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

使用定量 3D 光片显微镜评估组织工程颅面骨移植物的血管表型

• 批准号: 10064965
• 负责人: Alexandra Rindone
• 金额: $ 4.63万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064965
• 关键词: 3-Dimensional; 3D Print; Adipose tissue; Adult; Area; Autologous Transplantation; Blood Vessels; Bone Development; Bone Growth; Bone Regeneration; Bone Tissue; Bone Transplantation; Caliber; Calvaria; Cells; Color; Complex; Craniofacial Abnormalities; Data; Defect; Development; Fostering; Gold; Growth Factor; Homeostasis; Image; Imaging Techniques; Immunodeficient Mouse; Implant; Injury; Knowledge; Light; Maintenance; Methods; Microscopy; Morbidity - disease rate; Mus; Natural regeneration; Optics; Osteogenesis; Outcome; PECAM1 gene; Patients; Phenotype; Protocols documentation; Publishing; Reporting; Research; Resolution; Scientist; Site; Structure; Techniques; Technology; Thick; Three-Dimensional Imaging; Tissue Engineering; Tissue imaging; Work; angiogenesis; bone; bone cell; bone engineering; bone healing; clinical application; craniofacial; craniofacial bone; craniofacial complex; craniofacial tissue; endothelial-specific sialomucin; fluorescence imaging; high risk; image processing; imaging modality; implantation; improved; in vivo; insight; neovascularization; neovasculature; novel; osteogenic; osteoprogenitor cell; polycaprolactone; quantitative imaging; scaffold; spatial relationship; standard care; stem

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.

全世界每年有超过200万患者接受骨移植治疗，以填补关键尺寸的颅面缺损。 由于自体移植，目前的金标准治疗，引入了高风险的供区发病率， 组织工程骨移植物（TEBG）具有显著的几何限制， 有效再生几何形状复杂的血管化颅面组织的潜力。然而，在这方面， TEBG的翻译应用一直受到有关关系的重大知识差距的限制， 血管结构和骨再生之间的联系本研究的总体目标是开发和 实施新的定量3D成像技术，从根本上了解新技术是如何 脉管系统影响TEBG中的骨形成。虽然已经开发了促进血管生成的策略， 在再生骨中，已发表的报告表明，TEBG内形成的脉管系统的量 与再生骨的数量和质量无关。在原生骨中，微环境相互作用 血管和骨细胞之间的相互作用对骨的生长和维持至关重要。特别是，科学家们 最近鉴定了一种CD 31和内粘蛋白表达高的血管表型，称为“H型”， 与骨祖细胞密切相关，并且是骨稳态所必需的。要确定类型是否 H血管与TEBG中的骨再生有关，这项拟议的工作将整合整体安装 用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TEBG（> mm 3 体积）在3D中以单细胞分辨率。结合这些技术将实现前所未有的3D定量 表征血管表型和血管-骨细胞关系。血管和骨形成将是 用先前研究的用于治疗4-mm鼠临界尺寸缺损的TEBG进行评价。在目标1中， 用于整体包埋免疫染色、透明化和光片成像天然鼠颅骨的方案， 将开发植入的颅骨TEBG，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Aim 2中，这种3D定量成像技术将 用于确定特定血管表型是否与骨形成增强相关， TEBG。首先，血管和骨形成将在已知产生两个不同水平的骨的TEBG中进行比较 为了确定H型血管的发育是否有助于骨愈合，需要观察H型血管的再生。第二、 血管生成和成骨生长因子对TEBG中血管化骨形成的影响将被 评价以进一步阐明血管表型与骨再生的关系。这些发现将 使有针对性的策略，以促进血管化骨再生的发展。这项研究将 对开发改善颅面神经衰弱患者的治疗方法具有实质性的积极影响 受伤