Department of Medical Sciences, Unit of Dental Sciences and Biomaterials, University of Trieste, Italy.
J Dent Res. 2012 May;91(5):467-72. doi: 10.1177/0022034512439210. Epub 2012 Feb 21.
Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.
牙本质蛋白酶被认为在混合层（HL）的降解中起重要作用。本研究通过原位酶谱法和功能酶活性测定研究了 HL 的明胶酶活性。假设由酸蚀-冲洗型粘接剂形成的 HL 具有活性明胶酶活性，并且在粘接过程中牙本质中的 MMP-2 和 -9 活性增加。用 Adper Scotchbond 1XT 粘接并用复合树脂修复酸蚀牙本质标本。将粘接/牙本质界面切片放在载玻片上，用荧光素标记的明胶覆盖，24 小时后用多光子共聚焦显微镜观察。制备人牙本质粉末等分试样，并分配到以下处理中：A、未处理；B、用 10%磷酸酸蚀；或 C、用 10%磷酸酸蚀并与 Scotchbond 1XT 混合。用功能酶测定法测量牙本质粉末提取物中的 MMP-2 和 -9 活性。在 HL 的底部检测到强烈且连续的酶活性，而上部 HL 的酶活性则更为不规则。酸蚀和随后的粘接应用均显著增加了 MMP-2 和 -9 活性（p<0.05）。结果首次证明了 HL 中的内在 MMP 活性以及酸蚀和粘接应用均能强烈激活基质结合的 MMP 活性。
