Comparative investigation of hydroxyapatite/collagen composites prepared by CaCl₂ addition at different time points in collagen self-assembly process

Mineralization of collagen is a common combination process mainly involving collagen self-assembly and hydroxyapatite formation. Previous measurement of turbidity showed that collagen self-assembly follows the nucleation–growth model. In the present study, 1.0 M CaCl₂ was mixed with 0.6 mg/mL collagen solution in PBS (10 mM phosphate, 80 mM NaCl, pH 7.2, and T = 35 °C) at the beginning of the lag period, at the beginning of the growth period, at t_1/2 (the time to reach half of the total turbidity change), and during the plateau period. Four different hydroxyapatite/collagen (HAp/COL) composites were prepared [COL (1), COL (2), COL (3), and COL (4)]. The optical densities increased with CaCl₂ addition, and a wave trough appeared in the mineralization kinetic curves because of amorphous/crystalline conversion. HAp formation was confirmed by Fourier transform infrared spectroscopy and X-ray diffraction measurements. Energy-dispersive spectroscopy results showed that the calcium/phosphorus ratio of COL (2) is close to that of human bone. Images obtained by scanning electron microscopy revealed that nanosized plate-like HAp of COL (2) formed and became uniformly embedded in collagen, whereas HAp formed large clusters in COL (1), COL (3), and COL (4). Quantitation analyses of collagen and HAp incorporated into composites showed that mineralization at different time points promote collagen fibril generation and have little impact on the HAp content. These results suggest that the composite fabricated by addition of CaCl₂ at the beginning of the growth period is a promising material for bone repair and implantation.