Journal
BONE
Volume 41, Issue 6, Pages 1005-1016Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.bone.2007.08.036
Keywords
bone formation; mineralization front; calcospherulites; biomineralization; bone matrix proteins; type I collagen
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Funding
- NIAMS NIH HHS [AR052775, 1P30 AR-050953, AR045171, R01 AR045171-01A2, R01 AR052775-02, R01 AR045171, R01 AR052775, P30 AR050953] Funding Source: Medline
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Previous work has suggested that calcospherulites actively participate in the mineralization of developing and healing bone. This study sought to directly test this hypothesis by developing a method to isolate calcospherulites and analyzing their capacity to seed mineralization of fibrillar collagen. The periosteal surface of juvenile rat tibial diaphysis was enriched in spherulites of similar to 0.5-mu m diameter exhibiting a Ca/P ratio of 1.3. Their identity as calcospherulites was confirmed by their uptake of calcein at the tibial mineralization front 24 h following in vivo injection. Periosteum was dissected and unmineralized osteoid removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites were then released from the mineralization front by dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction analysis revealed they contained apatite crystals (c-axis length of 17.5 +/- 0.2 nm), though their Ca/P ratio of 1.3 is lower than that of hydroxyapatite. Much of their nonmineral phosphorous content was removed by ice-cold ethanol, elevating their Ca/P ratio to 1.6, suggesting the presence of phospholipids. Western blot analyses showed the presence of bone matrix proteins and type I collagen in these preparations. Incubating isolated calcospherulites in collagen hydrogels demonstrated that they could seed a mineralization reaction on type I collagen fibers in vitro. Ultrastructural analyses revealed crystals on the collagen fibers that were distributed rather uniformly along the fiber lengths. Furthermore, crystals were observed at distances well away from the observed calcospherulites. Our results directly support an active role for calcospherulites in inducing the mineralization of type I collagen fibers at the mineralization front of bone. (C) 2007 Elsevier Inc. All rights reserved.
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