The Size Exclusion Characteristics of Type I Collagen

Abstract

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.

Mineralization by Inhibitor Exclusion

“…We had previously hypothesized that calcification inhibitors that are small enough to penetrate the collagen fibril will prevent mineral growth inside the fibril, not selectively calcify the fibril (9). We tested this hypothesis using bone Gla protein (BGP; osteocalcin), a 6-kDa inhibitor of apatite growth (32) that is able to rapidly penetrate all of the water within the collagen FIGURE 4.…”

“…We have suggested that large inhibitors of apatite growth can paradoxically favor mineralization within the fibril by selectively preventing apatite growth in the solution outside of the fibril (9). We have also proposed that large nucleators of apatite formation may generate small crystals outside the collagen fibril and that some of these crystals can subsequently diffuse into the fibril and grow (9).…”

“…Fetuin is the most abundant serum inhibitor of apatite crystal growth (11,12), and with a molecular weight of 48 kDa fetuin is too large to penetrate the collagen fibril (9). Fetuin is also termed fetuin-A (to distinguish it from a recently discovered homologue, fetuin-B (13)) and is sometimes called ␣2-HS glycoprotein in humans.…”

“…The collagen fibril therefore provides the aqueous compartment in which mineral grows. We have recently shown that the physical structure of the collagen fibril plays an important additional role in mineralization, that of a gatekeeper allowing molecules smaller than a 6-kDa protein to freely access the water within the fibril while preventing molecules larger than a 40-kDa protein from entering the fibril (9).…”

“…We have suggested that large inhibitors of apatite growth can paradoxically favor mineralization within the fibril by selectively preventing apatite growth in the solution outside of the fibril (9). We have also proposed that large nucleators of apatite formation may generate small crystals outside the collagen fibril and that some of these crystals can subsequently diffuse into the fibril and grow (9). Because the size exclusion characteristics of the fibril allow rapid penetration of molecules the size of a 6-kDa protein, apatite crystals up to 12 unit cells in size should in principle be able to freely access all of the water within the fibril (9).…”