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Isolation of calcospherulites from the mineralization front of bone
- Source :
- Cells, tissues, organs. 189(1-4)
- Publication Year :
- 2008
-
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.
- Subjects :
- Male
Paper
Histology
Blotting, Western
chemistry.chemical_element
Bone Matrix
Calcium
Mineralization (biology)
Bone and Bones
Rats, Sprague-Dawley
chemistry.chemical_compound
Calcification, Physiologic
Dispase
Endopeptidases
medicine
Animals
Organic matter
Collagenases
chemistry.chemical_classification
Osteoid
Proteins
Flow Cytometry
Rats
Calcein
chemistry
Biochemistry
Collagenase
Biophysics
Anatomy
medicine.drug
Biomineralization
Subjects
Details
- ISSN :
- 14226421
- Volume :
- 189
- Issue :
- 1-4
- Database :
- OpenAIRE
- Journal :
- Cells, tissues, organs
- Accession number :
- edsair.doi.dedup.....d8d3928daf0854bef09f62df95cb7144