In vitro mineralization of dense collagen substrates: A biomimetic approach toward the development of bone-graft materials

Bone is an organic–inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group’s work is to use biomimetic processing techniques that lead to bone-like structures. In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway. The various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100 μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process.