Carbohydrate Polymers 267 (2021) 118167

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Hydroxyapatite-reinforced alginate fibers with bioinspired dually

aligned architectures
Fuqiang Wan a, Hang Ping a, Wenxuan Wang a, Zhaoyong Zou a, Hao Xie b, Bao-Lian Su c,
Dabiao Liu d, *, Zhengyi Fu a, *
a
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Luoshi Road No. 122, Wuhan 430070, China
b
School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Luoshi Road No. 122, Wuhan 430070, China
c
Laboratory of Inorganic Materials Chemistry, University of Namur, B-5000 Namur, Belgium
d
Department of Mechanics, School of Aerospace Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

A R T I C L E I N F O A B S T R A C T

Keywords: Biological materials have excellent mechanical properties due to their organized structures from nano- to macro-
Bioinspired scale. Artificial manufacture of materials with anisotropic microstructures still remains challenging. We
Spin-coating described a stress-induced method to fabricate anisotropic alginate fibers. Organic-inorganic composite fibers
Anisotropy
were obtained by incorporating aligned hydroxyapatite (HAP) nanowires into the alginate fiber. Detailed
Sodium alginate
Dual alignment
structural characterization revealed the bone-like structure of the HAP-reinforced alginate fibers. Tensile test
Hydroxyapatite results showed that the maximum Young's modulus and tensile strength were 4.3 GPa and 153.8 MPa, respec­
tively. A multiscale reinforcing mechanism is proposed after the discussion of the structure-property relationship:
highly ordered and compacted nanofibrils aligned along the longitudinal direction at the microscale, and two
kinds of alginate gels with different mechanical behaviors at the nanoscale coexisted (acidic alginate gel and
calcium-alginate gel). This work validates the effectiveness of the bioinspired fabrication strategy, which inspires
further manufacturing and optimization of materials for diverse applications.

1. Introduction
Biological structural materials, such as tendons, skeletal muscles and
bones, exhibit anisotropic mechanical properties due to their exquisite
nano- to macroscale hierarchical structures, all of which have evolved
for billions of years (Fratzl & Weinkamer, 2007; Meyers, McKittrick, &
Chen, 2013; Naleway, Porter, McKittrick, & Meyers, 2015; Wegst, Hao,
Eduardo, Tomsia, & Ritchie, 2015). Skeletal muscle, composed of
densely packed and highly oriented muscle fiber bundles, is responsible
for force generation and propagation (Jana, Levengood, & Zhang, 2016).
Bone is responsible for mechanical support and is a hybrid hard tissue in
which apatite nanoplates are embedded in aligned collagen fibrils to
form fibrillar arrangements. These highly-organized structures span
several length scales, endowing skeletal muscles and bones with excel­
lent mechanical strength, especially along the direction of fiber packing
(Naleway et al., 2015; Zhao, Fang, Rong, & Liu, 2017). Imitating these
biological structures is an efficient and ideal way to fabricate high-
performance materials. Many studies have considered the design and
fabrication of materials with specific functionalities and outstanding
mechanical properties (Kong et al., 2018; Liu, Zhang, & Ritchie, 2020;
Yu et al., 2020).
Recently, magnetic and electric fields have been widely used to align
inorganic objects in polymer matrices, and these inorganic objects are
fixed after hydrogenation triggered by light or thermal irradiation (Zhao
et al., 2017). A cartilage-like anisotropic hydrogel (Liu et al., 2015) was
fabricated by incorporating nanometer-scale sheets of titanium oxide
(TiNS). A parallel arrangement of the TiNS in a hydrogel precursor so­
lution was achieved under an applied magnetic field and fixed after
gelation, which endowed the anisotropic hydrogel with remarkable
anisotropic mechanical properties. Dielectrophoresis was proposed to
align carbon nanotubes (CNTs) to manufacture contractile muscle
myofibers with controllable electrical and mechanical properties
(Ramon-Azcon et al., 2013). For other applications, it is easy to create
CNT-based micropatterns inside scaffolds using this universal method.
These strategies were applied to the preparation of bulk hydrogel ma­
terials and a great diversity of functional nanoscale objects with varying
morphologies. Incorporating ordered building blocks into biological
materials is regarded as an efficient strategy for improving their

* Corresponding author.
E-mail addresses: dbliu@hust.edu.cn (D. Liu), zyfu@whut.edu.cn (Z. Fu).

https://doi.org/10.1016/j.carbpol.2021.118167
Received 15 February 2021; Received in revised form 12 April 2021; Accepted 4 May 2021
Available online 7 May 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.
F. Wan et al.
mechanical behaviors. However, the order arrangement is only confined
to magnetic or other inorganic nano-objects, and the organic polymer
matrix remains disordered. Developing advanced processing method­
ologies that can conveniently prepare organic-based organized struc­
tures without the need for a strong magnetic field or contact strategies
remains a challenge.
A microfluidic spinning method was proposed for fabricating
anisotropic hydrogels with various structures (Du, Li, Wu, & Chen, 2019;
Tian et al., 2017). Although hydrogels with anisotropic properties can be
easily prepared using this simple strategy, anisotropic structures can
only be realized at the macroscale due to the limitation of microfluidic
channels. Force plays a critical role in tissue morphogenesis and
patterning, especially in mechanical tissues, such as tendons, cartilage,
and bone (Forslund & Aspenberg, 2002; Heisenberg & Bellaiche, 2013;
Jana et al., 2016; Kim et al., 2013). Tseng et al. (2017) fabricated hi­
erarchical silk fibers based on a hydrogel with controlled nano fibrillar
architectures in a mold. The force generated by substrate deformation is
vital for the assembly of nanofibrils and the formation of a highly
organized network. By introducing external tensile forces along the
fibrous direction, Gong et al. (Matsuda, Kawakami, Namba, Nakajima, &
Gong, 2019; Mredha et al., 2018) fabricated alginate hydrogels with
aligned hierarchical fibrous structures and biomimetic self-growing
hydrogels with a double network. A repetitive force was applied to
break brittle network strands and generate mechanoradicals that trig­
gered reconstruction, facilitating the formation of new networks. These
studies indicate that mechanical force is beneficial for the formation of
highly ordered materials with remarkable mechanical properties. Un­
fortunately, maintaining the ordered structure after the removal of the
external force is challenging. It is significant to develop this novel
method to prepare high-performance materials with anisotropic struc­
tures from nano- to macroscale (Huang et al., 2019).
Hence, this work aims to improve the stress-induced method for
fabricating materials with bioinspired organized architectures and
enhanced properties. In this study, alginate fibers and HAP-reinforced
composite fibers with anisotropic architectures organized in a reminis­
cent of natural fibrous materials were prepared via a spin-coating
method. Subsequent structural characterization and analysis were per­
formed in detail, which revealed the bone-like microstructure of the
HAP-reinforced alginate fibers. Furthermore, tensile tests were per­
formed on these fibers, and their performance was compared with ma­
terials that have similar structures or components to verify the
effectiveness of the spin-coating method. Finally, a multiscale
strengthening mechanism was proposed for the bone-mimicking fiber
after a detailed discussion about the structure-property relationships.
This bone-mimicking fiber has potential applications in bioengineering
materials, and the novel method can inspire the design and preparation
of high-performance materials.
2. Materials and methods
2.1. Chemical reagents and materials
Calcium dichloride (CaCl2), calcium nitrate tetrahydrate (Ca
(NO3)2⋅4H2O), sodium silicate (Na2SiO3⋅9H2O), sodium phosphate
(Na3PO4), and acridine were purchased from Sinopharm Chemical Re­
agent Co., Ltd., China. Sodium alginate (Alginate) was purchased from
Aladdin Industrial Co. (Shanghai, China). The fraction FG of α-L-gulur­
onic acid and the molecular weight MW was determined according to the
previous work (Maki et al., 2011). The value of FG and MW are 0.5 and
140 kg mol− 1, respectively. Coverslips (20×20 mm) were purchased
from Sangon Biotech, China.
2.2. Fiber fabrication
Alginate solutions with different concentrations (1, 2, 3, and 4 wt%)
and a calcium dichloride solution (0.5 M) were alternately dropped onto
2
Carbohydrate Polymers 267 (2021) 118167
a coverslip to form a hydrogel, and the cover glass was attached to a
running spin coater (SC100-SE, China) (running at 2000, 3000, 4000
and 5000 rpm). Fibers began to form at the corner of the glass substrate
after excess hydrogels formed on the cover glass. We stopped adding
alginate and calcium dichloride solution onto the glass substrate when
the fibers were long enough for the tensile tests. Then, the cover glass
was taken off and left to dry at room temperature. Dried fibers were
collected from the cover glass by using a tweezer. HAP nanowires were
prepared by following the procedures introduced in the literature (Lin,
Liu, Chang, & Zhu, 2011). Alginate (4 wt%) powder was gently added
into a hydroxyapatite nanowire (0.1 wt%) slurry obtained after ultra­
sonic dispersion (KQ-50B, Kunshan Shumei, China) and continuously
stirred for 1 d under a stirring speed of 1000 rpm to achieve homoge­
neity. The hybrid suspension was used to fabricate the HAP-
strengthened fibers according to the above procedures.
2.3. Fiber characterization
Field emission scanning electron microscopy (FESEM, Hitach-
SU8020, Tokyo, Japan) images collected at a 5 kV acceleration volt­
ages. High-resolution transmission electron microscopy (HRTEM, JEM-
2100F, Japan) images were used to investigate the microstructure of
the fibers. To obtain the crystal structure of HAP nanowires, powder X-
ray diffraction (XRD) with the 2θ angles ranging from 10◦ to 80◦ was
performed on the X-ray diffractometer (D8 Advance, Bruker, German).
The fiber samples were frozen by using liquid nitrogen with a freezing
system (LN Ultra, RMC, America), and the ultrathin frozen sections were
obtained on an ultramicrotome (PowerTome-XL, RMC, America). Ul­
trathin frozen sections were transferred to the copper grids for HRTEM
investigation. Polarized optical microscopy (POM, BX51-p, Olympus,
Japan) was used to illustrate the anisotropy. A confocal laser scanning
microscope (CLSM, FV3000, Olympus, Japan) was used to obtain the
internal structure information for structural remodeling. Acridine (0.01
wt% in alginate solution), a water-soluble fluorescent dye with emission
wavelengths ranging from 510 to 550 nm, was excited by an Ar laser
(488 nm). Hundreds of fluorescent images indicating the internal
structure were obtained by setting a Z-dimension step depth of 10 nm
per slice. Small-angle X-ray scattering (SAXS) analysis was performed on
a small- and wide-angle X-ray scattering system (SAXSess mc^2, Anton
Paar, Austria), and the tube voltage of the X-ray generator was 40 kV.
The wavelength of the incident X-ray beam was 0.1542 nm. The scarred
X-rays were detected by a detector positioned at a distance of about
306.8 mm from the sample holder. The exposure time was held for 1000
s for each sample to ensure sufficient statistical accuracy. In the low-q
range (0.1 < q < 0.4 nm− 1), the SAXS data was analyzed according to
the relation I(q)~q− α (Draget, Stokke, Yuguchi, Urakawa, & Kajiwara,
2003). The values of α indicate different scattering behaviors of these
samples. For two-dimensional wide-angle X-ray scattering (2D-WAXS)
experiments, the NANOATAR (Bruker, German) system was used. 2D
scattering X-rays were detected with the 2θ angles ranging from 5◦ to
40◦ by a 2D detector (Vantec-2000) positioned with a sample-to-
detector distance set at 42 mm. Fiber bundles with a width of about 5
mm were used for SAXS and 2D-WAXS measurements. The fiber
micromorphology was observed with an inverted biological microscope
(MI52-N, Mshot, China), which was controlled by the microscopic dig­
ital measurement and analysis system (Mshot) and fiber diameters and
lengths were measured by the system. The average of nine test mea­
surements was taken as the actual diameter. Fourier transforms infrared
(FTIR) spectra of alginate fiber, and HAP reinforced fiber samples were
recorded on an FTIR spectrometer (Thermo Nicolet, USA) in the range
between 4000 and 400 cm− 1. These fibers were tightly glued in parallel
to form bundles on a sample card with a square window for the FTIR test.
The structure anisotropy analysis was performed by obtaining fast
Fourier transform (FFT) of SEM images using ImageJ.
F. Wan et al.
2.4. Mechanical test
Fibers were dried at room temperature. The tensile tests were con­
ducted on the fibers with a micro-tensile tester (Guo, He, Li, Lei, & Liu,
2019) equipped with a laser displacement sensor (Keyence, LK-G30),
which has a load resolution of 0.00695 mN. The fiber sample was
glued in the prepared sample card, and then the sample card was
clamped to two grips on the tester. All samples had a gauge length of
approximately 5 mm, and the travel velocity of the bottom grip was set
at 0.2 mm min− 1 to stretch these samples. All the mechanical tests were
performed at room temperature. At least three specimens fabricated
under each condition were tested to decrease the random error. The
testing time for each specimen was within 10 min. The micro-tensile
tester's operating system and the experimental data were collected and
analyzed in a system designed based on LabVIEW (Guo et al., 2019; Liu,
He, Hu, & Ding, 2017).
3. Results and discussion
3.1. Fabrication and characterization of fibers with anisotropic
nanostructure
Alginates are natural polysaccharides composed of β-D-mannuronate
Fig. 1. Fiber fabrication and microstructure characterization. (a) Schematic illustration of the fabrication of a highly anisotropic fiber. (b) SEM image of a knot of the
fiber. (c, d) Surface and internal morphology of the highly anisotropic fiber. (e) POM images of 1 wt%-2000. (f) A slice of the CLSM image and (g) a reconstruction
model of 1 wt%-2000.
3
Carbohydrate Polymers 267 (2021) 118167
(M) and α-L-guluronate residues (G) (Agulhon, Robitzer, David, &
Quignard, 2012). The fabrication method is shown in Fig. 1a. Alginate
solution forms hydrogels immediately upon contact with Ca2+ (Naka­
jima, Kawano, & Onoe, 2017), and fibers start to form after the glass
substrate is fully covered by excess hydrogel (see Fig. 1a). Excessive
water in the fiber is excluded during spin-coating, accompanied with
further gelation and structure fixation, which can effectively overcome
the difficulties of maintaining the structure during the fabrication of
anisotropic hydrogels. The surface morphology and internal organized
structure composed of compact nanofibrils shown in scanning electron
microscopy (SEM) images in Fig. 1b–d suggest good flexibility and
structural integrity. The mechanical tensions generated by the centrif­
ugal force along the longitudinal direction play a critical role in fiber
formation and lead to the formation of a highly ordered structure,
dehydration and a reduction of porosity before complete gelation. Be­
sides, the flow of HAP nanowires on the glass substrate and in a sodium
alginate solution along the fibers also contributes to the alignment
(Yang, Zhu, Chen, Qin, & Xiong, 2018). Nanofibrils are brought closer
together to form a compact structure via ionic or hydrogen bonding
among the molecular alginate chains.
These fibers are prepared under different spin-coating speeds at
varying alginate solution concentrations to regulate the centrifugal
force. The samples are labeled according to fabrication conditions, e.g.,
F. Wan et al.
/ / { }/
( ) ( ) ∑
n
I(q) = k1 2 2 2
1+a q + k2 2 2
1+ξ q + 2
qmi [J1 (qRi )/qRi ] + m3
i=1
a sample fabricated from a hydrogel with an alginate concentration of 1
wt% and a spin-coating speed of 2000 rpm is designated as 1 wt%-2000.
SEM images of the fracture morphology of 1 wt%-2000 and 1 wt%-3000
are shown in Fig. S1a–b and S1c–d, respectively. The organized struc­
tures of the aligned nanofibrils along the longitudinal direction are
clearly shown, as indicated by the arrows in these figures. Transmission
electron microscopy (TEM) photographs of ultrathin frozen sections of 1
wt%-2000 (Fig. S1e) and 1 wt%-3000 (Fig. S1f) provide further evidence
for the formation of the anisotropic structure. The texture distribution
directions are the same as the fiber length direction indicated by the
arrow. Polarizing optical microscopy (POM) is widely used to charac­
terize anisotropic structures according to the interference color (Linge
Johnsen, Bollmann, Lee, & Zhou, 2018; Sørensen, 2013; Tseng et al.,
2017). POM images of 1 wt%-2000 are shown in Fig. 1e. The right part
in Fig. 1e is captured between crossed polarizers, showing an evident
birefringence behavior that correlates with the highly anisotropic
structure. Generally, the faster the spin coating method is, the thinner
the fiber diameter is, which is indicated by POM images in Fig. S2. The
observed interference color is determined by the retardation value (R),
which increases with the specimen thickness (d) for different birefrin­
gence values (Δn). Although the fiber diameters are different, these fi­
bers show second-order turquoise blue or yellow colors under the 530
nm tint plate. It is concluded that the fiber with a smaller diameter
possesses a higher birefringence value due to the higher degree of order
at the nanoscale in the structures generated under different mechanical
constraints (associated with different spin-coating speeds). Confocal
laser scanning microscopy (CLSM) images of 1 wt%-2000 are shown in
Fig. 1f. The distinct anisotropic microstructure is indicated by the green
fluorescence distributed along the length direction. The reconstruction
of the partial structure of 1 wt%-2000 is shown in Fig. 1g after inte­
grating hundreds of sliced images to determine the anisotropic
microstructure.
We conducted SAXS on the highly anisotropic fiber to determine the
nanostructures. Fig. S3 shows plots of the scattering intensities versus
the scattering vector q on a log-log scale. The scattering vector q is
defined by Eq. (1). In this experiment, the maximum length probed by X-
rays is approximately 89 nm (Agulhon et al., 2012):
q = (4π/λ)sin(θ/2)
where λ is the wavelength of the incident X-ray (0.154 nm), θ is the
scattering angle, and α is the scattering exponent. The black lines in
Fig. S3 are linear fits of scattering plots in the low-q range (0.1 < q < 0.4
nm− 1). The values of α for all scattering plots are between 2 and 3, which
indicates that these fibers exhibit a scattering behavior similar to that of
the acid-alginate hydrogel system, which is dependent on pH (Agulhon
et al., 2012; Draget, Skjak-Braek, & Stokke, 2006). In this regard, we
employed the Debye-Bueche formula (to account for random aggre­
gates) and the Ornstein-Zernicke function (to account for single chains)
to fit the experimental data of the 1 wt%-2000 and 1 wt%-3000 samples
(Draget et al., 2003; Draget et al., 2006). The total scattering profile is
calculated by Eq. (2), which is referred to as the acidic two-component
model.
/( )2 /( )
I(q) = k1 1 + a2 q2 + k2 1 + ξ2 q2 (2)
where k1 and k2 are constants, ξ is the correlation length, which was set
to 1 nm in this work, and a is the average size of aggregates. In addition,
ξ is defined by ξ = 2π/qm, and qm is the scattering intensity peak position
Carbohydrate Polymers 267 (2021) 118167
q2 (3)
(Wang, White, Konno, Saito, & Nozawa, 1995). However, the fitting
results shown in Fig. S4a are unsatisfactory. Considering that the algi­
nate hydrogel is crosslinked due to the interaction between Ca2+ and
carboxylate groups, we concluded that rod-like structures might exist in
this system except for single chains that are not crosslinked and random
aggregates (Yuguchi, Hasegawa, Padoł, Draget, & Stokke, 2016). The
disagreement between the fitting results of Fig. S4a is possibly due to
rod-like structure. Hence, we employed an elaborate fitting model (Eq.
(3)) obtained by combining the acidic two-component model and the
one- or two-component rod model (Stokke et al., 2000):
where Ri is the cross-sectional radius of the rod-like structure, J1 is the
first-order Bessel function, mi and m3 are adjustable constants and n = 1
or 2. Fig. S4b shows the fitting results obtained by applying the one-
component rod model (n = 1). The model is of no physical signifi­
cance even though the fitting results are substantially improved because
a in equation 4 exhibits negative values. Therefore, we used the two-
component rod model (n = 2). Here, a was set to 5 nm, as in previous
studies (Draget et al., 2006). Fig. 2 shows good agreement between the
fitting model (black lines) and the scattering data of the fibers prepared
under different conditions. The estimated R1 and R2 are listed in
Table S1, which shows that these samples possess similar cross-sectional
radii of approximately 0.5 nm and 1.5 nm. We conclude that two kinds
of nanostructures may exist in these fibers, i.e., structures of an acidic
hydrogel stabilized due to intermolecular hydrogen bonds and a Ca-
alginate hydrogel with ionic crosslinking (see Fig. 2e).
To gain more insight into the nanostructures of these fibers, we
performed two-dimensional wide-angle X-ray scattering (2D-WAXS)
measurements. Based on the outcomes analyzed in the supplementary
materials (Fig. S5–S6, Table S2), the predictions of the model of the
nanostructures of the fibers agree with the SAXS fitting results in terms
of the two kinds of alginate gel coexisting in these fibers.
3.2. HAP-reinforced fiber with dually aligned nanostructures
HAP nanocrystals are widely used for strengthening polymers due to
their high mechanical performance (Chen, Gao et al., 2018; Wegst et al.,
(1)
2015). We introduced oriented HAP nanowires into the highly aniso­
tropic fiber to improve its mechanical performance. Such a structure
imitates the complex architecture of natural bones and brittle minerals
(elongated HAP nanoplatelets) distributed in soft proteins (highly
aligned collagen fiber matrices; He, Rajasekharan, Tehrani-Bagha, &
Andersson, 2015; Yang et al., 2018). The micromorphology and struc­
tural information of HAP nanowires are shown in Fig. 3a–e. These HAP
nanowires had a good dispersity, lengths of approximately 1.6 μm and
average diameters of approximately 16.7 nm. The detailed statistical
analysis can be seen in Fig. S7. Fig. 3e shows the excellent crystallinity of
HAP in the XRD pattern, which corresponds well to the standard data
(JCPDS No. 09-0432).
The samples are also named according to their fabrication condi­
tions, e.g., 4 wt%/HAP-3000. To obtain a stable and homogeneous HAP
nanowire suspension, we chosen the highest concentrated alginate so­
lution (4 wt%) to fabricate the HAP-strengthened fibers. As depicted in
Fig. 3f‑g, HAP nanowires are distributed anisotropically in the fibers.
Fig. 3g shows the longitudinal cross-section image of the 4 wt%/HAP-
3000 sample. The inset POM image of 4 wt%/HAP-3000 in Fig. 3f shows
distinct birefringence, which indicates that the incorporation of HAP
nanowires does not damage the anisotropic microstructure of the
4
F. Wan et al. Carbohydrate Polymers 267 (2021) 118167

Fig. 2. Fitting outcomes of the SAXS profiles of all fibers obtained by using the acidic two-component with the additional term of the two-component rod model. The
fibers were fabricated with different concentrations of alginate solution: (a) 1 wt%, (b) 2 wt%, (c) 3 wt% and (d) 4 wt%. (e) Illustration of the nanostructure of the
anisotropic fiber.

Fig. 3. Microstructure information for HAP nanowires and the fabrication of HAP-reinforced bone-mimicking fibers. (a, b) SEM and (c) TEM images of HAP
nanowires. (d) Selected area electron diffraction pattern of 4c. (e) XRD pattern of HAP nanowires. (f) Schematic illustration of the preparation of bone-mimicking
fibers. (g) Internal morphology along the length direction.

alginate fiber.
SEM images of cross-sections along the longitudinal direction of the
fibers are shown in Fig. 4a (4 wt%) and Fig. 4b (4 wt%/HAP). It is
evident that a higher spin-coating speed (from 3000 to 5000 rpm)
resulted in a denser structure composed of thinner nanofibrils. After the
incorporation of HAP nanowires, the organized structure became more
distinct. More details of internal fiber structures at lower magnification
can be found in Fig. S8. Fig. 4c–d shows the anisotropy analysis of the
SEM images of the HAP-strengthened fibers processed by FFT (Kiriya
et al., 2012). Gray value-azimuth angle curves are obtained with the FFT
images, which indicate that the nanofibrils are oriented perpendicular to
the peaks in these curves. Taking the 4 wt%/HAP-5000 sample as an
example, one may find that the nanofibrils and HAP nanowires are
oriented perpendicularly to the 90◦ and 270◦ directions.

5
F. Wan et al. Carbohydrate Polymers 267 (2021) 118167

Fig. 4. Internal structure of the fibers before and after incorporating the HAP nanowires. SEM images of cross-sections along the longitudinal direction of the (a) 4 wt
% and (b) HAP-strengthened fibers fabricated at different spin-coating speeds. (c, d) The anisotropy analysis of the HAP-reinforced fibers.

as shown in the stress-strain curves in Fig. S10 denser and more com­
Table 1
pacted structure (see Table 1 for details). The stress-strain curves of the
Mechanical properties of fibers prepared under different parameters.
different HAP nanowire-strengthened bone-mimicking fibers in Fig. 5a
Sample Young's Tensile Tensile Work of exhibit the same tendency as the anisotropic fibers without HAP. As
modulus, strength, strain, extension,
characterized by the work of extension (W), the toughness exhibits a
E [GPa] σt [MPa] εt [%] W [MJ m− 3]
similar tendency. The tensile strain (εt) decreased as the spin-coating
1 wt%-2000 0.6 ± 0.1 38.2 ± 13.4 10.2 ± 3.4 3.2 ± 0.9
speed increased, indicating that an increased spin-coating speed led to
1 wt%-3000 1.2 ± 0.0 74.5 ± 1.2 6.9 ± 0.6 2.8 ± 0.4
2 wt%-2000 1.7 ± 1.0 60.6 ± 7.0 12.5 ± 3.4 5.8 ± 1.2
a tenser fiber as the stretching degree of the nanofibrils in the fibers
2 wt%-3000 3.3 ± 0.1 102.0 ± 17.0 12.0 ± 0.7 8.7 ± 1.7 increased. Residual disorder may still have existed in the untensed fi­
2 wt%-4000 3.2 ± 0.2 92.0 ± 14.3 10.2 ± 3.0 4.7 ± 1.8 bers. It is well-known that the tensile force along the longitudinal di­
2 wt%-5000 3.4 ± 0.9 136.4 ± 32.7 10.8 ± 1.9 10.1 ± 2.2 rection of fiber is beneficial to forming an anisotropic and compacted
3 wt%-3000 1.8 ± 0.2 89.6 ± 4.96 14.6 ± 1.9 9.0 ± 1.2
structure (Mredha et al., 2018).
3 wt%-4000 2.0 ± 0.4 95.4 ± 21.7 13.2 ± 1.7 8.2 ± 3.4
3 wt%-5000 2.5 ± 0.1 110.0 ± 25.8 12.8 ± 1.1 9.6 ± 3.2 Similarly, the strength of fibers increases as the concentration of
4 wt%-3000 1.1 ± 0.4 63.4 ± 22.4 37.1 + 7.0 17.9 ± 8.9 hydrogels increases. The fracture morphologies (Fig. S11) are listed in
4 wt%-4000 2.2 ± 0.5 89.1 ± 6.1 15.7 ± 0.5 9.8 ± 0.6 the supplementary information and in Fig. 5b. The compact internal
4 wt%-5000 2.3 ± 0.9 116.5 ± 4.9 17.8 ± 5.7 13.2 ± 2.5
structure and ductile fracture behavior were confirmed by the cross-
4 wt%/HAP- 1.8 ± 0.1 – – –
3000
section images taken after the tensile test (Yu et al., 2020). As ex­
4 wt%/HAP- 2.1 ± 0.6 116.6 ± 25.0 19.6 ± 3.8 16.0 ± 5.5 pected, both the tensile strength and Young's modulus of the fibers were
4000 significantly improved after incorporating the HAP nanowires, as shown
4 wt%/HAP- 3.8 ± 0.5 141.9 ± 12.8 15.9 ± 4.7 15.2 ± 3.3 in Table 1 and Fig. 5c. Among all the samples, the 4 wt%/HAP-5000
5000
exhibited the maximum Young's modulus (E, 4.3 GPa) and tensile
strength (σ t, 153.8 MPa). The tensile strength is more significant than
3.3. Mechanical properties and reinforcement mechanism that for bone-mimicking materials (tensile strength: <60 MPa) (He, Xu,
Zhang, & Qu, 2020) and many other HAP/polymer-based composites
Tensile tests were conducted on a specially designed micro-tensile (Akindoyo, Beg, Ghazali, Heim, & Feldmann, 2018; Arundhati et al.,
tester at room temperature to evaluate the effect of the anisotropic 2018; Chen, Sun et al., 2018). Moreover, the tensile properties of the
structures on the mechanical properties. Thoroughly dried fibers sample HAP-reinforced fibers are comparable to those of human compact bones
were glued onto the sample card with epoxy with a rectangular window, in the longitudinal direction (78.8–151 MPa) (Yaszemski, Payne, Hayes,
as shown in Fig. S9. More details about the tester and experimental setup Langer, & Mikos, 1996) More details about the comparison of the me­
can be found in our previous reports (Guo et al., 2019; Liu et al., 2017). chanical properties of these materials are shown in Fig. 5d and Table S3
Different specimens were obtained by varying the fabrication conditions in the supplemental materials.
and incorporating HAP nanowires, as listed in Table 1. Fibers fabricated The enhancement of the mechanical properties after the incorpora­
with the same concentration of alginate solution exhibited a significant tion of HAP nanowires is obvious. The cross-sectional images of 4 wt
enhancement in the tensile strength as the spin-coating speed increased, %/HAP-4000 (Fig. 5b-i, i-1) and 4 wt%/HAP-5000 (Fig. 5b-ii, ii-1) after

6
F. Wan et al.
Fig. 5. Mechanical test results of the fibers and a comparison of the mechanical properties with their counterparts. (a) Stress-strain curves of the HAP-reinforced
fibers. (b) Cross-sectional images of 4 wt%/HAP-4000 (i, i-1) and 4 wt%/HAP-5000 (ii, ii-1) after the tensile tests. (c, d) A comparison of the mechanical proper­
ties with HAP/polymer composites in the literature.
the tensile tests show that the HAP nanowire pull-out along the tensile
direction, as indicated by black arrows, is critical for improving the
strength (Chen, Gao, et al., 2018; Gao et al., 2017). To verify the bone-
mimicking properties, tensile tests were conducted on the swellon fiber
(4 wt%/HAP-3000) after swelling in water for 24 h (Fig. S12a). After
absorbing water, fibers become thicker (Fig. S12b). Fibers fabricated
under higher spin-coating speed exhibit larger swelling ration of diam­
eter due to more compact structure. The mechanical properties of
swollen fiber decreased dramatically (Fig. S12c–d). Given the bone-
mimicking structure and similar mechanical property, we speculate
that there are analogous deformation mechanisms in bones during ten­
sile tests. It includes the slippage of chains and HAP in the polymer
matrix, the stretching of alginate chains, bond breakage, and chain
disruption. The organic nanofibers bear the stress initially and then
transfer the stress to the HAP nanowires embedded in the fibers. The
interaction between the HAP nanowires and the molecular alginate
chains could account for the strengthening effects, as suggested by the
FTIR results (Fig. S13) obtained in reflection mode. The bands at 1420
cm− 1 and 820 cm− 1 of pure alginate correspond to the stretching vi­
brations of the asymmetric carboxylate group and characteristic bands
of PO3−4 respectively (Lin et al., 2011; Yu et al., 2020). The shift to low
wavenumbers is due to the interaction between Ca2+ and the carbox­
ylate group, restricting the vibrations (Gao et al., 2017).
It is widely acknowledged that acidic alginate gels are much more
brittle than ionic alginate gels and break at a deformation degree far
below that of ionic gels (Draget et al., 2006). These random aggregates,
which easily deform, leading to the brittleness and plasticity of acidic
gels. The total energy required to break the fiber is increased because it
is necessary to break these bonds in Ca2+-crosslinked alginate gels. The
rod-like junction zone contributes significantly to the tensile strength
due to its strain-hardening response during the tensile tests (Zhang,
7
Carbohydrate Polymers 267 (2021) 118167
Daubert, & Foegeding, 2007). In addition to the ordered arrangement of
the nanofibers and the HAP reinforcement at the microscale, the coex­
istence of the two kinds of alginate gel with different mechanical be­
haviors at a finer length scale in these fibers contributes significantly to
the remarkable mechanical performance at the nanoscale.
4. Conclusions
The anisotropic hierarchical structures of biological materials have
inspired the design and fabrication of materials. In this study, HAP-
reinforced fibers with a bone-mimicking architecture were prepared
using a spin-coating method. Mechanical force plays a critical role in
forming the highly anisotropic structure and the alignment of the HAP
nanowires. In addition to the coexistence of two kinds of alginate gels,
incorporating aligned inorganic building blocks (HAP nanowires) into
the anisotropic fiber improved its mechanical performance. A multi-
scale reinforcing mechanism was proposed, which provided a deep un­
derstanding of the formation principles and mechanical properties of the
bioinspired alginate fibers. This bioinspired mechanical force-directed
fabrication strategy is enlightening for designing high-performance
materials with anisotropic structures. To manufacture other polymer-
based materials with specific structure-function properties, further
research is needed to develop the versatility and scalability of the
method. For instance, it has potential to improve the spin-coating
method by learning from centrifugal spinning technology, which is ex­
pected to realize the preparation of anisotropic high-performance fiber
materials at a large scale.
CRediT authorship contribution statement
Fuqiang Wan: Conceptualization, Methodology, Investigation, Data
F. Wan et al.
Analysis, Writing. Hang Ping, Wenxuan Wang, Zhaoyong Zou: Meth­
odology, Investigation. Hao Xie, Bao-lian Su: Conceptualization, Anal­
ysis. Dabiao Liu: Formal analysis, Writing. Zhengyi Fu: Supervision,
Funding acquisition, Project administration.
Declaration of competing interest
The authors report no declarations of interest.
Acknowledgment
The authors acknowledge funding support from the National Natural
Science Foundation of China (51521001, 51832003, 51902236,
31771032, and 11972168).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2021.118167.
References
Agulhon, P., Robitzer, M., David, L., & Quignard, F. (2012). Structural regime
identification in ionotropic alginate gels: Influence of the cation nature and alginate
structure. Biomacromolecules, 13, 215–220.
Akindoyo, J. O., Beg, M. D. H., Ghazali, S., Heim, H. P., & Feldmann, M. (2018). Impact
modified PLA-hydroxyapatite composites–thermo-mechanical properties. Composites
Part A: Applied Science and Manufacturing, 107, 326–333.
Arundhati, B., Sovan, L., Banerjee, N., et al. (2018). Organically modified clay supported
chitosan/hydroxyapatite-zinc oxide nanocomposites with enhanced mechanical and
biological properties for the application in bone tissue engineering. International
Journal of Biological Macromolecules Structure Function & Interactions, 106, 11–19.
Chen, C., Sun, X., Pan, W., Hou, Y., Liu, R., Jiang, X., et al. (2018). Graphene oxide-
templated synthesis of hydroxyapatite nanowhiskers to improve the mechanical and
osteoblastic performance of poly(lactic acid) for bone tissue regeneration. ACS
Sustainable Chemistry & Engineering, 6, 3862–3869.
Chen, S. M., Gao, H. L., Zhu, Y. B., Yao, H. B., Mao, L. B., Song, Q. Y., et al. (2018).
Biomimetic twisted plywood structural materials. National Science Review, 5,
703–714.
Draget, K. I., Skjak-Braek, G., & Stokke, B. T. (2006). Similarities and differences between
alginic acid gels and ionically crosslinked alginate gels. Food Hydrocolloids, 20,
170–175.
Draget, K. I., Stokke, B. T., Yuguchi, Y., Urakawa, H., & Kajiwara, K. (2003). Small-angle
x-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels.
Biomacromolecules, 4, 1661–1668.
Du, X. Y., Li, Q., Wu, G., & Chen, S. (2019). Multifunctional micro/nanoscale fibers based
on microfluidic spinning technology. Advanced Materials, 31.
Forslund, C., & Aspenberg, P. (2002). CDMP-2 induces bone or tendon-like tissue
depending on mechanical stimulation. Journal of Orthopaedic Research, 20,
1170–1174.
Fratzl, P., & Weinkamer, R. (2007). Nature’s hierarchical materials. Progress in Materials
Science, 52, 1263–1334.
Gao, H. L., Chen, S. M., Mao, L. B., Song, Z. Q., Yao, H. B., Colfen, H., et al. (2017). Mass
production of bulk artificial nacre with excellent mechanical properties. Nature
Communications, 8.
Guo, S., He, Y., Li, Z., Lei, J., & Liu, D. (2019). Size and stress dependences in the tensile
stress relaxation of thin copper wires at room temperature. International Journal of
Plasticity, 112, 278–296.
He, W. X., Rajasekharan, A. K., Tehrani-Bagha, A. R., & Andersson, M. (2015).
Mesoscopically ordered bone-mimetic nanocomposites. Advanced Materials, 27,
2260–2264.
He, Y., Xu, W. H., Zhang, H., & Qu, J. P. (2020). Constructing bone-mimicking high-
performance structured poly(lactic acid) by an elongational flow field and facile
annealing process. ACS Appllied Materials and Interfaces, 12, 13411–13420.
Heisenberg, C. P., & Bellaiche, Y. (2013). Forces in tissue morphogenesis and patterning.
Cell, 153, 948–962.
Huang, W., Restrepo, D., Jung, J. Y., Su, F. Y., Liu, Z., Ritchie, R. O., et al. (2019). Multi-
scale toughening mechanisms in biological materials and bioinspired designs.
Advanced Materials, 31, Article e1901561.
Jana, S., Levengood, S. K., & Zhang, M. (2016). Anisotropic materials for skeletal-muscle-
tissue engineering. Advanced Materials, 28, 10588–10612.
Carbohydrate Polymers 267 (2021) 118167
Kim, H. N., Jiao, A., Hwang, N. S., Kim, M. S., Kang, D. H., Kim, D. H., et al. (2013).
Nanotopography-guided tissue engineering and regenerative medicine. Advanced
Drug Delivery Reviews, 65, 536–558.
Kiriya, D., Ikeda, M., Onoe, H., Takinoue, M., Komatsu, H., Shimoyama, Y., et al. (2012).
Meter-long and robust supramolecular strands encapsulated in hydrogel jackets.
Angewandte Chemie, International Edition, 51, 1553–1557.
Kong, W. Q., Wang, C. W., Jia, C., Kuang, Y. D., Pastel, G., Chen, C. J., et al. (2018).
Muscle-inspired highly anisotropic, strong, ion-conductive hydrogels. Advanced
Materials, 30.
Lin, K. L., Liu, X. G., Chang, J., & Zhu, Y. J. (2011). Facile synthesis of hydroxyapatite
nanoparticles, nanowires and hollow nano-structured microspheres using similar
structured hard-precursors. Nanoscale, 3, 3052–3055.
Linge Johnsen, S., Bollmann, J., Lee, H., & Zhou, Y. (2018). Accurate representation of
interference colours (Michel–Lévy chart): From rendering to image colour
correction. Journal of Microscopy, 269, 321–337.
Liu, D., He, Y., Hu, P., & Ding, H. (2017). Characterizing torsional properties of
microwires using an automated torsion balance. Experimental Mechanics, 57,
297–311.
Liu, M. J., Ishida, Y., Ebina, Y., Sasaki, T., Hikima, T., Takata, M., et al. (2015). An
anisotropic hydrogel with electrostatic repulsion between cofacially aligned
nanosheets. Nature, 517, 68–72.
Liu, Z. Q., Zhang, Z. F., & Ritchie, R. O. (2020). Structural orientation and anisotropy in
biological materials: Functional designs and mechanics. Advanced Functional
Materials, 30, Article 1908121.
Maki, Y., Ito, K., Hosoya, N., Yoneyama, C., Furusawa, K., Yamamoto, T., et al. (2011).
Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-
ray scattering measurements. Biomacromolecules, 12, 2145–2152.
Matsuda, T., Kawakami, R., Namba, R., Nakajima, T., & Gong, J. P. (2019).
Mechanoresponsive self-growing hydrogels inspired by muscle training. Science, 363,
504–508.
Meyers, M. A., McKittrick, J., & Chen, P. Y. (2013). Structural biological materials:
Critical mechanics-materials connections. Science, 339, 773–779.
Mredha, M. T. I., Guo, Y. Z., Nonoyama, T., Nakajima, T., Kurokawa, T., & Gong, J. P.
(2018). A facile method to fabricate anisotropic hydrogels with perfectly aligned
hierarchical fibrous structures. Advanced Materials, 30.
Nakajima, S., Kawano, R., & Onoe, H. (2017). Stimuli-responsive hydrogel microfibers
with controlled anisotropic shrinkage and cross-sectional geometries. Soft Matter, 13,
3710–3719.
Naleway, S. E., Porter, M. M., McKittrick, J., & Meyers, M. A. (2015). Structural design
elements in biological materials: Application to bioinspiration. Advanced Materials,
27, 5455–5476.
Ramon-Azcon, J., Ahadian, S., Estili, M., Liang, X. B., Ostrovidov, S., Kaji, H., et al.
(2013). Dielectrophoretically aligned carbon nanotubes to control electrical and
mechanical properties of hydrogels to fabricate contractile muscle myofibers.
Advanced Materials, 25, 4028–4034.
Sørensen, B. E. (2013). A revised Michel-Lévy interference colour chart based on first-
principles calculations. European Journal of Mineralogy, 25, 5–10.
Stokke, B. T., Draget, K. I., Smidsrød, O., Yuguchi, Y., Urakawa, H., & Kajiwara, K.
(2000). Small-angle x-ray scattering and rheological characterization of alginate
gels. 1. Ca− alginate gels. Macromolecules, 33, 1853–1863.
Tian, Y., Zhu, P. G., Tang, X., Zhou, C. M., Wang, J. M., Kong, T. T., et al. (2017). Large-
scale water collection of bioinspired cavity-microfibers. Nature Communications, 8.
Tseng, P., Napier, B., Zhao, S. W., Mitropoulos, A. N., Applegate, M. B., Marelli, B., et al.
(2017). Directed assembly of bio-inspired hierarchical materials with controlled
nanofibrillar architectures. Nature Nanotechonology, 12, 474–480.
Wang, Z. Y., White, J. W., Konno, M., Saito, S., & Nozawa, T. (1995). A small-angle X-ray-
scattering study of alginate solution and its sol-gel transition by addition of divalent-
cations. Biopolymers, 35, 227–238.
Wegst, U. G. K., Hao, B., Eduardo, S., Tomsia, A. P., & Ritchie, R. O. (2015). Bioinspired
structural materials. Nature Materials, 14, 23–36.
Yang, R. L., Zhu, Y. J., Chen, F. F., Qin, D. D., & Xiong, Z. C. (2018). Bioinspired
macroscopic ribbon fibers with a nacre-mimetic architecture based on highly
ordered alignment of ultralong hydroxyapatite nanowires. ACS Nano, 12,
12284–12295.
Yaszemski, M. J., Payne, R. G., Hayes, W. C., Langer, R., & Mikos, A. G. (1996). Evolution
of bone transplantation: Molecular, cellular and tissue strategies to engineer human
bone. Biomaterials, 17, 175–185.
Yu, Y. D., He, Y., Mu, Z., Zhao, Y. Q., Kong, K. R., Liu, Z. M., et al. (2020). Biomimetic
mineralized organic-inorganic hybrid macrofiber with spider silk-like
supertoughness. Advanced Functional Materials, 30.
Yuguchi, Y., Hasegawa, A., Padoł, A. M., Draget, K. I., & Stokke, B. T. (2016). Local
structure of Ca2+ induced hydrogels of alginate–oligoguluronate blends determined
by small-angle-X-ray scattering. Carbohydrate Polymers, 152, 532–540.
Zhang, J. H., Daubert, C. R., & Foegeding, E. A. (2007). A proposed strain-hardening
mechanism for alginate gels. Journal of Food Engineering, 80, 157–165.
Zhao, Z., Fang, R., Rong, Q., & Liu, M. (2017). Bioinspired nanocomposite hydrogels with
highly ordered structures. Advanced Materials, 29.