Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong Special Administrative Region.
Melbourne Dental School, The University of Melbourne, Australia.
Arch Oral Biol. 2017 Dec;84:29-36. doi: 10.1016/j.archoralbio.2017.09.012. Epub 2017 Sep 18.
Proanthocyanidin (PA) is a natural collagen cross-linker that has been used in dentine matrix biomodification for reparative and preventive therapies. This study evaluated the ultrastructure of collagen after its interaction with PA. Furthermore, the mineralization of PA-biomodified collagen matrix was observed.
Ten freshly extracted sound human molars were sectioned into 0.5mm×1.7mm×7mm beams for ultrastructural evaluation of PA and dentine matrix under Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Specimens for TEM were completely demineralized and divided into three groups according to PA treatments: deionized water, 2% PA and 6.5% PA. The specimens were fixed, dehydrated, sectioned and examined using TEM. Specimens for FESEM were lightly conditioned with EDTA and similarly divided into the three groups for observation using FESEM. Type I collagen from calf skin was used to analyse the mineral interaction after treatment with 6.5% PA. Formvar- and carbon-coated 400-mesh Ni grids (EMS, Hatfiels, PA, USA) were placed over a 2mg/mL collagen solution prepared from calf skin-derived Type I collagen to achieve self-assembly of collagen fibrils. Grids were treated with 6.5% PA and divided into two groups. One group was floated over a remineralization solution containing 20mM HEPES, 2.25mM CaCl-2HO, 1.35mM KHPO4, 3.08mM NaN3 and 130mM KCl and the other group was over a CPP-ACP solution (Tooth mousse 1:100 dilution with deionized water). The floating samples were kept in a 37°C and 100% humidity chamber. Grids were taken out at selected time durations (24h, 48h and 72h for mineralization solution/24h for CPP-ACP) and observed under TEM without staining. Selected area electron diffractions (SAEDs) were performed at 110kV.
Following treatment of demineralized dentine collagen matrix with PA, the size and number of interfibrillar spaces were reduced. The collagen fibrils aggregated together with a reduction in porosity. A characteristic banding pattern of collagen fibrils was observed under TEM. Treatment of PA-biomodified collagen fibrils with remineralization solution increased mineral aggregation along its long axis, when compared to the control group. Furthermore, treatment of PA-biomodified collagen fibrils with CPP-ACP solution enhanced mineral uptake and deposition as well as initiated apatite formation within 24h.
Proanthocyanidin alters the ultrastructure of demineralized dentine collagen matrix. The PA-biomodified collagen matrix promotes remineralization.
原花青素（PA）是一种天然的胶原蛋白交联剂，已被用于牙本质基质生物修饰以进行修复和预防治疗。本研究评估了 PA 与胶原蛋白相互作用后的胶原超微结构。此外，还观察了 PA 修饰的胶原蛋白基质的矿化情况。
从 10 颗新鲜拔出的健康人磨牙中切取 0.5mm×1.7mm×7mm 的小梁，用于场发射扫描电子显微镜（FESEM）和透射电子显微镜（TEM）下的 PA 和牙本质基质的超微结构评估。用于 TEM 的标本完全脱矿，并根据 PA 处理分为三组：去离子水、2%PA 和 6.5%PA。标本固定、脱水、切片，用 TEM 检查。用于 FESEM 的标本用 EDTA 轻度调理，并同样分为三组，用 FESEM 观察。从小牛皮肤中提取的 I 型胶原蛋白用于分析用 6.5%PA 处理后的矿物质相互作用。将 Formvar 和碳涂覆的 400 目 Ni 网格（EMS，Hatfiels，PA，USA）置于从小牛皮肤衍生的 I 型胶原蛋白制备的 2mg/ml 胶原蛋白溶液上，以实现胶原蛋白纤维的自组装。网格用 6.5%PA 处理，并分为两组。一组在含有 20mM HEPES、2.25mM CaCl-2HO、1.35mM KHPO4、3.08mM NaN3 和 130mM KCl 的再矿化溶液上漂浮，另一组在 CPP-ACP 溶液（牙慕斯 1：100 稀释去离子水）上漂浮。浮动样品保持在 37°C 和 100%湿度室中。在选定的时间段（24h、48h 和 72h 用于矿化溶液/24h 用于 CPP-ACP）从网格中取出，并在 TEM 下观察，无需染色。在 110kV 下进行选区电子衍射（SAED）。
在用 PA 处理脱矿牙本质胶原蛋白基质后，纤维间空间的大小和数量减少。胶原纤维聚集在一起，孔隙率降低。TEM 下观察到胶原纤维的特征带纹模式。与对照组相比，用再矿化溶液处理 PA 修饰的胶原蛋白纤维增加了其长轴上的矿物质聚集。此外，用 CPP-ACP 溶液处理 PA 修饰的胶原蛋白纤维在 24 小时内增强了矿物质的摄取和沉积，并在其中引发了磷灰石的形成。
原花青素改变了脱矿牙本质胶原蛋白基质的超微结构。PA 修饰的胶原蛋白基质促进再矿化。
Zotero 插件