Evaluating blood vessel phenotype in tissue-engineered craniofacial bone grafts using quantitative 3D light-sheet microscopy

全球每年有200多万患者接受骨移植治疗，以填补临界大小的颅面缺损。 由于自体移植是目前的黄金标准治疗，引入了供区发病率的高风险，并 显著的几何约束，组织工程骨移植(TEBGs)提供了一种有前途的替代方案 有效地再生几何复杂的、带血管的头面部组织的潜力。然而， TEBGs的翻译应用一直受到关于该关系的重大知识差距的限制 血管结构和骨再生之间的关系。这项研究的总体目标是发展和 实施新的定量3D成像技术，以获得对近地天体如何 血管系统影响TEBG中的骨形成。虽然已经开发了促进血管生成的策略 在再生骨骼方面，已发表的报告表明，在TEBG中形成的血管数量 与再生骨的数量或质量无相关性。在天然骨骼中，微环境相互作用 血管和骨细胞之间的联系对于骨骼的生长和维持是必不可少的。特别是，科学家们已经 最近发现了一种高CD31和内粘蛋白表达的血管表型，称为“H型”，即 与骨祖细胞密切相关，是维持骨骼内环境稳定所必需的。要确定是否键入 在TEBG中，H血管与再生骨有关，这项拟议的工作将整合整体安装 用一种新的光学透明方法和光片显微镜进行免疫染色以成像整个TEBGs(&gt；mm3 体积)，以单单元分辨率的3D图像。结合这些技术将实现史无前例的3D定量 血管表型和血管-骨细胞关系的特征。血管和骨的形成将是 用以前研究过的用于治疗4 mm小鼠临界大小缺陷的TEBGs进行评估。在目标1中， 天然小鼠颅骨整体免疫染色、透明和光片成像方案 植入的颅骨TEBG将被开发出来，以实现血管表型的3D定量表征 以及血管和骨细胞之间的空间关系。在目标2中，这种3D定量成像技术将 用于确定特定的血管表型是否与增强的骨形成相关 TEBGS。首先，血管和骨的形成将在已知的两个不同水平的骨的TEBG中进行比较。 以确定H型血管发育是否有助于骨愈合。第二, 血管生成因子和成骨生长因子对TEBGs血管化骨形成的影响 评估以进一步阐明血管表型与骨再生的关系。这些发现将 能够制定有针对性的策略，促进血管化的骨再生。这项研究将 对改进颅面部衰弱患者的治疗方法产生实质性的积极影响 受伤。

Over 2 million patients worldwide are treated annually with bone grafts to fill critical-sized craniofacial defects. Since autografts, the current gold standard treatment, introduce a high risk of donor site morbidity and have significant geometric constraints, tissue-engineered bone grafts (TEBGs) present a promising alternative with the potential to effectively regenerate geometrically complex, vascularized craniofacial tissues. However, translational application of TEBGs has been limited by significant knowledge gaps regarding the relationship between vascular structure and bone regeneration. The overall objective of this study is to develop and implement novel quantitative 3D imaging techniques to gain a fundamental understanding of how neo- vasculature impacts bone formation in TEBGs. While strategies have been developed to promote angiogenesis in regenerating bone, published reports demonstrate that the amount of vasculature formed within TEBGs has no correlation with the quantity or quality of regenerated bone. In native bone, microenvironmental interactions between vessels and bone cells are essential to bone growth and maintenance. In particular, scientists have recently identified a vessel phenotype high in CD31 and endomucin expression, termed “Type H”, that is intimately associated with osteoprogenitors, and necessary for bone homeostasis. To determine whether Type H vessels are related to regenerating bone in TEBGs, this proposed work will integrate whole-mount immunostaining with a novel optical clearing method and light-sheet microscopy to image entire TEBGs (>mm3 volume) in 3D at single-cell resolution. Combining these technologies will enable unprecedented 3D quantitative characterization of vessel phenotypes and vessel-bone cell relationships. Vessel and bone formation will be evaluated with previously investigated TEBGs used to treat 4-mm murine critical-sized defects. In Aim 1, protocols for whole-mount immunostaining, clearing, and light-sheet imaging native murine calvaria and implanted calvarial TEBGs will be developed to enable 3D quantitative characterization of vessel phenotypes and spatial relationships between vessels and bone cells. In Aim 2, this 3D quantitative imaging technique will be applied to determine whether specific vessel phenotypes are correlated with enhanced bone formation in TEBGs. First, vessel and bone formation will be compared in TEBGs known to yield two distinct levels of bone regeneration in order to determine whether Type H vessel development contributes to bone healing. Second, the effects of angiogenic and osteogenic growth factors on vascularized bone formation in TEBGs will be evaluated to further elucidate the relationship of vessel phenotypes to bone regeneration. These findings will enable the development of targeted strategies to promote vascularized bone regeneration. This research will have a substantial positive impact on developing improved treatments for patients with debilitating craniofacial injuries.