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Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

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Related in: MedlinePlus

Detection of polyP in a bone resorption site of an EDTA-decalcified, 3-month-old murine vertebra.(A, left) Confocal fluorescence image (400–700 nm) from a 5–10 micron bone section stained with DAPI and exposed to multiphoton excitation (787 nm). (A, right) Spectral scans of imaged region (A, left) were acquired in 20 nm bins. Emission intensity was plotted for each of the indicated ROI. Blue ROI: DAPI-DNA emission. Yellow ROI: DAPI-polyP emission. (B, left) The 580 nm bin emission for the same image captured in (A) spatially resolves DAPI-polyP distribution. (B, right) Schematic identifies relevant fluorescent regions (A, left) within the resorption zone. (C) The same bone section was subsequently stained for TRAP and counterstained with haematoxylin (an aqueous process, thought to accelerate hydrolytic degradation of polyP) to confirm the presence of osteoclasts (red staining) at the resorption site (left and right images correspond to high and low magnification, respectively).
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pone-0005634-g002: Detection of polyP in a bone resorption site of an EDTA-decalcified, 3-month-old murine vertebra.(A, left) Confocal fluorescence image (400–700 nm) from a 5–10 micron bone section stained with DAPI and exposed to multiphoton excitation (787 nm). (A, right) Spectral scans of imaged region (A, left) were acquired in 20 nm bins. Emission intensity was plotted for each of the indicated ROI. Blue ROI: DAPI-DNA emission. Yellow ROI: DAPI-polyP emission. (B, left) The 580 nm bin emission for the same image captured in (A) spatially resolves DAPI-polyP distribution. (B, right) Schematic identifies relevant fluorescent regions (A, left) within the resorption zone. (C) The same bone section was subsequently stained for TRAP and counterstained with haematoxylin (an aqueous process, thought to accelerate hydrolytic degradation of polyP) to confirm the presence of osteoclasts (red staining) at the resorption site (left and right images correspond to high and low magnification, respectively).

Mentions: Figure 2A shows an example of the spectrum-wide emission from DAPI-stained vertebral bodies using laser-scanning confocal microscopy. Fluorescence was collected between 400–700 nm; after scanning across the same spectral range in 20 nm bins, we selected various regions of interest and plotted the emission spectrum (Figure 2A, right). DAPI-polyP complexes were identified within granules located in regions of bone resorption by their specific emission wavelength at 520 nm (yellow) [62], [63], whereas regions lacking polyP featured a prominent peak emission near 460 nm—a characteristic emission wavelength for DAPI-DNA (blue) (Figure 2A, vertical lines). The image in Figure 2B (left) was recorded at the 580 nm emission bin, and spatially shows the regions with a DAPI-polyP complex emission. Regions that fluoresced at 580 nm included granules set back from a resorption pit, as well as regions associated with bone resorption (see diagram in Figure 2B, right). Regions of bone resorption were confirmed by subsequent staining of the same section for tartrate-resistant acid phosphatase (TRAP, red), which is a marker for osteoclasts [64], and counterstaining with haematoxylin (Figure 2C).


Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

Detection of polyP in a bone resorption site of an EDTA-decalcified, 3-month-old murine vertebra.(A, left) Confocal fluorescence image (400–700 nm) from a 5–10 micron bone section stained with DAPI and exposed to multiphoton excitation (787 nm). (A, right) Spectral scans of imaged region (A, left) were acquired in 20 nm bins. Emission intensity was plotted for each of the indicated ROI. Blue ROI: DAPI-DNA emission. Yellow ROI: DAPI-polyP emission. (B, left) The 580 nm bin emission for the same image captured in (A) spatially resolves DAPI-polyP distribution. (B, right) Schematic identifies relevant fluorescent regions (A, left) within the resorption zone. (C) The same bone section was subsequently stained for TRAP and counterstained with haematoxylin (an aqueous process, thought to accelerate hydrolytic degradation of polyP) to confirm the presence of osteoclasts (red staining) at the resorption site (left and right images correspond to high and low magnification, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683572&req=5

pone-0005634-g002: Detection of polyP in a bone resorption site of an EDTA-decalcified, 3-month-old murine vertebra.(A, left) Confocal fluorescence image (400–700 nm) from a 5–10 micron bone section stained with DAPI and exposed to multiphoton excitation (787 nm). (A, right) Spectral scans of imaged region (A, left) were acquired in 20 nm bins. Emission intensity was plotted for each of the indicated ROI. Blue ROI: DAPI-DNA emission. Yellow ROI: DAPI-polyP emission. (B, left) The 580 nm bin emission for the same image captured in (A) spatially resolves DAPI-polyP distribution. (B, right) Schematic identifies relevant fluorescent regions (A, left) within the resorption zone. (C) The same bone section was subsequently stained for TRAP and counterstained with haematoxylin (an aqueous process, thought to accelerate hydrolytic degradation of polyP) to confirm the presence of osteoclasts (red staining) at the resorption site (left and right images correspond to high and low magnification, respectively).
Mentions: Figure 2A shows an example of the spectrum-wide emission from DAPI-stained vertebral bodies using laser-scanning confocal microscopy. Fluorescence was collected between 400–700 nm; after scanning across the same spectral range in 20 nm bins, we selected various regions of interest and plotted the emission spectrum (Figure 2A, right). DAPI-polyP complexes were identified within granules located in regions of bone resorption by their specific emission wavelength at 520 nm (yellow) [62], [63], whereas regions lacking polyP featured a prominent peak emission near 460 nm—a characteristic emission wavelength for DAPI-DNA (blue) (Figure 2A, vertical lines). The image in Figure 2B (left) was recorded at the 580 nm emission bin, and spatially shows the regions with a DAPI-polyP complex emission. Regions that fluoresced at 580 nm included granules set back from a resorption pit, as well as regions associated with bone resorption (see diagram in Figure 2B, right). Regions of bone resorption were confirmed by subsequent staining of the same section for tartrate-resistant acid phosphatase (TRAP, red), which is a marker for osteoclasts [64], and counterstaining with haematoxylin (Figure 2C).

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Show MeSH
Related in: MedlinePlus