Tracking Strain‐Specific Morphogenesis and Angiogenesis of Murine Calvaria with Large‐Scale Optoacoustic and Ultrasound Microscopy

Skull bone development is a dynamic and well‐coordinated process playing a key role in maturation and maintenance of the bone marrow (BM), fracture healing, and progression of diseases such as osteoarthritis or osteoporosis. At present, dynamic transformation of the growing bone (osteogenesis) as well as its vascularization (angiogenesis) remain largely unexplored due to the lack of suitable in vivo imaging techniques capable of noninvasive visualization of the whole developing calvaria at capillary‐level resolution. We present a longitudinal study on skull bone development using ultrasound‐aided large‐scale optoacoustic microscopy (U‐LSOM). Skull bone morphogenesis and microvascular growth patterns were monitored in three common mouse strains (C57BL/6J, CD‐1, and Athymic Nude‐Foxn1nu) at the whole‐calvaria scale over a 3‐month period. Strain‐specific differences in skull development were revealed by quantitative analysis of bone and vessel parameters, indicating the coupling between angiogenesis and osteogenesis during skull bone growth in a minimally invasive and label‐free manner. The method further enabled identifying BM‐specific sinusoidal vessels, and superficial skull vessels penetrating into BM compartments. Our approach furnishes a new high‐throughput longitudinal in vivo imaging platform to study morphological and vascular skull alterations in health and disease, shedding light on the critical links between blood vessel formation, skull growth, and regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).