Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jun 19;284(25):17092-101.
doi: 10.1074/jbc.M109.007013. Epub 2009 May 4.

Mineralization by Inhibitor Exclusion: The Calcification of Collagen With Fetuin

Affiliations
Free PMC article

Mineralization by Inhibitor Exclusion: The Calcification of Collagen With Fetuin

Paul A Price et al. J Biol Chem. .
Free PMC article

Abstract

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism "mineralization by inhibitor exclusion," the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.

Figures

FIGURE 1.
FIGURE 1.
The recalcification of bone by using fetuin to selectively inhibit mineral growth outside the collagen fibril: time course of supernatant calcium. The test matrix was a 1-cm segment cut from the midshaft region of a rat tibia and demineralized in EDTA for 72 h (26). The solutions for the calcification test were prepared as described (24) and contained 2 ml HEPES, pH 7.4, with 5 mm calcium and phosphate and either 5 mg/ml fetuin or no fetuin. A single demineralized tibia was added immediately after mixing to create the 5 mm conditions, and the solutions were mixed end-over-end at room temperature. There were three tubes per experimental group. Aliquots of each solution were removed at the indicated times and analyzed for calcium. Each time point is the average calcium level in the three replicate solutions.
FIGURE 2.
FIGURE 2.
The recalcification of bone by using fetuin to selectively inhibit mineral growth outside the collagen fibril: analysis for mineral calcium and phosphate. The experiment described in the legend for Fig. 1 was terminated at 24 h, and the mineral that precipitated outside of the tibia was separated from the tibia. Then the mineral precipitate and tibia were both analyzed for calcium and phosphate. The results show the mean ± S.D. of the measurements made on the three replicate bone samples at each condition.
FIGURE 3.
FIGURE 3.
Evidence that the capacity of bone collagen for mineral is limited. Either 4 mg of demineralized bone sand or an amount of non-demineralized bone sand with the same collagen content (18 mg) was added to a 50-ml volume of 0.2 m HEPES, pH 7.4, containing 5 mg/ml fetuin, 5 mm calcium, and 5 mm phosphate, and the solution was mixed end-over-end at room temperature for 2 days. For subsequent recalcification cycles, the spent solution was replaced with fresh calcification solution, and the bone sand was mixed for another 2 days. The bone sand was then analyzed for calcium and phosphate. The results show the mean ± S.D. of the measurements made on the three replicate bone samples at each condition.
FIGURE 4.
FIGURE 4.
FTIR and powder x-ray diffraction spectra of bone that has been recalcified by using fetuin to selectively inhibit mineral growth outside the collagen fibril. Demineralized bovine bone sand was recalcified with fetuin as described in the legend for Fig. 3, and samples of the recalcified bone and of non-demineralized bone were each ground to a fine powder. The graph shows the FTIR spectrum of each sample, and the insets show the powder x-ray diffraction spectrum (see ”Experimental Procedures“ for details).
FIGURE 5.
FIGURE 5.
Dependence of bone collagen calcification on fetuin concentration when homogeneous crystal formation is driven by 5 mm calcium and phosphate. Four mg of demineralized bone sand was added to a 2-ml volume of 0.2 m HEPES, pH 7.4, containing 5 mm calcium, 5 mm phosphate, and the indicated concentration of fetuin. The solution was mixed end-over-end at room temperature for 2 days, and the bone sand was then analyzed for calcium and phosphate (see ”Experimental Procedures“ for details).
FIGURE 6.
FIGURE 6.
Evidence that fetuin sustains conditions that calcify bone collagen. Two-ml volumes of 0.2 m HEPES, pH 7.4, were prepared that contained 5 mm calcium, 5 mm phosphate, and 5 mg/ml fetuin. Four mg of demineralized bone sand was added at the indicated times after mixing calcium and phosphate. The solution was then mixed end-over-end at room temperature for 2 days, and the bone sand was analyzed for calcium and phosphate. The results show the mean ± S.D. of the measurements made on the three replicate bone samples at each condition (see ”Experimental Procedures“ for details).
FIGURE 7.
FIGURE 7.
Calcification of tendon collagen by using fetuin to selectively inhibit mineral growth outside the collagen fibril: analysis for mineral calcium and phosphate. The solutions for the calcification test were prepared as described (24) and contained 2 ml of HEPES, pH 7.4, with 5 mm calcium and phosphate and either 5 mg/ml fetuin or no fetuin. Hydrated rat tail tendon (4 mg dry weight) was added immediately after mixing to create the 5 mm conditions, and the solutions were mixed end-over-end for 24 h at room temperature. There were three tubes per experimental group. Mineral that precipitated in the solution outside of the tendon was separated from the tendon, and the mineral precipitate and tendon were both analyzed for calcium and phosphate. The results show the mean ± S.D. of the measurements made on the three tendon samples at each condition.
FIGURE 8.
FIGURE 8.
Scanning electron microscopy shows that mineral is located within the collagen fibers of tendon that has been calcified using fetuin. The procedure described in the legend for Fig. 3 was used to calcify 4 mg of rat tail tendon (dry weight). The calcified collagen was washed with 0.05% KOH, dehydrated in ethanol, and dried. Samples were then sputter-coated with an ultra-thin layer of gold/palladium and examined with a scanning electron microscope at 20 kV. The bottom two panels show the results of the elemental analysis performed on the ×60,000 field immediately above: carbon is green; calcium is blue; phosphorus is red; and areas containing calcium and phosphorus are purple. (The electron dispersive x-ray spectra of these ×60,000 fields are shown in supplemental Fig. C.) Bars are 20 μm for the top row of images and 1 μm for the middle and bottom rows (see ”Experimental Procedures“).
FIGURE 9.
FIGURE 9.
Calcification of Sephadex G25 by using fetuin to selectively inhibit mineral growth outside the gel beads: time course of supernatant calcium. The solutions prepared for the calcification test contained 2 ml of HEPES, pH 7.4, with 5 mm calcium and phosphate and fetuin only, Sephadex G25 only, fetuin plus Sephadex G25, and fetuin plus Sephadex G75. Each solution was placed into a 10 × 75-mm tube and mixed end-over-end at room temperature; there were three tubes per experimental group. Aliquots of each solution were removed at the indicated times and analyzed for calcium. Each time point is the average calcium level in the three replicate solutions.
FIGURE 10.
FIGURE 10.
Calcification of Sephadex G25 by using fetuin to selectively inhibit mineral growth outside the gel beads: analysis for mineral calcium and phosphate. The experiment described in the legend for Fig. 7 was terminated at 24 h, the mineral that precipitated outside of the Sephadex was separated from the Sephadex using a 20-μm sieve, and the mineral precipitate and Sephadex were both analyzed for calcium and phosphate. The results show the mean ± S.D. of the measurements made on the three replicate Sephadex samples tested at each condition.

Similar articles

See all similar articles

Cited by 41 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback