Influence of molecular weight and concentration of carboxymethyl chitosan on biomimetic mineralization of collagen.

The objective of the present study was to systematically investigate the influence of molecular weight (MW) and concentration of carboxymethyl chitosan (CMC), which served as non-collagenous protein (NCP) surrogates, on biomimetic mineralization of type I collagen. Supersaturated CMC-stabilized amorphous calcium-phosphate (CMC-ACP) dispersions containing different MWs (20 kDa, 60 kDa, 150 kDa) and concentrations (25, 50, 100, 200, 400 μg ml) of CMC were prepared. After mineralization in the aforementioned dispersions for 7 days, the pattern and extent of biomimetic mineralization of collagen scaffolds were investigated. Our study showed that increasing CMC concentration resulted in increasing stability and decreasing particle size of CMC-ACP dispersions. Images from scanning and transmission electron microscopy revealed that intrafibrillar mineralization of collagen was obtained with 20k-200, 60k-100, 60k-200 and 150k-200 CMC-ACP dispersions, with hydroxyapatite (HAp) formation confirmed by Fourier transform infrared spectroscopy and X-ray diffraction measurements, whereas HAp formed extrafibrillar clusters in other collagen scaffolds. Thermogravimetric analysis showed that the combined effect of MW and concentration of CMC contributed to different extents of biomimetic mineralization, and was correlated with the stability and particle size of CMC-ACP dispersions, and the size-exclusion characteristics of type I collagen. The results of this work support the effective function of CMC as NCP analogs, and provide parameters of MWs and concentrations of CMC for applications in hard tissue engineering as well as insights into intersections of mechanisms in biomimetic mineralization.