Page 14 - IJB-8-4

Silk Fibroin and Calcium Phosphate 3D Scaffolds Promote in vitro Osteogenesis
Table 1. Primer sequences of Runx2, OPN, OCN, OSX, and Col1a.
Gene Primer sequences
Runx2 Forward: 5’-ATGCTTCATTCGCCTCACAAA-3’
Reverse: 5’-GCACTCACTGACTCGGTTGG-3’
OPN Forward: 5’-AGCAAGAAACTCTTCCAAGCAA-3’
Reverse: 5’-GTGAGATTCGTCAGATTCA TCCG-3’
OCN Forward: 5’-CGCTCTGTCTCTCTGACCTCAC-3’
Reverse: 5’-CACTACCTTATTGCCCTCCTGC-3’
OSX Forward: 5’-ATGGCGTCCTCTCTGC TTG-3’
Reverse: 5’-TGAAAGGTCAGCGTATGGCTT-3’
COL1A1 Forward: 5’-GCTCCTCTTAGGGG CCACT-3’
Reverse: 5’-CCACGTCTCACCATTGGGG-3’

3. Results and discussion monofilaments showed a significant Barus “swelling”
effect after extrusion. The diameter of the filament in SF
3.1. Viscosity characterization of SF-based inks scaffolds decreased from 386 ± 48 μm for Scaffold 1 to
As mentioned earlier, SF aqueous solutions exhibit 293 ± 32 μm for Scaffold 3. It suggests that increasing
low viscosity compared with other natural polymers, the SF content can suppress the swelling effect due to
especially SA solution. Thus, SA was introduced to thicken its less elastic or retractable conformations compared
the SF-based inks. Three formulations were prepared by with SA. The monofilament exhibits a lamellar structure,
carefully mixing the two solutions of varied weights, which could be attributed to the freezing process and
pure SF solution (15 wt%) and SA solution (3 wt%). The similar morphologies were reported in the literature .
[44]
three mixture inks have slightly varied solid contents The thickness of the lamella appeared to increase from
ranging from 10.2 wt% to 11.0 wt% and SF content from Scaffold 1 to Scaffold 3.
9.0 wt% to 10.0 wt%. The viscosity-shear rate curves of Printing speed is one critical parameter for extrusion
SF solution, SA solution, and the three inks from steady- printing. As shown in Figure 2B, the increase in the
flow tests are shown in Figure 1B. SA solution shows printing speed from 6 mm/s to 10 mm/s did not appear to
stable viscosity of 6.5 Pa·s at low shear rate and an change the mean size of the monofilaments for the Scaffold
obvious shear-thinning behavior at shear rate >10 s . In 3, but the shape fidelity of the cross-sections became poor
-1
contrast, SF solution shows initial shear-thinning at low with the increasing speed. Although the micromorphology
shear rate <1 s , which may suggest a disassociation or of the SF scaffolds was not affected by the printing speed in
-1
disentanglement process of SF chains under shear. The this range, the mechanical tests in Figure S2 revealed that
viscosity of SF solution was ~0.1 Pa·s across the 1–10 s increasing the printing speed led to dramatically reduced
3
-1
shear rate range, a typical Newtonian liquid. The mixed compressive modulus of the scaffolds, suggesting changes
inks exhibit intermediate viscosities between pure SF and in the microstructure or conformation structure of the
SA solutions and also a shear-thinning behavior at both scaffolds. Thus, the lowest speed 6 mm/s was chosen. In
low and high shear rates, combining the characteristics of addition, lowering the substrate temperatures from −10°C
the two solutions. The viscosity of mixture inks falls in to −15°C did not appear to affect the filament morphology
a printable range, and the three inks were applied in the (Figure 2C). Although the diameters of the filaments were
extrusion printing for fabricating scaffolds. The properties more consistent at −15°C, indicating a faster freezing
of ink in oscillation mode as shown in Figure S1 reveal process and shape fixation, we chose −10°C for easier
that the relationships between the storage modulus (G’)/ temperature control in an open-air printing environment.
loss modulus (G”) with frequency for SA and SF inks After a stabilization treatment of the frozen
are similar, but the printable frequency range of inks scaffolds in the methanol solution and rinse out of the
gradually narrows with increasing content of SF. SA using citric acid, the 3D SF scaffolds were further

3.2. Morphology and structure of SF scaffolds processed with post-mineralization. Methanol and
with or without mineralization ethanol can instantly induce the β-sheetconformation
transition in SF and led to a robust physically cross-
Figure 2A shows the micromorphology of the extrusion- linked morphology [45,46] . Moreover, the calcium ions
printed 3D SF scaffolds from the three mixture inks. can coordinate with carboxylic groups in SA chains
The details in the surface and the cross-section of a and could result in a crosslinking structure in SA. The
single filament can be seen. The needle with an inner content of mineralized calcium phosphate in the scaffold
diameter of 200 μm was used. However, the printed can be controlled by the number of deposition cycles as

6 International Journal of Bioprinting (2022)–Volume 8, Issue 4