Anti-proteolytic capacity and bonding durability of proanthocyanidin-biomodified demineralized dentin matrix

摘要

Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin–dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolytic activity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability. Keywords: bonding durability, collagenolysis, crosslinking agents, dentin, proanthocyanidinsIntroductionThere is common consensus that resin–dentin bonds created with contemporary hydrophilic dentin bonding systems deteriorate over time.~(1,2) Collagen disorganisation in the incompletely resin-infiltrated demineralized dentin matrix is one of the main degradation patterns found in unsuccessful adhesive restorations.~(3) Continuing efforts have focused on preservation of the integrity of the collagen matrix of the hybrid layer in an attempt to achieve durable dentin bonding. Considering the key role of matrix metalloproteinases (MMPs) in interfacial ageing over time, inhibiting the activity of host-derived MMPs and reducing the susceptibility of collagen matrices to MMP-induced degradation via bonding procedures may thus be a rational and effective approach for the improvement of bonding durability.~(2)Many attempts have been made to apply MMP inhibitors to acid-etched dentin prior to the application of adhesives.~(4,5,6) The only such inhibitor that has proved to be effective for reducing the degradation of resin–dentin bonds after in vivo ageing is chlorhexidine (CHX), a classic nonspecific MMP inhibitor.~(7) However, its MMP-inhibitory effect lasts only 9–12 months, and then weakens over time as CHX slowly leaches from the resin/dentin interface.~(8) Proanthocyanidins (PAs), a group of naturally occurring polyphenolic compounds, have the potential to control MMP-mediated diseases, such as periodontitis, by inhibiting both MMP production and activity.~(9) Epasinghe et al. ~(10) demonstrated that PA can inactivate more than 90% of soluble recombinant MMP-2, -8 and -9 and approximately 70%–80% of cysteine cathepsin B and K; these results are significantly better those derived from the use of CHX.Apart from being a potential non-selective MMP inhibitor, PA has gained much attention over the past few years because of its crosslinking capabilities, which are beneficial for enhancing demineralized dentin matrix and improving resin-dentin bond strength. Castellan et al. ~(11,12,13) reported that PA pre-treatment increased the immediate elastic modulus of dentin matrix and was effective even after bacterial collagenase challenge or 1-year of storage in artificial saliva. Additionally, it provided enhanced immediate adhesion and long-term stabilisation to demineralized dentin after 1-year of ageing in water. Hechler et al. ~(14) evaluated the long-term performance of PA application both as an additive to the adhesive and as a primer in an extra bonding step. They found that after 52 weeks' exposure to collagenase digestion, the bond strength of the PA-primer group was significantly higher than that of the control, whereas no significant difference was found between the PA-adhesive group and the control. This observation strongly supports the use of PA as a primer. However, all of the above-mentioned studies had a common limitation: the PA priming time was over 10?min, which is not clinically practical. An extra bonding step in PA biomodification for a clinically relevant duration improved the mechanical properties of demineralized dentin collagen~(15) as well as the immediate resin-dentin bond strength to dentin,~(16) and was effective at reducing the interfacial ageing caused by thermal