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Shi, et al.
templates , suggesting that the SF scaffold does not mass of scaffolds at the end of 4 weeks was 30.0 ± 8.5%,
[34]
have much template effect on the crystalline formation 41.0 ± 10.0%, and 43.0 ± 9.8%, respectively. Because
of calcium phosphate under general thermodynamics. protease XIV acts on the hydrophilic region of SF, it easily
Thus, the crystalline phase of calcium phosphate on SF attacked the hydrophilic sites that were more exposed to
scaffolds can be designed and controlled using common water and led to the initial fast degradation. Obviously,
mineralization parameters and conditions. the silk II structure that was much less hydrophilic was
Figures 3C and D show the details of the crystalline difficult to degrade in SF. Thus, the SF scaffold with
morphology under varied mineralization conditions. The more SF and hence more silk II structure appeared the
crystals from the organic solvent environment were more slowest to degrade and maintained the greatest mass at a
uniform and in slightly greater sizes than those from the similar time point. In contrast with pure SF scaffolds, the
aqueous solution. Most crystalline morphologies were mineralized scaffolds showed much slower degradation
plate-like and the exception was the amorphous phase rate in the 1 week and exhibited a slight mass increase
st
under pH 10–11 and aqueous environment. It is worth in the later 3 weeks. It is proposed that the mass increase
noting that the amorphous phase of calcium phosphate may be the dissolution and recrystallization of DCPD
was believed to be better resorbed compared with other crystals into HAp crystals, which was observed in another
forms, thus providing a better calcium source, and exerting research . The mineralized scaffolds with the minerals/
[22]
a better effect of bone-induction . This mineralization crystals covering the SF scaffolds were more stable under
[50]
condition deserves further exploration. in vitro degradation, which may provide longer support as
an implant material.
3.3. Water retention and in vitro degradation
behavior of SF scaffolds 3.4. Mechanical properties

Water retention of scaffold is a basic characteristic Before evaluating the mechanical performance of the
of porous scaffolds, reflecting the porosity and the scaffolds, we extrusion-printed the monofilaments from
hydrophilicity of scaffold materials. Figure S4A shows three inks and tested their tensile properties. Figure 4A
the mass change of scaffolds after water absorption for shows the tensile stress-strain curves of various filaments.
24 h. For SF scaffolds, the equilibrium water contents It is interesting to note that the tensile breaking stress of
are in the range of 860%–880%. The first 1 h was the the monofilament increased from 0.24 MPa for Ink 1 to
fastest water absorption period, followed by a slow- 0.94 MPa for Ink 3 (Figure 4B). Similarly, the tensile
increase period. The scaffold reached saturation of water modulus showed a marked increase from 0.82 ± 0.17
absorption after only 1 h, which is appropriate for the MPa to 2.82 ± 0.43 MPa. With only 1% increase in the SF
scaffold to be applied in tissue engineering. content, both tensile strength and modulus were elevated
Porosity is also an important parameter of scaffolds. by ~3 times, confirming that the conformation structure
High porosity promotes the transportation of nutrients, in Ink 3-printed scaffolds was different and contained a
oxygen, metabolites, etc. The drainage method was used significantly greater content of Silk II structure.
to estimate the porosity of the scaffold. According to The compressive stress-strain curves of the SF
Equation 1, despite the increase of SF in the scaffolds, scaffolds are compared in Figure 4C. The compressive
the porosity values P of 68 ± 5%, 67 ± 7% and 70 ± 2% modulus derived from the initial linear region between
1
did not show obvious difference. According to Equation 1% and 3% strain in Figure 4D are 166 ± 16 kPa,
2, the porosity P of the scaffolds can be calculated as 91 242 ± 36 kPa, and 241 ± 16 kPa for Ink 1, Ink 2, and
2
± 1%, 87 ± 1% and 90 ± 1%. For the drainage method, Ink 3-based scaffolds, respectively. The compressive
water may not enter the closed pores or small pores of the properties of the scaffolds were consistent with that of
scaffold, resulting in smaller amount of absorbed water. monofilaments. Our SF scaffolds were significantly stiffer
On the other hand, the density used to calculate P may than previously reported SF/SA scaffolds based on inks
2
[51]
be greater than the actual density in the pore walls of the with lower SF concentration, for example, ~5% . After
scaffolds, leading to greater porosity values. Therefore, mineralization, the compressive modulus of mineralized
the actual porosity of the scaffolds should be between P SF scaffolds increased to 698 ± 62 kPa, 1611 ± 273 kPa,
1
and P . There was no significant difference among the and 1188 ± 347 kPa after 15 deposition cycles for the three
2
scaffolds of varied SF contents. inks, exhibiting a five-fold increase as shown in Figure 4G.
The degradation behavior of the scaffold must also These results agreed with Morris’s “eggshell” theory,
be considered in tissue engineering. Figure S4B shows suggesting the hard and stiff mineral deposit can markedly
the mass change of the scaffolds during the in vitro enhance the modulus of the mineralized SF scaffolds.
degradation in the solution of protease XIV and PBS. The As discussed above, with increasing pH, the
scaffolds degraded the fastest in the 1 week, and reached crystalline phase from mineralization would change from
st
a slower degradation rate in the later 3 weeks. The residual DCPD at acidic pH to DCP and HA at basic pH. The DCPD
International Journal of Bioprinting (2022)–Volume 8, Issue 4 9