Investigation of Postnatal Craniofacial Bone Development with Tissue Clearing-Based Three-Dimensional Imaging

Traditional two-dimensional histological sections and microcomputed tomography remain to be the major tools for studying craniofacial bones despite the complicated spatial organization of craniofacial organs. Recently, our laboratory developed the Poly(Ethylene Glycol) Associated Solvent System (PEGASOS) tissue clearing method, which can efficiently render hard tissues, including bones and teeth fully transparent without losing endogenous fluorescent signals. Complete tissue transparency enables us to acquire three-dimensional (3D) images of craniofacial bone vasculature, osteogenesis utilizing various labeling strategies, thus to investigate the spatial relationship among different tissues during postnatal craniofacial development. We found out that during the early stage of postnatal development, craniofacial osteogenesis occurs throughout the entire craniofacial bones, including the periosteum, dura, bone marrow, and suture. After 3-4 weeks, craniofacial osteogenesis is gradually restricted to the suture region and remaining bone marrow space. Similarly, craniofacial bone vasculature gradually restricts to the suture region. Osteogenesis is spatially associated with vasculature during the entire postnatal development. Importantly, we demonstrated that in adult calvarial bones, Gli1+ mesenchymal stem cells were also spatially associated with the vasculature. These findings indicate that craniofacial bones share similar osteogenesis mechanism as the long bone despite their distinct osteogenic mechanisms. In addition, the PEGASOS tissue clearing method-based 3D imaging technique is a useful new tool for craniofacial research.