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.

中文翻译：

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用于牙髓再生研究的管模型中 DPSC-EC 的新生血管形成

基于细胞的牙髓再生过程中的新血管形成过程很难研究。在这里，我们开发了一个模拟根管空间并允许直接可视化体外血管化过程的管模型。使用源自引导人牙髓干细胞（DPSC）表达内皮细胞标记物 CD144、vWF、VEGFR1 和 VEGFR2 的内皮样细胞（EC）。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 14 mm，顶端开口直径 1 或 2 mm）中，较大端用 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变化。然后固定样品并进行组织学分析以确定血管形成。细胞接种后 1 至 3 天，在培养物中观察到血管样网络。 96 孔板中的细胞/凝胶可保存长达 25 天。组织学上，96孔板或管中的hMVEC和DPSC-EC均显示细胞内液泡形成。一些细胞显示出合并的大液泡，表明内腔化。还观察到类似血管的管状结构。除了冠状三分之一处的一些样本（顶端直径为 1 毫米）外，整个管中的细胞看起来都很健康。组织学分析还显示，整个管样品中存在纸浆样软组织，具有血管样结构。 hMVEC 形成的血管腔尺寸比 DPSC-EC 更大，而后者往往具有更多的管腔和管状结构计数。我们得出的结论是，DPSC-EC 可以在体外形成血管结构并维持在 3 维纤维蛋白凝胶系统中。管模型似乎是模拟根管空间以进行血管形成和牙髓再生研究的适当且简单的系统。