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Silk Fibroin and Calcium Phosphate 3D Scaffolds Promote in vitro Osteogenesis
μm. The extrusion pressure was 1.2 – 1.5 bar. A typical 2.7. Fourier transformed infrared spectroscopy
3D intersecting grid structure was designed. The three (FTIR)
mixture inks were extrusion-printed to fabricate scaffolds
at the printing speeds of 6 – 10 mm/s. As shown in The conformation structure of SF scaffolds was analyzed
Figure 1A, the scaffolds were frozen at various platform using FTIR. The scaffolds were ground into powder/flakes
temperatures (−10°C, −13°C and −15°C). Then, the and mixed with potassium bromide at a mass ratio of
frozen scaffolds were carefully transferred to a solution 1:100, we then pressed the mixture to obtain a transparent
containing 80% methanol, 10% deionized water, 5% film. The spectra were recorded in the wavenumber range
-1
ethanol, and 5% calcium chloride for 24 h to complete of 650–4000 cm through the accumulation of 32 scans
conformation transition of SF and reach stabilization. at 25°C. PeakFit (Version 4.12) was used to perform the
-1
SA was dissolved and removed by putting the scaffolds peak deconvolution of Amide I region (1600–1700 cm )
in 1 M citric acid solution, which were then soaked and and to estimate the conformation contents. For peak
washed with deionized water. deconvolution, set parameters are Gaussian type, the
peak width at half height (full width at half peak) of
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2.4. Post-mineralization of SF scaffolds 5 cm , 11 peaks in total, and the peak position specified
according to ref. .
[41]
SF scaffolds from Ink 3 with a SF concentration
of 10.0 wt% were chosen to fabricate hybrid SF 2.8. X-ray diffraction (XRD)
scaffolds. As shown in Figure 1C, for the post-
mineralization of SF scaffolds, calcium acetate and Crystalline structure of the SF scaffolds and the hybrid
diammonium phosphate solutions were first prepared scaffolds was analyzed using an X-ray diffractometer
with three pH values (4 – 5, 7 – 8, and 10 – 11) and two (D/Max2200PC, Rigaku, Beijing, China) with Cu Kα
solvent systems, aqueous-based and methanol-based, radiation (wavelength of 0.1542 nm) in a step-scan mode
respectively. About 25% ammonia and 25% acetic acid in the 2θ range of 10–70° at a scanning speed of 6°/min.
solution were used to adjust the pH. The scaffolds were The wet scaffolds were firstly cut into 1–2 mm thick
cut into cuboid shapes with 4 mm × 4 mm at the bottom slices, which were then compressed for the test.
side, and then dipped in calcium acetate solution and 2.9. Porosity measurement and calculation
diammonium phosphate solution alternately, each time
for 30 s. The excess solution on the scaffolds was gently For metallic or inorganic porous materials, the Archimedes
absorbed by filter paper after taking out from the first drainage method is convenient to measure the porosity
solution and before immersing in the next solution. The of the scaffolds. However, for polymer materials water
procedure was repeated for 5, 10, and 15 times. For could go in not only the voids, but also the cell wall/
each mineralization group, more than five samples were polymer structure, causing swelling. Therefore, the
prepared. measurement of porosity can be inaccurate. Here, we used
the Archimedes drainage method to measure the porosity
2.5. Rheological test (P ) of the SF scaffolds using Equation 1. It was repeated
1
Rheology tests of the inks were performed on a 5 times to obtain the average porosity for each scaffold.
Rheometer (DHR-2, TA Instruments, New Castle, DE, P = ( M − M ) /( 1 1 (1)
V )
1
1
2
USA). For all the experiments, 25 mm 2° cone plate
was used. The viscosity-shear rate curves were obtained M is the mass of the dry scaffold; M is the mass
2
1
under the steady flow mode with the shear rate range of the scaffold saturated with water; ρ is the density of
1
of 0.1 – 1000 s . Strain sweep tests were conducted to water; V is the apparent volume of the scaffold.
-1
1
obtain the linear viscoelastic region and the limit of the In addition, the theoretical porosity was also
elastic shear strain. calculated using Equation 2 and taking an average density
value of 1.3 g.cm for SF solids.
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2.6. Scanning electron microscopy (SEM) P M /( V ) (2)
1
The microstructure and morphology of freeze-dried 2 1 21
scaffolds were observed on a SEM (Quanta 250 FEG) at M is the mass of the dry scaffold; ρ is density of
2
1
an accelerating voltage of 20 kV. The wet SF scaffolds SF; V is the apparent volume of the scaffold.
1
were first cut and frozen at −40°C for 12 h before 2.10. Mechanical tests
transferring to a freeze-dryer at −50°C for 24 h. The dry
scaffolds were sputter-coated with gold. The external and Quasi-static compression tests were conducted on
internal cross-sectional morphologies of the scaffolds a dynamic mechanical analyzer/DMA Q800 (TA
were observed. Instruments, Waters Ltd.). The scaffolds were firstly
4 International Journal of Bioprinting (2022)–Volume 8, Issue 4