Isolation of Calcospherulites from the Mineralization Front of Bone

Abstract

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

Human bone mesoscale 3D structure revisited by plasma focused ion beam serial sectioning

“…These findings are supported by cryo electron microscopy studies, which observe the accumulation of mineral on the exterior of the fibril in in vitro conditions (Nudelman et al, 2013). Similar spherical particles 'calcospherulites' have been postulated to be related to the mineralization front only (Midura et al, 2008) and early bone mineralization from in vitro culture of MC3T3-E1 osteoblast cells show comparable spherical particles (mineralization foci), and also notes these in mouse calvarial bone with osteopontin closely associated with their margin (Addison et al, 2014).…”

Phosphorylated Proteins and Control over Apatite Nucleation, Crystal Growth, and Inhibition

“…It appears that the aggregates, in the in vivo milieu specifically accumulate the BSP and alkaline phosphatase components as they increase in size, and act together to nucleate the apatite within the BMF. In a recent publication271, Midura et al show that the mineralized BMF, called calcospherulites, seed the formation of apatite crystals within the BMF/calcospherulites, but also demonstrate that incubation of the calcospherulites in a collagen fibril hydrogel also seeds the mineralization of neighboring collagen fibrils “at distances well away from the observed calcospherulites.” Gorski et al272 have shown that enzymatic processing of the BAG-75 and DSP takes place within the BMF releasing 45-50 kDa fragments of each protein, When the enzymatic activity is inhibited, the BMF lose their ability to nucleate apatite crystal formation in the matrix. These are very interesting data which have implications for a system for delivery of, in the case of bone, BSP and other soluble factors to the mineralization front, where they may be released to bind to the collagen fibrils and initiate the collagen-related nucleation.…”

Biomineralization of bone: a fresh view of the roles of non-collagenous proteins

“…Midura et al (29, 30) recently isolated calcospherulites (from the mineralization front of tibial periosteum) and used them to induce the mineralization of type I collagen in vitro . Calcospherulites were identified as 0.5 um diameter particles which had taken up calcein within the 24 h following in vivo injection.…”