Multiphase intrafibrillar mineralization of collagen.

In the past, the two major biomineralization motifs, biosilicification and biocalcification, were considered as two discrete processes. However, there is increasing evidence of the existence of an inextricable relationship between biosilica and calcium-based biominerals. The recent discovery of a unique silica–chitin–aragonite biocomposite in one genus of demosponges (Verongida) introduces a novel mechanism of multiphase hierarchical biomineralization. Considerable effort has been devoted to the development of silica-/ calcium-based organic–inorganic hybrids; however, none of the techniques used could demonstrate the composite nature of their natural counterparts. Herein, we report a bottom-up, biomimetic biomineralization strategy that results in the intrafibrillar mineralization of collagen with hierarchically arranged silica–apatite multiphase minerals. The mineralization mechanism involves the precipitation and crystal growth of polymer-induced amorphous calcium phosphate precursors within the intrafibrillar spaces of hierarchically silicified collagen. Silicified-collagen-templated intrafibrillar apatite formation provides a model for the formation of multiphase-mineralized skeletons in invertebrates and also results in a biocomposite with increased fatigue resistance and resilience owing to the interpenetrating arrangement of amorphous silica, collagen, and crystalline apatite; the biocomposite also demonstrates enhanced bioactivity, biocompatibility, and potential for the correction of bone defects as a result of the presence of these multiphase components. 7] Nature has destined each organism to receive one mineral (silica, calcium carbonate, or calcium phosphate) as their primary skeletal building block. Nevertheless, genuine multiphase mineralization does exist. Examples include silica– chitin–aragonite skeletons in Demosponges, opal–chitin– goethite radulae in molluscs, silica–chitin–apatite shells in Brachiopods, and silica–chitin–willenite teeth in Copepods. These rare natural multiphase biominerals provide evolutionary insight into biomineralization and inspiration for the development of novel multiphase-mineralized biocomposites. A unique biocomposite from the order Verongida (V. gigantea) is a representative example of natural multiphase mineralization, with a three-dimensional matrix of silicified chitin fibrils infiltrated by regularly distributed aragonite crystals within the siliceous construct. We previously reported a multiphase-mineralized eggshell membrane (ESM) created by introducing nanostructured calcium phosphate or silica into the different compartmental niches of the biopolymer membrane by the use of amorphous precursor phases of the corresponding mineral. The differential distribution of calcium phosphate and silica in the ESM is attributed to the different organic composition of the fiber cores and mantles and represents a different phenomenon from the multiphase mineralization in Verongida, in which two different minerals are arranged hierarchically within one organic template. As the in vitro biomimetic intrafibrillar calcification and intrafibrillar silicification of type I collagen were both described recently, we examined the possibility of the intrafibrillar hierarchical multiphase mineralization of type I collagen through a biomimetic strategy with the expectation that novel multiphase biocomposites could result. In the present multiphase biomimetic mineralization strategy, poly(allylamine hydrochloride)-stabilized silicic acid (PAH–SA) and poly(aspartic acid)-stabilized amorphous calcium phosphate (PAsp–ACP) were used for the in vitro intrafibrillar silicification and calcification, respectively, of type I collagen through stepwise bottom-up approaches (see section S2 in the Supporting Information). Collagen