Do collagen cross-linkers improve dentin's bonding receptiveness?

OBJECTIVES: Dentin biomodification using collagen cross-linkers has been proposed as one of the strategies to improve bond durability of adhesives to dentin. However, literature is not very consistent regarding their benefit, in particular when cross-linkers are applied in clinically realistic application times. This study investigated the effect of three cross-linkers on the mini-interfacial fracture toughness (mini-iFT) of four adhesives bonded to dentin following either etch&rinse (E&R) or self-etch (SE) modes. METHODS: 60 molars were randomly divided in accordance with the three variables: cross-linker, adhesive and bonding mode (n=5). The cross-linkers glutaraldehyde (5wt%; GA), proanthocyanidin (6.5wt%; PA), or UVA-activated riboflavin (0.5wt%; RB), and distilled water (control) were applied on dentin for 60s after acid-etching (E&R) or before self-etching (SE). The 3-step E&R adhesive (3E&Ra) OptiBond FL (Kerr), the 2-step SE adhesive (2SEa) Clearfil SE Bond 2 (Kuraray Noritake) and the universal adhesives G-Premio Bond (GC) and Prime&Bond Active (Dentsply), the latter two employed in both E&R and SE modes, were applied following the respective manufacturer's instructions. Composite buildups (8×8×8mm) were made using Filtek Supreme XTE (3M) prior to 1-week storage in artificial saliva. After the teeth were sectioned into mini-specimens (1.5×2.0×18mm), a single notch was prepared at the adhesive-dentin interface. Half of the specimens were immediately loaded until failure by 4-point bending to determine the mini-iFT, while the remaining specimen set was tested upon 6-month aging. Data were statistically analyzed with a linear model (p<0.05). RESULTS: No significant decrease in mini-iFT was noted only for PA (p<0.05), while the mini-iFT decreased for both other cross-linkers and in quite a similar way as when solely water (Wa) was applied. SIGNIFICANCE: The cross-linker proanthocyanidin (PA) applied in clinically relevant conditions was able to maintain a stable mini-iFT after 6-month aging. The incorporation of UVA-activated riboflavin (RB) and glutaraldehyde (GA) in the dentin-bonding protocol appeared not effective to improve the stability of adhesive-dentin interfaces.

Effects of Composites Containing Bioactive Glasses on Demineralized Dentin.

The aim of this study was to evaluate the degradation of completely demineralized dentin specimens in contact with a filler-free or 2 ion-releasing resins containing micrometer-sized particles of Bioglass 45S5 (BAG) or fluoride-containing phosphate-rich bioactive glass (BAG-F). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also used to evaluate the remineralization induced by the experimental ion-releasing resin-based materials. Dentin beams were totally demineralized in H3PO4 (10%) and placed in direct contact with a filler-free (RESIN) or 2 experimental ion-releasing resins (BAG or BAG-F) and immersed in artificial saliva (AS) up to 30 d. Further specimens were also processed and submitted to FTIR and SEM analysis to evaluate the remineralization induced by such ion-releasing resins before and after AS immersion. BAG and BAG-F alkalinized the incubation media. A significant decrease of the dry mass was observed between the specimens of all groups stored for 3 and 30 d in AS. However, the fluoride-containing phosphate-rich bioactive glass incorporated into a resin-based material (BAG-F) showed greater ability in reducing the solubilization of C-terminal cross-linked telopeptide (ICTP) and C-terminal telopeptide (CTX) after prolonged AS storage. Moreover, after 30 d of AS storage, BAG-F showed the greatest remineralizing effect on the stiffness of the completely demineralized dentin matrices. In conclusion, fluoride-containing phosphate-rich bioactive glass incorporated as micrometer-sized filler in dental composites may offer greater beneficial effects than Bioglass 45S5 in reducing the enzyme-mediated degradation and remineralization of demineralized dentin.

Use of crosslinkers to inactivate dentin MMPs.

OBJECTIVES: This 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. METHODS: Six 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. RESULTS: MMP 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. SIGNIFICANCE: The 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.

Effect of pretreatment with collagen crosslinkers on dentin protease activity.

OBJECTIVES: This study evaluated the effect of dentin pretreatment with collagen crosslinkers on matrix metalloproteinase (MMP) and cathepsin K mediated collagen degradation. METHODS: Dentin beams (1mm×2mm×6mm) were demineralized in 10% H3PO4 for 24h. After baseline measurements of dry mass, beams were divided into 11 groups (n=10/group) and, were pretreated for 5min with 1% glutaraldehyde (GA); 5% GA; 1% grape-seed extract (GS); 5% GS; 10% sumac (S); 20µM curcumin (CR); 200µM CR; 0.l% riboflavin/UV (R); 0.5% R; 0.1% riboflavin-5-phosphate/UV (RP); and control (no pretreatment). After pretreatment, the beams were blot-dried and incubated in 1mL calcium and zinc-containing medium (CM, pH 7.2) at 37°C for 3, 7 or 14 days. After incubation, dry mass was reassessed and aliquots of the incubation media were analyzed for collagen C-telopeptides, ICTP and CTX using specific ELISA kits. Data were analyzed by repeated-measures ANOVA. RESULTS: The rate of dry mass loss was significantly different among test groups (p<0.05). The lowest 14 day mean dry mass loss was 6.98%±1.99 in the 200µM curcumin group compared to control loss of dry mass at 32.59%±5.62, p<0.05, at 14 days. The ICTP release over the incubation period (ng/mg dry dentin) ranged between 1.8±0.51 and 31.8±1.8. CTX release from demineralized beams pretreated with crosslinkers was significantly lower than CM (5.7±0.2ng/mg dry dentin). SIGNIFICANCE: The results of this study indicate that collagen crosslinkers tested in this study are good inhibitors of cathepsin K activity in dentin. However, their inhibitory effect on MMP activity was highly variable.

Effect of polyacrylic acid on dentin protease activities.

OBJECTIVE: This study tested whether treatment of demineralized dentin with polyacrylic acid (PAA) has any activatory or inhibitory activity on dentin matrix metalloproteinases (MMP)s or cathepsin K (CAT-K). METHODS: Dentin beams (1mm×2mm×6mm; n=10) were completely demineralized with EDTA. After initial dry mass assessment, the beams were dipped into 37% phosphoric acid (PA), PA+2% benzalkonium chloride (BAC), PA+2% chlorhexidine digluconate (CHX), 10% PAA, PAA+BAC or PAA+CHX for 20s. Demineralized beams without treatment served as control. All beams were incubated in simulated body fluid (SBF) for 1 week and the dry mass loss was evaluated. Aliquots of SBF were used to analyze solubilized telopeptide fragments using ICTP as indicator of MMP-mediated collagen degradation and CTX for CAT-K-mediated degradation. Additional demineralized beams (n=10) were used to measure the influence of different chemical treatments on total MMP activity of EDTA-demineralized dentin using generic MMP assay. Data were analyzed by ANOVA (α=0.05). RESULTS: Dry mass loss ranged from 6% (PA) to 2% for (PA-BAC) or (PAA-BAC) (p<0.05). ICTP release of PAA-treated group was significantly higher (p<0.05) than the control, and not significantly different from the PA group (p>0.05). PA+CHX or PAA+CHX and PAA+BAC showed significantly lower ICTP than PA or PAA groups (p<0.05). CAT-K activity increased significantly after 10% PAA treatment compared to control (p<0.05) or to PA postreatment. SIGNIFICANCE: Demineralized dentin treated with 10% polyacrylic acid activated CAT-K more than 37% phosphoric acid; 2% chlorhexidine digluconate seems to be a better inhibitor of MMPs and CAT-K than 2% benzalkonium chloride.

Zoledronate and ion-releasing resins impair dentin collagen degradation.

This study analyzed the amounts of solubilized telopeptides cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and C-terminal crosslinked telopeptide of type I collagen (CTX) derived from matrix-metalloproteinases (MMPs) and cysteine cathepsins (CTPs) subsequent to application of a filler-free (Res.A) or an ion-releasing resin (Res.B) to ethylenediaminetetraacetic acid (EDTA)-demineralized dentin with or without zoledronate-containing primer (Zol-primer) pre-treatment. The chemical modification induced following treatments and artificial saliva (AS) storage was also analyzed through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Totally EDTA-demineralized specimens were infiltrated with Res.A or Res.B with or without Zol-primer pre-treatment, light-cured, and immersed in AS for up to 4 wk. ICTP release was reduced following infiltration with Res.B and further reduced when Res.B was used with Zol-primer; remarkable phosphate mineral uptake was attained after AS storage. CTX release was increased in Res.A- and Res.B-treated dentin. However, when Zol-primer was used with Res.A, the CTX release fell significantly compared to the other tested resin-infiltration methods. In conclusion, zoledronate offers an additional inhibitory effect to the ion-releasing resins in MMP-mediated collagen degradation. However, Zol-primer induces a modest reduction in CTX release only when used with resin-based systems containing no ion-releasing fillers.

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices.

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin-dentin bonds. The current work studied the size-exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than ∼1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives in water-saturated matrices.

Effects of etch-and-rinse and self-etch adhesives on dentin MMP-2 and MMP-9.

Auto-degradation of collagen matrices occurs within hybrid layers created by contemporary dentin bonding systems, by the slow action of host-derived matrix metalloproteinases (MMPs). This study tested the null hypothesis that there are no differences in the activities of MMP-2 and -9 after treatment with different etch-and-rinse or self-etch adhesives. Tested adhesives were: Adper Scotchbond 1XT (3M ESPE), PQ1 (Ultradent), Peak LC (Ultradent), Optibond Solo Plus (Kerr), Prime&Bond NT (Dentsply) (all 2-step etch-and-rinse adhesives), and Adper Easy Bond (3M ESPE), Tri-S (Kuraray), and Xeno-V (Dentsply) (1-step self-etch adhesives). MMP-2 and -9 activities were quantified in adhesive-treated dentin powder by means of an activity assay and gelatin zymography. MMP-2 and MMP-9 activities were found after treatment with all of the simplified etch-and-rinse and self-etch adhesives; however, the activation was adhesive-dependent. It is concluded that all two-step etch-and-rinse and the one-step self-etch adhesives tested can activate endogenous MMP-2 and MMP-9 in human dentin. These results support the role of endogenous MMPs in the degradation of hybrid layers created by these adhesives.

Effect of phosphoric acid on the degradation of human dentin matrix.

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We "acid-etched" experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

Flexural strength,water sorption and solubility of a methylmethacrylate-free denture base polymer reinforced with glass fibre reinforcement.

A methylmethacrylate-free denture base polymer (Eclipse) in comparison to a conventional denture base polymer (Palapress vario) was evaluated after water saturation and Stick glass fibre reinforcement. The data were analysed with ANOVA at a = 0.05. Water-storage caused a decrease in the flexural strength and stiffness of the materials (p > 0.05). Conventional denture base material with fibre reinforcement gave highest flexural strength (201.1 MPa) compared to fibre reinforced Eclipse (79.1 MPa) (p < 0.05). Water sorption after 76 days was 2.08% (Palapress vario) and 1.55% (Eclipse). Fibre-reinforcement of methylmethacrylate-free material was not as successful as conventional denture base and needs to be further optimized.

Carbodiimide cross-linking inactivates soluble and matrix-bound MMPs, in vitro.

Matrix metalloproteinases (MMPs) cause collagen degradation in hybrid layers created by dentin adhesives. This in vitro study evaluated the feasibility of using a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to inactivate soluble rhMMP-9, as an example of dentin MMPs, and matrix-bound dentin proteases. The inhibitory effects of 5 EDC concentrations (0.01-0.3 M) and 5 incubation times (1-30 min) on soluble rhMMP-9 were screened with an MMP assay kit. The same EDC concentrations were used to evaluate their inhibitory effects on endogenous proteinases from completely demineralized dentin beams that were incubated in simulated body fluid for 30 days. Decreases in modulus of elasticity (E) and dry mass of the beams, and increases in hydroxyproline content of hydrolysates derived from the incubation medium were used as indirect measures of matrix collagen hydrolysis. All EDC concentrations and pre-treatment times inactivated MMP-9 by 98% to 100% (p < 0.05) compared with non-cross-linked controls. Dentin beams incubated in 0.3 M EDC showed only a 9% decrease in E (45% decrease in control), a 3.6% to 5% loss of dry mass (18% loss in control), and significantly less solubilized hydroxyproline when compared with the control without EDC cross-linking (p < 0.05). It is concluded that EDC application for 1 min may be a clinically relevant and effective means for inactivating soluble rhMMP-9 and matrix-bound dentin proteinases if further studies demonstrate that EDC is not toxic to pulpal tissues.

The inhibitory effects of quaternary ammonium methacrylates on soluble and matrix-bound MMPs.

Matrix metalloproteinases (MMPs) bound to dentin contribute to the progressive degradation of collagen fibrils in hybrid layers created by dentin adhesives. This study evaluated the MMP-inhibiting potential of quaternary ammonium methacrylates (QAMs), with soluble rhMMP-9 and a matrix-bound endogenous MMP model. Six different QAMs were initially screened by a rhMMP-9 colorimetric assay. For the matrix-bound endogenous MMPs, we aged demineralized dentin beams for 30 days in calcium- and zinc-containing media (CM; control), chlorhexidine, or QAMs in CM to determine the changes in dry mass loss and solubilization of collagen peptides against baseline levels. The inhibitory effects of QAMs on soluble rhMMP-9 varied between 34 and 100%. Beams incubated in CM showed a 29% decrease in dry mass (p < 0.05), whereas beams incubated with QAMs showed only 0.2%-6% loss of dry mass. Significantly more solubilized collagen was detected from beams incubated in CM (p < 0.05). It is concluded that QAMs exhibited dentin MMP inhibition comparable with that of chlorhexidine, but required higher concentrations.

Incremental layers bonding of silorane composite: the initial bonding properties.

OBJECTIVES: Lack of oxygen inhibition layer of silorane composite with cationic polymerization raises the question of the bonding of incremental layers of the composite. This study aimed to evaluate the bond strength of the silorane composite layers. METHODS: Fresh, 20 s, 5 min aged silorane composite (Silorane, 3M-ESPE) was used as substrate to adhere new silorane composite. For a comparison, dimethacrylate-based composite resin (Z250, 3M-ESPE) was adhered to the silorane composite with or without intermediate adhesive resin. As a control, dimethacrylate composite with oxygen inhibition layer was attached to fresh dimethacrylate composite. The bonded specimens (n=12/group) were water stored for 24 h. The shear bond strengths (SBS) were measured with a crosshead speed of 1.0 mm/min. Failure modes were assessed. Data were analysed by ANOVA, Tukey's post hoc tests and Chi-square tests (p=0.05). RESULTS: Dimethacrylate-dimethacrylate composite resin combination showed the highest mean SBS (33.0 MPa) values with no adhesive failures. Fresh silorane-silorane SBS was slightly lower (26.7 MPa) and was further decreased by aging the substrate for 20s (25.4 MPa) and 5 min (22.4 MPa). The percent of adhesive failures increased from 25% to 75%, respectively. The failure modes were significantly different (Chi-square, p<0.001). Silorane-dimethacrylate composite showed the lowest (4.0 MPa) SBS among the groups, which was increased significantly by use of phosphate-methacrylate-based intermediate resin (p<0.05). CONCLUSION: In order to bond dimethacrylate composite to silorane composite, a phosphate-methacrylate-based intermediate resin is required. The silorane composite showed slightly lower incremental bonding properties than conventional dimethacrylate composites.