Peritubular dentin, a highly mineralized, non-collagenous, component of dentin: isolation and capture by laser microdissection

We demonstrate the capability and technique to perform microdissection and isolation of select regions of untreated, mineralized dentin using laser capture. Dentin is a complex, non-homogeneous tissue comprised of a mineralized collagenous matrix (intertubular dentin [ITD]), odontoblastic processes (ODPs), a void space (tubules) that forms within the ITD left behind by the retraction of ODPs during dentin maturation, and a highly mineralized non-collagenous component that exists at the interface between the tubules and ITD known as peritubular dentin (PTD). PTD forms as the dentin matures. The ODPs retract toward the direction of the pulp; leaving very little PTD at either the DEJ or near the pulp. Statistical analysis of thin cross-sections of coronal bovine dentin imaged by light microscopy reveal that the area occupied by PTD >50%. To examine the nature of PTD and its relation to both the tubules and ITD, we devised a series of steps to carefully prepare sections of coronal bovine dentin so that areas of the dentin tissue could be cut and isolated for further analysis. We demonstrate that it is possible to selectively isolate targeted regions of dentin for analysis and that high resolution analysis of such sections can be performed using electron microscopy. Results show that the mineralized PTD has a different texture than mineralized ITD and that there is a distinct boundary between the PTD and the ITD. Selective isolation of mineralized tissue components for further analytical study opens the door for the investigation of similar enigmatic mineralized structures.

本研究展示了利用激光捕获（laser capture）技术对未处理的矿化牙本质的特定区域进行显微切割与分离的方法与能力。牙本质是一种复杂的非均质组织，由矿化胶原基质（管间牙本质，intertubular dentin, ITD）、成牙本质细胞突起（odontoblastic processes, ODPs）、牙本质成熟过程中ODPs回缩后在ITD内部形成的空隙（牙本质小管），以及位于牙本质小管与ITD界面处的高度矿化非胶原组分——管周牙本质（peritubular dentin, PTD）共同构成。管周牙本质随牙本质成熟过程而形成。成牙本质细胞突起向牙髓方向回缩，因此在牙釉质牙本质界（dentinoenamel junction, DEJ）或牙髓附近几乎不会形成管周牙本质。通过光学显微镜成像对牛冠部牙本质的薄切片进行统计分析后发现，管周牙本质所占面积超过50%。为探究管周牙本质的本质及其与牙本质小管和管间牙本质的关联，本研究设计了一系列步骤，以谨慎制备牛冠部牙本质切片，从而实现牙本质组织特定区域的切割与分离，用于后续分析。本研究证实，可选择性分离牙本质的目标区域用于分析，且可通过电子显微镜对此类切片开展高分辨率分析。研究结果显示，矿化的管周牙本质与矿化的管间牙本质具有不同的组织结构特征，且二者之间存在清晰的分界。对矿化组织组分进行选择性分离以开展后续分析研究，为探究其他类似的神秘矿化结构开辟了新路径。