Fernando Nato

Chlorhexidine stabilizes the adhesive interface: a 2 year in vitro study

OBJECTIVES This study evaluated the role of endogenous dentin MMPs in auto-degradation of collagen fibrils within adhesive-bonded interfaces. The null hypotheses tested were that adhesive blends or chlorhexidine digluconate (CHX) application does not modify dentin MMPs activity and that CHX used as therapeutic primer does not improve the stability of adhesive interfaces over time. METHODS Zymograms of protein extracts from human dentin powder incubated with Adper Scotchbond 1XT (SB1XT) on untreated or 0.2-2% CHX-treated dentin were obtained to assay dentin MMPs activity. Microtensile bond strength and interfacial nanoleakage expression of SB1XT bonded interfaces (with or without CHX pre-treatment for 30s on the etched surface) were analyzed immediately and after 2 years of storage in artificial saliva at 37 degrees C. RESULTS Zymograms showed that application of SB1XT to human dentin powder increases MMP-2 activity, while CHX pre-treatment inhibited all dentin gelatinolytic activity, irrespective from the tested concentration. CHX significantly lowered the loss of bond strength and nanoleakage seen in acid-etched resin-bonded dentin artificially aged for 2 years. SIGNIFICANCE The study demonstrates the active role of SB1XT in dentin MMP-2 activation and the efficacy of CHX inhibition of MMPs even if used at low concentration (0.2%).

Use of a specific MMP-inhibitor (galardin) for preservation of hybrid layer.

OBJECTIVE Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging. METHODS Dentin powder obtained from human molars was treated with galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with galardin as additional primer for 30s and (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturers instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukeys tests (alpha=0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed (chi(2) test, alpha=0.05). RESULTS The inhibitory effect of galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year (p<0.05). Interfacial nanoleakage expression after aging revealed reduced silver deposits in galardin-treated specimens compared to controls (p<0.05). CONCLUSIONS This study confirmed that the proteolytic activity of dentinal MMPs was inhibited by the use of galardin in a therapeutic primer. Galardin also partially preserved the mechanical integrity of the hybrid layer created by a two-step etch-and-rinse adhesive after artificial aging.

Final Rinse Optimization: Influence of Different Agitation Protocols

INTRODUCTION This study examined the effect of different root canal irrigant agitation protocols in the penetration of an endodontic irrigant into dentinal tubules. METHODS Fifty-six human single-rooted teeth were shaped with nickel-titanium instruments, and a final rinse of 5% sodium hypochlorite labeled with 0.2% alizarin red was performed. Specimens were assigned to 7 groups (N = 8) and submitted to the following rinse activation protocols: no agitation (control group), K-File or gutta-percha agitation, or different sonic (EndoActivator [Advanced Endodontics, Santa Barbara, CA] and Plastic Endo, Lincolnshire, IL) and ultrasonic (Satelec [Acteongroup, Merignac, France] and EMS, Nyon, Switzerland) agitations. Specimens were sectioned at 1, 3, and 5 mm from the apex in 1-mm-thick slabs, ground, and prepared for fluorescence microscopy at 100x with a wavelength of 450 milliseconds. Irrigant penetration into dentinal tubules was analyzed by using Kruskal-Wallis analysis of variance followed by post-hoc comparisons. RESULTS Groups were ranked in the following order: control = K-file = gutta-percha < EndoActivator = Plastic Endo < Satelec = EMS. At 1 mm from the apex, the highest score was found for the EMS group compared with the control, K-file, gutta-percha, EndoActivator, and Plastic Endo groups, whereas no difference was found with the Satelec group. CONCLUSION The results support the use of an ultrasonic agitation to increase the effectiveness of the final rinse procedure in the apical third of the canal walls.

Effect of UVA-activated Riboflavin on Dentin Bonding

Recent studies have reported collagen cross-linking after exposure to riboflavin followed by ultraviolet-A (UVA) exposure. This study is the first to investigate the effect of a riboflavin-containing primer on adhesive interface stability and dentinal matrix metalloproteinase activity. Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Adhesive was applied to control specimens without riboflavin/UVA. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy. To investigate dentinal matrix metalloproteinase activity, we performed correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond. Ultraviolet-activated riboflavin treatment increased the immediate bond strength to dentin at all aging intervals (p < 0.05 vs. control) and decreased interfacial nanoleakage in aged specimens (1 yr; p < 0.05). Zymograms revealed that riboflavin/UVA pre-treatment inhibited dentinal matrix metalloproteinase activity (especially MMP-9). In conclusion, dentinal collagen cross-linking induced by riboflavin/UVA increased immediate bond strength, stabilized the adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing the durability of resin-dentin bonds.

Immunohistochemical and biochemical assay of MMP-3 in human dentine

OBJECTIVE The function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays. METHODS Powdered dentine from extracted human teeth was prepared and (1) partially demineralised with 1% H(3)PO(4) for 10min or (2) untreated (control). The presence of MMP-3 was measured using a colorimetric assay system (QuantiSir™, Epigentek, USA). Additional cryo-fractured dentine fragments were processed for immunohistochemical identification of MMP-3 under FEI-SEM. Casein-zymography was used to investigate MMP-3 activity. RESULTS MMP-3 detected level was 2.732ng/μL in partially demineralised dentine powder, whilst it increased to 3.280ng/μL in mineralised dentine. The FEI-SEM analysis revealed positive immunolabelling patterns for MMP-3, predominantly localized on the intertubular collagen fibrillar network showing MMP-3 directly or indirectly bound to the collagen fibrils. Casein-zymograms showed positive proteolytic activity for MMP-3 in demineralised dentine powder. CONCLUSION The results of the study clearly revealed the presence and distribution of MMP3 in human sound dentine. Whilst the presence was verified, its role is still unclear. Future studies are needed to investigate the possible involvement of MMP-3 in physiological and pathological condition of the dentine-pulp complex.

Analysis of differential artificial ageing of the adhesive interface produced by a two‐step etch‐and‐rinse adhesive

This study was performed to evaluate the effects of different in vitro ageing techniques on the dentine-bonded interface produced by a two-step etch-and-rinse adhesive. Composite build-ups were bonded to sectioned human molars using XP BOND and cut into non-trimmed dentine-composite beams for microtensile testing. Beams were assigned to one of the following storage conditions: (i) artificial saliva, 24 h (control); (ii) 10% sodium hypochlorite (NaOCl), 1 h; (iii) 10% NaOCl, 3 h; (iv) 60,000 thermal cycles, 2 months; (v) artificial saliva, 2 months; (vi) 60,000 thermal cycles, 6 months; and (vii) artificial saliva, 6 months. Beams were then pulled until failure and bond strength was calculated. Additional specimens were examined to investigate interfacial nanoleakage expression. NaOCl solution significantly reduced bonding compared with the control (group 2 = group 3 < group 1); and thermocycling reduced the bond strength in comparison to specimens stored for the same time-period in artificial saliva (group 4 < group 5; group 6 < group 7). Artificial ageing affected bond strength only after 6 months of storage (group 7 < group 5 = group 1). Increased nanoleakage was found under all ageing conditions in comparison with controls. NaOCl solution is a rapid and reliable in vitro ageing method for examining the durability of the adhesive interface produced by two-step etch-and-rinse adhesive systems.

Role of preliminary etching for one-step self-etch adhesives.

The aim of this study was to analyse the effect of preliminary phosphoric acid etching of enamel and dentine before the application of two, one-step self-etch adhesive systems. The systems were applied onto acid-etched or smear-layer-covered enamel and dentine. The treatment groups were as follows: group 1, Adper Easy Bond (3M ESPE) on etched substrate; group 2, Adper Easy Bond (control); group 3, iBond Self-Etch (Heraeus Kulzer) on etched substrate; and group 4, iBond Self-Etch (control). Enamel and dentine bond strengths were calculated using microshear and microtensile bond-strength tests. Additional specimens were prepared to evaluate nanoleakage at the dentine-adhesive interface and were investigated using light microscopy or transmission electron microscopy. Both adhesives demonstrated higher microshear bond strengths when enamel was pre-acid-etched with phosphoric acid (Adper Easy Bond 28.7 ± 4.8 MPa; iBond Self-Etch 19.7 ± 3.6 MPa) compared with controls (Adper Easy Bond 19.2 ± 3.3 MPa; iBond Self-Etch 17.5 ± 2.7 MPa) and increased microtensile bond strength when applied on acid-etched (Adper Easy Bond 35.8 ± 5.7 MPa; iBond Self-Etch 24.3 ± 7.9 MPa) vs. smear-layer-covered dentine (Adper Easy Bond 26.9 ± 6.2 MPa; iBond Self-Etch 17.6 ± 4.3 MPa). Adper Easy Bond showed lower nanoleakage than iBond Self-Etch, irrespective of preliminary etching. The results of this study support the use of phosphoric acid etching before the application of one-step self-etch adhesive systems.

Immunohistochemical localization of dentin matrix protein 1 in human dentin

Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.

Effects of pH, ionic strength, and applied voltage on migration of dental monomers in an organic matrix

OBJECTIVES The application of an electric field has been shown to positively influence the bonding of dentin bonding systems (DBS) by improving adhesive impregnation into dentin. However, the mechanism responsible for this phenomenon has not been completely elucidated. The aim of this study was to clarify the effects of pH, matrix ionic strength, and applied voltage on the migration of commonly used DBS monomers in a model matrix (agarose gel). METHODS Some common monomers examined were bis-GMA (2,2-bis[4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl] propane); HEMA (2-hydroxyethyl methacrylate); 2-MP (bis[2-(methacryloyloxy) ethyl] phosphate); TCDM [di(hydroxyethyl methacrylate) ester of 5-(2,5,-dioxo tetrahydrofurfuryl)-3-methyl-3-cyclohexenyl-1,2-dicarboxylic acid]; and TEGDMA (triethylene glycol dimethacrylate). Agarose gels poured into a horizontal 10-well electrophoretic cell were used to mimic the collagen fibrils of the dentin organic matrix. The role of pH, matrix ionic strength, and voltage on monomer migration was assayed by modifying the experimental conditions. RESULTS Results of experiments performed at pH 3.1, 6.3, 8.5, and 12.3; at low, medium, and high ionic strength; and at 50 and 100 V clearly showed that DBA monomer migration toward both the anode and the cathode can be affected by each of these parameters. SIGNIFICANCE Migration of acrylic monomers toward the anode or cathode can be achieved as desired by selective choice of pH, ionic strength, and applied voltage. Additional studies are needed to evaluate the synergistic effects of DBS monomer blends on migration in an electric field.