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Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures
- Source :
- Cells, tissues, organs. 194(2-4)
- Publication Year :
- 2011
-
Abstract
- The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.
- Subjects :
- Mineralized tissues
Paper
Histology
Tissue Fixation
Freeze Substitution
Microscopy, Energy-Filtering Transmission Electron
Mineralogy
chemistry.chemical_element
Calcium
Spectrum Analysis, Raman
Mineralization (biology)
chemistry.chemical_compound
Calcification, Physiologic
medicine
Pressure
Humans
Cells, Cultured
Bone mineral
Osteoblasts
Water
Osteoblast
Phosphorus
Phosphate
medicine.anatomical_structure
chemistry
Freeze substitution
Biophysics
Solvents
Anatomy
Biomineralization
Subjects
Details
- ISSN :
- 14226421
- Volume :
- 194
- Issue :
- 2-4
- Database :
- OpenAIRE
- Journal :
- Cells, tissues, organs
- Accession number :
- edsair.doi.dedup.....f6d7303ab9587a3abc07f188c1803d38