PDF(Pubmed)
Abstract：
The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.
摘要：
基于细胞的牙髓再生过程中的新生血管形成过程难以研究。在这里，我们开发了一种模拟根管空间的管模型，并允许在体外直接可视化血管化过程。内皮样细胞（ECs）来源于引导人牙髓干细胞（DPSC）表达内皮细胞标志物CD144，vWF，使用VEGFR1和VEGFR2。人微血管内皮细胞(hMVEC)用作阳性对照。DPSC-EC在基质胶上形成类似于hMVEC的小管。将细胞在纤维蛋白原/凝血酶或小鼠血液中混合,并接种到96孔板的孔中或注射到锥形塑料管(长度为14mm,尖端开口的直径为1或2mm)中,其中较大的末端用MTA密封以模拟根管空间。将孔或管中的细胞/凝胶在体外孵育不同时间,并在显微镜下观察形态学变化。然后将样品固定并处理用于组织学分析以确定血管形成。细胞接种后1至3d，在培养物中观察到血管样网络。将96孔板中的细胞/凝胶维持长达25天。96孔板或试管中的hMVEC和DPSC-EC均显示细胞内液泡形成。一些细胞显示合并的大液泡，表明管腔形成。还观察到类似血管的管状结构。除了冠状部分中的一些样品(顶端直径Imm)之外,细胞在整个管中看起来是健康的。组织学分析还显示整个具有血管样结构的管样品中的浆状软组织。hMVEC比DPSC-EC形成更大的血管腔尺寸，而后者倾向于具有更多的腔和管状结构计数。我们得出结论，DPSC-EC可以形成血管结构，并在体外维持在3维纤维蛋白凝胶系统中。管模型似乎是模拟根管空间的适当且简单的系统，用于血管形成和牙髓再生研究。
