A novel bi-template induced co-assembly method was employed to fabricate biomimetic bone substitute materials, collagen (COL)-fibroin/hydroxyapatite (COL-SF/HA) composite by using a combination of Type I COL and silk fibroin (SF) molecular templates. As a control, COL/HA and SF/HA composites were also synthesized via single-template assembly technology. The structure and morphology of the resulting assembly composites were investigated by X-ray diffractometer, Fourier transform infrared spectra, transmission electron microscope, and thermogravimetric analysis. Their sizes and size distributions were measured by DLS. The results indicated that the mineral phases in COL-SF/HA, COL-HA, and SF-HA composites were needle-like nano-HA crystals. In comparison to those in COL-HA and SF-HA, the mineral phase in COL-SF/HA displayed smaller size and more narrow distribution. Of all above biomimetic composites, the HA was well assembled with molecular template(s), and the organic content of the composite was about 12%-20%, which was quite similar to the natural bone in composition. CD and SDS-PAGE were used to examine the secondary structure and subunit composition of template proteins. The results revealed that the spatial structure of co-assembly template proteins played a pivotal role in controlling and regulating HA crystal nucleation and growth. Based on the experimental results above, a possible co-assembly mechanism for the HA growing on fibrous bi-template proteins was suggested.
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