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.