Multiscale 3D Whole Joint Cellular and Molecular Mapping Reveals Disease‐Specific Neurovascular Plasticity Underlying the Structure‐Pain Relationship

Abstract Understanding musculoskeletal joints from a 3D multiscale perspective, from molecular to anatomical levels, is essential for resolving the confounding relationships between structure and pain, elucidating mechanisms regulating joint health and diseases, and developing new treatment strategies. Here, a musculoskeletal joint immunostaining and clearing (MUSIC) method specifically developed to overcome key challenges of immunostaining and optical clearing of intact joints are introduced. Coupled with large‐field light sheet microscopy, this approach achieves 3D high‐resolution, microscale neurovascular mapping within the context of whole‐joint anatomy without the need for image coregistration across various joints, including temporomandibular joints, knees, and spines, and multiple species, including mouse, rat, and pig. These results reveal 3D heterogeneous neurovascular distributions and previously uncharacterized neurovascular pathways within joints. Using two complementary models of joint disease, degeneration and injury, disease‐specific microscale neurovascular alterations are identified. These findings extend beyond conventional macroscale assessments of joint morphology and provide a framework to link structural changes with pain. Importantly, our results show that the relationship between joint structure and pain is not universal but disease‐dependent, underscoring distinct pain mechanisms in different disease contexts. This platform offers a powerful tool for multiscale 3D analysis, advancing understanding of joint pathophysiology and intricate interplay among joint tissues.