[136–146]. The principle of the porogen leaching and gas foaming techniques is the
use of porogens, such as sodium chloride and ammonium percarbonate acting as a
template and gas-forming agent, respectively. After treatment of fibroin/porogen
composites with alcohols to induce β-sheet formation, the porogen is leached out
with water to form the pores of the scaffolds. To produce fibroin scaffolds by
freeze-drying, aqueous fibroin solutions are mixed with alcohol to obtain a gel
precipitate following freezing at a low temperature, and are finally freeze-dried
[137]. It was shown that the porogen leaching and gas foaming techniques produce
scaffolds having larger pores (100–200 μm) than the scaffolds formed by freezedrying (10–50 μm). The compressive moduli of the scaffolds vary, depending on
the preparation conditions between 10 kPa and 3 MPa.
An alternative simple route for the production of 3D highly porous fibroin
networks is the cryogelation technique. Silk fibroin cryogels with remarkable
properties were recently obtained from frozen fibroin solutions (4.2–12.6 %) at
subzero temperatures between À5 and À22
C [50]. This was achieved by the
addition of EGDE to the cryogelation system. It was shown that the cryogelation
reactions conducted in the absence of EGDE did not lead to gel formation after
1 day of reaction time, indicating that cryo-concentration alone does not induce
fibroin gelation. Experiments indicate that the presence of EGDE triggers the
conformational transition of fibroin from random coil to β-sheet structure and,
hence, fibroin gelation [49, 50]. Figure 18a shows the amide I band region of
ATR-FTIR spectra, presenting the carbonyl stretching vibration of amide groups
on silk fibroin. The spectrum of fibroin before gelation (dotted curve in Fig. 18a) is
characterized by a peak at 1,640 cm
À1 , which indicates the presence of primarily
random coil and/or β-helix conformations [147, 148]. After cryogelation, all samples display a main peak at 1,620 cm
À1 , which was assigned to the β-sheet
conformation [148]. In addition to the main peak, shoulders at 1,660 and
1,698 cm
À1 are seen, which can be assigned to α-helix and β-turn conformations,
respectively. This indicates the occurrence of a conformational transition from
random coil to β-sheet structure in frozen fibroin solutions. Further evidence for
the β-sheet formation comes from the X-ray profiles of freeze-dried cryogels
(Fig. 18b). Silk fibroin before gelation (dotted curve in Fig. 18b) exhibits a broad
peak at around 22
o , indicating an amorphous structure [149]. After gelation, all the
cryogels show a distinct peak at 20.9
o and two minor peaks at 9.8 and 24.5
o . These
are the characteristic peaks of the β-sheet crystalline structure of silk fibroin,
corresponding to β-crystalline spacing distances of 4.3, 9.0, and 3.6 A
o , respectively
[149, 150]. The results of β-sheet content analysis show that fibroin chains before
gelation have 12 Æ 2 % β-sheet structures, while their contribution increases to
33 Æ 2 % [50].
One of the unique features of fibroin cryogels is their elasticity, which allows
them to resist compression without any crack development. All the cryogel samples
were very tough and could be compressed up to about 99.8 % strain without any
crack development; during compression, water inside the cryogel was removed
(Fig. 9). Upon unloading, the compressed cryogel immediately swelled to recover
its original shape.
Synthesis and Structure–Property Relationships of Cryogels
141
use of porogens, such as sodium chloride and ammonium percarbonate acting as a
template and gas-forming agent, respectively. After treatment of fibroin/porogen
composites with alcohols to induce β-sheet formation, the porogen is leached out
with water to form the pores of the scaffolds. To produce fibroin scaffolds by
freeze-drying, aqueous fibroin solutions are mixed with alcohol to obtain a gel
precipitate following freezing at a low temperature, and are finally freeze-dried
[137]. It was shown that the porogen leaching and gas foaming techniques produce
scaffolds having larger pores (100–200 μm) than the scaffolds formed by freezedrying (10–50 μm). The compressive moduli of the scaffolds vary, depending on
the preparation conditions between 10 kPa and 3 MPa.
An alternative simple route for the production of 3D highly porous fibroin
networks is the cryogelation technique. Silk fibroin cryogels with remarkable
properties were recently obtained from frozen fibroin solutions (4.2–12.6 %) at
subzero temperatures between À5 and À22
C [50]. This was achieved by the
addition of EGDE to the cryogelation system. It was shown that the cryogelation
reactions conducted in the absence of EGDE did not lead to gel formation after
1 day of reaction time, indicating that cryo-concentration alone does not induce
fibroin gelation. Experiments indicate that the presence of EGDE triggers the
conformational transition of fibroin from random coil to β-sheet structure and,
hence, fibroin gelation [49, 50]. Figure 18a shows the amide I band region of
ATR-FTIR spectra, presenting the carbonyl stretching vibration of amide groups
on silk fibroin. The spectrum of fibroin before gelation (dotted curve in Fig. 18a) is
characterized by a peak at 1,640 cm
À1 , which indicates the presence of primarily
random coil and/or β-helix conformations [147, 148]. After cryogelation, all samples display a main peak at 1,620 cm
À1 , which was assigned to the β-sheet
conformation [148]. In addition to the main peak, shoulders at 1,660 and
1,698 cm
À1 are seen, which can be assigned to α-helix and β-turn conformations,
respectively. This indicates the occurrence of a conformational transition from
random coil to β-sheet structure in frozen fibroin solutions. Further evidence for
the β-sheet formation comes from the X-ray profiles of freeze-dried cryogels
(Fig. 18b). Silk fibroin before gelation (dotted curve in Fig. 18b) exhibits a broad
peak at around 22
o , indicating an amorphous structure [149]. After gelation, all the
cryogels show a distinct peak at 20.9
o and two minor peaks at 9.8 and 24.5
o . These
are the characteristic peaks of the β-sheet crystalline structure of silk fibroin,
corresponding to β-crystalline spacing distances of 4.3, 9.0, and 3.6 A
o , respectively
[149, 150]. The results of β-sheet content analysis show that fibroin chains before
gelation have 12 Æ 2 % β-sheet structures, while their contribution increases to
33 Æ 2 % [50].
One of the unique features of fibroin cryogels is their elasticity, which allows
them to resist compression without any crack development. All the cryogel samples
were very tough and could be compressed up to about 99.8 % strain without any
crack development; during compression, water inside the cryogel was removed
(Fig. 9). Upon unloading, the compressed cryogel immediately swelled to recover
its original shape.
Synthesis and Structure–Property Relationships of Cryogels
141