Lorenzo Breschi

Collagen intrafibrillar mineralization as a result of the balance between osmotic equilibrium and electroneutrality

Mineralisation of fibrillar collagen with biomimetic process-directing agents has enabled scientists to gain insight into the potential mechanisms involved in intrafibrillar mineralisation. Here, by using polycation- and polyanion-directed intrafibrillar mineralisation, we challenge the popular paradigm that electrostatic attraction is solely responsible for polyelectrolyte-directed intrafibrillar mineralisation. Because there is no difference when a polycationic or a polyanionic electrolyte is used to direct collagen mineralisation, we argue that additional types of long-range non-electrostatic interactions are responsible for intrafibrillar mineralisation. Molecular dynamics simulations of collagen structures in the presence of extrafibrillar polyelectrolytes show that the outward movement of ions and intrafibrillar water through the collagen surface occurs irrespective of the charges of polyelectrolytes, resulting in the experimentally verifiable contraction of the collagen structures. The need to balance electroneutrality and osmotic equilibrium simultaneously to establish Gibbs-Donnan equilibrium in a polyelectrolyte-directed mineralisation system establishes a new model for collagen intrafibrillar mineralisation that supplements existing collagen mineralisation mechanisms.

Academy of Dental Materials guidance on in vitro testing of dental composite bonding effectiveness to dentin/enamel using micro-tensile bond strength (μTBS) approach

OBJECTIVEnAn ideal dental adhesive should provide retentive strength, marginal seal, be relatively simple to achieve and demonstrate clinical durability. Future improvements in adhesive bonding to tooth structure require in vitro test methods that provide reliable data for materials development and/or evaluation of experimental variables. The objective of this project was to identify a test method that is relatively easy to perform, repeatable and ultimately useful for predicting clinical outcomes.nnnMETHODSnThe Academy of Dental Materials initiated a project to develop and distribute guidance documents on laboratory test methods that are useful for the evaluation of dental adhesives and cements, composite resins and ceramics.nnnRESULTSnThe dental adhesive sub-group has identified the micro-tensile bond strength test, especially after subjecting the specimens to a durability challenge, as currently the best practical surrogate measure of dental composite restoration retention.nnnCONCLUSIONnThe following μTBS guidance is meant to aid the researcher in conducting the μTBS test. The authors, while recognizing the limitations of a static, strength-based test method, welcome comments and suggestions for improvements of this guidance document in future revisions.

Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin

It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one.

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

OBJECTIVESnEfforts 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.nnnMETHODSnThis 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.nnnRESULTSnThe 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.nnnSIGNIFICANCEnAlthough 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.

Effect of a one-step self-etch adhesive on endogenous dentin matrix metalloproteinases

Degradation of the hybrid layer created in dentin by dentin adhesives is caused by enzyme activities present within the dentin matrix that destroy unprotected collagen fibrils. The aim of the present study was to evaluate the effect of a one-step self-etch adhesive system on dentinal matrix metalloproteinases 2 and 4 (MMP-2 and MMP-9, respectively) using in situ zymography and an enzymatic activity assay. The null hypothesis tested was that there are no differences in the activities of dentinal MMPs before and after treatment with a one-step adhesive system. The MMP-2 and MMP-9 activities in dentin treated with the one-step adhesive, Adper Easy Bond, were quantified using an enzymatic activity assay system. The MMP activities within the hybrid layer created by the one-step adhesive tested were also evaluated using in situ zymography. The enzymatic assay revealed an increase in MMP-2 and MMP-9 activities after treatment with adhesive. In situ zymography indicated that gelatinolytic activity is present within the hybrid layer created with the one-step self-etch adhesive. The host-derived gelatinases were localized within the hybrid layer and remained active after the bonding procedure. It is concluded that the one-step self-etch adhesive investigated activates endogenous MMP-2 and MMP-9 with the dentin matrix, which may cause collagen degradation over time.

Cross-linking effect on dentin bond strength and MMPs activity

OBJECTIVEnThe 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.nnnMETHODSnFreshly 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.nnnRESULTSnThe 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.nnnSIGNIFICANCEnThe 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

OBJECTIVEnThe 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.nnnMETHODSnNon-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.nnnRESULTSnA 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).nnnSIGNIFICANCEnBecause 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.

No-waiting dentine self-etch concept—Merit or hype

OBJECTIVEnA recently-launched universal adhesive, G-Premio Bond, provides clinicians with the alternative to use the self-etch technique for bonding to dentine without waiting for the adhesive to interact with the bonding substrate (no-waiting self-etch; Japanese brochure), or after leaving the adhesive undisturbed for 10s (10-s self-etch; international brochure). The present study was performed to examine in vitro performance of this new universal adhesive bonded to human coronal dentine using the two alternative self-etch modes.nnnMETHODSnOne hundred and ten specimens were bonded using two self-etch application modes and examined with or without thermomechanical cycling (10,000 thermal cycles and 240,000 mechanical cycles) to simulate one year of intraoral functioning. The bonded specimens were sectioned for microtensile bond testing, ultrastructural and nanoleakage examination using transmission electron microscopy. Changes in the composition of mineralised dentine after adhesive application were examined using Fourier transform infrared spectroscopy.nnnRESULTSnBoth reduced application time and thermomechanical cycling resulted in significantly lower bond strengths, thinner hybrid layers, and significantly more extensive nanoleakage after thermomechanical cycling. Using the conventional 10-s application time improved bonding performance when compared with the no-waiting self-etch technique. Nevertheless, nanoleakage was generally extensive under all testing parameters employed for examining the adhesive.nnnCONCLUSIONnAlthough sufficient bond strength to dentine may be achieved using the present universal adhesive in the no-waiting self-etch mode that does not require clinicians to wait prior to polymerisation of the adhesive, this self-etch concept requires further technological refinement before it can be recommended as a clinical technique.nnnCLINICAL SIGNIFICANCEnAlthough the surge for cutting application time to increase user friendliness remains the most frequently sought conduit for advancement of dentine bonding technology, the use of the present universal adhesive in the no-waiting self-etch mode may not represent the best use of the adhesive.

Effect of pH on dentin protease inactivation by carbodiimide

A water-soluble crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), has been used as a pretreatment of acid-etched dentin to inactivate matrix-bound endogenous dentin proteases. The aim of this study was to evaluate the effect of pH on the inactivation capacity of EDC. Demineralized dentin beams (1xa0×xa02xa0×xa06xa0mm) were divided into six groups (nxa0=xa08 per group). Then, EDC (0.3xa0M) was solubilized in distilled water with pH of 2, 4, 6, 7, 9, or 11. Control EDC was solubilized in 0.1xa0M 2-(N-morpholino) ethanesulfonic acid (MES) buffer and its pH was adjusted to 6. The dentin beams were pretreated for 1xa0min with EDC at each pH or with EDC in MES buffer at pHxa06.0 and then incubated in 1xa0ml of simulated body fluid (pHxa07.2) for 1, 3, 7, or 14xa0d. Untreated beams served as controls. At each study time-point, the dry mass of dentin beams was assessed and the incubation media were analyzed for carboxyterminal telopeptide of type-I collagen (ICTP) and C-terminal telopeptide of type I collagen (CTX) using specific ELISAs. Data were subjected to repeat-measures anova. The results of the study indicated that specimens pretreated with EDC in MES buffer showed the lowest collagen degradation in terms of mass loss and release of telopeptides, while specimens pretreated in alkaline media showed the highest collagen degradation. This study indicates that the pH of the EDC solution plays an important role in the stability of dentin protease inactivation.

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.