Valeria Angeloni

Effect of carbodiimide (EDC) on the bond stability of etch-and-rinse adhesive systems.

OBJECTIVE Recent studies supported the use of protein cross-linking agents during bonding procedures to inactivate endogenous dentin proteases, preventing dentin collagen degradation thus improving bond durability. The aim of this study was to evaluate the effect of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-containing conditioner on the stability of the adhesive interface created by two etch-and-rinse adhesives. METHODS Human dentin was etched with 35% phosphoric acid, treated with 0.3M EDC-containing conditioner followed by a three-step or a two-step etch-and-rinse adhesive. Adhesives were applied to control specimens without EDC pre-treatment. Specimens were subjected to microtensile bond strength test and pulled to failure after 24h or 1 year of storage and interfacial nanoleakage expression was evaluated and quantified by light microscopy. Additionally, to investigate endogenous dentin matrix metalloproteinase activity a zymographic assay was performed on protein extracts obtained from phosphoric-acid-etched dentin powder with or without EDC treatment. RESULTS The use of the EDC-containing conditioner did not affect immediate bond strength to dentin but contributed to preserve the bond strength after 1 year (p<0.05) for both tested adhesives. No difference was found in the interfacial nanoleakage expression that increased after aging irrespective from the treatment. EDC pre-treatment inhibited dentin endogenous MMPs as assayed with the zymography. SIGNIFICANCE In conclusion, the results of the study provide proof that EDC can produce long-term inactivation of MMPs in acid-etched dentin matrices contributing to bond strength preservation over time. Future studies are needed to support the use of EDC in vivo.

Carbodiimide Inactivation of MMPs and Effect on Dentin Bonding

The use of protein cross-linking agents during bonding procedures has been recently proposed to improve bond durability. This study aimed to use zymography and in situ zymography techniques to evaluate the ability of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linker to inhibit matrix metalloproteinase (MMP) activity. The hypotheses tested were that: (1) bonding procedures increase dentin gelatinolytic activity and (2) EDC pre-treatment prevents this enzymatic activity. The zymographic assay was performed on protein extracts obtained from dentin powder treated with Optibond FL or Scotchbond 1XT with or without 0.3M EDC pre-treatment. For in situ zymography, adhesive/dentin interfaces were created with the same adhesives applied to acid-etched dentin slabs pre-treated or not with EDC conditioner. Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesive application, while the use of EDC as a primer inactivated dentin gelatinases. Results of in situ zymograpy showed that hybrid layers of tested adhesives exhibited intense collagenolytic activity, while almost no fluorescence signal was detected when specimens were pre-treated with EDC. The correlative analysis used in this study demonstrated that EDC could contribute to inactivate endogenous dentin MMPs within the hybrid layer created by etch-and-rinse adhesives.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE (P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls (P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage (P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls (P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.

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

OBJECTIVE The 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. METHODS Freshly 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. RESULTS The 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. SIGNIFICANCE The 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.

Role of chlorhexidine on long-term bond strength of self-adhesive composite cements to intraradicular dentin

PURPOSE To examine the effect of CHX pre-treatment on long-term bond strength of fiber posts luted with self-adhesive resin cements. MATERIALS AND METHODS Seventy-two single-rooted teeth were selected for root canal treatment and post space preparation. The tested self-adhesive cement/post combinations were (N = 36): 1. RelyX Fiber-Posts luted with RelyX Unicem; 2. Rebilda Posts luted with Bifix SE Cement. For both self-adhesive cements, half of the specimens (experimental groups) were luted after the application of a solution of 2% CHX, while no CHX application was performed for the remaining specimens (control groups). Luted specimens were cut and used for push-out bond strength evaluation immediately, and after storage in artificial saliva for 6 months or 1 year. Additional specimens were processed for quantitative interfacial nanoleakage analysis. RESULTS ANOVA showed that the variable times of storage had a significant influence on the results (p < 0.05), while no influence of the luting procedure (cements with or without CHX) on the final outcome (p > 0.05) was found. Tukeys pairwise post-hoc test showed that the radicular bond strength decreased with time of storage. In particular, a significant difference was found between T0 and T1y, but not between T0 and T6m. In contrast, in terms of pretreatment, no significant reduction in push-out bond strength was observed, irrespective of the aging time. CONCLUSION CHX pretreatment did not prevent bond strength degradation of fiber posts luted with self-adhesive cements.

Experimental use of an acrolein-based primer as collagen cross-linker for dentine bonding

OBJECTIVES The objective of the present study was to investigate the long-term effect of 0.01% acrolein (ACR) aqueous solution, employed as an additional primer, on the mechanical durability and enzymatic activity of resin-dentine interfaces created with a simplified etch-and-rinse adhesive. METHODS Dentine surfaces were etched with 35% phosphoric acid for 15s, rinsed and blot-dried. Specimens were then assigned to: Group 1: dentine pre-treated with 0.01% ACR aqueous solution for 1min and bonded with Adper Scotchbond 1 XT (SB1XT), a 2-step etch-and-rinse adhesive; Group 2: SB1XT was applied on untreated acid-etched dentine (control). Resin composite build-ups were made using Filtek Z250. Microtensile bond strength was tested by stressing sectioned specimens to failure immediately or after 1year of storage in artificial saliva at 37°C. Zymography and in-situ zymography assays were performed for examining dentine matrix metalloproteinase (MMP) activities. RESULTS The use of 0.01% ACR as conditioning primer appeared to have contributed better to preservation of bond strength over time without affecting immediate bond strength. Zymography and in-situ zymography showed reduction in MMP activities after the application of ACR. CONCLUSION Dentine collagen cross-linking produced by an ACR-based primer increases the longevity of resin-dentine bonds by reinforcement of the adhesive interface and reduction of dentine MMP activities. Further studies are required to evaluate the potential in vivo and in vivo cytotoxicity of ACR. CLINICAL SIGNIFICANCE The acrolein-based primer is a potentially useful clinical bonding tool because it demonstrates good collagen cross-linking ability within a clinically-acceptable working time. Although a low ACR concentration was employed in the present study, the cytotoxicity of ACR should be tested prior to clinical use.