Osteocytes Play a Key Role in the Formation and Maintenance of Mineralized Bone

The theory that osteoblasts (Obs) regulate mineralized bone formation is a cornerstone of bone biology. However, the role of osteocytes (Ocy), the most abundant bone cell, in bone formation and remodeling still remains speculative. Thus, we tested if the Ocy does, in fact, have a role in this vital function. Using SEM and FITC (a fluorescent dye filling in Oycs and canalicular networks), we documented a close association between Ocy maturation and the gaining of bone mineral content. Quantitative data revealed that osteocytes reduce their volumes by 50% as they mature, and the volume vacated by the reduced cell body in the matrix was replaced by mineral overtime. Confocal microscopic examination of DAPI and double labeling (calcein/alizarin red injection to label newly formed matrix) in bone revealed that not only new bone/mineral is deposited at the mineralization front of bone edges, but also surrounding Ocy lacuna and along canalicular networks, indicating direct matrix synthesis by osteocytes. Access to mineral source is critical in order for the Ocy to play such a vital and direct role in formation of mineralized bone. Yet, it is widely believed that bone possesses limited number of blood vessels. In our study using high resolution acid‐etching SEM and confocal imaging, we demonstrated high vessel density in the bone and particularly Ocy maintained very rich contact with vessels through their dendrites. Moreover, a dye injection into the rabbit abdominal aorta revealed dye appearance in the Ocy bodies and their canalicular networks in < 2 minutes, indicating a rapid exchange and delivery of mineral between capillaries and Ocys. The vital and pathological role of osteocytes in mineralization was further demonstrated in Dmp1 mice, where Obs failed to form Ocys and b‐catenin levels are >10‐fold higher than WT, leading to an osteomalacia bone phenotype. Moreover, in the offspring of mice obtained by crossing Dmp1 ‐Cre and Catnb Δex3 mice, the elevated b‐catenin levels recaptured an osteomalacia phenotype similar to Dmp1 KO mice except without the FGF‐23 abnormalities. Currently, imbalances of Obs and osteoclasts are implicated in osteoporosis, although neither cell is embedded in bone matrix where Ocys likely play a key pathological role. To study the potential role of Ocys in maintaining balanced bone homeostasis, we evaluated patients with osteoporosis and ovariectomized (OVX) rats demonstrating that distinct Ocy changes from a spindle to a round shape with >50% increase in Ocy volumes (indicating mineral loss) and reductions of Ocy numbers and canaliculus. Unexpectedly, there were also sharp reduction of blood vessels in Ovx rat bones. Administration of Sclerostin antibody to Ovx rats reversed these changes, leading to recovered bone formation. Our studies suggest that the Ocys are the key cells building and maintaining bone mineralization, as such, Ocy may serve as a novel potential therapeutic target in treatment of bone diseases such as osteomalacia and osteoporosis. Support or Funding Information Supported by NIH R01 DE018486 and R01DE025014 to Dr. Jian. Q. Feng 1 Bone mineralization is directly linked to osteocyte(a). Acid‐etched SEM images show mouse calvaria osteocyte maturation from newborn to 5 months of age. As it matured, the osteocyte volume was reduced by nearly 50%, but its surface area increased by >50%, due to an increased number of dendrites, which led to well‐defined mineral filling the decreased cell volume, (b). Double labeling + Dapi staining revealed by confocal microscopy shows mineral deposition in the matrix surrounding the Ocys, and at the mineralization front surrounding the Ocy canaliculi.2Double labeling revealed a dual mineralization pattern in osteon (dynamic, including active, inactive, and newly formed) and non‐osteon area: (synchronized) within dog cortical bone (a , left); which is confirmed by the backscatter SEM imaging technique on the same bone slide (a, right ) ; H&E stains showed increased micro‐cracks in a 98‐yr‐old osteoporosis patient compared to a 30‐yr‐old male cortical bone (b) ; The confocal FITC stained images revealed much enlarged Haversian canals with reduced Ocy number in each osteon in 98‐yr‐old bone (c) ; and the backscatter SEM image revealed highly active remodeling in the same 30‐yr‐old male cortical bone, as reflected by a mixed pattern of light shade (less active) and dark (active, dominant) (d, left ) in comparison with the 98‐yr‐old bone, in which most osteons were in light shade, indicating a lack of bone remodeling (d, right ) .