Biomimetic mineral layer over layer-by-layer assemblies: Growth mechanism directed by collagen fibrils

In this study, we describe a promising biomimetic method for growing a hybrid calcium phosphate (CaP) - collagen coating designed following the same principles that govern biomineralization of hard tissues. For this purpose, we explore the versatility of layer-by-layer (LbL) method to incorporate type I collagen and alkaline phosphatase (ALP), while controlling their supramolecular organization and bioactivity. This provides a way to generate in situ CaP ion precursors in the vicinity of collagen proteins, which are self-assembled into fibrils at the solid/liquid interface and further crosslinked. The catalytic properties of ALP, including initial activity and operational stability, is evaluated when the enzymes are embedded within the LbL film. Moreover, a systematic study of the intermediate states of the mineralization is performed by means of atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Results show that the presence of collagen fibrils within the LbL film plays a pivotal role in the growth mechanism of the biomimetic mineral layer. The main findings support that the role of fibrils consists in favoring the adsorption of enzymes and providing a suitable environment for CaP nucleation and growth. Interestingly, early-stage AFM observations reveal the preferential formation of colloidal particles of CaP onto the collagen fibrils, followed by a significant increase of their surface coverage, yielding particles onto and in-between the fibrils. Further mineralization leads to a coating fully covering the support and mainly exhibiting flat and continuous layer covered with a dense network of mineralized collagen fibrils. The approach described in this work provides a straightforward way for the design of biomimetic hybrid coatings with the perspective of tuning cell-material interaction in biomedical applications dealing with hard mineralized tissues.