Fig. 2: Vascular and bone connectivity during calvarial bone healing.

a Intravital multiphoton microscopy showing newly formed calvarial bone matrix and regenerating microvasculature at PLD14. Maximum intensity projections (left) of SHG⁺ (white) injured calvarial bone (cb), new SHG⁺ bone (nb) and Flk1-GFP⁺ (green). White arrowheads indicate the growing bone edge. Separating gap (yellow arrowhead) between newly formed and pre-existing calvarial bone. Note that Flk1-GFP⁺ microvessels are aligned with the surface of calvarial bone without connections to vessels inside the calcified tissue. Single planes on the right. Corresponding 3D representation is shown in Supplementary Movie 1. b Cross section showing Flk1-GFP⁺ (green) microvasculature and SHG⁺ (white) calvarial bone at PLD14 (maximum intensity projection). Arrow points to sinusoidal capillaries in the adjacent BM cavity without vascular connections to the regenerating vasculature (arrowheads). c Cross sections showing Flk1-GFP⁺ (green) microvasculature and SHG⁺ (blue) calvarial bone at PLD9, PLD14 and PLD21, and control without lesion. Arrowheads point to gaps separating injured calvarial bone (cb) and new bone (nb). d Overview cross sections showing SHG⁺ (white) calvarial bone with lesions at PLD9, PLD14 and PLD21. Lesions were cut centrally (left) and peripherally (right). Arrowheads point to gaps separating calvarial (cb) and new bone (nb). Arrows indicate new BM cavities. e Schematic cross sections at three stages of calvarial bone healing. Sprouting vessels (sv) derived from meningeal vessels (mv) form an early vascular plexus. Sprouts grow outward and connect to periosteal vessels (pv). The newly formed microvasculature remodels and adapts to the expanding new bone (nb). Progressive ossification results in lesion closure and formation of early BM cavities. cb: calvarial bone. Reproducibility was ensured by n = 3 or more biologically independent experiments.