Three-dimensional visualization of nerves in bones of the human hand in osteoarthritis

Bone is innervated by nerves that have important roles in signaling bone pain, regulating bone homeostasis and remodeling, control of bone vasculature and hematopoiesis. However, a detailed knowledge of how nerves influence bone function is lacking, which has contributed to significant challenges for clinicians in successfully managing bone disease. In this study, we used tissue clearing and light-sheet microscopy to visualize and explore the 3D distribution of nerves in human bone with a view to better understand how nerves are distributed throughout different parts of the small bones of the hand. Human hand bones were obtained from patients undergoing excision due to advanced osteoarthritis. Bones were fixed and cleared using an optimized solvent-based clearing protocol. Nerves in the bone were immunolabeled using an antibody directed against a pan-neuronal marker (protein gene product 9.5). Samples were imaged using light-sheet microscopy (Ultramicroscope Blaze, Miltenyi Biotec). Three-dimensional visualization and analyses were performed with Imaris (Bitplane). There is extensive innervation of the small bones of the human hand. Immunolabeled nerves followed blood vessels into each bone via nutrient foramina and branched extensively within trabecular bone and marrow cavity spaces. Many axons had a typical corkscrew morphology associated with the innervation of the vasculature, and many terminated as free nerve endings, including in osteochondral channels beneath articular surfaces. Nerve entry points and their distribution through each of the different bones varied. There was evidence of a greater density of innervation in areas where cartilage had degenerated, relative to those where cartilage was intact. This is the first detailed 3D visualization of the neuroanatomical distribution of nerves in intact human bone. This approach to visualizing nerves in bone will be useful in informing the rational design of approaches to the management of a variety of skeletal pathologies, including pain management for osteoarthritis of the hand joints.