Intrafibrillar Mineralization of Collagen using a Liquid-Phase Mineral Precursor
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Intrafibrillar Mineralization of Collagen using a Liquid-Phase Mineral Precursor Matthew J. Olszta, Elliot P. Douglas, Laurie B. Gower Dept. of Materials Science and Engineering, 212 Rhines Hall, Gainesville, FL, 32611 ABSTRACT Intrafibrillar mineralization of type-I collagen with hydroxyapatite (HA) is the basis of the complex biological composite known as bone, which from a material science perspective is a fascinating example of an interpenetrating bioceramic composite. Using a polymer-induced liquid-precursor (PILP) process, collagen substrates were highly infiltrated with a liquid-phase mineral precursor to calcium carbonate (CaCO3). At sections of partially mineralized collagen, banded mineral patterns were observed perpendicular to the collagen fibrils, while other fibrils were completely mineralized. An acid etch, used to preferentially remove superficial mineral, further revealed such banded patterns in fully mineralized samples. Removal of the collagen matrix with a dilute hypochlorite solution showed an interpenetrating mineral phase, with mineral disks that spanned the diameter of the pre-existing collagen fibrils, supporting our hypothesis that intrafibrillar mineralization can be achieved via capillary action applied to a liquid-phase mineral precursor. INTRODUCTION Intrafibrillar mineralization of type-I collagen, which occurs during secondary bone formation, leads to a nanostrutured architecture that affords mammalian bone with excellent mechanical properties. This primary level of structure of bone is comparable to an interpenetrating network, in which nanoscopic crystals of hydroxyapatite (HAP) are dispersed throughout the collagen matrix. The accepted model for intrafibrillar mineralization suggests that nucleation begins in the holes zones between the self-assembled tropocollagen molecules, yet the research community has generally been unsuccessful in recreating this mechanism in vitro [1-3]. Limited success has been shown by Bradt et al. [3], where a few crystals appear to nucleate in the hole zones of the fibril, but a highly mineralized interpenetrating composite structure has not been achieved. Most commonly, in vitro mineralization of type-I collagen yields heterogeneous surface nucleation of the inorganic constituent. As with any process within the body, formation of bone is mediated by complex biological mechanisms, and thus, such straightforward attempts at recreating mineralized collagenous tissues seems to be missing some key ingredients. One indication that mineralization may be more than just a simple nucleation phenomenon is work done on early mineralizing bone, which demonstrates that an amorphous prescursor exists, referred to as the “inorganic substance in bands” (ISB), because the electron dense regions correlate with the banding pattern of collagen [4]. This seems to suggest that the mineral phase is first formed intrafibrillarly within the gap zones as an amorphous phase, subsequently crystallizing to create a highly mineralized composite. This possibility still leads
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