L. Breschi

Role of Dentin MMPs in Caries Progression and Bond Stability

Dentin can be described as a biological composite with collagen matrix embedded with nanosized hydroxyapatite mineral crystallites. Matrix metalloproteinases (MMPs) and cysteine cathepsins are families of endopeptidases. Enzymes of both families are present in dentin and collectively capable of degrading virtually all extracellular matrix components. This review describes these enzymes and their presence in dentin, mainly focusing on their role in dentin caries pathogenesis and loss of collagen in the adhesive hybrid layer under composite restorations. MMPs and cysteine cathepsins present in saliva, mineralized dentin, and/or dentinal fluid may affect the dentin caries process at the early phases of demineralization. Changes in collagen and noncollagenous protein structure may participate in observed decreases in mechanical properties of caries-affected dentin and reduce the ability of caries-affected dentin to remineralize. These endogenous enzymes also remain entrapped within the hybrid layer during the resin infiltration process, and the acidic bonding agents themselves (irrespective of whether they are etch-and-rinse or self-etch) can activate these endogenous protease proforms. Since resin impregnation is frequently incomplete, denuded collagen matrices associated with free water (which serves as a collagen cleavage reagent for these endogenous hydrolase enzymes) can be enzymatically disrupted, finally contributing to the degradation of the hybrid layer. There are multiple in vitro and in vivo reports showing that the longevity of the adhesive interface is increased when nonspecific enzyme-inhibiting strategies are used. Different chemicals (i.e., chlorhexidine, galardin, and benzalkonium chloride) or collagen cross-linker agents have been successfully employed as therapeutic primers in the bonding procedure. In addition, the incorporation of enzyme inhibitors (i.e., quaternary ammonium methacrylates) into the resin blends has been recently promoted. This review will describe MMP functions in caries and hybrid layer degradation and explore the potential therapeutic role of MMP inhibitors for the development of improved intervention strategies for MMP-related oral diseases.

Adhesive performance of a multi-mode adhesive system: 1-Year in vitro study

OBJECTIVESnThe aim of this study was to investigate the adhesive stability over time of a multi-mode one-step adhesive applied using different bonding techniques on human coronal dentine. The hypotheses tested were that microtensile bond strength (μTBS), interfacial nanoleakage expression and matrix metalloproteinases (MMPs) activation are not affected by the adhesive application mode (following the use of self-etch technique or with the etch-and-rinse technique on dry or wet dentine) or by ageing for 24h, 6 months and 1year in artificial saliva.nnnMETHODSnHuman molars were cut to expose middle/deep dentine and assigned to one of the following bonding systems (N=15): (1) Scotchbond Universal (3M ESPE) self-etch mode, (2) Scotchbond Universal etch-and-rinse technique on wet dentine, (3) Scotchbond Universal etch-and-rinse technique on dry dentine, and (4) Prime&Bond NT (Dentsply De Trey) etch-and-rinse technique on wet dentine (control). Specimens were processed for μTBS test in accordance with the non-trimming technique and stressed to failure after 24h, 6 months or 1 year. Additional specimens were processed and examined to assay interfacial nanoleakage and MMP expression.nnnRESULTSnAt baseline, no differences between groups were found. After 1 year of storage, Scotchbond Universal applied in the self-etch mode and Prime&Bond NT showed higher μTBS compared to the other groups. The lowest nanoleakage expression was found for Scotchbond Universal applied in the self-etch mode, both at baseline and after storage. MMPs activation was found after application of each tested adhesive.nnnCONCLUSIONSnThe results of this study support the use of the self-etch approach for bonding the tested multi-mode adhesive system to dentine due to improved stability over time.nnnCLINICAL SIGNIFICANCEnImproved bonding effectiveness of the tested universal adhesive system on dentine may be obtained if the adhesive is applied with the self-etch approach.

High resolution SEM evaluation of dentin etched with maleic and citric acid

OBJECTIVESnThis study evaluated the ultra-morphological effects of maleic and citric acid on human dentin by means of a field emission in-lens scanning electron microscope (FEISEM). Both acids were tested on human dentin at pH 0.7 and 1.4 in aqueous solutions.nnnMETHODSnEach of 12 dentin disks were divided into four groups and exposed to either maleic acid at pH 0.7, maleic acid at pH 1.4, citric acid at pH 0.7 and citric acid at pH 1.4. All samples were then fixed and dehydrated in a critical point drying apparatus. Observations were carried out by means of a FEISEM (JEOL 890) after coating with a carbon-platinum film.nnnRESULTSnBoth acids removed smear layer and partially removed smear plugs. Details of fine structures measuring from 5 to 15 nm were shown on the intertubular demineralized dentin. Maleic acid at pH 0.7 showed the highest depth of demineralization of all the tested samples; citric acid, showed a higher depth of demineralization values when tested at pH 1.4 than at pH 0.7.nnnSIGNIFICANCEnThe FEISEM reveals ultra-structural aspects of the demineralization process of the dentin tissue of the both acids tested. Differences related to the pH of the acids were found. Images obtained at high magnification clarify the dentin collagen structure of both peritubular and intertubular dentin. Small periodic structures associated with collagen fibrils were also imagined.

Water distribution in dentin matrices: bound vs. unbound water.

OBJECTIVEnThis work measured the amount of bound versus unbound water in completely-demineralized dentin.nnnMETHODSnDentin beams prepared from extracted human teeth were completely demineralized, rinsed and dried to constant mass. They were rehydrated in 41% relative humidity (RH), while gravimetrically measuring their mass increase until the first plateau was reached at 0.064 (vacuum) or 0.116 gH2O/g dry mass (Drierite). The specimens were then exposed to 60% RH until attaining the second plateau at 0.220 (vacuum) or 0.191 gH2O/g dry mass (Drierite), and subsequently exposed to 99% RH until attaining the third plateau at 0.493 (vacuum) or 0.401 gH2O/g dry mass (Drierite).nnnRESULTSnExposure of the first layer of bound water to 0% RH for 5 min produced a -0.3% loss of bound water; in the second layer of bound water it caused a -3.3% loss of bound water; in the third layer it caused a -6% loss of bound water. Immersion in 100% ethanol or acetone for 5 min produced a 2.8 and 1.9% loss of bound water from the first layer, respectively; it caused a -4 and -7% loss of bound water in the second layer, respectively; and a -17 and -23% loss of bound water in the third layer. Bound water represented 21-25% of total dentin water. Chemical dehydration of water-saturated dentin with ethanol/acetone for 1 min only removed between 25 and 35% of unbound water, respectively.nnnSIGNIFICANCEnAttempts to remove bound water by evaporation were not very successful. Chemical dehydration with 100% acetone was more successful than 100% ethanol especially the third layer of bound water. Since unbound water represents between 75 and 79% of total matrix water, the more such water can be removed, the more resin can be infiltrated.

Can quaternary ammonium methacrylates inhibit matrix MMPs and cathepsins

OBJECTIVEnDentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are known to be produced by MMPs. CTX fragments are thought to come from cathepsin K activity. The purpose of this study was to determine if quaternary methacrylates (QAMs) can inhibit matrix MMPs and cathepsins.nnnMETHODSnDentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24h at pH 7.4 to identify which cathepsins release CTX at neutral pH. Beams were dipped in ATA, an antimicrobial QAM to determine if it can inhibit dentin matrix proteases. Other beams were dipped in another QAM (MDPB) to determine if it produced similar inhibition of dentin proteases.nnnRESULTSnOnly beams incubated with cathepsin K lost more dry mass than the controls and released CTX. Dentin beams dipped in ATA and incubated for 1 week at pH 7.4, showed a concentration-dependent reduction in weight-loss. There was no change in ICTP release from control values, meaning that ATA did not inhibit MMPs. Media concentrations of CTX fell significantly at 15wt% ATA indicating that ATA inhibits capthesins. Beams dipped in increasing concentrations of MDPB lost progressively less mass, showing that MDPB is a protease-inhibitor. ICTP released from controls or beams exposed to low concentrations were the same, while 5 or 10% MDPB significantly lowered ICTP production. CTX levels were strongly inhibited by 2.5-10% MDPB, indicating that MDPB is a potent inhibitor of both MMPs and cathepsin K.nnnSIGNIFICANCEnCTX seems to be released from dentin matrix only by cathepsin K. MMPs and cathepsin K and B may all contribute to matrix degradation.

Phosphoric acid esters cannot replace polyvinylphosphonic acid as phosphoprotein analogs in biomimetic remineralization of resin-bonded dentin

Polyvinylphosphonic acid (PVPA), a biomimetic analog of phosphoproteins, is crucial for recruiting polyacrylic acid (PAA)-stabilized amorphous calcium phosphate nanoprecursors during biomimetic remineralization of dentin collagen matrices. This study tested the null hypothesis that phosphoric acid esters of methacrylates in dentin adhesives cannot replace PVPA during bimimetic remineralization of resin-dentin interfaces. Human dentin specimens were bonded with: (I) XP Bond, an etch-and-rinse adhesive using moist bonding; (II) XP Bond using dry bonding; (III) Adper Prompt L-Pop, a self-etching adhesive. The control medium contained only set Portland cement and a simulated body fluid (SBF) without any biomimetic analog. Two experimental Portland cement/SBF remineralization media were evaluated: the first contained PAA as the sole biomimetic analog, the second contained PAA and PVPA as dual biomimetic analogs. No remineralization of the resin-dentin interfaces could be identified from specimens immersed in the control medium. After 2-4 months in the first experimental medium, specimens exhibited either no remineralization or large crystal formation within hybrid layers. Only specimens immersed in the second remineralization medium produced nanocrystals that accounted for intrafibrillar remineralization within hybrid layers. The null hypothesis could not be rejected; phosphoric acid esters in dentin adhesives cannot replace PVPA during biomimetic remineralization of adhesive-bonded dentin.

Cusp fracture resistance in composite–amalgam combined restorations

OBJECTIVEnTo evaluate the in vitro resistance to fracture and microleakage in composite-amalgam combined restorations.nnnMETHODSnSeventeen Class I cavities with unsupported enamel walls prepared in extracted permanent molar teeth were treated with a bonding agent (Scotchbond MP, 3M Dental), the buccal cusps were reinforced with a composite resin (Z-100, 3M Dental) and the cavities were then restored with amalgam (Permite, Oral B). All teeth were load tested using a special fatigue-stress apparatus, immersed in a dye solution and then sectioned for examination by stereomicroscopy and scanning electron microscopy (SEM).nnnRESULTSnFractures in enamel supported by composite were present in 11 cases while 12 specimens included fractures in enamel supported by bonded amalgam. Microleakage was observed in 16% of the enamel-amalgam interfaces, 10% of the dentin-amalgam interfaces, 7% of the amalgam-composite interfaces, 4% of the composite-enamel interfaces and 11% of the composite-dentin interfaces.nnnCONCLUSIONSnBonded amalgam appears to be as effective as bonded composite in supporting undermined enamel in terms of resistance to fracture, but composite may have better marginal adaptation to enamel compared to bonded amalgam. Good marginal adaptation may be observed between amalgam and composite in composite-amalgam restorations.

Use of crosslinkers to inactivate dentin MMPs.

OBJECTIVESnThis study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity.nnnMETHODSnSix different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography.nnnRESULTSnMMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release.nnnSIGNIFICANCEnThe result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.

Field emission in-lens SEM study of enamel and dentin.

This investigation used field emission in-lens scanning electron microscopy (FEISEM) for the study of tooth surfaces, with particular reference to adhesive bonding and acid conditioning. Dentin wafers with an intact enamel periphery were treated by either ethylenediaminetetraacetic acid (EDTA) (pH 7.4) or phosphoric acid (pH 0.7). The samples were then fixed, sequentially dehydrated in alcohol, and either air- or critical point-dried. After coating, surfaces were examined by FEISEM. For enamel, intraprismatic crystals were clearly recognizable, with the crystals showing both a longitudinal and parallel orientation to the long axis of the prisms. For dentin, the surface ultrastructure (mineral crystals and collagen banding) for the both untreated and treated samples was observed. Fine structures measuring on the order of 6 nm were also observed on samples treated by EDTA. We conclude that FEISEM can routinely provide high-resolution images of enamel and dentin, and that it has the capability of revealing the defined distribution of crystals and collagen fibers in dental tissues.

Ultrastructural Aspects of the DNA Polymerase α Distribution During the Cell Cycle

We studied the nuclear topography of the replicating enzyme DNA polymerase α in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase α antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase α observed in G1 - and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase α complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase α. The evidence of DNA polymerase α circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.