Effect Of Collagenase And Its Inhibitors On The Durability Of Dentin Bond

Dental defects caused by caries and exogenous injuries are frequently seen in clinic. With the advantages as no mercury contamination, less grinding of tooth structure, esthetic, good biocompatibility, contemporary adhesive restorative technique for dental defects has been widely used in clinical practice. Dentin has a special structure that contains massive organic substances. Additionally as the resin composite with the features of polymerization shrinkage, it's difficult to form a stable bonding on the dentin, especially to maintain the durability of dentin bond. The basic mechanism of bonding to dentin is the formation of a hybrid layer. As the collagen fibrils are the main organic substance in hybrid layer, its integrity plays an important role to maintain the longevity of bonding. Recently researches indicate that dentin contains endogenous gelatinolytic/collagenolytic activity. The integrity of the hybrid layer will be destroyed once this kind of enzyme activity induces the degradation of the dentin collagen fibrils. Therefore the durability of dentin bond will be impacted negatively. The bonded interface formed by the dentin organic matrix, hydroxyapatite crystallites, and resin monomers remains the weakest area of the adhesive restorations. So, proper treatment of this interface is critical to the success of dentin adhesive restorations.Firstly, this paper uses the method of Atomic Force Microscopy (AFM) to observe the surface morphology of the organic substance layer (mainly collagen fibrils) of the acid-etched dentin. On the basis of this morphology observation, AFM in situ observation is performed to observe the enzymatic degradation of dentin collagen fibrils exposed to exogenous collagenase in order to simulate the activity of endogenous enzyme activity. Then another study was conducted to detect the inhibitory effects of different exogenous collagenase inhibitors on the enzymatic reaction mentioned above. Secondly, human third molars were used as the research objects to study the effect of the exogenous collagenase inhibitors on the durability of dentin bond in vitro, using the methods of micro-tensile bond strength test (μTBS) and Scan Electron Microscope (SEM).The target is to make clear the acting factors and mechanisms that influence the stability and durability of dentin bond.Objectives and Methods:1. To image the dentin collagen fibrils with AFM. After proper treatment the prepared dentin specimen was exposed the network of collagen fibrils, and AFM was used to make clear images of the collagen fibrils. From this method, the morphology of the dentin matrix (mainly collagen fibrils) was detected, which would provide a platform for the further experimental study.2. To observe the enzymatic degradation process of dentin collagen fibrils under the action of collagenase. The acid-etched dentin specimen was put into the AFM fluid cell, then, the mixed solution of collagenaseⅠand collagenaseⅡwas perfused into the cell. AFM, in contact mode, was used in situ to continuously monitor the enzymatic degradation of the dentin organic matrix (mainly collagen fibrils) by collagenase. The microscopic morphology changes of dentin collagen fibrils were recorded in order to explore the features of that enzymatic degradation reaction.3. To observe the inhibition of enzymatic degradation reaction by different collagenase inhibitors with AFM. Chlorhexidine and Captopril were used as the inhibitors of collagenase. The inhibition effect of collagenase inhibitors on the enzymatic degradation reaction between collagenaseⅡand collagen fibrils was detected by AFM. From experimental results, the inhibitory effects of the collagenase inhibitors were deducted.4. To study the effect of the collagenase inhibitors on the durability of dentin bond. Human third molars were used in vitro. With the coronal enamel removed, the tooth was treated to produce a standard dentin surface for bonding application. After acid-etching, the dentin surface was applied with Chlorhexidine gluconate before bonding and filled with resin composite layer by layer. The bonded teeth were then prepared to make micro-tensile specimens and subjected to water-aging test. Micro-tensile bond strength test was used to test the immediate bond strength and the bond strength after six months'aging. Additionally SEM test was used to observe the changes of the components of fracture surface for comparative study. The effects of collagenase inhibitors on dentin bond were finally evaluated and the mechanism for bond degradation was deduced.Results:1. The method of imaging dentin collagen fibrils with AFM was established, and the clear AFM images of dentin collagen fibrils were obtained. Dentin collagen fibrils interweaved into a network and the characteristic periodic cross-banding structures with an axial repeat distance of about 67nm were clearly seen.2. The enzymatic degradation process of human dentin collagen fibrils exposed to exogenous collagenase was observed using AFM in situ. It has been shown that the topographic images of dentin specimens obtained with the AFM gradually changed with time after being treated with the mixed solution of collagenase I and II. The network of collagen fibrils became very sparse during the last period of collagenase treatment, and the micro-structures of dentin substrate combined with single or bunched collagen fibrils were exposed.3. CollagenaseⅡcan degrade dentin organic substance (mainly collagen fibrils) effectively. Massive collagen fibrils interweaved into a network before the treatment of CollagenaseⅡ. After 6 hours of CollagenaseⅡtreatment, the collagen fibrils become significantly less and the topographic images of massive minerals on the dentin substrate were revealed.4. Chlorhexidine and Captopril can inhibit the activities of collagenaseⅡ. The morphology of dentin organic matrix (mainly collagen fibrils) has no significant change after being exposed to the Chlorhexidine-contained collagenaseⅡsolution and Captopril-contained collagenaseⅡfor 6 hours respectively.5. The application of collagenase inhibitor (Chlorhexidine digluconate) can improve the durability of dentin bond. The bond strength of the micro-tensile specimen in the control group without the application of collagenase inhibitor decreased with a higher degree compared with the experimental group after aging in water for 6 months, showing significant difference (p<0.05). In the results of SEM test, the fractural surface of the specimens in experimental and control group shows the different fracture types. Most of the fractural surfaces of the control specimens without the application of collagenase inhibitor are at the bottom of the hybrid layer, and the collagenase fibrils become significantly less.Conclusions:1. AFM is an effective method for real-time observation of dentin collagen fibrils. It is possible is to make real-time observation on the enzymatic degradation process of dentin collagen fibrils exposed to exogenous collagenase using AFM.2. CollagenaseⅡcan degrade dentin organic substance (mainly collagen fibrils), and the exogenous collagenase inhibitors (Chlorhexidine and Captopril) can inhibit the activities of collagenaseⅡ.3. The application of collagenase inhibitor (Chlorhexidine digluconate) in the performance of dentin bonding can improve the durability of dentin bond.