A Novel Self-Assembled Collagenous Matrix Which Serves as a Template for Oriented Growth of Hydroxyapatite Crystal
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Dentistry of New Jersey - Robert Wood Johnson Medical School, Dept. Pathology - Division of Biomaterials, 675 Hoes Lane, Piscataway, NJ 08854, [email protected] ABSTRACT
Collagen fibers self-assembled from solutions of molecular type I collagen were mineralized at pH 9.5, by exposure to super-saturated solutions of calcium and phosphate for a one week period in a double diffusion chamber. Uniaxial tensile mechanical properties increased with mineralization and electron microscopy of the mineral formed within the fiber was morphologically similar to the mineral phase of calcified tissues. Selected area electron diffraction confirms the presence of hydroxyapatite crystal. Further, the aligned fibrillar substructure serves as a template for the orientation of the c-axis diffraction maxima of the hydroxyapatite. These results indicate that an aligned system composed exclusively of selfassembled type I collagen fibrils serves as a scaffold for oriented growth of mineral analogous to calcification in vertebrate bone. INTRODUCTION
Early investigations of bone formation suggested that mineralization is a process of heterogeneous nucleation of hydroxyapatite crystal within the pores or "hole" region of the collagen fibrillar scaffold of the tissue1 . Subsequent in-vitro studies found that a collagenous matrix with a characteristic 64 nm repeat pattern enhanced mineral formation and the mineral formed in association with the periodicity of the fibril 2 ,3 . However, the early methods of protein extraction and purification employed in these investigations were not as accurate as current standards 4 , and as a result the role of type I collagen in the process of matrix mineralization is undetermined. In vertebrate hard tissues it is currently believed that an interaction between anionic proteins and type I collagen fibrils provide a specific architectural arrangement of negatively charged groups which act as nucleators of hydroxyapatite and mediate crystal orientation with respect to 4 the fibril axis . This oriented growth of an inorganic phase in association with a organic
substrate is a feature common to a large variety of organisms employing mineralized matrices for structural support 5 . In mammalian bone the organic substrate consists of approximately 90% type I collagen and 10% of non-collagenous proteins 6 . Ultra-structural observations of this mineralized tissue indicate a distinct orientation of the mineral crystal c-axis with respect to the fibril axis as well as a periodic deposition of the mineral in register with the period substructure of the fiber 7 , which clearly implies the importance of collagen fibrils in the process of mineralization. To further investigate the role collagen in matrix mineralization, we have developed an in-vitro double diffusion system for the mineralization of reconstituted collagen fibers 8 . Previous results indicated that mineralization of an insoluble collagen fiber over a broad range of incubating pH's increased the mechanical properties of the fiber 8 . Further, electron
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