Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna-Alma Mater Studiorum, Via San Vitale 59, Bologna, Italy.
Department of Restorative Dentistry and Cariology, Adhesive Dentistry Research Group Institute of Dentistry, University of Turku, FI-20014 Turun Yliopisto, Turku Finland; Turku University Hospital, Kiinamyllynkatu 4-8 Turku, Finland.
J Dent. 2018 Dec;79:90-95. doi: 10.1016/j.jdent.2018.10.008. Epub 2018 Oct 24.
Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin.
Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles.
WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (∼20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix.
This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network.
It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.
基质金属蛋白酶（MMPs）是牙本质内源性酶，据称在牙本质有机基质降解中起关键作用。本研究旨在研究 MMP-7 在正常人类牙本质中的存在、定位和分布。
将牙本质粉末化、脱矿和溶解在等电聚焦缓冲液中。将分离的蛋白质转移到硝酸纤维素膜上进行 Western 印迹（WB）分析。为了进行酶谱分析，将牙本质蛋白等分试样在含有荧光标记明胶的 12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中电泳。此外，使用人 MMP-MAP 多重试剂盒的荧光微球免疫测定法测量牙本质 MMP 的浓度。使用预嵌入和后嵌入免疫标记技术研究 MMP-7 在牙本质中的定位和分布。对牙本质进行冷冻断裂，将部分脱矿的牙本质片段用单克隆抗 MMP-7 进行标记，并与金纳米颗粒偶联的二级抗体进行标记。在脱矿牙本质基质中，用高度电子致密的分散金颗粒鉴定 MMP-7 标记。
提取牙本质蛋白的 WB 和酶谱分析显示 MMP-7 的存在（约 20-28 kDa）。此外，使用荧光微球免疫测定法在孵育牙本质梁的上清液中发现了 MMP-7。FEI-SEM 和 TEM 分析确立了 MMP-7 是人类牙本质有机基质的固有成分。
本研究表明 MMP-7 是人类牙本质纤维网络的内源性成分。
了解牙本质基质重塑和降解背后的潜在过程对于开发最优化的临床方案和确保牙修复体的长期效果至关重要。
