1 Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, PR China.
2 Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna-Alma Mater Studiorum, Bologna, Italy.
J Dent Res. 2018 Apr;97(4):409-415. doi: 10.1177/0022034517747264. Epub 2018 Jan 2.
A chelate-and-rinse extrafibrillar calcium chelation dentin bonding concept has recently been developed and investigated for its effectiveness in improving resin-dentin bonding by bridging the gap between wet and dry dentin bonding. The objective of the present study was to evaluate the gelatinolytic activity of hybrid layers (HLs) created using the chelate-and-rinse bonding technique. Gelatinolytic activity within the HL was examined using in situ zymography and confocal laser-scanning microscopy after 24-h storage or after thermomechanical cycling. Dentin specimens were bonded with Prime&Bond NT (Dentsply Sirona) after conditioning with 15 wt% phosphoric acid for 15 s (control) or 15 wt% polymeric chelators (sodium salt of polyacrylic acid; PAAN) of 2 different molecular weights for 60 s. For each reagent, bonding was performed using dry-bonding and wet-bonding techniques ( n = 10). Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and examined with a confocal laser-scanning microscope. Fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was quantified. Gelatinolytic activity was expressed as the percentage of green fluorescence emitted within the HL. After storage for 24 h, enzymatic activity was only detected within the completely demineralized phosphoric acid-etched dentin, with values derived from dry bonding higher than those from wet bonding ( P < 0.05). Almost no fluorescence signals were detected within the HL when dentin was conditioned with PAANs compared with the controls ( P < 0.05). After thermomechanical cycling, enzymatic activities significantly increased for the phosphoric acid-conditioned, drying-bonding group compared with 24-h storage ( P < 0.05). The present study showed that the use of the chelate-and-rinse bonding concept for both dry-bonding and wet-bonding approaches results in the near absence of matrix-bound collagenolytic activities in the HL even after aging. This may be attributed to fossilization of endogenous proteases via preservation of intrafibrillar minerals within the dentin collagen matrix.
一种螯合-冲洗型细胞外纤维钙螯合牙本质粘结概念最近已经被开发并研究，其目的是通过桥接湿粘结和干粘结之间的间隙来改善树脂-牙本质粘结，以提高其有效性。本研究的目的是评估使用螯合-冲洗粘结技术形成的混合层（HL）的明胶酶活性。通过在 24 小时储存或热机械循环后使用原位酶谱法和共焦激光扫描显微镜检查 HL 内的明胶酶活性。牙本质标本用 Prime&Bond NT（登士柏西诺德）粘结，预处理用 15wt%磷酸 15 秒（对照组）或 2 种不同分子量的聚合螯合剂（聚丙烯酸钠盐；PAAN）60 秒。对于每种试剂，使用干粘结和湿粘结技术进行粘结（n=10）。含有粘结-牙本质界面的切片用荧光素标记的明胶覆盖，并用共焦激光扫描显微镜检查。水解荧光素标记的明胶发出的荧光强度进行定量。明胶酶活性表示为 HL 内发出的绿色荧光的百分比。储存 24 小时后，仅在完全脱矿的磷酸酸蚀牙本质内检测到酶活性，干燥粘结的数值高于湿粘结（P<0.05）。与对照组相比，用 PAAN 预处理牙本质时，HL 内几乎没有荧光信号（P<0.05）。热机械循环后，与 24 小时储存相比，磷酸酸处理、干燥粘结组的酶活性显著增加（P<0.05）。本研究表明，即使在老化后，螯合-冲洗粘结概念用于干粘结和湿粘结方法都几乎不存在 HL 内的基质结合胶原酶活性。这可能是由于牙本质胶原基质内的纤维内矿物质的保存导致内源性蛋白酶的石化。
