Regeneration of orofacial tissues is hampered by the lack of adequate vascular supply. Implantation of in vitro engineered, prevascularized constructs has emerged as a strategy to allow the rapid vascularization of the entire graft. Given the angiogenic properties of dental pulp stem cells, we hereby established a preclinical model of prevascularized constructs loaded with stem cells from human exfoliating deciduous teeth (SHED) in a 3-dimensional-printed material and provided a functional analysis of their in vivo angiogenesis, vascular perfusion, and permeability. Three different cell-loaded collagen hydrogels (SHED–human umbilical vein endothelial cell [HUVEC], HUVEC with SHED-conditioned medium, and SHED alone) were cast in polylactic acid (PLA) grids and ectopically implanted in athymic mice. At day 10, in vivo positron emission tomography (PETscan) revealed a significantly increased uptake of radiotracer targeting activated endothelial cells in the SHED-HUVEC group compared to the other groups. At day 30, ex vivo micro–computed tomography imaging confirmed that SHED-HUVEC constructs had a significantly increased vascular volume compared to the other ones. Injection of species-specific lectins analyzed by 2-photon microscopy demonstrated blood perfusion of the engineered human vessels in both prevascularized groups. However, in vivo quantification showed increased vessel density in the SHED-HUVEC group. In addition, coinjection of fluorescent lectin and dextran revealed that prevascularization with SHED prevented vascular leakage, demonstrating the active role of SHED in the maturation of human-engineered microvascular networks. This preclinical study introduces a novel PLA prevascularized and implantable construct, along with an array of imaging techniques, to validate the ability of SHED to promote functional human-engineered vessels, further highlighting the interest of SHED for orofacial tissue engineering. Furthermore, this study validates the use of PETscan for the early detection of in vivo angiogenesis, which may be applied in the clinic to monitor the performance of prevascularized grafts.

中文翻译：

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用于监测 SHED 支持的功能性工程船舶的多尺度成像


缺乏足够的血管供应阻碍了口面部组织的再生。体外工程预血管化构建体的植入已成为一种允许整个移植物快速血管化的策略。鉴于牙髓干细胞的血管生成特性，我们在此建立了一个预血管化构建体的临床前模型，该模型在 3 维打印材料中加载了来自人类脱落乳牙 (SHED) 的干细胞，并对其体内血管生成进行了功能分析，血管灌注和通透性。将三种不同的细胞负载胶原水凝胶（SHED-人脐静脉内皮细胞 [HUVEC]、含有 SHED 条件培养基的 HUVEC 和单独的 SHED）浇注在聚乳酸 (PLA) 网格中，并异位植入无胸腺小鼠体内。第 10 天时，体内正电子发射断层扫描 (PETscan) 显示，与其他组相比，SHED-HUVEC 组中针对活化内皮细胞的放射性示踪剂的摄取显着增加。第 30 天时，离体微计算机断层扫描成像证实，与其他构建体相比，SHED-HUVEC 构建体的血管体积显着增加。通过双光子显微镜分析的物种特异性凝集素注射证明了两个预血管化组中工程化人类血管的血液灌注。然而，体内定量显示 SHED-HUVEC 组的血管密度增加。此外，荧光凝集素和葡聚糖的共注射表明，SHED 的预血管化可防止血管渗漏，证明 SHED 在人体工程微血管网络成熟中的积极作用。 这项临床前研究引入了一种新型 PLA 预血管化和可植入结构，以及一系列成像技术，以验证 SHED 促进功能性人体工程血管的能力，进一步凸显 SHED 对口面部组织工程的兴趣。此外，本研究验证了 PETscan 在体内血管生成早期检测中的应用，可应用于临床监测预血管化移植物的性能。