Removal of water binding proteins from dentin increases the adhesion strength of low-hydrophilicity dental resins.

OBJECTIVES: To investigate the role of proteoglycans (PGs) on the physical properties of the dentin matrix and the bond strength of methacrylate resins with varying hydrophilicities. METHODS: Dentin were obtained from crowns of human molars. Enzymatic removal of PGs followed a standard protocol using 1 mg/mL trypsin (Try) for 24 h. Controls were incubated in ammonium bicarbonate buffer. Removal of PGs was assessed by visualization of glycosaminoglycan chains (GAGs) in dentin under transmission electron microscopy (TEM). The dentin matrix swelling ratio was estimated using fully demineralized dentin. Dentin wettability was assessed on wet, dry and re-wetted dentin surfaces through water contact angle measurements. Microtensile bond strength test (TBS) was performed with experimental adhesives containing 6% HEMA (H6) and 18% HEMA (H18) and a commercial dental adhesive. Data were statistically analyzed using ANOVA and post-hoc tests (α = 0.05). RESULTS: The enzymatic removal of PGs was confirmed by the absence and fragmentation of GAGs. There was statistically significant difference between the swelling ratio of Try-treated and control dentin (p < 0.001). Significantly lower contact angle was found for Try-treated on wet and dry dentin (p < 0.002). The contact angle on re-wet dentin was not recovered in Try-treated group (p = 0.9). Removal of PGs significantly improved the TBS of H6 (109% higher, p < 0.001) and H18 (29% higher, p = 0.002) when compared to control. The TBS of commercial adhesive was not affected by trypsin treatment (p = 0.9). SIGNIFICANCE: Changing the surface energy of dentin by PGs removal improved resin adhesion, likely due to more efficient water displacement, aiding to improved resin infiltration and polymerization.

Long-term Evaluation of Dentin Matrix Stability and Gelatinolytic Activity after Dentin Pretreatment with Caffeic Acid Phenethyl Ester.

PURPOSE: To investigate the long-term effect of 0.05% or 0.1% caffeic acid phenethyl ester (CAPE) on dentin matrix stability and hybrid layer stability, using an etch-and-rinse (Adper Scotchbond Multipurpose/ASB) or a self-etch adhesive (Clearfil SE Bond/CSE). MATERIALS AND METHODS: Dentin matrix specimens were assigned to five groups: 0.05% or 0.1% CAPE, green tea (GT), and the controls distilled water (DW) and dimethyl sulfoxide (DMSO). Following immersion of specimens for 1 h, modulus of elasticity (ME) and dentin mass change (MG) were determined at 3 post-treatment time points: immediately afterwards and at 3 and 6 months. Collagen solubilization (CS) was estimated by hydroxyproline (HYP) quantification. Resin-dentin interfaces with both adhesives were assessed with in situ zymography tests to evaluate gelatinolytic activity (GA). The dentin pretreatments were actively applied for 60 s. The sealing ability of aged resin-bonded slices was assessed by nanoleakage tests. RESULTS: GT increased immediate ME, which decreased significantly after 3 months (p < 0.0001). The CAPE groups did not differ from the control groups. GT provided a significant increase in dentin matrix mass after treatment (p < 0.0001). No significant differences regarding MG were observed for CAPE 0.1%, CAPE 0.05%, DW, and DMSO groups after 3 and 6 months. Cumulative HYP release revealed that CAPE groups and GT were statistically similar to DW and DMSO; the GT group exhibited statistically significantly less HYP release than did CAPE groups (p = 0.0073). Treatment with 0.05% or 0.1% CAPE presented lower GA when applied to ASB before acid conditioning (p < 0.05), but no differences were detected when the CAPE groups were applied to CSE. CAPE at 0.1% significantly reduced nanoleakage for CSE, and 0.05% CAPE with CSE presented levels of nanoleakage similar to those of the CSE control group. CONCLUSION: CAPE at 0.05% or 0.01% did not influence ME, MG, or CS, but reduced GA when applied to ASB before acid conditioning. CAPE at 0.1% with CSE promoted adhesive layer integrity.

Long-term nanomechanical properties and gelatinolytic activity of titanium tetrafluoride-treated adhesive dentin interface.

OBJECTIVE: This study investigated the effects of dentin pretreatment with 2.5% titanium tetrafluoride (TiF4) on nanomechanical properties, and the in situ gelatinolytic activity of the dentin-resin interface, for up to 6 months. METHODS: Twenty-four human teeth were prepared by exposing occlusal flat dentin surfaces, and were randomly assigned to experimental groups, according to application or non-application of a TiF4 pretreatment, and to the adhesive systems (Clearfil SE Bond or Scotchbond Universal). Resin composite (Filtek Supreme Ultra) was built up incrementally on the teeth in all the groups. Then, the specimens were sectioned and randomly selected for evaluation at 24h, 3 months and 6 months of storage time. The reduced modulus of elasticity (Er) and the nanohardness of the underlying dentin, as well as the hybrid layer and the adhesive layer were measured using a nanoindenter. Gelatinolytic activity at the dentin-resin interfaces was assessed by in situ zymography using quenched fluorescein-conjugated gelatin at 24h and 6 months. Statistical analyses were performed with ANOVA and Tukey's tests. RESULTS: There were no differences in Er and nanohardness values between adhesives systems and pretreatment (p=0.1250). In situ zymography showed significantly higher gelatinolytic activity after 6 months for all the experimental groups (p=0.0004), but no differences between the adhesive systems (p=0.7708) and the surface pretreatment (p=0.4877). SIGNIFICANCE: Dentin pretreatment with 2.5% TiF4 followed by self-etching adhesive systems did not influence nanomechanical properties or gelatinolytic activity of the adhesive-dentin interface layers, over time.

Role of proteoglycans on the biochemical and biomechanical properties of dentin organic matrix.

OBJECTIVE: Proteoglycans (PGs) are multifunctional biomacromolecules of the extracellular matrix of collagen-based tissues. In teeth, besides a pivotal regulatory role on dentin biomineralization, PGs provide mechanical support to the mineralized tissue and compressive strength to the biosystem. This study assessed enzymatic protocols for selective PGs removal from demineralized dentin to determine the roles of these biomacromolecules in the bulk mechanical properties and biostability of type I collagen. METHODS: Selective removal of glycosaminoglycans chains (GAGs) and PGs from demineralized dentin was carried out by enzymatic digestion protocols using chondroitinase ABC (c-ABC) and trypsin (Try). A comprehensive study design included assessment of dentin matrix mass loss, biodegradability of the PGs/GAGs-depleted dentin matrix, ultimate tensile strength (UTS) and energy to fracture tests. Quantitative data was statistically analyzed by two-way and one-way ANOVA followed by the appropriate post hoc tests (α=0.05). RESULTS: Transmission electron microscopy images show effective GAGs removal by c-ABC and Try and both enzymatic methods released statistically similar amounts of GAGs from the demineralized dentin. Try digestion resulted in about 25% dentin matrix mass loss and increased susceptibility to collagenolytic digestion when compared to c-ABC (p=0.0224) and control (p=0.0901). Moreover, PGs digestion by Try decreased the tensile strengths of dentin. Statistically lower energy to fracture was observed in c-ABC-treated dentin matrix. CONCLUSIONS: GAGs plays a pivotal role on tissue mechanics and anisotropy, while the core protein of PGs have a protective role on matrix biostability.

Encapsulation of grape seed extract in polylactide microcapsules for sustained bioactivity and time-dependent release in dental material applications.

OBJECTIVE: To sustain the bioactivity of proanthocyanidins-rich plant-derived extracts via encapsulation within biodegradable polymer microcapsules. METHODS: Polylactide microcapsules containing grape seed extract (GSE) were manufactured using a combination of double emulsion and solvent evaporation techniques. Microcapsule morphology, size distribution, and cross-section were examined via scanning electron microscopy. UV-vis measurements were carried out to evaluate the core loading and encapsulation efficiency of microcapsules. The bioactivity of extracts was evaluated after extraction from capsules via solvent partitioning one week or one year post-encapsulation process. Fifteen human molars were cut into 7mm×1.7mm×0.5mm thick mid-coronal dentin beams, demineralized, and treated with either encapsulated GSE, pristine GSE, or left untreated. The elastic modulus of dentin specimens was measured based on three-point bending experiments as an indirect assessment of the bioactivity of grape seed extracts. The effects of the encapsulation process and storage time on the bioactivity of extracts were analyzed. RESULTS: Polynuclear microcapsules with average diameter of 1.38µm and core loading of up to 38wt% were successfully manufactured. There were no statistically significant differences in the mean fold increase of elastic modulus values among the samples treated with encapsulated or pristine GSE (p=0.333), or the storage time (one week versus one year storage at room temperature, p=0.967). SIGNIFICANCE: Polynuclear microcapsules containing proanthocyanidins-rich plant-derived extracts were prepared. The bioactivity of extracts was preserved after microencapsulation.

Experimental primers containing synthetic and natural compounds reduce enzymatic activity at the dentin-adhesive interface under cyclic loading.

OBJECTIVE: To evaluate the effect of experimental primers (chlorhexidine, enriched mixture of proanthocyanidins, and doxycycline) on the adhesive properties and gelatinolytic activity at dentin-resin interfaces of occlusal Class I restorations. METHODS: The inactivation of enzymes by the experimental primers was assessed by fluorescence assay and gelatin zymography. To assess the adhesive properties, occlusal Class I cavities were prepared in sound human molars, etched with phosphoric acid and restored with one of the primers and an etch-and-rinse adhesive system (Adper Single Bond Plus-3M ESPE). After the restorative procedures, specimens were divided into two subgroups (n=6) consisting of storage in incubation buffer or axial cyclic loading at 50N and 1,000,000 cycles. Then, the specimens were sectioned and slices were assigned to in situ zymography assay and microtensile bond strength (TBS) test. RESULTS: Fluorescence assay and gelatin zymography revealed that the experimental primers inactivated rMMPs. In situ zymography (2-way ANOVA, Tukey, p<0.05) showed that cyclic loading increased the gelatinolytic activity at the resin-dentin interface and the experimental primers decreased the gelatinolytic activity at the adhesive interface. The experimental primers had no significant effects on dentin-adhesive bond strengths with or without cyclic loading (2-way ANOVA, p>0.05). SIGNIFICANCE: The use of experimental primers impaired the enzymatic activity at the dentin-adhesive interface after cyclic loading and the activity of rMMPs. Cyclic loading did not have a significant effect on the bond strength.