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RESEARCH ARTICLE
www.advhealthmat.de
3D Printing Unique Nanoclay-Incorporated Double-Network
Hydrogels for Construction of Complex Tissue Engineering
Scaffolds
Zhongwei Guo, Lina Dong, Jingjing Xia, Shengli Mi, and Wei Sun*
The development of new biomaterial inks with good structural formability and
mechanical strength is critical to the fabrication of 3D tissue engineering
scaffolds. For extrusion-based 3D printing, the resulting 3D constructs are
essentially a sequential assembly of 1D filaments into 3D constructs. Inspired
by this process, this paper reports the recent study on 3D printing of
nanoclay-incorporated double-network (NIDN) hydrogels for the fabrication of
1D filaments and 3D constructs without extra assistance of support bath. The
frequently used “house-of-cards” architectures formed by nanoclay are
disintegrated in the NIDN hydrogels. However, nanoclay can act as physical
crosslinkers to interact with polymer chains of methacrylated hyaluronic acid
(HAMA) and alginate (Alg), which endows the hydrogel precursors with good
structural formability. Various straight filaments, spring-like loops, and
complex 3D constructs with high shape-fidelity and good mechanical strength
are fabricated successfully. In addition, the NIDN hydrogel system can easily
be transformed into a new type of magnetic responsive hydrogel used for 3D
printing. The NIDN hydrogels also supported the growth of bone marrow
mesenchymal stem cells and displayed potential calvarial defect repair
functions.
Dr. Z. Guo, Prof. S. Mi, Prof. W. Sun
Tsinghua Shenzhen International Graduate School
Tsinghua University
Shenzhen , China
E-mail: weisun@mail.tsinghua.edu.cn
Dr. Z. Guo, L. Dong, Prof. W. Sun
Precision Medicine and Healthcare Research Center
Tsinghua-Berkeley Shenzhen Institute
Tsinghua University
Shenzhen , China
J. Xia, Prof. W. Sun
Department of Mechanical Engineering and Mechanics
Tsinghua University
Beijing , China
Prof. W. Sun
Department of Mechanical Engineering
Drexel University
Philadelphia, PA , United States
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/./adhm.
DOI: 10.1002/adhm.202100036
1. Introduction
The development of 3D scaffolds is cru-
cial for tissue engineering and regenera-
tive medicine applications.[1–3] Compared
with traditional scaffold materials (bioce-
ramics, medical metals, and thermoplas-
tic polymers), hydrogels are a promising
type of biomaterial due to their resemblance
to extracellular matrices as well as their
broad range of mechanical strengths and
biological functions.[4–6 ] The emergence of
3D printing technology enables the fabri-
cation of hydrogel scaffolds with precise
control over scaffold structure and prop-
erties for the customized repair of tissue
defects, including cartilage, bone, and soft
tissues.[7–11 ] Although some progress has
been made in this field, challenges re-
main in applying 3D printing technology
in biomedicine. The development of vari-
ous biomaterial inks with good structural
formability and mechanical strength for di-
verse tissue engineering scenarios is partic-
ularly important.
Among the available 3D printing technologies, extrusion-
based 3D printing is widely used due to its easy manipula-
tion and high efficiency in the field of tissue engineering. For
extrusion-based 3D printing, a qualified biomaterial ink must be
able to stably extrude and maintain printed structures during the
whole printing process. To satisfy this requirement, extrusion-
based 3D printing often employs the following two methodolo-
gies to maintain hydrogel structural formability: support bath
fabrication[12–14 ] and self-supporting fabrication.[15–19 ] Consider-
ing that the former methodology is not a straightforward strat-
egy and that the removal of the support bath material may lead
to difficulties on specific occasions, printing a structure directly
in air without the requirement for a support bath would be
more convenient.[4,20–22 ] The resulting 3D structures fabricated
by extrusion-based 3D printing are essentially a sequential as-
sembly of 1D filaments. Herein, the extrusion-based 3D printing
technique could be utilized as a fabrication strategy for 1D fila-
ments and 3D constructs simultaneously.
Naturally derived hydrogels are beneficial for cellular pro-
cesses due to their better mimicking of ECM than synthetic
hydrogels.[23–26 ] However, the poor mechanical properties of
most naturally derived hydrogels limit their application as
tissue substitutes. The emergence of double-network (DN)
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Figure 1. Design strategy for the fabrication of D filaments and D constructs based on the unique, mechanically strong NIDN hydrogels.
hydrogels has attracted considerable attention because of
their excellent mechanical performance and easy preparation
process.[27–30 ] Hyaluronic acid (HA) and alginate (Alg) possess
good biocompatibility and nontoxic degradation products and
are widely used in hydrogel synthesis. However, the mechan-
ical strength of these reported HA/Alg hydrogels tends to be
weak compared with that of most DN hydrogels. Our previous
work presented a mussel-inspired DN hydrogel composed of HA
and Alg, which exhibited good mechanical strength.[31] Benefit-
ting from its two-step synthesis strategy, these HA/Alg hydrogels
could be tamed into printable biomaterials. However, the hydro-
gels are partially covalently crosslinked before printing, which
leads to potential instability and discontinuity throughout the
printing process. Therefore, the key issue relies on the choice of
printing material and related crosslinking mechanisms to main-
tain the balance between good structural formability and high
mechanical strength.
Nanoclay (Laponite XLG) is a member of the smectite mineral
family and has a positively charged edge and a negatively charged
area on the top and bottom surfaces. Reports have demon-
strated that nanoclay could support the printing of different ma-
terials, such as PEGDA, Alg, gelatin, pluronics, NIPAAm, and
PNAGA.[21,22,32–34 ] However, nanoclay solutions exhibit complex
phase transform behavior, and the inclusion of polymers further
complicates the sol-gel transform behavior, thus presenting chal-
lenges in determining precise interactions between nanoclay and
polymer chains. Besides, new formulations of nanoclay-polymer
composite and their potential applications in 3D printing and tis-
sue engineering still remain to be fully explored.
In this work, we developed a novel self-supporting, self-
recovery, and 3D printable nanoclay-incorporated double-
network (NIDN) hydrogel biomaterial ink, which could be used
in the fabrication of mechanically strong 1D filaments and
3D constructs. The work presented in this paper is outlined
as follows. In Section 1, we focused on the characterization
and analysis of the potential interaction mechanisms among
nanoclay, methacrylated hyaluronic acid (HAMA), and Alg to
illustrate the reasons for the good structural formability and
unusual disintegration of the “house-of-cards” architectures. In
Section 2, the internal crosslinking networks of NIDN hydrogels
after solidification were characterized. In Section 3, diverse 1D
filaments and 3D structures were fabricated directly in air based
on the good structural formability and high mechanical strength.
Finally, the NIDN hydrogel was further used as supporting ma-
terial for magnetic microparticles to satisfy applications such
as 3D printing or magnetic guided movement. The potential
biocompatibility and calvarial defect repair functions were also
evaluated.
2. Results and Discussion
As shown in Figure 1, nanoclay, HAMA, Alg, and Irgacure 2959
were first combined to produce a novel self-supporting and self-
recovery biomaterial ink. Through the solidification path, the ink
was crosslinked by UV light and Ca2+ions, which induced the
covalent crosslinking of HAMA and Alg-Ca2+ionic crosslink-
ing, respectively. With the interactions of nanoclay, this physical-
chemical double crosslinking network resulted in mechanically
strong NIDN hydrogels. Through the extrusion path, the hydro-
gel could form different 1D filaments and 3D constructs with the
assistance of an extrusion-based 3D printing technique. The in-
clusion of HAMA in our work disintegrated the “house-of-cards”
architecture. Latter addition of Alg transforms the mixture into
a gel-like substance with dynamic fracture-recovery behaviors,
which ensures the printing effect. Finally, these resulting struc-
tures could acquire excellent mechanical properties after further
solidification and thus could be applied as potential tissue engi-
neering scaffolds.
2.1. Synthesis and Characterization of Nanoclay-Incorporated
Double-Network (NIDN) Hydrogel Precursors
As shown in Figure 2, the nanoclay was first fully dissolved in
ultrapure water, which formed a homogenous and transparent
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Figure 2. Synthesis process and rheological properties of the NIDN hydrogel precursors. a) Representative images of the i) % Clay sol-like mixture,
ii) % Clay-% HAMA mixture, and iii) % Clay-% HAMA-% Alg gel-like mixture. b) Oscillatory frequency sweep of NIDN hydrogel precursors with
dierent compositions (G′: storage modulus; G″: loss modulus).
Figure 3. SEM images of NIDN hydrogel precursors with dierent compositions.
sol-like mixture. This change was benefited from the nanoclay’s
unique “house-of-cards” structure, which could serve as an inter-
nal scaffold of hydrogel composites.[21,22,35 ] However, the addition
of HAMA turns the sol-like mixtures into a turbid liquid solution,
which is unusual. The further inclusion of Alg transforms the
mixture into a gel-like substance. These physical changes were
also confirmed by the oscillatory frequency sweep (Figure 2b). By
regulating the nanoclay concentration from 1 to 7%, the storage
modulus of the mixtures increased obviously, which was consis-
tent with the higher crosslinking density of the nanoclay. Then,
the mixtures transformed into a viscous liquid (G′<G″)within-
creasing amounts of HAMA. The final addition of Alg endows
the mixtures with elastic properties again (G′>G″).
Nanoclay (Laponite XLG) possesses a large surface area and
an anisotropic surface charge distribution, including a positively
charged area along the edge and a negatively charged area on
the top and bottom surfaces.[22,35 ] In our work, the addition
of HAMA disintegrated the already formed “house-of-cards”
network, which transforms the sol-like mixture into a liquid
mixture. We hypothesize that the addition of negatively charged
HAMA polymers interacts with the positively charged edge
of the nanoclay, which disrupts the “house-of-cards” network
based on electrostatic interactions between their surface and
edge. Therefore, the “house-of-cards” network collapsed. Further
inclusion of Alg increases the viscosity of the mixtures through
physical entanglement with HAMA and interactions with nan-
oclay, which helps to reacquire gel-like properties. To test this
hypothesis, SEM was first used to give a detailed description
of the morphology change through the preparation process
(Figure 3). Before dissolving in water, the nanoclay exhibited
a blocky angular structure, which was the result of a single
nanoclay platelet layer. When the layered nanoclay is completely
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Figure 4. Interactions between NIDN hydrogel precursors with dierent compositions. a) Optical image, b) UV–vis spectra, and c) FT-IR spectra of the
hydrogel precursors.
and randomly dispersed, flat and smooth exfoliated clay is ob-
tained. This morphological change indicated that an exfoliation
process transformed layered clay into exfoliated clay, which was
also demonstrated by the XRD curves (Figure S1, Supporting
Information). Compared with layered clay, amorphous exfoliated
clay has fewer diffraction peaks.[36,37 ] The addition of HAMA
leads to a partially connected platelet structure with a rough
edge, which indicates that nanoclay with positive edges was likely
enveloped by negatively charged HAMA. Further incorporation
of Alg into the Clay-HAMA mixtures exhibited a rough and
irregular surface with larger areas than Clay-HAMA composites,
which could be the result of Alg polymers forming physical
entanglement with HAMA and interactions with nanoclay.
The interactions among nanoclay, HAMA, and Alg were also
investigated through UV–vis spectra and zeta potential measure-
ments. As shown in Figure 4a, solutions of nanoclay, HAMA, and
Alg were transparent. However, the addition of HAMA into the
nanoclay solutions leads to a slightly opaque liquid. Further inclu-
sion of Alg aggravated this phenomenon. This phenomenon was
confirmed by the UV–vis spectra, which showed that the trans-
mittance of the mixture weakened after the addition of HAMA
and Alg (Figure 4b, Table S1, Supporting Information). As shown
in Table S1, Supporting Information, aqueous solutions of nan-
oclay exhibited negative zeta potential values (−10.67 mV), which
is reasonable because the number of positive charges on its
edge is approximately 10% of the number of negative charges
on its faces.[38,39 ] Electrostatic interactions were expected if the
two components possess opposite charges. Upon the addition of
HAMA, the zeta potential value decreased to −36.93 mV, con-
firming that the negatively charged HAMA polymer wrapped
around the nanoclay’s positive edges, thereby increasing the per-
centage of negative charges versus positive charges. Further in-
clusion of Alg decreased the zeta potential value to −70.57 mV.
The FT-IR spectra of the NIDN hydrogel precursors with dif-
ferent compositions are depicted in Figure 4c. The characteristic
peaks of nanoclay, HAMA, and Alg show some differences. The
characteristic peaks of nanoclay situated at 963 cm−1(-Si–O–Si-,
stretching vibration) were observed in the FT-IR spectrum. Com-
pared with the FT-IR spectrum of the nanoclay, the spectra of the
Clay-HAMA and Clay-HAMA-Alg hydrogel precursors exhibited
a shift in the Si–O–Si stretching vibration peak towards higher
frequencies (990 cm−1), which can be attributed to the interac-
tion between the HAMA, Alg, and nanoclay through the Si-OH
groups.[40,41 ] A broad band at 3300 cm−1was assigned to the vibra-
tion of the hydroxyl groups of HA. The obvious broadening of the
hydroxyl band (3300 cm−1) in the spectrum of the Clay-HAMA
hydrogel precursors might be due to the hydrogen bonding in-
teractions between the HAMA backbone and nanoclay. Other
peaks at 3610 and 3402 cm−1were reduced or shifted to lower
wavenumbers. In addition, new vibrations were not observed,
indicating that no new covalent bonds were formed among the
nanoclay, HAMA, and Alg. These results confirmed that nanoclay
acted as a physical crosslinker to interact with HAMA and Alg.
2.2. Solidification Path: Characterization of the Internal
Crosslinking Networks of NIDN Hydrogels
With the assistance of UV light and Ca2+ions, the NIDN hydrogel
precursors could be solidified to enhance structural integrity and
strength. To better understand the internal crosslinking mecha-
nism, three kinds of hydrogels were compared: HAMA SN hydro-
gels, HAMA-Alg DN hydrogels, and NIDN hydrogels (Figure 5).
The proposed crosslinking mechanism and physical appearance
of these three kinds of hydrogels are shown in Figure 5a,b.
First, HAMA SN hydrogels were formed by the photopoly-
merization of HAMA solutions. The synthetic route for HA-MA
is described in Figure S2a, Supporting Information. An NMR
analysis confirmed methacrylate conjugation to the HA back-
bone based on the appearance of vinyl proton peaks (shaded
red) at 𝛿=5.8 ppm and 𝛿=6.25 ppm (Figure S2b, Supporting
Information).[42] The 1H-NMR spectra show that the modifica-
tion ratio of HAMA was ≈25%. The failure stress of the HAMA
SN gels was increased by increasing the polymer concentration
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Figure 5. Characterization of the NIDN hydrogels. a) Schematic illustration of the crosslinking mechanism of the i) HAMA SN hydrogels, ii) HAMA-Alg
DN hydrogels, and iii) NIDN hydrogels. b) Photographs of the hydrogels. c) Swelling property of the hydrogels. Data are shown as the mean ±standard
deviation (n =). d) Compressive stress-strain curves of hydrogels with dierent compositions. e) Tensile stress-strain curves of hydrogels with dierent
compositions.
(3, 4, and 5%) (Figure S3, Supporting Information). However,
the mechanical strength of HAMA SN hydrogel is much weaker
than the requirement for tissue engineering applications. Two
different strategies were incorporated to enhance the mechani-
cal strength of covalently crosslinked HAMA SN hydrogels, in-
cluding a reversible Alg-Ca2+ionically crosslinked network and
nanoclay as a matrix filler. The resulting NIDN hydrogels could
withstand 500 g of weight or lift a 500 g steel block (Figure S4,
Supporting Information).
Cryo-SEM was utilized to demonstrate the structural differ-
ence between these hydrogels. As shown in Figure S5, Support-
ing Information, we can observe the distribution of the polymer
network within the frozen gels. The frozen HAMA SN hydro-
gels exhibit a very fine/porous structure. The frozen HAMA-Alg
DN hydrogels, quite differently, show two obvious phases, with
one composed of a fine/porous polymer network and the other
composed of a dense polymer network. The distinct properties
and distributions of polymer networks should be closely related
to the different mechanical performances of the hydrogels. The
covalently crosslinked HAMA SN hydrogel is fragile and cannot
withstand large localized stress. However, the incorporation of
Alg chains will interpenetrate within the covalently crosslinked
HAMA network, and the Alg chains will be ionically crosslinked
by Ca2+. These two crosslinking networks have a cooperative
effect, which will enhance the mechanical properties. Further-
more, the incorporation of nanoclay as a matrix filler distributed
through the whole polymer network made the hydrogel structure
much denser, and its internal pores could not even be seen.
The swelling properties of the three hydrogels were investi-
gated by measuring the wet weight during 5 days of incubation in
ultrapure water at 37 °C (Figure 5c). All three hydrogels reached
an equilibrium state after one day of incubation, which remained
constant during the rest of the incubation period. There was an
obvious increase in the swelling ratio of the HAMA SN hydrogels
compared with the other two hydrogels. However, the swelling
ratio of the NIDN hydrogels remained almost unchanged dur-
ing the incubation period. This anti-swelling behavior would be
beneficial for implanted biomaterials in tissue engineering ap-
plications due to the decreased swelling pressure towards the
surrounding tissues.[43] A possible reason is that the formation
of the double-crosslinked network and the incorporation of nan-
oclay matrix fillers made the hydrogel structure more compact
and mechanically strong, which prevented the penetration of wa-
ter molecules and maintained the internal structure.
The mechanical data obtained from the compressive and ten-
sile tests is shown in Figure 5d,e. The compressive failure stress
of the HAMA-Alg DN hydrogels reached 1200 kPa, which was
more than 12.6 and 3.4 times higher than those of the HAMA SN
hydrogels (95 kPa) and Alg SN hydrogels (353 kPa), respectively
(Figure S7, Supporting Information). The compressive modulus
of the HAMA-Alg DN hydrogels reached 350 kPa, which was
9.7 and 4.7 times higher than that of the HAMA SN hydrogels
(36 kPa) and Alg SN hydrogels (74 kPa). In addition, the results
of the tensile test exhibited a similar mechanical reinforcement
trend. The combination of the above two single networks led to
significantly higher failure stress and modulus of the HAMA-Alg
DN gels compared with the SN gels. This improvement was bene-
fit from the combination of a covalently crosslinked network and
a reversible Alg-Ca2+ionic crosslinked network. The reversible
Alg-Ca2+cross-linking networks can bear stress and unzip from
ionically cross-linked points, thus supplying an energy dissipa-
tion mechanism. Therefore, the HAMA-Alg DN hydrogels exhib-
ited improved mechanical performance.
The inclusion of nanoclay in hydrogel matrices is an ef-
fective method of enhancing the mechanical properties of
hydrogels.[21,32 ] In our work, the incorporation of nanoclay into
HAMA-Alg DN hydrogels also increased the compressive failure
stress and modulus from 1200 kPa and 350 kPa to 4000 kPa and
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Figure 6. Self-supporting and self-recovery behaviors of the NIDN hydrogel precursors. a) Images showing the injection of the hydrogel precursors
through a syringe to form self-supporting filaments. b) Continuous flow curves of % HAMA, % Alg, % Clay,%Clay-%HAMA, and %Clay-%HAMA-
%Alg hydrogel precursors. c) G′and G″from continuous strain sweep with alternate high strain (%, shaded green) and low strain (%) conditions
showing self-recovery ability.
640 kPa, respectively (Figure S7, Supporting Information). The
tensile stress-strain test also exhibited much better mechanical
performance for the NIDN hydrogels. The resilient properties
were investigated quantitatively with repetitive loading (Figure
S8, Supporting Information, 60% strain). One prominent fea-
ture in the repeated stress-strain curves was the emergence of
hysteresis loops between the loading-unloading curves. The de-
formed hydrogel can achieve 88.1% hysteresis in the first loading-
unloading cycle, indicating partial recovery of the dissipative
properties. Based on the above results, the NIDN hydrogels pos-
sess anti-swelling properties and high mechanical strength that
make them suitable for biomaterial applications.
2.3. Extrusion Path: Synthesis and Characterization of the 1D
Filaments and 3D Constructs
With the assistance of nanoclay, the NIDN hydrogel precursors
acquired self-supporting and self-recovery abilities simultane-
ously, which ensured good structural formability (Figure 6).
The NIDN hydrogel precursors could be loaded into a medical
syringe and then extruded into self-supporting filaments or
different shaped molds (Figure 6a, Movie S1, Supporting In-
formation). The rheological properties of hydrogel precursors
play a crucial role in their application as injectable bioinks in 3D
printing.[31,44 ] As shown in Figure 6b, the viscosity of HAMA and
Alg was lower than that of nanoclay at a low shear rate. However,
the viscosity of the nanoclay and its mixture with HAMA and
Alg decreased significantly with increasing shear rate, thus
demonstrating typical shear-thinning properties. Moreover, the
step-strain time-sweep measurement revealed rapid and full re-
covery of the hydrogel structure following repeated high strains
(Figure 6c). This rapid and reversible transition from solid-like to
liquid-like behavior when an external force is applied makes the
NIDN hydrogel precursors highly suitable for extrusion-based
3D printing. The rheological behavior was enabled by the phys-
ical crosslinks between nanoclay particles and polymers, which
could form and break dynamically. Upon exposure to UV light
and Ca2+solution, covalent and ionic crosslinks were induced to
increase the structural integrity and mechanical strength (Figure
S9, Supporting Information).
As a fiber fabrication strategy, the traditional wet-spinning
technique usually needs a “coagulating bath” to allow the materi-
als to solidify fast enough to obtain the fibers. This liquid-to-solid
transition process requires a complex and time-consuming reg-
ulation of the material compositions and coagulating bath.[45,46 ]
Benefitting from the good structural formability, the NIDN
hydrogels could be made into continuous 1D filaments and 3D
constructs simultaneously (Figure 7,Figure 8). As illustrated in
Figure 7a, we could fabricate meter-long, straight NIDN hydro-
gel filaments with uniform diameters, which could be collected
like normal threads (Movie S2, Supporting Information). With
different nozzle gauges, straight filaments with diverse diame-
ters such as 100, 200, and 300 µm were fabricated successfully
(Figure 7b). Good flexibility was demonstrated by easily making
knots by hand. Benefitting from the flexibility, a two-ply yarn
with clear and regular boundary was also produced by wrapping
two straight filaments tightly (Figure 7c). The element mapping
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Figure 7. Fabrication of continuous D filaments based on NIDN hydrogels. a) Representative images of a straight filament (. m long, m
diameter) and straight filament rotating around the collecting rod. b) Optical images of straight filaments with diameters of , , and m,
respectively. A knot made from a straight filament showing flexibility. (Scale bar: m) c) SEM images of a two-ply yarn made from a straight filament.
Element mappings of the two-ply yarn. d) Tensile stress-strain curves of a straight filament (filament diameter: m). e) Filaments could be made
into spiral shapes that could be stretched to large strains, and then a handmade clover was fabricated by using spiral shape filament.
results demonstrate that Si, Mg, Cl, and Ca elements were well
colocalized in the two-ply yarn. Figure 7d is the typical tensile
stress-strain curve of the hydrogel filament. The tensile fracture
strength can reach several MPa, which could satisfy many appli-
cation scenarios. In addition to the straight filaments, a spinning
rod was used to make spiral-shaped filaments (Figure 7e, Movie
S3, Supporting Information). These spiral-shaped filaments
could be manually stretched from an initial 2 cm to a final length
of 8 cm. A handmade clover was also prepared. The resulting
spiral-shaped filaments did not fracture through the whole
stretching or operation process, which was benefit from the
good flexibility and mechanical properties of NIDN hydrogels.
With the assistance of 3D models, complex structures were
printed layer by layer and then an integral architecture was estab-
lished (Figure 8a). 3D constructs with various geometries were
successfully printed, including pyramid structures, vascular-like
structures, human nose-like structures, and human ear-like
structures. Due to the effect of gravity, 3D printing of dangling
structures is always a challenge. To further demonstrate the
printing fidelity of the NIDN hydrogel, we printed several tubu-
lar structures with different incline angles (90°, 60°, and 45°).
As shown in Figure 8b, the predesigned height of the inclined
tube is 4.5 mm, while the resulting height is only slightly lower
than the theoretical value. The injection of green dye solution
into the hollow vascular-like structure before solidification is
clearly shown in Figure 8c. No leakage was observed because the
deposited layers connected very well to ensure that the wall was
seamless. This result demonstrates that a tubular architecture
can be printed with high fidelity and maintain its integrity
throughout the printing process. Once the printing process was
finished, UV irradiation and Ca2+-induced ionic crosslinking
were employed to solidify the printed structures. As shown in
Figure 8d, the printed nose or ear structures could resist large
bending and twisting deformation due to their good mechanical
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Figure 8. D printing of complex architectures based on the NIDN hydrogels. a) Representative images of a printed pyramid, vascular-like structure,
nose-like structure, and ear-like structure. b) Maximum height of D printed inclined tubes as a function of incline angle. Data are shown as the mean
±SD (n =). c) Green dye solution injection through the hollow vascular-like structure. d) Bend and twist behavior of the D printed human ear and
nose. Scale bars: mm.
properties. Based on the above results, NIDN hydrogels could
be used as excellent printing materials with high shape fidelity
and good mechanical performance.
2.4. Preparation and Characterization of Magnetic Responsive
NIDN (M-NIDN) Hydrogels and Their Application in 3D Printing
3D printing of stimuli-responsive hydrogels is of great signif-
icance, and magnetically responsive hydrogels have attracted
much attention because of their unique remote controllable
properties.[47–49 ] The proposed NIDN hydrogel system in our
work can easily be transformed into a new type of magnetic re-
sponsive hydrogel used for 3D printing. As shown in Figure 9a,
magnetic Fe3O4microparticles were incorporated in the NIDN
hydrogel precursors. With increasing Fe3O4concentrations (w/v)
from 2 to 12%, the maximum actuation distances extended from
4 to 27 mm (Figure S10a, Figure S10b, Movie S4, Supporting In-
formation). As shown in Figure S10c, Supporting Information,
the magnetic Fe3O4microparticles were tightly adhered to by the
surrounding polymer networks, which might be due to the sur-
face Fe ions interacting with the numerous carboxyl and hydroxy
groups in HA or Alg. In addition, the impact of Fe3O4content on
the rheological properties of the NIDN hydrogel precursors was
investigated (Figure 9b). With increasing Fe3O4content, the stor-
age modulus (G′) and loss modulus (G″) were elevated. These
results suggested that the dispersion of magnetic microparti-
cles enhances the rheological properties of the hydrogel precur-
sors, which is probably due to the electrostatic interactions be-
tween magnetic microparticles and NIDN hydrogel precursors.
The step-strain time sweep measurement revealed rapid and full
recovery of the magnetic hydrogel structures following repeated
high strains (Figure 9c). This rheological behavior demonstrated
that the incorporation of magnetic Fe3O4microparticles had no
adverse effect on the self-recovery properties of the NIDN hydro-
gel system. Benefitting from the above rheological properties, the
prepared M-NIDN hydrogel ferrofluids could be extruded into
a self-supporting, uniform filament (Figure 9d). A lattice struc-
ture with magnetic responsiveness was also printed (Movie S5,
Supporting Information). Slic3r was used to translate the 3D
dolphin model into a layer-by-layer fiber deposition pathway. As
shown in Figure 9e, better shape fidelity of the printed dolphin
was achieved when the fill density of 3D printing elevated (Fig-
ure 9e). In addition, the magnetic dolphin robot achieved rota-
tional movement when the magnetic field (NdFeB magnet, N35)
was programmed to rotate.
In nature, many tissues are composed of numerous repeat-
ing functional units.[50] Therefore, the control movement of re-
peating functional units plays a significant role. Compared with
other strategies, magnetically driven navigation or assembly is
rapid and cost-effective.[51,52 ] In our work, the M-NIDN hydro-
gel filament could be cut into a series of repeating, magnetic
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Figure 9. Preparation and characterization of magnetic NIDN (M-NIDN) hydrogels for D printing and magnetic guided movement. a) Schematic illus-
tration of the preparation of M-NIDN hydrogel ferrofluids and the resulting extrusion filament. MMPs represent magnetic microparticles. b) Oscillatory
frequency sweep of the NIDN hydrogel precursors before and after the inclusion of magnetic microparticles. c) G′and G″from continuous strain sweep
with alternate high strain (%, shaded green) and low strain (%) conditions. d) Digital images of the magnetic ferrofluids, extrusion filament and
D printed lattice structure with magnetic responsive behavior. e) Top view of a D printed, magnetic dolphin robot with low or high filling density. The
magnetic dolphin robot could be actuated by an outer programmed magnetic field (NdFeB magnet, N).
cylindrical mini-gels (Figure S11a, Supporting Information). Af-
ter the subsequent solidifying process, these magnetic mini-gels
acquired integrity and stability. By controlling the magnetic field,
they were navigated and moved forward from one end of the
tube to another successfully (Figure S11b, Supporting Informa-
tion). In addition, a spheroid-like construct assembled by mag-
netic mini-gels was also produced by utilizing a magnetized rod.
To collect the magnetic mini-gels, the distance between the rod
and mini-gels should be shorter than the maximum actuation
distance. Further works are needed to improve the controllability
and precision of the system as a potential biotechnology tool.
2.5. Biocompatibility of NIDN Hydrogels and Their Application in
Calvarial Defect Reconstruction
Bone marrow derived mesenchymal stem cells (BMSCs) were
used to assess the biocompatibility of the NIDN hydrogels. As
shown in Figure S12a, Supporting Information, BMSCs gradu-
ally spread and covered the scaffold surface and exhibited irreg-
ular, filopodia-like structures. Cell attachment was further con-
firmed by the F-actin/DAPI staining image shown in Figure S13,
Supporting Information, in which obvious cytoskeleton and cel-
lular junctions were observed after 7 days of incubation. Further-
more, we used the Cell Counting Kit-8 (CCK-8) to investigate
the cell proliferation ability on the hydrogel scaffolds. As shown
in Figure S12b, Supporting Information, BMSCs seeded on the
scaffolds maintained a high proliferation rate throughout the cul-
ture period, thereby confirming that the hydrogels were bene-
ficial for cell attachment and growth. All these results demon-
strated that our hydrogel system could support cell attachment
and proliferation.
ALP is an early indicator of the osteogenic phenotype and
plays an important role in the initial stages of bone matrix
mineralization.[53] The level of ALP is related to the differenti-
ation of osteoblasts, and greatly affects the process of cell-matrix
mineralization of osteoblasts. We performed ALP staining to
evaluate the osteogenic differentiation ability of BMSCs on NIDN
hydrogel scaffolds at 3, 7, and 14 days (Figure S14, Supporting In-
formation). With the extension of incubation time, the ALP posi-
tive area gradually became larger and denser, which indicates that
ALP activity is gradually increasing. Along with the proliferation
and differentiation process, cells gradually began to contact each
other and formed cell clusters. Then, the cell boundary became
unclear and a large amount of ALP and ECM were produced after
culturing for 14 days. This result is in accordance with the ALP
activity assay.
We also investigated the bone regeneration ability of the NIDN
hydrogels in a rat calvarial defect model (Figure 10a, Figure 10b).
The non-porous scaffold and 3D printed porous scaffold (pore
size: 300 µm) were used to evaluate the repair efficiency. During
8 weeks of the experiment, none of the rats showed any defect
complications such as inflammatory soft tissue swelling or ec-
topic bone formation. The Micro-CT scanning images and quan-
titative analyses were utilized to evaluate the regenerated bones
(Figure 10c,d). Newly generated bone in the original cylindrical
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Figure 10. The calvarial bone regeneration after the implantation of hydrogel scaolds for and weeks in vivo. a) Macroscopic and fluorescence
images of the non-porous scaold and D printed porous scaold based on the NIDN hydrogels. b) Schematic of the surgical operation that places the
scaolds to seal o the mm diameter cylindrical calvarial defects of rats. c) Micro-CT reconstructed images of calvarial defects after implantationfor
weeks and weeks. d) Quantitative statistics of bone tissue volume/total tissue volume (BV/TV), bone mineral density (BMD), trabecular thickness
(Tb.Th), and trabecular number (Tb.N) (n =, *p<., **p<.).
defect was observed. The non-porous and porous scaffolds all ex-
hibited better bone regeneration effects than the control group.
The porous scaffold group was more efficient at promoting new
bone regeneration than the non-porous group. Quantitative anal-
yses of the bone tissue volume/total tissue volume (BV/TV), bone
mineral density (BMD), trabecular thickness (Tb.Th), trabecular
number (Tb.N) also confirmed the above findings. Our results
demonstrated that nanoclay incorporated DN hydrogels could
promote bone regeneration of calvarial defect. In addition, pre-
vious studies have demonstrated that cells prefer scaffolds with
pore sizes between 200 and 500 µm for better proliferation and
differentiation during osteogenesis.[27,54 ] Therefore, porous scaf-
folds with uniform 300 µm pore sizes were fabricated through
extrusion-based 3D printing to improve the bone regeneration
effect.
The degradability of scaffold materials is crucial for materi-
als used in bone tissue engineering. To understand the degra-
dation process of the NIDN hydrogels in vitro, the hydrogel sam-
ples were incubated in a culture medium for 1, 3, 7, and 14 days
with the addition of hyaluronidase (100 U mL−1,Sigma).As
shown in Figure S15, Supporting Information, the remaining
weight of NIDN hydrogels was reduced by 37% after 14 days
of incubation. Besides, the compressive modulus of the NIDN
hydrogels declined from 640 to 73 kPa after 14 days of incu-
bation. The NIDN hydrogel system is composed of three types
of crosslinking networks including a nanoclay-based physically
crosslinked network, a covalently crosslinked HAMA network,
and an ionically crosslinked Alg-Ca2+network. One explanation
for the decreased remaining weight and compressive modulus
could be that the ions in the culture medium diffuse and inter-
change within the hydrogel network during incubation, which
leads to the disintegration of ionic Alg-Ca2+crosslinking. Eventu-
ally, this activity degrades the hydrogel structure and causes the
hydrogel to lose its components and mechanical strength over
time. This phenomenon has been commonly observed in hydro-
gels based on ionic crosslinking networks.[31] Besides, the ad-
dition of hyaluronidase could degrade the backbone of HAMA,
which leads to the disintegration of covalent HAMA crosslinking
networks.[55]
Furthermore, Van Gieson’s staining of calvarial undecalcified
sections was performed to determine the degradation of NIDN
hydrogels in vivo and tissue regeneration induced by this process
(Figure S16, Supporting Information). After 2 weeks of implan-
tation, a small quantity of new bone formed in the defect site
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for both the hydrogel-treated group and the non-treated group.
In addition, only a small portion of hydrogel scaffold materials
could be observed at the defect site and the original structure was
already disintegrated. Nearly all the defect space in the hydrogel-
treated group was filled with a large amount of connective tissues
remarkably larger than that in the control group. The boundary
between the new bone and connective tissues was in close contact
without any gap. These connective tissues successfully provided
a sealed space to allow bone regeneration. After 4 weeks of im-
plantation, the newly formed bone generated from the hydrogel-
treated group was obviously thicker and more abundant than that
of the control group. The residual bone defect was filled with or-
derly arranged collagen fibers and connective tissues, which may
serve as an osteoconductive ECM. The implanted hydrogel scaf-
fold was not distinguishably observed in the defect site, which in-
dicates that the hydrogel was largely degraded in the cylindrical
calvarial defect. Compared with the control group, the hydrogel
scaffold showed an improved healing effect with new bone and
soft tissue formation.
From the perspective of composition, bone tissue is
largely composed of organic components and inorganic
components.[55,56 ] Recent studies report that nanoclay (Laponite),
an effective rheological enhancer, can be incorporated into var-
ious polymers including polyethylene glycol,[57] gelatin,[58]
chitosan,[59,60 ] and Alg[61] to improve their physical and mechan-
ical properties. Although each nanoclay-based material exhibits
beneficial effects for its specific purpose, new formulations
combined with appropriate manufacturing methods are still
attractive. The details of different nanoclay-polymer interaction
mechanisms and potential repair effects in vivo remain to be
fully explored.
Y. Kim et al. previously synthesized a bisphosphonate func-
tionalized HA to increase the interactions with nanoclay par-
ticles, thus generating nanoclay-HA composite hydrogels with
enhanced mechanical properties and slow release of bone mor-
phogenetic protein-2 (BMP-2).[62,63 ] B. O. Okesola et al. de-
veloped a co-assembling system that integrates HA tyramine
(HA-Tyr), bioactive peptide amphiphiles (GHK-Cu2+), and nan-
oclay (Laponite) to engineer hydrogels with physical, mechani-
cal, and biomolecular signals that can be tuned to enhance bone
regeneration.[64] Nevertheless, the mechanical strength of these
hydrogels remains weak, and proper manufacturing techniques
are neglected, which may further improve the repair effect of
bone defects.
In our study, the combination of organic HAMA, Alg, and in-
organic nanoclay served as biocompatible and osteoconductive
ECM mimics, which displayed potential calvarial defect repair
functions. HAMA hydrogels could provide organic microenvi-
ronments for bone formation, because their backbone polymer
could interact with mesenchymal stem cells (MSCs) through sev-
eral cell surface receptors expressed by MSCs, including CD44
and CD168.[65,66 ] However, it is still difficult to efficiently induce
bone regeneration with HAMA hydrogels alone due to the lack
of mechanical strength and inorganic components.[55] There-
fore, an Alg-Ca2+ionic crosslinking network and nanoclay were
added to improve the mechanical properties and mineralization
of the formed tissues, thus better mimicking native bone tissue.
Benefitting from its good structural formability and mechanical
strength, our system could be utilized as a biomaterial ink to
print different hydrogel-based tissue engineering scaffolds. How-
ever, the high degradability of NIDN hydrogels limits their long-
term application in bone tissue engineering. Further works will
be needed to enhance their stability, which may improve the re-
pair effect of bone defects.
3. Conclusions
In this work, mechanically strong NIDN hydrogels with good
structural formability and osteoinductivity for calvarial bone
regeneration are reported. The NIDN hydrogel system is com-
posed of three types of crosslinking networks including a
nanoclay-based physically crosslinked network, a covalently
crosslinked HAMA network, and an ionically crosslinked Alg-
Ca2+network. The interaction mechanism between nanoclay
and polymers is carefully investigated through rheological
tests, SEM, UV–vis spectra, FT-IR spectra, and zeta potential
tests. Driven by the electrostatic interactions and other physi-
cal interactions among nanoclay, HAMA, and Alg, the NIDN
hydrogel precursors still acquired good structural formability
even without the “house-of-cards” internal scaffolds formed by
nanoclay. With the incorporation of magnetic microparticles,
NIDN hydrogels can easily be transformed into a new type of
magnetic responsive hydrogel used for 3D printing. Finally,
Micro-CT and Van Gieson’s staining results demonstrated that
3D printed NIDN hydrogel scaffold possesses biocompatibility
and improved healing effect with new bone and soft tissue
formation. This study provides a promising biomaterial ink that
offers an opportunity to customize personalized scaffolds for
precision therapy of tissue defects. It also provides a scientific
and application basis for hydrogel design and 3D bioprinting to
promote the advanced manufacturing of biomaterials.
4. Experimental Section
Materials and Reagents:HA (MW – kDa) was purchased
from Lifecore Biomedical (Chaska, MN, USA). Methacrylic anhy-
dride (MA), Irgacure , and ultrapure Alg were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Nanoclay (Laponite XLG,
[Mg.Li. SiO(OH)]Na. ) (BYK, Wesel, Germany) and FeO
microparticles (.%, m, Beijing DK Nanotechnology Co. LTD) were
used as received. Other analytical grade chemicals were purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure
water was obtained using a NANO pure Infinity system (Barnstead
Thermolyne, Sigma-Aldrich).
Preparation of Nanoclay-Incorporated Double-Network (NIDN) Hydro-
gels:For the preparation of NIDN hydrogels, Nanoclay (Laponite XLG,
% w/v) was first fully dissolved in ultrapure water under vigorous stirring,
followed by HAMA (% w/v) and Irgacure (.% w/v). HAMA was
synthesized according to the previous report.[] Second, Alg (% w/v)
was added to the above solution. The mixture was then stirred vigorously
and allowed to homogenize for h to acquire the NIDN hydrogel precur-
sors, which could be used directly in extrusion-based D printing. Third,
the NIDN hydrogel precursors were irradiated by UV light (wavelength:
nm, light intensity: mW cm−) for min to induce the forma-
tion of a covalent crosslinking network among HAMA. After UV irradia-
tion, the hydrogels were immersed in a . Caclsolution for min to
form Alg-Ca+ionically crosslinked network. To illustrate the mechanical
properties of the dierent hydrogels, HAMA single network (HAMA SN)
hydrogels, Alg single network (Alg SN) hydrogels, and HAMA-Alg double-
network (HAMA-Alg DN) hydrogels without the addition of nanoclay were
also prepared.
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Characterization of the Hydrogels:The physical and mechanical prop-
erties of the hydrogels were characterized according to the previous
report.[] Three replicates were used for each hydrogel composition. Rhe-
ological measurements were performed at °C on a rheometer (MCR,
Anton Paar, Austria) using a plate-plate geometry ( mm diameter).
All tests were performed immediately after transferring the gel sample
onto the sample stage. Frequency sweeps were performed at % strain
from .– Hz. The shear viscosity was measured at shear rates from
– s−.
To measure the equilibrium swelling ratio, all samples were completely
lyophilized. Then, the dried samples were incubated in ultrapure water
at °C. The wet weight of the hydrogels was measured at several time
points (days , , , , , and ) during incubation. The swelling ratio was
calculated using the equation (Ws−Wo)/Wo×, where Wsrepresents
the weight of the swollen hydrogel at each time point and Worepresents
the weight of the dried hydrogel on day . To determine the degradation
rate of the NIDN hydrogels, the hydrogel samples were incubated in cul-
ture medium (DMEM) for , , , and days with hyaluronidase treat-
ment ( U mL−, Sigma). The remaining weight and compressive mod-
ulus were measured.
To elucidate the interior crosslinked network morphology of the hydro-
gels, cryo-SEM images were obtained with an FEI Quanta microscope
(FEI, USA). The hydrogel sample was frozen in nitrogen slush at − °C.
Then, the sample was transferred under vacuum to a chamber of the
cryo-fracture apparatus (Quorum PPT Cryo Transfer System), where
it was fractured at − °C. After sputter coating with gold, the sample
was transferred into the SEM chamber, where it remains frozen during
imaging on another cold stage and cooled by liquid nitrogen.
The zeta potentials of the NIDN hydrogel mixture solutions with dif-
ferent compositions were determined with a Zetasizer NanoZS appa-
ratus (Malvern Instruments) at °C. The zeta potential value was the
average of at least three successive measurements. The transparency
of the solution was detected with a UV–vis spectrophotometer (Hitachi
U-) at °C. All samples were properly diluted in ultrapure water
(Milli-Q).
Fourier transform infrared spectroscopy (FT-IR, Nicolet iS) was used
to characterize the lyophilized hydrogel samples. The morphology of the
nanoclay was measured by wide-angle X-ray diraction (XRD). XRD was
performed using an Advance wide angle X-ray diractometer with Cu K𝛼
radiation and 𝜃ranging from ° to ° at a scanning rate of ° min−on
a Bruker D ADVANCE instrument.
3D Printing of the NIDN Hydrogels into 1D Filaments and 3D Constructs:
First, the NIDN hydrogel precursors were loaded into a syringe, which was
fixed at the extrusion printhead. Slicr was used to translate the D model
into a layer-by-layer fiber deposition pathway. Second, the printing material
was extruded on a moving platform (D printer: SunP CPD/BioMaker)
based on the model. During printing, the hydrogel precursors experienced
shear-thinning inside the extrusion needle and quickly recovered their vis-
cosity upon exiting. Therefore, these mixtures could be used to print var-
ious D filaments and D structures without requiring a support bath.
Once the whole printing process finished, UV light (wavelength: nm,
light intensity: mW cm−) was directed at the printing scaold for
min. After UV irradiation, the scaolds were immersed in a . Cacl
solution for min. Unless otherwise stated, the printing process was per-
formed at room temperature with the extrusion speed and moving speed
of the nozzle set to mm s−and a needle gauge of G (inner diameter:
m).
Preparation of the Magnetic NIDN (M-NIDN) Hydrogels:The NIDN
hydrogel precursors were prepared as described above. FeOmicroparti-
cles were added into the NIDN hydrogel precursors with vigorous stirring
to generate magnetic homogenous ferrofluid. After vigorous stirring, the
mixtures were allowed to homogenize for h and degassed in a vacuum
chamber for – min. To measure the maximum actuation distance,
FeOmicroparticles with dierent concentrations (, , , and % w/v)
were added to the hydrogel precursors. Oscillatory frequency sweeps of
the hydrogel precursors with dierent FeOcontents (, , , , and %)
were performed at % strain from .– Hz. To perform D printing, mix-
tures of FeOmicroparticles (% w/v) with the NIDN hydrogel precursor
were used. A NdFeB magnet (N) was used to remotely guide the mag-
netic hydrogel.
BMSCs Seeding on the NIDN Scaffold:
Cell Culture:All cell and animal procedures were performed accord-
ing to a protocol approved by the Animal Ethics Committee of Tsinghua
University. BMSCs were isolated from whole bone marrow aspirates of the
distal femur of Sprague–Dawley (SD) rats ( weeks old). The isolated BM-
SCs were cultured with DMEM supplemented with % FBS ( °C, %
CO). BMSCs in the third passage were employed in the following cellular
assays. Before seeding cells, the NIDN hydrogel scaolds were disinfected
by immersion in % ethanol overnight and then irradiation with UV light
for h. Then, the scaolds were washed three times with PBS and incu-
bated in the culture media for h. The cell suspensions ( ×cells
mL−) were pipetted onto the top side of each scaold (one × mm
lattice scaold, with ×cells/scaold). After h of initial adhesion,
mL cell culture medium was added to each sample. Then, the cell-laden
scaolds were cultured in an incubator at °C with an atmosphere of
% CO, with the culture media replaced every day. The nonadherent cells
were rinsed by replacing the culture media on day .
Cell Morphological Analysis:The cell-laden hydrogel scaolds were
incubated in the calcein-AM solution for min at °C and % CO
under dark conditions. Living cells were stained green with the fluorescent
marker calcein-AM and observed with a fluorescence microscope (Leica,
DMi). In addition, samples were fixed with % (v/v) paraformaldehyde
for cytoskeleton/nuclei staining on day . The cell-laden hydrogel scaolds
were stained with Alexa Fluor Phalloidin for F-actin and ,-diamidino-
-phenylindole (DAPI) for cell nuclei. After fixing and staining, cells were
imaged using a fluorescence microscope (Leica, DMi) and the images
were merged using ImageJ.
Cell Proliferation Analysis:Cell proliferation ability was determined
using a Cell Counting Kit- (CCK-, Dojindo) according to the manufac-
turer’s instructions. After incubation with % CCK- solution at °C for
h, cell proliferation was quantified by measuring the optical density of
the CCK- solution at nm. To reduce the eect of nanoclay adsorbing
the staining solution, scaolds without cell seeding were regarded as the
blank group.
Cell Dierentiation Analysis:The quantification of Alkaline phos-
phatase (ALP) activity and ALP staining was used to evaluate the osteo-
genesis of BMSCs.[] After , , and days of culture, the cell-laden
hydrogels were rinsed three times with PBS and then lysed in lysis buer
( L, radioimmunoprecipitation assay (RIPA) buer, Beyotime Biotech-
nology, Shanghai, China) for min at °C. Cell debris was removed by
centrifugation at rpm, at °C for min, and then the supernatant
( L) was added to the chromogenic substrate ( L) in a -well plate
and incubated at °C for h. Then, a stop buer ( L) was added
to stop the reaction. The ALP activity was determined by measuring the
absorbance at nm using a microplate reader (Spectra III; SLT-Lab In-
struments, Salzburg, Austria). Each sample was analyzed in triplicate and
the total protein content was used to normalize the ALP activity by a BCA
assay kit (BCA Protein Assay Kit, Thermo Fisher Scientific, Massachusetts,
USA). To visualize the dierentiation ability of BMSCs, the ALP staining
method was applied based on a BCIP/NBT ALP Color Development Kit
(Beyotime Biotechnology, Shanghai, China).
Calvarial Defect Repairment Model:To identify the bone repair ability of
the NIDN hydrogels, two full-thickness craniotomy defects ( mm diam-
eter) were created in the parietal bone of weeks old male SD rats using
a micro bone drill. Two intervals of and weeks after surgery were con-
sidered to evaluate the healing process in dierent groups (n =ineach
group). The rat calvaria defect without any treatment was regarded as a
control, while the calvaria defect treated with a nonporous scaold or a D
printed porous scaold (pore size ≈ m) was regarded as a sample.
After or weeks, the rats were sacrificed and fixed in % (w/v) buered
paraformaldehyde for h and analyzed by micro-CT assay (SkyScan ,
Bruker, Kontich, Belgium) to evaluate bone repair. A cylindrical region of
mm in diameter with a height covering the entire thickness of the calvar-
ial bone was chosen as the region of interest (ROI). The scan conditions
included an X-ray tube potential of kV, an X-ray intensity of A, and
an exposure time of ms. Reconstruction was accomplished by Nrecon.
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D images were obtained from contoured D images by methods based
on distance transformation of the grayscale original images (CTvox). D
and D analyses were performed using CT Analyzer software. After D con-
struction, the calvarial bone regeneration ability was evaluated by the bone
volume density (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular
number (Tb.N, mm−), and bone mineral density (BMD, g cm−).
After micro-CT scanning, the specimens were dehydrated in ascending
grades of ethanol and embedded in methyl-methacrylate polymer (Sigma,
Chemical Co, St. Louis, USA). The specimens were cut and ground from
the center of the defects perpendicular to the sagittal suture with a Leika
system (HistoCore AUTOCUT, Hamburg, Germany) to a thickness of
m. Van Gieson’s picric acid-fuchsin staining was carried out by the
conventional method and examined under an optical microscope (Nikon
Eclipse E, Japan).
Statistical Analysis:All experiments were repeated at least three times.
Statistical analyses were performed using SigmaPlot . software. The
data are shown as the means ±standard deviation. Dierences between
the values were evaluated using one-way analysis of variance (ANOVA).
Significant dierences are indicated at *p<. and **p<..
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors acknowledge financial support from the Na-
tional Key Research and Development Program of China (No.
YFC) and the Project of Basic Research of Shenzhen,
China (JCYJ & JCYJ).
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
Data sharing is not applicable to this article as no new data were created
or analyzed in this study.
Keywords
D printing, double-network hydrogels, nanoclays, nanocomposite hydro-
gels, tissue engineering scaolds
Received: January ,
Revised: March ,
Published online:
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