Bone is an interpenetrating inorganic/organic composite that consists of mineralized collagen fibrils, which is hierarchically organized into various structures. The structure of mineralized collagen fibril, in which nano-crystals of hydroxyapatite are embedded within the collagen fibrils, provides remarkable mechanical and bio-resorptive properties. Therefore, there have been many attempts to produce collagen-hydroxyapatite composites having a bone-like structure. However, duplication of even the most fundamental level of bone structure has not been easily achieved by conventional nucleation and growth techniques, which are based on the most widely accepted hypothesis of bone mineralization.
In nature, the collagen fibril is mineralized via intrafibrillar mineralization, which produces preferentially oriented hydroxyapatite nano-crystals occupying the interstices in collagen fibrils. Our group has demonstrated that intrafibrillar mineralization can be achieved by using a new method based on the Polymer-Induced Liquid-Precursor (PILP) mineralization process. In the PILP process, a poly-anionic additive can produce an amorphous calcium phosphate precursor which enables us to achieve intrafibrillar mineralization of collagen. It is thought that the precursor is pulled into the interstices of the collagen fibrils via capillary forces, and upon solidification and crystallization of the precursor produces an interpenetrating composite with the nanostructured architecture of bone.
In this dissertation, to demonstrate the effectiveness of the PILP process on the intrafibrillar mineralization of collagen fibril, various collagen scaffolds, such as turkey tendon, bovine tendon and synthetic collagen sponge, were mineralized by the PILP process. Various poly-aspartates with different molecular weight were also used for the optimization of the PILP process for the mineralization of the collagen scaffolds. With the systematic researches, we discovered that the molecular weight of poly-aspartic acid affects the degree of intrafibrillar mineralization of collagen scaffolds. High molecular weight poly-aspartic acid could produce a stable and dispersed amorphous precursor, leading to a high degree of intrafibrillar mineralization. The mineral content of the collagen sponge mineralized using high molecular weight poly-aspartic acid was equivalent to the mineral content of bone. According to X-ray diffraction analysis of the mineralized collagen, the size and composition of the intrafibrillar hydroxyapatite produced by the PILP process were almost identical to carbonated hydroxyapatite in bone. The selective area electron diffraction patterns indicated that the  direction of hydroxyapatite is roughly aligned along the c-axis of collagen fibril, leading to the formation 002 arcs. Using dark field imaging, it was possible to visualize the preferentially oriented hydroxyapatite in TEM. Thermal analysis of mineralized collagen also showed a reduction in the thermal stability of collagen, which is similar to that observed in the collagen in bone, due to the presence of intrafibrillar hydroxyapatite.
Now, we confidently suggest that the PILP process can provide a new way to develop synthetic bone-like composites whose nano-structure is very close to the nano-structure of natural bone. Moreover, we hope that our successful intrafibrillar mineralization of collagen via the precursor mechanism revives discussion of hypothesis of bone mineralization via the amorphous calcium phosphate phase.