Project Summary/Abstract: Intramembranous bone regeneration plays an important role in several orthopaedic and dental procedures such as implant placement, fracture healing, and distraction osteogenesis. Prior studies have examined different processes during intramembranous bone regeneration, such as inflammation, angiogenesis, and bone regeneration. However, an under explored aspect of intramembranous bone regeneration is pain sensing neurons, such as their distribution and morphological changes over the course of intramembranous bone regeneration. A rigorous examination of pain sensing neurons can be achieved by tissue clearing and light sheet microscopy, which allows three-dimensional visualization and quantitation of tissues. However, implementing these approaches have been met with significant technical challenges in musculoskeletal tissues, such as significant autofluorescence and insufficient depth of field due to collagen-rich extracellular matrix. Our preliminary data demonstrate several approaches that overcome these challenges, but further refinement and comparison studies to conventional histological approaches are needed. Therefore, we are proposing this supplement that aims to develop musculoskeletal tissue specific tissue clearing light sheet microscopy that will examine periostin expressing cells and pain sensing neurons during intramembranous bone regeneration. The supplement will address the strategic goal “Goal B: Expand rheumatic, skin, and musculoskeletal disease pain research capacity (cohorts, technologies or data) beyond current research base/collaborators” with a specific scientific area in novel pain imaging technologies for musculoskeletal tissues.