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We have put forth the hypothesis that collagen is mineralized during bone formation by means of a polymer-induced liquid-precursor (PILP) process, in which a liquid-phase mineral precursor could be drawn into the gaps and grooves of the collagen fibrils by capillary action, and upon solidification, leave the collagenous matrix embedded with nanoscopic crystallites of hydroxyapatite. This hypothesis is based upon our observations of capillarity seen for liquid-phase mineral precursors generated with calcium carbonate. Here, we demonstrate proof-of-concept of this mechanism by mineralizing Cellagen sponges (type I reconstituted bovine collagen) in the presence of a liquid-precursor phase to calcium carbonate. Scanning electron microscopy (SEM) was used to examine the mineralized collagen, which in combination with selective etching studies, revealed the extent to which the mineral phase infiltrated the collagenous matrix. A roughly periodic array of disk-like crystals was found to be embedded within the collagen fibers, demonstrating that the mineral phase spans across the diameter of the fibers. Some of the morphological features of the mineralized fibers in our in vitro model system are similar to those seen in natural bone (albeit of a different mineral phase), lending support to our hypothesis that these non-equilibrium morphologies might be generated by a PILP process. SEM provides a different perspective on the morphology of bone, and has been useful here for examining the extent of mineralization in composite structures generated via the PILP process. However, further investigation is needed to examine the nanostructural arrangement of the crystallites embedded within the collagenous matrix.

Biologically mineralized tissues are well recognized for their unusual crystal morphologies and hierarchically organized composite structures. The soluble acidic macromolecules associated with biominerals are thought to play an important role in modulating the mineral morphology. Our in vitro studies, which use acidic polypeptide additives to modify crystal growth of calcium-based minerals, have demonstrated a crystallization mechanism that proceeds via a liquid-phase mineral precursor. Various features of the crystals produced via this mechanism, such as "extruded" mineral fibers and mineralized collagen composites, have led us to propose the hypothesis that an amorphous, liquid-phase precursor could play a fundamental role in the morphogenesis of calcium-based biominerals. Although in vivo evidence of this process remains to be determined, we demonstrate crystallization features that mimic bone and dental enamel and suggest that this process could be relevant to biomineralization in both vertebrates and invertebrates.