Quantitative 3D Imaging of the Cranial Microvascular Environment at Single-Cell Resolution

Vascularization is critical for skull development, maintenance, and healing. Yet, there remains a significant knowledge gap in the relationship of blood vessels to cranial skeletal progenitors during these processes. Here, we introduce a quantitative 3D imaging platform to enable visualization and analysis of high-resolution datasets (>100 GB) throughout the entire murine calvarium. Using this technique, we provide single-cell resolution 3D maps of vessel phenotypes and skeletal progenitors in the frontoparietal cranial bones. Through these high-resolution datasets, we demonstrate that CD31hiEmcnhi vessels are spatially correlated with both Osterix+ and Gli1+ skeletal progenitors during postnatal growth, healing, and stimulated remodeling, and are concentrated at transcortical canals and osteogenic fronts. Interestingly, this relationship was weakened in mice with a conditional knockout of PDGF-BB in TRAP+ osteoclasts, suggesting a potential role for osteoclasts in maintaining the native cranial microvascular environment. Our findings provide a foundational framework for understanding how blood vessels and skeletal progenitors spatially interact in cranial bone, and will enable more targeted studies into the mechanisms of skull disease pathologies and treatments. Additionally, our technique can be readily adapted to study numerous cell types and investigate other elusive phenomena in cranial bone biology.

血管化对于颅骨的发育、稳态维持与损伤修复至关重要。然而，在上述过程中，血管与颅骨骨骼祖细胞之间的关联仍存在显著的认知空白。本研究构建了一套定量三维成像平台，可实现对整个小鼠颅盖骨的高分辨率数据集（单数据集规模超100 GB）的可视化与全流程分析。借助该技术，我们获得了额顶颅骨中血管表型与骨骼祖细胞的单细胞分辨率三维图谱。通过这些高分辨率数据集，我们证实：在出生后发育、损伤修复及诱导性重塑过程中，CD31hiEmcnhi血管（CD31高表达、内皮黏蛋白Emcn高表达的血管表型）与Osterix阳性（Osterix+）及Gli1阳性（Gli1+）的骨骼祖细胞均存在空间相关性，且这类血管集中分布于穿皮质管道与成骨前沿。值得注意的是，在抗酒石酸酸性磷酸酶阳性（TRAP+）破骨细胞中条件性敲除血小板衍生生长因子BB (PDGF-BB) 的小鼠模型中，这种关联出现显著减弱，这提示破骨细胞在维持颅骨原生微血管微环境中可能发挥潜在作用。我们的研究结果为理解血管与骨骼祖细胞在颅骨内的空间相互作用提供了基础框架，也将为针对颅骨疾病病理机制与治疗方案的精准研究提供重要支撑。此外，本研究建立的技术可便捷地适配多种细胞类型的研究，用于探索颅骨生物学中诸多尚未阐明的现象。