Adhesion to chondroitinase ABC treated dentin

Abstract

Dentin bonding relies on complete resin impregnation throughout the demineralised hydrophilic collagen mesh. Chondroitin sulphate-glycosaminoglycans are claimed to regulate the three-dimensional arrangement of the dentin organic matrix and its hydrophilicity. The aim of this study was to investigate bond strength of two etch-and-rinse adhesives to chondroitinase ABC treated dentin. Human extracted molars were treated with chondroitinase ABC and a double labeling immunohistochemical technique was applied to reveal type I collagen and chondroitin 4/6 sulphate distribution under field emission in-lens scanning electron microscope. The immunohistochemical technique confirmed the effective removal of chondroitin 4/6 sulphate after the enzymatic treatment. Dentin surfaces exposed to chondroitinase ABC and untreated specimens prepared on untreated acid-etched dentin were bonded with Adper Scotchbond Multi-Purpose or Prime and Bond NT. Bonded specimens were submitted to microtensile testing and nanoleakage interfacial analysis under transmission electron microscope. Increased mean values of microtensile bond strength and reduced nanoleakage expression were found for both adhesives after chondroitinase ABC treatment of the dentin surface. Adper Scotchbond Multi-Purpose increased its bond strength about 28%, while bonding made with Prime and Bond NT almost doubled (92% increase) compared to untreated specimens. This study supports the hypothesis that adhesion can be enhanced by removal of chondroitin 4/6 sulphate and dermatan sulphate, probably due to a reduced amount of water content and enlarged interfibrillar spaces. Further studies should validate this hypothesis investigating the stability of chondroitin 4/6 and dermatan sulphate-depleted dentin bonded interface over time.

Figure 1

FEISEM images of dentin resulting from mixing a back scattered image, that reveals the gold nanoparticles conjugated with secondary antibodies as 15 nm diameter white spots for chondroitin 4/6 sulphate and 30 nm for type I collagen fibrils, with a secondary electron image, that shows the dentin morphology. Dentin specimens exposed to the enzymatic treatment are represented in Fig. 1a and 1c, while untreated dentin surfaces are shown on Fig. 1b and 1d. Fig. 1a: after 24 h chondroitinase ABC pretreatment, 30 nm gold particles identifying type I collagen fibrils are clearly visible while no 15 nm gold labeling selective for chondroitin 4/6n sulphate can be detected. Fig. 1b: Dentin surface etched with phosphoric acid and exposed to the double immunolabelling technique. Chondroitin 4/6 sulphate appears as globular structures clearly labeled by 15 nm colloidal gold particles, localized along branching minor fibrils (arrows). Collagen fibrils are positively labeled by 30 nm gold particles. Fig. 1c. High magnification view of chondroitinase ABC treated dentin showing enlarged matrix mesh (asterisks), negative labeling for chondroitin 4/6 sulphate but positive for type I collagen fibrils. Fig. 1d: high magnification FEI-SEM micrograph revealing the complex network of major collagen fibrils and globular chondroitin 4/6 sulphate structures (arrows) in untreated dentin specimens.

Figure 2

TEM micrographs of un-demineralized and unstained specimens showing the interfacial nanoleakage expression of Adper Scotchbond Multi-Purpose (Fig. 2a,2b) and Prime&Bond NT (Fig. 2c,2d). Fig. 2a: Low magnification image of Adper Scotchbond Multi-Purpose applied on chondroitin 4/6 sulphate-depleted dentin due to Chondroitinase ABC treatment showing minimal nanoleakage expression characterized by small scattered silver nitrate particles; dentin tubule (T); hybrid layer (HL). Fig. 2b: Adper Scotchbond MP applied on untreated control dentin: nanoleakage expression of the bonded interface is similar to chondroitinase ABC pretreated sample with small silver aggregates (arrows), i.e. no influence on nanoleakage expression was found due to the enzymatic treatment for the three-step etch-and-rinse adhesive; Dentin tubule (T); hybrid layer (HL). Fig. 2c: The use of chondroitinase ABC pretreatment before the application of Prime & Bond NT resulted in a strong reduction of silver nitrate expression within the hybrid layer created on intertubular dentin; Dentin tubule (T); hybrid layer (HL). Fig. 2d: application of Prime & Bond NT on untreated dentin showed spotted nanoleakage expression close to the tubular lumen (T) exposed by acid etching. Silver grains (arrows) are also located at the top of the hybrid layer (HL) forming an uniformly distributed electron-dense layer. Dentin tubule (T); hybrid layer (HL). All images were taken at the same magnification (Bar = 1μm).