The study investigates the relationship between angiogenesis and osteogenesis during calvarial bone regeneration using advanced longitudinal intravital multiphoton microscopy. Key findings include: 1. **Vascular Regeneration and Bone Healing**: Early vascular sprouting is not directly coupled to osteoprogenitor invasion. Vessels grow inside the lesion without association with osteoprogenitors, while osteoprogenitors from the periosteum give rise to bone-forming osteoblasts at the injured edge. 2. **Osteogenic Cell Invasion**: Osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, advancing regenerative ossification. 3. **Dynamic Blood Flow**: Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate growing bone. 4. **Single-Cell Profiling**: Single-cell RNA sequencing (scRNA-seq) reveals mesenchymal stromal cell heterogeneity and expression of genes related to angiogenesis and hypoxia, reflecting the heterogeneity of bone stromal cells. 5. **Clinical Implications**: The findings have implications for bone regeneration and bioengineering approaches, highlighting the need to understand the complex interactions between angiogenesis and osteogenesis in bone repair. 6. **Notch and VEGF Signaling**: Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification process. The study provides a detailed understanding of the dynamic cellular and molecular events during calvarial bone regeneration, emphasizing the uncoupling of angiogenesis and osteogenesis.The study investigates the relationship between angiogenesis and osteogenesis during calvarial bone regeneration using advanced longitudinal intravital multiphoton microscopy. Key findings include: 1. **Vascular Regeneration and Bone Healing**: Early vascular sprouting is not directly coupled to osteoprogenitor invasion. Vessels grow inside the lesion without association with osteoprogenitors, while osteoprogenitors from the periosteum give rise to bone-forming osteoblasts at the injured edge. 2. **Osteogenic Cell Invasion**: Osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, advancing regenerative ossification. 3. **Dynamic Blood Flow**: Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate growing bone. 4. **Single-Cell Profiling**: Single-cell RNA sequencing (scRNA-seq) reveals mesenchymal stromal cell heterogeneity and expression of genes related to angiogenesis and hypoxia, reflecting the heterogeneity of bone stromal cells. 5. **Clinical Implications**: The findings have implications for bone regeneration and bioengineering approaches, highlighting the need to understand the complex interactions between angiogenesis and osteogenesis in bone repair. 6. **Notch and VEGF Signaling**: Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification process. The study provides a detailed understanding of the dynamic cellular and molecular events during calvarial bone regeneration, emphasizing the uncoupling of angiogenesis and osteogenesis.