Fig. 1: Longitudinal intravital multiphoton imaging of calvarial bone healing after lesion injury.

a Schematic of a chronic cranial window for intravital imaging of calvarial bone repair. A drill hole lesion is inserted in the parietal calvarial bone. Coverslip and titanium fixation ring are mounted over the lesion. The lesion area of the is visualized using multiphoton laser microscopy. Modified from Stewen J, Bixel MG. Intravital Imaging of Blood Flow and HSPC Homing in Bone Marrow Microvessels. Methods Mol Biol. 2019;2017:109-121⁸⁴ © 2019 Springer Science+Business Media, LLC, part of Springer Nature. b Schematic showing a drill hole lesion the parietal calvarial bone. c Longitudinal intravital multiphoton microscopy showing dynamic sprouting and remodeling of GFP⁺ (green) microvasculature and regenerating SHG⁺ (blue) bone matrix in Flk1-GFP⁺ reporter mice after calvarial bone injury. Overview images show maximum intensity projections of Flk1-GFP⁺ microvasculature, injured SHG⁺ calvarial bone (cb) and newly formed SHG⁺ bone matrix (nb). Zoom-in views show early Flk1-GFP⁺ sprouts (white arrow heads) at PLD6, Flk1-GFP⁺ microvessels aligning the injured calvarial bone (top) and early sprouts (yellow arrow head, bottom) reaching over the outer bone edge at PLD9, remodeled Flk1-GFP⁺ microvessels connecting to periosteal vessels (yellow arrow heads) at PLD12, and matured Flk1-GFP⁺ microvessels enclosed by new SHG⁺ bone matrix at PLD30. d Intravital multiphoton microscopy showing SHG⁺ fibers of new bone (nb) originating from the injured calvarial bone (cb) in close association with Flk1-GFP⁺ blood vessels at PLD15. Arrow heads point to the front of the growing bone. e Histogram showing new microvasculature and new bone in the lesion (in % of total lesion area). Data are presented as mean values ± SD with n = 5 (for PLD0-PLD21), n = 4 (for PLD24-33) and n = 3 (for PLD36-42) biologically independent animals. Source data are provided as a Source Data file. f Schematic showing various stages of calvarial bone repair. New vascular sprouts (sv) originate from the meningeal vasculature (mv) and form an early vascular plexus (vp) that connects with the periosteal vasculature (pv). Formation of new bone (nb) initiates at the injured calvarial bone surface (cb) and progressively grows into the vascularized lesion gradually remodeling or enclosing blood vessels.