Biomaterials Research Unit, Faculty of Dentistry, University of Sydney, Sydney Dental Hospital, 2 Chalmers Street, Surry Hills, NSW 2010, Australia.
Micron. 2012 Feb;43(2-3):229-36. doi: 10.1016/j.micron.2011.08.003. Epub 2011 Aug 12.
Dentin is a mineralized dental tissue underlying the outer enamel that has a peculiar micro morphology. It is composed of micrometer sized tubules that are surrounded by a highly mineralized structure, called peritubular dentin (PTD), and embedded in a collagen-rich matrix, named intertubular dentin. The PTD has been thought to be composed of a highly mineralized collagen-free organic matrix with unknown composition. Here we tested the hypothesis that proteoglycans and glycosaminoglycans, two important organic structural features found in dentin, are key participants in the microstructure and composition of the PTD. To test this hypothesis dentin blocks were demineralized with 10 vol% citric acid for 2 min and either digested with 1mg/ml TPCK-treated trypsin with 0.2 ammonium bicarbonate at pH 7.9 (TRY) or 0.1 U/mL C-ABC with 50mM Tris, 60mM sodium acetate and 0.02% bovine serum albumin at pH 8.0 (C-ABC). TRY is known to cleave the protein core of dentin proteoglycans, whereas C-ABC is expected to selectively remove glycosaminoglycans. All specimens were digested for 48 h in 37°C, dehydrated in ascending grades of acetone, immersed in HMDS, platinum coated and imaged using an FE-SEM. Images of demineralized dentin revealed a meshwork of noncollagenous fibrils protruding towards the tubule lumen following removal of the peritubular mineral and confirmed the lack of collagen in the peritubular matrix. Further, images revealed that the peritubular organic network originates from a sheet-like membrane covering the entire visible length of tubule, called lamina limitans. Confirming our initial hypothesis, after the digestion with C-ABC the organic network appeared to vanish, while the lamina limitans was preserved. This suggests that glycosaminoglycans are the main component of the PTD organic network. Following digestion with TRY, both the organic network and the lamina limitans disappeared, thus suggesting that the lamina limitans may be primarily composed of proteoglycan protein cores. In summary, our results provide novel evidence that (1) PTD lacks collagen fibrils, (2) PTD contains an organic scaffold embedded with mineral and (3) the PTD organic matrix is manly composed of glycosaminoglycans, whereas the lamina limitans is primarily made of proteoglycans protein cores.
牙本质是一种矿化的牙齿组织，位于外层釉质下方，具有独特的微观形态。它由微米大小的小管组成，这些小管被高度矿化的结构包围，称为管周牙本质（PTD），并嵌入富含胶原蛋白的基质中，称为管间牙本质。一直以来，人们认为 PTD 由一种组成未知的高度矿化无胶原蛋白的有机基质组成。在这里，我们测试了这样一个假设，即糖胺聚糖和蛋白聚糖，牙本质中两种重要的有机结构特征，是 PTD 微观结构和组成的关键参与者。为了验证这一假设，用 10 体积%柠檬酸将牙本质块脱矿 2 分钟，然后用 1mg/ml TPCK 处理的胰蛋白酶（TRY）或 0.1U/ml C-ABC 在 pH8.0 下（含 0.2 氯化铵碳酸氢盐）进行消化。TRY 已知能切割牙本质蛋白聚糖的蛋白核心，而 C-ABC 预计能选择性地去除糖胺聚糖。所有标本在 37°C 下消化 48 小时，用递增浓度的丙酮脱水，浸入 HMDS 中，镀铂并用 FE-SEM 成像。脱矿牙本质的图像显示，在去除管周矿化物质后，向小管腔突出的非胶原蛋白纤维网格状结构证实了管周基质中缺乏胶原蛋白。此外，图像显示管周有机网络源自覆盖整个可见小管长度的片状膜，称为限界层。证实了我们最初的假设，在用 C-ABC 消化后，有机网络似乎消失了，而限界层得以保留。这表明糖胺聚糖是 PTD 有机网络的主要成分。在用 TRY 消化后，有机网络和限界层都消失了，这表明限界层可能主要由蛋白聚糖蛋白核心组成。总之，我们的结果提供了新的证据，证明（1）PTD 缺乏胶原纤维，（2）PTD 含有嵌入矿物质的有机支架，（3）PTD 有机基质主要由糖胺聚糖组成，而限界层主要由蛋白聚糖蛋白核心组成。
