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The Size exclusion characteristics of collagen and elastin fibers and role of fetuin in their calcification


The diffusion of molecules into a mineralizing matrix may affect the process of serum-initiated mineralization. We theorized that biological matrices that mineralize in serum must have molecular exclusion characteristics due to the large number of inhibitors of mineralization present in serum. For our initial tests, we developed a gel filtration-like procedure using columns packed with collagen from tendon or bone. The elution volumes of test molecules from these columns show the volume within each column that is accessible to the test molecules, and reveal the size exclusion characteristics of the collagen within the column. Molecules smaller than a 6 kDa protein are shown to diffuse into all of the space occupied by water within the collagen fibrils, while molecules larger than a 40 kDa protein are excluded from this space. We hypothesized that fetuin, a 48 kDa serum calcification inhibitor, promotes fibril calcification by selectively inhibiting apatite growth outside the fibril. This hypothesis was tested by examining the impact of removing fetuin on serum's ability to mineralize collagen. The presence of fetuin in serum is shown to determine the location of serum-driven mineralization- in fetuin's presence, mineral forms only within the collagen fibril; in fetuin's absence, mineral forms only outside the fibril. Calcification of the elastin fibers of the artery media is also a serum driven process. We hypothesize that elastin's mineralization is dependant on fetuin preventing mineral growth outside, but not inside the elastin fiber. This hypothesis was tested by examining the impact of removing fetuin on serum's ability to mineralize elastin. By subsequently running molecules over a column packed with elastin fibers, we show that the fiber has size exclusion characteristics comparable to those of collagen, and is dependant on fetuin for mineralization in serum. These observations show that serum calcification activity consists of at least two large proteins. One unidentified protein generates crystal nuclei outside of the collagen fibril or elastin fiber, which then diffuse in. The other protein, fetuin, binds crystal nuclei outside the collagen or elastin matrices, inhibiting their growth, and paradoxically promoting matrix mineralization. This mineralization mechanism provides insight into the ways biological matrices calcify in vivo

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