The aim of this research is to fabricate biomimetic keratin scaffold via forming porous scaffold by freeze-drying with subsequent rapid mineralization through electrodeposition. Figure 1L schematically illustrate the keratin porous scaffold preparation and electrodeposition process. Figure 1 demonstrates SEM and crystal structure of the mineralized keratin scaffold that deposited at 4V for 1h at different electrode. When the keratin scaffold is fixed on the stainless steel electrode, the deposited calcium phosphate salts completely cover the whole scaffold surface as shown in Fig. 1A and B. In contrast, when the keratin scaffold is mineralized on the copper electrode, the calcium phosphate salt formed is not compactly arranged and the aggregates did not have a complete and consistent shape as in Fig. 1C and D. From Fig. 2E we could observe blank areas between adjacent bulges, which indicated that the calcium phosphate salt crystals grew haphazardly and could not completely envelop the scaffold.
Figure 1I presents the XRD pattern of the apatite deposited on the surface of keratin scaffold under different deposition electrodes. When using Cu, Ni, Ti electrode, well defined peaks at (020), (021), and (041) were clearly observed and match well with calcium phosphate dihydrate crystalline data (DCPD). While, adopting SS electrode, the XRD pattern of the deposited mineral showed some new peak at (211) that corresponding with the diffraction planes of HAp, indicating that the scaffold contained HAp in the deposited crystals only when mineralization is performed on SS electrodes. As shown in Fig. 1K, the mineralized layer is found to be composed mainly of calcium phosphates (O, Ca and P), with a molar ratio of Ca/P of 1.41, indicating that the mineralized layer contains a variety of calcium phosphate crystals.
It can be seen from Fig. 2A that calcium phosphate crystals grown on the surface of the scaffold at lower voltage (3V), forming a complete and uniform mineralized layer with thin thickness. A uniform and nest-like coating could be observed when the mineralization voltage increase to 4 V as shown in Fig. 2B. When the voltage continues to rise to 5V, the scaffold is covered with multiple layers of calcium phosphate, and a large number of spherical aggregates appeared in the uppermost layer (Fig. 2C). On the other hand, the mass of the scaffold also increased when using higher deposition voltages as shown in Fig. 2G.
Figure 2H presents the pattern of the mineral crystals decorated on the surface of keratin scaffold at different voltages. When the mineralization voltage is 3V, there is a clear diffraction peak of (020) crystal plane at 2θ of 11.7°, which is a typical characteristic peak of DCPD crystals. This indicates that higher deposition voltage could result in some deposited crystal being converted from DCPD to HA.
The mineralization duration is an important factor affecting the quality of the deposited layer. Effect of electrolyte duration on the mineralization of keratin scaffold was investigated by varying the mineralization time (0.5 h, 1.0 h, 1.5 h and 2.0 h) at 4V using SS electrode. Tiny granule shape of mineral crystal could be observed deposited on the scaffold from Fig. 3A. The coatings obtained at 1.5h was plate-like, similar in shape to flower clusters as can be seen from Fig. 3E. When the mineralization time reached 2.0 h, the calcium phosphate crystals grown into complete spheres and gathered densely on the surface of the previous layer of the deposited body.
The diffraction peaks of the (020) and (021) crystallographic planes of DCPD appeared near 2θ of 11° and 2θ of 20° when the mineralization time is short (0.5h and 1.0h). As the mineralization time is extended to 1.5h and 2h, the diffraction peaks on the crystalline surface of DCPD disappeared and the diffraction peak of HA at 31.9° became more pronounced. This indicates that the mineralization time is long enough to induce the transformation of crystalline DCPD to HA.
The mechanism of the electrodeposition of surface of keratin scaffold was proposed in Fig. 3K. When the deposition voltage is applied, the pH value in the vicinity around the cathode will increase and resulted the amphoteric keratin molecules becoming negatively charged. Firstly, the Ca2+ in the electrolyte migrated to the cathode, and then the anions around the cathode, including PO43−, HPO42−,H2PO4−,OH−, combined with Ca2+ to form various Ca–P deposits onto the surface of porous scaffolds. The nucleation and growth of calcium phosphate crystals on the template surface resulted forming different types of crystals.