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Regulating the stemness of mesenchymal stem cells by tuning
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ARTICLE

Fabrication of high performance silk fibroin fibers via stable jet

electrospinning for potential use in anisotropic tissue
Published on 14 May 2018. Downloaded by University of Windsor on 15/05/2018 03:18:24.

Received 00th January 20xx,

Accepted 00th January 20xx
regeneration
DOI: 10.1039/x0xx00000x Bingcheng Yia, Huilan Zhanga, Zhepao Yua, Huihua Yuana,d, Xianliu Wanga and Yanzhong
Zhanga,b,c,e*
www.rsc.org/

Regenerated silk fibroin (SF) from Bombyx mori silkworm cocoon is a highly regarded natural protein-biomaterial suitable
for engineering a variety of biological tissues. Electrospinning offers a unique approach to fiber formation that can readily
produce micro- and nano-scale fibers recapitulating the ultrastructure of the native extracellular matrix. However, SF
fibers from conventional electrospinning suffer from the problem of poor mechanical properties for load-bearing relevant
tissue regeneration applications. In this study, highly aligned high-strength SF fibers were fabricated by a recently emerged
stable jet electrospinning (SJES) approach, with the aid of high molecular weight poly(ethylene oxide) (PEO) acting as a
fiber-forming ingredient to increase control over the jetting instability during electrospinning. Results showed that 90% of
the collected SF/PEO (mass ratio 88:12) fiber assembly via SJES oriented unidirectionally with an angle variation < 1°, and
displayed obvious anisotropic wettability. Mechanically, the as-electrospun highly aligned SF/PEO fibers exhibited a 22.0-
fold increase in ultimate tensile strength (50.85 ± 1.13 MPa) and a 49.3-fold increase in Young’s modulus (1185.99 ±
164.56 MPa) compared with the randomly oriented SF fibers. A subsequent methanol treatment further boosted up
remarkably the tensile strength to 73.91 ± 5.15 MPa and Young's modulus to 2426.13 ± 86.67 MPa. Mechanical
performance of the SF fibers via SJES was also impressive even tested in wet state. The substantial improvement in
mechanical properties of the electrospun SF fibers is attributed to the SJES-enabled higher molecular orientation and
contents of secondary structure (α-helix and β-pleated sheet), as well as the high degree of fiber alignment. Moreover,
biological tests verified that these SF-based fibrous scaffolds supported the induced pluripotent stem cell derived
mesenchymal stem cells to adhere, migrate and grow in a manner of orienting along the fiber axis. We speculate these
high-performance biomimicking SF fibers might give rise to improved efficacy while being utilized to regenerate
architecturally anisotropic load-bearing tissues (e.g., tendon, ligament, and blood vessel).

1. Introduction nanofiber bundles in meniscus [3], just to mention a few. As the

unique biological functions of each anisotropic tissue depends
Anisotropic tissues refer to those biological tissues that exhibit
critically on its structural organization [4], recapitulating the
non-isotropic attributes (e.g., mechanical properties) when
architectural anisotropy via controlling the nanofiber alignment
measured along different directions. Structurally, such kind of
have become the focus of much research in the tissue
tissues is usually characterized by the typical feature of a highly
engineering and regenerative medicine community. Nanofibers
oriented underlying nanofibrous matrix (and associated cellular
from electrospinning with a fineness scale similar to the
orientation), which determines anisotropic load-bearing or other
elements (e.g., collagen) of the native extracellular matrix
functionalities. Examples include the uniaxially aligned
(ECM) demonstrated to offer a tremendous possibility for
collagen nanofibers in tendon/ligament [1], highly ordered
improving the efficacy of engineered tissue substitutes. Thus
collagen nanofibrils arranged in a pseudohexagonal lattice in
produced nanofibrous scaffolds have been used in engineering a
cornea [2], circumferentially and radially aligned collagen
wide range of tissues, including the hierarchically organized
anisotropic tendon [5], knee meniscus [6], annulus fibrosus [7],
blood vessel [8] and articular cartilage [9]. However, there
remain significant challenges in generating nanofibers with
high degree of alignment and mechanical robustness,
particularly while engineering load-bearing tissues with the
anisotropic ultrastructure.
Silk fibroin (SF), extracted from Bombyx mori silkworm
cocoons by degumming the coating layer of sericin, is a highly
regarded natural protein-biomaterial suitable for engineering a
variety of load-bearing biological tissues (e.g., tendon/ligament
[10], arterial wall [11], heart valve [12]), in view of its

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remarkable mechanical strength and toughness, demonstrated under stirring for 5-8 h at 72 °C, yielding a 10 wt% solution.
biocompatibility, and controllable degradation rates [13]. Many The solution was then dialyzed with dialysis cassettes
experiments have solidly corroborated that the regenerated SF (molecular weight cut-off, 14,000 Da) against distilled water
can be successfully converted to nanoscaled fibers by for 4 days to remove the salt ions. Lastly, the resultant solution

Journal of Materials Chemistry B Accepted Manuscript

dissolving in solvents of formic acid [14], hexafluoroacetone was filtered, cast on glass dishes, and lyophilized to obtain the
[15], hexafluoro-2-propanol [16] or simply water [17] for regenerated SF sponges.
electrospinning. However, the SF fibers from conventional 2.2 Preparation of regenerated silk fibroin
electrospinning are well-known to have much lower mechanical
properties than its pristine fiber forms because of structure High molecular weight PEO (Mw > 5,000,000, Avocado,
changes in the process of regeneration as well as random fiber London, UK) was used to formulate a series of SF/PEO blends
orientation and low crystallinity within the fibers. Poor (SF/PEO = 100:0, 96:4, 94:6, 92:8, 90:10, 88:12, and 86:14,
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mechanical strength tends to preclude their use as principal w/w) dissolved in formic acid at the concentration of 20% (w/v).
components or deteriorate efficiency in the construction of Viscosity and conductivity of the solutions were measured with
more organized tissue structures (i.e., anisotropic tissues). a LVDV-1viscometer (Shanghai Fangrui, Shanghai, China) and
While post-electrospinning treatment with methanol [18-20] a DDSJ-308A conductivity meter (Shanghai Jingke, Shanghai,
and/or stretching [21-23] and co-electrospinning with China), respectively. Then, the spinning dope was transferred to
reinforcing nanocomponents such as carbon nanotubes [21, 24], 5 mL syringe with 18 G blunt needle for stable jet
nanohydroxyapatite [25-27] and graphene [28] have been electrospinning [30] (Figure S1) at ambient conditions (20-
demonstrated to potentiate the mechanical properties 25 °C, 25-30% humidity). Solution feeding rate, applied
effectively, some know-hows to substantially improve the voltage and roller (10 cm in diameter) speed were varied to
orientation uniformity on both the fiber and molecular levels optimize the process. For the purpose of comparison, randomly
could afford fundamental solutions to the noted problem from arrayed SF/PEO (98:2, w/w) fibers, denoted as ‘SF (random)’,
the viewpoint of processing-structure-property relationships of were also prepared through conventional electrospinning [18].
the fiber formation. All the electrospun fibrous mats were dried in vacuo at room
Generation of anisotropy in nanofibrous SF scaffolds with temperature for 3 days.
high degree of fiber alignment and adequate mechanical 2.3 Post-spinning treatments
performance is a basic step to confer orientation on cells for To enable water insolubility of the SF per se, the electrospun
regenerating those load-bearing anisotropic tissues. Herein we aligned SF/PEO fibrous mats were treated by a methanol/water
investigated the fabrication of high performance SF fibers via (90/10, v/v) solution for 10 min, from which an amorphous silk
employing a recently emerged approach termed stable jet to β-sheet conformational transition can be achieved (denoted
electrospinning (SJES) [29-31] for generating highly aligned as ‘M-SF/PEO’) [18]. For the purpose of comparison, randomly
ultrafine SF fibers for potential uses in engineering anisotropic arrayed SF/PEO (98:2, w/w) fibers were also treated by the
tissues. In this study, jet whipping motion in conventional same protocol and denoted as ‘M-SF (random)’ after the
electrospinning was eliminated by formulating the SF spinning treatment. To remove the water-soluble component of PEO, the
dope with a high molecular weight poly(ethylene oxide) (PEO) M-SF/PEO mats were immersed in distilled water with water-
to enable facile collection of highly aligned SF fiber arrays. We changing every 2 h at room temperature. The PEO-extracted
characterized the processing parameters-dependent fiber fibrous mats were denoted as ‘W-M-SF/PEO’.
orientation, conformational transition upon different post-
spinning treatments, and physical and mechanical properties of 2.4 Characterization
thus produced fiber structures. Lastly, murine induced 2.4.1 Fiber morphology. Fiber morphology was observed by
pluripotent stem cells-derived mesenchymal stem cells (iPS- either a Hitachi TM-1000 scanning electron microscope (SEM)
MSCs) were selected to evaluate the capacity of SJES-enabled or a Hitachi S-4800 field emission scanning electron
high alignment of SF fibers in supporting cellular recruitment, microscope (FESEM) at an acceleration voltage of 8-10 kV. All
growth and orientation. the fibrous mats were sputter-coated for 40 s with gold to
increase conductivity prior to SEM imaging. Fiber diameters
were analyzed using Image J software (NIH, Bethesda,
2. Materials and methods Maryland, USA), and fiber alignment degree |A| was evaluated
2.1 Preparation of regenerated silk fibroin from the equation |A| = |Ar - Aa|, where Ar is the real intersection
Regenerated SF was prepared as follows. Firstly, 20 g of angle between the vertical axis of an SEM image and an
domestic Bombyx mori silkworm cocoons (Anguo Yuyanfang individual filament, and Aa is the average intersection angle
Chinese Medicine, Hebei, China) were degummed with 1 L of measured from the filament population (n > 50) in an SEM
aqueous Na2CO3 solution (0.5 wt%) at 100 °C for 30 min and image.
then rinsed thoroughly with distilled water. To degum 2.4.2 Molecular structure. Polarized Fourier transform
thoroughly this process was repeated three times. Subsequently, infrared (PFTIR) spectroscopy was employed to detect the
the degummed SF after air-drying was dissolved in a ternary molecular orientation in different electrospun SF fibers [32].
solvent system of CaCl2/CH3CH2OH/H2O (1:2:8 in mole ratio) Dichroic ratio (R) of a particular characteristic peak was

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calculated by using the formula R = A///A⊥, where A// and A⊥ grow around the fibrous scaffolds for 12, 24 and 48 h and
are the absorbances measured with incident beam polarized unattached cells were removed by rinsing with PBS. After
parallel and perpendicular to the fiber-array axis, respectively. adding fresh medium, the cells growing on tissue culture plate
To confirm the conformational transition of SF after the (TCP) around specimen was observed with an inverted

Journal of Materials Chemistry B Accepted Manuscript

methanol/water treatment in 2.3, FTIR spectra of the fluorescence microscope (Eclipse Ti-S, Nikon, Japan). The
degummed SF, PEO powder, and SF-based fibers (i.e., SF/PEO, number and spreading area of the attached cells around
M-SF/PEO and W-M-SF/PEO) were measured in the range of specimen were quantified.
4000-500 cm-1 with the 670 FTIR spectrometer (Nicolet-Nexus, 2.5.3 Cell proliferation assay. After culturing the iPS-MSCs
Thermo Fisher Scientific, Waltham, MA). Data collection was for 1, 4 and 7 days, cell proliferation was determined by the
performed by the accumulation of 128 scans with a resolution CCK-8 assay. In brief, at the predetermined time points cell-
of 4 cm-1. A quantitative analysis on the contents of random
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scaffold constructs were incubated in CCK-8 (Dojindo, Japan)

coil, α-helix and β-sheet conformation in fibers was performed solution (5%, v/v) at 37 °C for 3 h. Then the absorbance of the
as described previously [33]. forming orange formazan derivative was measured at 450 nm
2.4.3 Wettability. Wettability of the aligned SF-based fiber with a microplate reader (Multiskan MK3, Thermo Electron
mats post treatments (i.e., M-SF/PEO and W-M-SF/PEO) was Corporation, MA, USA). To observe the cytoskeletal
assessed by the sessile drop method [34] with a contact angle organization of the iPS-MSCs cultured on the aligned fiber
meter (VCA-optima, AST Inc.). During the measurements, 2 scaffolds, at 1, 4 and 7 days the cell-scaffold constructs were
µL of deionized water was dropped onto each fiber mat, and the fixed with a fresh-prepared paraformaldehyde solution (4%, v/v)
droplet was photographed in the directions of parallel and for 30 min followed by being soaked in PBS containing Triton
perpendicular to the fiber axis, respectively X-100 (0.1%, v/v) for 20 min. Then, filamentous actin (F-actin)
2.4.4 Tensile properties. Tensile properties of the aligned and cell nucleus were stained with 5 µg mL-1 Rhodamine-
fibrous mats (SF/PEO at dry state, M-SF/PEO at dry state and labeled phalloidin (Invitrogen, USA) for 20 min and 1 µg mL-1
W-M-SF/PEO at dry or wet state with prior immersing in DAPI (Invitrogen, USA) for 10 min, respectively. Afterwards,
deionized water for 48 h), collected at a drum rotation speed of the cell morphology was observed under the fluorescence
600 rpm for 2 h, and randomly arrayed SF fibers (SF at dry microscope.
state, M-SF at dry or wet state with prior immersing in 2.6 Statistical analysis
deionized water for 2 h) were determined using a tabletop Data are expressed as mean ± standard deviations of at least
tensile tester (HY-940FS, Hengyu Instrument, Shanghai, China) three samples. Statistical analysis was performed using Origin
equipped with a 50 N load cell at ambient conditions. All software, and Tukey’s HSD post hoc tests were used to make
samples were stretched at a cross-head speed of 10 mm/min pair-wise comparisons between groups. Statistically significant
from the initial length of 3 cm (gauge length). Ultimate tensile values were defined as *p< 0.05 or **p< 0.01 based on one-
strength, Young’s modulus, elongation at break and energy at way analysis of variance (ANOVA).
break were derived from the generated stress-strain curves. At
least 8 samples were tested for each type of the fiber mats.
2.5 Biological tests 3. RESULTS AND DISCUSSION
2.5.1 Generation of iPS-MSCs. Murine iPS-MSCs were 3.1 Electrospinning of highly-aligned SF fibers via SJES
generated as described in our previous work [35]. Briefly, the 3.1.1 Formation of a stable jet with adequate length.
murine iPSCs (Sidansai Biotechnology, Shanghai, China) were Stable jet electrospinning (SJES), featured with the absence of
cultured in a feeder layer of mouse embryonic fibroblast for jet whipping phenomenon in electrospinning, is an effective
proliferation. Then the iPSCs were transferred onto non-coated way to fabricate highly-aligned ultrafine polymer fibers [29-32,
6-well plate for suspension culture to form embryoid bodies 36]. To enable SJES, physical properties of the spinning
(EB), followed by an induction to form mesoderm of EB with solution, in particular polymer solution viscosity and
retinoic acid (RA, Sigma). Eventually, well-developed EB conductivity, play important roles in the formation of a linear
colonies were seeded onto 0.1% gelatin-coated 24-well plate jet with adequate length for easy collection (Figure 2A). In
for adherent culture to obtain iPS-MSCs. The generated iPS- general, spinning dopes with high viscosity and low
MSCs between passages 4 and 7 were utilized for subsequent conductivity favor the formation of longer stable jet. In our
biological tests. current study, pure silk solutions failed to be electrospun into
2.5.2 Cellular affinity. Cellular affinity was evaluated using fibrous form even at the highest concentration (34 wt%).
a modified method as described previously [18]. Briefly, 0.5 However, upon introduction of small amounts of high
mL of the iPS-MSCs suspension (2×104 cells/mL) was seeded molecular weight PEO (4-14 wt%), often acting as a fiber-
around the aligned fiber scaffolds of M-SF/PEO and W-M- forming promoter [18, 37, 38], to the SF solutions, varied
SF/PEO (Figure 1). Before cell seeding, all the fiber scaffolds lengths of a stable linear jet can be generated due to the
were incubated with 75% alcohol for 2 h, followed by an resultant increase in viscosity and decrease in conductivity of
extensive washing with sterile PBS and then immersed in cell the SF dominant solutions (Figure 2B). By adjusting the
culture medium overnight. The iPS-MSCs were allowed to distance between rotating collector and needle tip, a critical

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length of stable jet (up to 23 cm long), beyond which jet- noted in previous studies [40, 41, 47]. High degree of molecular
whipping occurs, can be determined for each SF/PEO orientation in the SJES is therefore associated with the better
formulation (Figure 2C). However, as the stable jet lengths macroscopic orientation of fibers.
below 20 cm, corresponding to the formulations of SF/PEO Moreover, characteristic absorption bands relating to random
coils at about 1645 (amide I, C-O stretching) and 1537 cm-1

Journal of Materials Chemistry B Accepted Manuscript

94:6 and 92:8, gave rise to adhesion between fibers, likely due
to incomplete solvent volatilization; and if also considering the (amide II, N-H bond bending associated with C-N stretching),
fiber size uniformity, the SF/PEO formulation 88:12 appeared and β-sheet conformation at about 1628 (amide I) and 1514 cm-
1
to be the optimum formulation system for generation of highly (amide II) [21, 48] were identified to verify conformational
aligned ultrafine SF fibers (Figure 2D). Table S1 gives a transition of the SF/PEO fibers after the methanol treatment
summary on the electrospinnability of the SF/PEO blends with (Figure 4B). The M-SF/PEO showed obvious β-sheet transition
varying mass ratios in SJES. with strong absorption peaks at 1628 and 1514 cm-1, and a high
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3.1.2 Effects of SJES parameters on fibers morphology. β-sheet content up to 95.43% was observed due to the
Based on the SF/PEO 88:12 formulation, typical methanol-induced rearrangement of SF chains and the
electrospinning variables, including feed rate, applied voltage formation of distinct hydrogen bonds [14]. Interestingly, it was
and collecting speed, were varied to investigate their influences also noted that there appears absorption peaks at 1514 cm-1 with
on diameter and orientation of the fibers collected at the critical the SJES-produced SF fibers only, indicating a portion of β-
stable jet length. As showed in Figure 3A, the formation of sheet conformation (about 11.38%) formed when compared to
aligned fibers without fiber-fiber bonding requires a feed rate of the SF (random) fibers, mainly consisted of α-helix
less than 0.4 mL/h. Increasing feed rate from 0.2 mL/h to 0.6 conformation (63.68%) [49]. The unidirectional pulling force
mL/h resulted in formation of thick fibers with diameters during the SJES process is believed to play a part in promoting
ranging from 1.2 to 2.2 µm, but higher degree of the fiber the β-sheet conversion of SF [50].
orientation (the |A| value is reduced nearly half from 0.830° to Water treatment of the M-SF/PEO fibers to leach out PEO,
0.417°). While increasing the fiber collecting speed from 200 to which is a water soluble and biocompatible component but
1000 rpm, the diameter of the as-received aligned fibers were unfavourable for protein adsorption and cell attachment [18],
attenuated (from 2.4 to 1.5 µm); and only at 600 rpm was a demonstrated that soaking the M-SF/PEO fibers in water for 48
better fiber orientation formed (Figure 3B). Applying higher h (in good accordance with that reported previously [18]) is
voltages benefited for generating finer fibers; however, a adequate to remove almost all the PEO (11.57%, slightly lower
remarkable effect on fiber orientation was observed as the jet than the theoretical value of 12%, see Figure S2A-B) out of the
stability is vulnerable to the voltage applied (Figure 3C) [39]. aligned M-SF/PEO fibers. Worthy of note, there is a slight
Taken together, the optimal parameters for electrospinning morphological alteration on the treated fiber surfaces by
highly aligned ultrafine SF/PEO fibers are: SF/PEO mass ratio exhibiting shallow grooves along the fiber axis as a result of the
of 88:12, feed rate of 0.2 mL/h, applied voltage of 8 kV, PEO extraction (Figure S2C). Such kind of groove-like traces
collecting speed of 600 rpm and the gap distance of 22 cm. further affirms the advantage of the SJES method in
introducing molecular orientation. Furthermore, water
3.2 Structural characterization treatment of the M-SF/PEO fibers to extract PEO also entailed
As the mechanical performance of fibers is well-known to be slightly more β-sheet conversion (Figure 4B).
closely connected with the molecular orientation, orientation Electrospinning of the SF/PEO blend system has been well-
characteristics of the as-electrospun SF/PEO fibers at molecular studied previously [11, 47, 51-53]. As a compatible hybrid
level were therefore examined by PFTIR (Figure 4A). When system, it was demonstrated that the PEO phase in the SF/PEO
the electric vector of the incident IR coincides with the blend was dispersed as small, elongated islands within the silk
transition moment vector of a particular bond vibration, fibroin matrix and oriented along the fiber direction [47], which
characteristic peaks of the bi-component SF/PEO fibers, i.e., is partially consistent with our above observation. To verify
amide II at 1514 cm-1 and amide III at 1234 cm-1 for SF as well whether the PEO component of our M-SF/PEO fibers might
as C-O-C symmetric stretch for PEO at 1101 cm-1, all show localize on surfaces of fibers more than cores of fibers and as a
higher intensities than that in SF (random) fibers. It thereby complementary evidence to Wang’s work concerning the PEO
implies the presence of molecular orientation in the as- distribution [47], shell-core structured composite fibers (i.e.,
electrospun highly-aligned fibers of SF/PEO. The molecular PEO (shell)/SF (core) fibers) were prepared via the SJES for
orientation within fiber matrix depends on the stretching forces- soaking treatment as well in distilled water for varied periods of
resulted jet elongation during electrospinning. This has been time. This further study (Fig. S3) showed that within the first 6
well-documented in previous studies [40-44]. In our case, as the h of immersion, PEO leaching from the M-SF/PEO fibrous
charged fluid jet in SJES always keeps the extensional forces mats was reached to 88.77%. And after 24 h of leaching
stretching polymer chains along one uniaxial direction, it may treatment almost all of the PEO was extracted out of the fibers.
consequently promote molecular chain orientation by Therefore, although we are not able to determine whether PEO
preventing the occurrence of molecule relaxation, in favour of was uniformly distributed within the SF fiber matrix or more
enhancing crystallization [45]; whereas the conventional localization on the fiber surfaces, evidences support the
electrospinning is inflicted by the intrinsic jet bending situation where some portions of the PEO were presented on
instability [46], leading to poor molecular orientation as also the SF/PEO fiber surface (as demonstrated in Fig. S2C, by

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showing grooved surface) and some portions of the PEO were functioned as a plasticizer in the process of molecular
embedded within the SF matrix. stretching. And despite of the remarkable decreases in tensile
In combination with the SJES-enabled macroscopic high strength and modulus, the measured data suggest its mechanical
alignment of fiber assemblies (Figure 3), otherwise results in adequateness of the W-M-SF/PEO in engineering some

Journal of Materials Chemistry B Accepted Manuscript

Figure 4A, B microscopically provide evidences the formation representative anisotropic load-bearing tissues. For instances,
of prominent secondary structure (e.g., the highly oriented SF the ultimate tensile strength and Young's modulus of native
molecular chains and the high content of β-sheet) within the SF coronary artery were at 1.5-6.3 MPa and 4.5-23.7 MPa [39]
fibers generated by the SJES approach (Figure 4C). As a result, while those of tendon were at 15.9-29.0 MPa and 140-210 MPa
improved mechanical properties with the SJES-produced SF [45]. The enhancement in β-sheet content and molecular
fibers are reasonably expected. orientation of the SF-based fibers via SJES in Figure 4 thus
correlated well with very marked improvements in the
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3.3 Wettability & mechanical properties

mechanical properties.
In agreement with previous observation [31, 36], our highly-
aligned fibrous mats of M-SF/PEO and W-M-SF/PEO both 3.4 Biological tests
displayed anisotropic wettability, in which the wetting tendency 3.4.1 Cellular affinity. Cellular affinity test by examining
in the direction parallel to the fiber axis is usually better than cell growth around a scaffold provides a direct means to assess
that in the perpendicular direction (Figure 5A-B). This unique the suitability of a scaffold in supporting attachment and
attribute would enhance the cell spreading and oriented growth growth of the recruited cells for particularly in situ tissue
through contact guidance along the fiber direction, a highly- regeneration [18, 62], in which the recruitment of host
desired cell orientation phenomenon in regenerating anisotropic stem/progenitor cells into an implanted cell-free scaffold to
tissues [31, 36]. It is also noted that W-M-SF/PEO fibers support cell population is essential. As shown in Figure 6A,
exhibited superior wetting ability than the M-SF/PEO fibers. visually there are always large numbers of the spindle-shaped
Such an enhanced wettability can be ascribed to the increased iPS-MSC cells around the W-M-SF/PEO scaffolds, in contrast
surface roughness of the W-M-SF/PEO fibers after PEO to that observed with the M-SF/PEO ones. Significant
extraction, given the intrinsic hydrophilicity nature of the SF differences are also evident from detection of the cell numbers
protein [54]. around the W-M-SF/PEO scaffolds (Figure 6B). The poor
In terms of mechanical properties, at dry state the as- cellular affinity with the M-SF/PEO scaffolds is most likely due
electrospun random silk mat from conventional electrospinning to the release of the soluble PEO, which inhibited the cells from
had an ultimate tensile strength and Young’s modulus of 2.31 attaching to the vicinity of fibers during incubation, due to the
MPa and 24.05 MPa, respectively. These weak mechanical hydrophilic nature of the PEO that limits protein adsorption
properties compare quite similar to the results obtained in [18]. Influence of PEO on cell attachment can also be noted
previous studies [14, 24, 55-58]. On the contrary, the from evaluating the cell spreading behavior (Figure 6C). While
mechanical properties of the SF/PEO fibrous mats from SJES the iPS-MSCs around the M-SF/PEO scaffolds had spread out
were considerably improved. As showed in Figure 5C-D and completely after 24 h, the cells with respect to the W-M-
Table 1, the averaged ultimate tensile strength and Young's SF/PEO group still presented increasing spreading tendency.
modulus of the as-electrospun highly-aligned SF/PEO are 50.85 Presence of PEO significantly reduced the ultimate cell
MPa and 1185.99 MPa respectively, which are both spreading area when compared to the PEO-free scaffolds of W-
significantly improved nearly by 22.0-fold and 49.3-fold more M-SF/PEO.
than that of the electrospun random SF mats. After methanol 3.4.2 Cellular growth. Apart from indirect evaluation of
treatment, its tensile strength and Young's modulus are further cellular affinity around scaffolds in vitro (Figure 6), cellular
enhanced by 32.0-fold and 100.9-fold (i.e., for the case of M- attachment and growth on the SF-based fibrous scaffolds were
SF/PEO) due to the remarkably increase of the β-sheet contents also assessed by seeding cells directly onto the aligned fiber
(Figure 4). However, after PEO extraction from the M-SF/PEO substrates for cultivation up to 7 days (Figure 7). This allows
mats, mechanical properties of the PEO-free W-M-SF/PEO for determining whether the aligned SF fiber scaffolds could
fibers were largely decreased as leaching out of PEO from the support cell population and directed growth post cell-migrating
M-SF/PEO fibers undermined the structural integrity by onto the scaffolds from adjacent tissues in the setting of in situ
forming internal defects. This is evident from the relationship tissue engineering. As expected, both the M-SF/PEO and W-M-
of tensile properties vs. leaching time, showing a progressive SF/PEO scaffolds supported the iPS-MSCs to grow and spread
declining tendency in mechanical properties with the PEO loss along the fiber direction. Cells in navicular long and thin shape
(Figure S4). Nevertheless, with a tensile strength of 41.45 ± on the aligned SF scaffolds is different from the polygonal
7.77 MPa and Young’s modulus of 1824.63 ± 614.50 MPa, morphology of the cells on the TCP control. Cellular
such a mechanical performance is still significantly higher than penetration below the scaffolds is also observed, likely due to
the result of the as-spun random SF fibers and other enhanced the slight disturbance of fiber alignment during the culture
SF-based mats as reported previously [57-61]. More process (Figure 7A). Moreover, the quantified cellular area and
interestingly, at wet state the W-M-SF/PEO fibers exhibited proliferation results (Figure 7B-C) both substantiated the
superior resilience with an elongation at break of 125.06% and potential impact of PEO on cell adhesion and expansion.
energy at break of 337.26 ± 36.94 kJ/m2 as water molecules are Nevertheless, with prolonging the culturing time, the

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suppressed effects were alleviated to some extent by having the Conflicts of interest
biocompatible PEO component leached out completely with
There are no conflicts of interest to declare.
medium changing. This is consistent with the previous
observation reported elsewhere [18].

Journal of Materials Chemistry B Accepted Manuscript

In cell therapies and/or regenerative medicine, derivation of Acknowledgements
iPS-MSCs from induced pluripotent stem cells [29, 63],
represents an effective alternative to obtain larger populations This work was partially supported by the National Key
of MSCs with the attributes of self-renewal, pluripotent Research and Development Program of China
differential potential, patient-specificity, and bypass the ethical (2016YFC1100203), the Fundamental Research Funds for the
concerns associated with ESCs. Therefore, iPSC-MSCs have Central Universities (CUSF-DH-D-2018066), the National
been considered to be a very promising cell source for Natural Science Foundation of China (51073032, 31570969,
Published on 14 May 2018. Downloaded by University of Windsor on 15/05/2018 03:18:24.

engineering different types of tissues or as a cell model for and 31771050), and the Key Basic Research Foundation of
evaluating cell-scaffold interactions. In our current study, to Shanghai Committee of Science and Technology
simulate a situation of in situ tissue regeneration with the SJES- (14JC1490100).
produced fibrous scaffolds, cellular affinity was firstly
examined by seeding the iPS-MSCs around the SF scaffolds for
culturing for 12, 24 and 48 h. Large numbers of the spindle-
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Figure 1 Schematic illustration of cellular affinity evaluation

Figure 2 (A) Schematic diagram of the SJES process for fabricating highly-aligned ultrafine
fibers. (B, C) Effects of PEO contents on viscosity and conductivity of the SF/PEO solutions, as
well as critical stable jet lengths in SJES. (D) Morphologies of the aligned SF/PEO fibers
collected at varied critical stable jet lengths. The electrospinning parameters used are as follows:
8.5 kV, 0.2 mL/h and 700 rpm.
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Figure 3 Effects of (A) feed rates (0.2-0.6 mL/h, other parameters: 8 kV, 22 cm and 600 rpm), (B)
collecting speeds (200-1000 rpm, other parameters: 7 kV, 22 cm and 0.2 mL/h) and (C) applied
voltages (6-10 kV, other parameters: 600 rpm, 22 cm and 0.2 mL/h) on the orientation and
fineness of the electrospun SF/PEO (88/12, w/w) fibers via SJES. Scale bar = 3 µm.
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Figure 4 (A) Polarized FTIR spectra and dichroic ratio of the electrospun fibrous mats of the
aligned SF/PEO and random SF fibers. (B) FTIR spectra of SF-based fibers and quantification on
the contents of their secondary structure, including random coil, α-helix and β-sheet. (C)
Schematic of the multi-level orderliness of SF fibers by SJES, dotted downward arrows indicate
the direction of electric field.
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Figure 5 (A-B) Contact angle of deionized water as a function of time for M-SF/PEO and
W-M-SF/PEO fibers. (C-D) Mechanical properties of the SF/PEO, M-SF/PEO, W-M-SF/PEO and
random SF fibers with the stretching direction along with fibers.

Figure 6 Cellular affinity test by seeding the iPS-MSCs around the fiber scaffolds of M-SF/PEO
and W-M-SF/PEO for 12, 24 and 48 h. (A) Cell morphology observed with an inverted
microscope (the dark area refers to the fiber scaffolds). Scale bars represent 100 µm. (B) The
quantified cell number around the fiber scaffolds (n = 6). (C) The measured area of cells around
the fiber scaffolds (n = 6). *p < 0.05, **p < 0.01.
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Figure 7 (A) Fluorescence microscopy images of the iPS-MSCs cultured on TCP and SF-based
substrates for 1, 4 and 7 days. The F-actin (red) and nuclei of cells (blue) were stained by
phalloidin and DAPI, respectively. Blue arrows indicate the direction of fibers and yellow dotted
arrows show the infiltration of cells into the scaffolds. Scale bar is 100 µm. (B) Cellular spreading
area (n = 6). (C) Proliferation of the iPS-MSCs (n = 8 each). *p < 0.05, **p < 0.01.

Table1 Tensile properties of different fibrous mats tested at dry and/or wet conditions
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：
Table of contents entry：

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The high performance SF fibers are attributed to the high fiber alignment,
molecular orientation and contents of β-pleated sheet.