The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO₂), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

颅骨骨缺损的愈合是一个紧迫的临床问题，涉及成骨生态位内血管生成和成骨之间的动态相互作用。尽管成骨生态位中的结构和功能血管重塑（即血管生成进化）是氧合、炎症和骨前体细胞的关键调节剂，但大多数临床和临床前研究仅限于表征脉管系统和骨的结构变化。因此，我们开发了一种新的多模态成像方法，首次能够对临床前颅骨缺损中重塑的脉管系统中的多个体内功能参数及其对从头成骨的影响进行纵向（即超过四个星期）和动态表征模型。我们采用多波长固有光信号（IOS）成像来评估微血管重塑、血管内氧合（SO ₂）和成骨作用。激光散斑对比（LSC）成像评估血流和血管成熟度的伴随变化；和微计算机断层扫描（μCT）来验证颅骨的体积变化。我们发现血管生成进化与颅骨再生紧密相关，并且对应于骨愈合的不同阶段，例如损伤、血肿形成、血运重建和重塑。前三个阶段发生在骨愈合的最初两周内，其特征是体内血管形态、血流、氧合和成熟度发生显着变化。总体而言，血管生成进化先于成骨，而成骨仅在骨愈合结束时（即四个星期）趋于稳定。总的来说，这些数据表明血管生成进化在成骨中的关键作用。我们相信，这种多模态成像方法有可能为更有效的组织工程颅骨缺损治疗的设计提供信息。