Figure 2.

Mineralization is directly linked to osteocytes (Ocys) but not osteoblasts (Obs) (Representative data from 2-5 animals for each assay). (a). Von Kossa staining showed newly formed mineral particles surrounding an Ocy site, far away from the Ob layers in the periosteum (n = 5); (b). Backscattered SEM (BSEM) image of the same bone exhibited (n = 3): lack of minerals in osteoblast layer; low mineral levels in the osteoid-layer, as indicated by calcium content (Ca²⁺) of 0.25±0.02% by EDX; a poorly formed mineral layer with individual sphere-like mineral structures (Ca²⁺, 18±0.5%); a mature mineral-layer (Ca²⁺, 24.6±0.3%), in which Ocys were surrounded by dense mineralized rings, suggesting that mineralization is initiated from Ocys and not Obs. (c). The acid-etched SEM images of the osteoid layer in a 2-month-old mouse femur displayed numerous Ocy dendrites surrounded by many empty spaces created by acid removal of mineralized matrix. An Ob is buried in the bone marrow filled with resin (n = 4). (d). Acid-etched SEM images of murine calvaria revealed an absence of minerals and very few Ocy dendrites in newborns (upper left), poor mineralization, and inadequate Ocy maturation in 10-day-old mice (upper right), whereas calvaria at 3 weeks (lower left) and 5 months of ages (lower right) display resistance to acid-etching due to enhanced matrix mineralization (n = 1). (e). The acid-etched SEM image of a 2-month-old mouse long bone showed poor mineralization in the early layer, which progresses to well-established mineralization and Ocy maturity (spindle-shaped cells with reduced volume) (n = 4). (f). The acid-etched SEM image of a 2-month-old mouse mandible displayed “thin mineral sheets” directly linked to Ocy dendrites (n = 1). (g) VolumeScope 3D imaging data showed immature dendrites (thick and short) of Ocys close to bone surfaces and mature dendrites (thin and long) of Ocys in deep bone areas (n =2).