (P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage (P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls (P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.

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.

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.

How Stable is Dentin As a Substrate for Bonding

Purpose of ReviewThis paper reviews the complexity of the composition of dentin, and the repercussions of this composition on the stability of dentin over time, particularly in relation to adhesive dental procedures.Recent FindingsDentin is a complex and dynamic structure that comprises the major part of the tooth. Most adhesive procedures in dentistry involve bonding to dentin. The hybrid layer (HL) created on the very variable and dynamic organic dentin phase may fail over time, leading to the failure of dental restorations. Literature showed that the collagen fibers in the HL are prone to hydrolysis and mechanical strain, as well as endogenous proteolytic activity (collagenolytic activity of matrix metalloproteinases and cysteine cathepsins). Hydrolysis of the resin phase of the HL also occurs over time.SummaryAdvancements in the area of dental adhesion have been huge. Silencing of collagenolytic enzymes (protease inhibitors and cross-linking agents) is one of the main strategies to decrease the degradation of the HL. In the following years, new techniques will also probably be available, and efficacy of some of the available techniques will perhaps be further clarified.

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.

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.

Influence of restorative procedures on endodontically treated premolars: Finite element analysis of a CT-scan based three-dimensional model

An endodontically treated tooth with mesial-occlusal-distal (MOD) cavity is often restored with composite resin. Palatal and buccal cusp reduction (MODP, MODPB), and/or fiber-reinforced composite posts (P), are used in an attempt to improve the longevity of the restoration. The aim of this study was to determine the effects of these procedures on von Mises stress values and distribution in dental tissues and restorative materials using finite element analysis. Based on CT scans of an extracted second upper premolar, six 3D endodontically treated tooth models (MOD, MODP, MODPB, MOD+P, MODP+P, MODPB+P) were created. Each model was subjected to a summary force of 150 N on the occlusal surface simulating the normal biting pattern and maximal von Mises stresses were calculated. MODP seems to reduce von Mises stress values in dental tissues and P seems to transfer some of the stresses from dental tissues to the composite filling.