Collagen degradation and preservation of MMP-8 activity in human dentine matrix after demineralization
Collagen degradation and preservation of MMP-8 activity in human dentine matrix after demineralization
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DOI：
10.1016/j.archoralbio.2016.04.003
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发表时间：
2016-08-01
影响因子：
3
通讯作者：
Ericson, Dan
中科院分区：
文献类型：
--
作者：
Hedenbjork-Lager, Anders;Hamberg, Kristina;Ericson, Dan
目的：龋病是由口腔微生物产生的酸性物质驱动，蛋白水解酶降解牙本质胶原基质的过程。基质金属蛋白酶(MMPs)通过降解胶原蛋白参与龋病的发生。本研究的目的是开发一种产生脱矿牙本质基质底物(DDM)的方法，以保持基质金属蛋白酶-8的生物活性，并不干扰后续的检测。这样的底物可以研究不同处理对脱矿牙本质中基质金属蛋白酶-8活性和胶原降解的影响。设计：将人牙本质在组织研磨机中粉碎并冷冻(-80摄氏度)。用EDTA或醋酸在透析管中对粉末进行脱矿。取脱矿牙本质基质(DDM)，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定胶原含量。DDM随后被暂停在PBS或Tesca缓冲器中。结果：EDTA或酸脱矿牙本质形成富含胶原蛋白的底物，同时保持基质金属蛋白酶8的活性。胶原在室温下降解，1wk后DDM的可溶组分中有较高的HYP含量，表明该方法在脱矿过程中保留了DDM的活性成分。结论：所提出的脱矿方法都提供了适合于进一步干预研究的不溶性DDM底物。然而，根据脱矿方法和使用的缓冲液的不同，底物也不同。这需要进一步的研究，以找到一种最佳的技术，以产生DDM的保留蛋白和酶的生物活性。(C)2016爱思唯尔有限公司。保留所有权利。
Objective: Dental caries is a process driven by acids produced by oral microorganisms followed by degradation of the dentine collagen matrix by proteolytic enzymes. Matrix metalloproteinases (MMPs) have been suggested to contribute to caries by degrading collagen. The aim of this study was to develop a method for generating demineralized dentine matrix substrate (DDM) maintaining MMP-8 bioactivity and no interference with later assays. Such a substrate would allow study of the effects of various treatments on MMP-8 activity and collagen degradation in demineralized dentine.Design: Human dentine was powderized in a tissue grinder and frozen (-80 degrees C). The powder was demineralized in dialysis tubes, using EDTA or acetic acid. The demineralized dentine matrix (DDM) was harvested and analyzed for collagen content using SDS-PAGE. The DDM was subsequently suspended in PBS or TESCA buffer. Protein, MMP-8 (ELISA) and collagen (HYP) was analyzed directly or after 1 wk.Results: EDTA or acid demineralization of dentine using dialysis yielded a substrate rich in collagen coupled with preserved MMP-8 activity. Collagen degraded in room temperature, assessed by higher HYP amounts in the soluble fraction of DDM after one wk, indicating that the methods used preserved active DDM-components after the demineralization process.Conclusions: The presented demineralization methods both provided insoluble DDM substrates suitable for further intervention studies. However, it was found that the substrates differed depending on the demineralization method and buffers used. This needs further study to find an optimal technique for generating DDM with retained proteins as well as enzymatic bioactivity. (C) 2016 Elsevier Ltd. All rights reserved.