-
Preparation and Properties of Dopamine Modified
Alginate/Chitosan-Hydroxyapatite Scaffolds with Gradient
Structure for
Bone Tissue Engineering
Dongjian Shia*
, Jiali Shena, Zhuying Zhang
a, Chang Shi
a, Mingqing Chen
a, Yanglin Gu
b*, Yang Liu
b
a Key Laboratory of Synthetic and Biological Colloids, Ministry
of Education, School of Chemical and Material Engineering,
Jiangnan University, Wuxi, China
b The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical
University
Corresponding Author:
Dr. Dongjian Shi, E-mail: [email protected]
Dr. Yanglin Gu, E-mail: [email protected]
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ABSTRACT: Three-dimensional (3D) homogenous scaffolds composed
of natural biopolymers have been
reported as superior candidates for bone tissue engineering.
There are still remaining challenges in
fabricating the functional scaffolds with gradient structures to
similar with natural bone tissues, as well as
high mechanical properties and excellent affinity to surround
tissues. Herein, inspired by the natural bone
structure, a gradient-structural scaffold composed of functional
biopolymers was designed to provide an
optimized 3D environment for promoting cell growth. To increase
the interactions among the scaffolds,
dopamine (DA) was employed to modify alginate (Alg) and
needle-like nano-hydroxyapatite (HA) was
prepared with quaternized chitosan as template. The obtained
dopamine-modified alginate (Alg-DA) and
quaternized chitosan-templated hydroxyapatite (QCHA) were then
used to fabricate the porous gradient
scaffold by ‘‘iterative layering’’ freeze-drying technique with
further crosslinking by calcium ions (Ca2+
).
The as-prepared Alg-DA/QCHA gradient scaffolds were possessed
seamlessly integrated layer structures
and high levels of porosity at around 77.5%. Moreover, the
scaffolds showed higher compression modules
(1.7 MPa) than many other biopolyermic scaffolds. The gradient
scaffolds showed appropriate degradation
rate to satisfy with the time of the bone regeneration. Both
human chondrocytes and fibroblasts could
adhesive and growth well on the scaffolds in vitro. Furthermore,
excellent osteogenetic activity of the
gradient scaffold can effectively promote the regeneration of
the bone tissue and accelerate the repair of the
bone defects in vivo, compared with that of the scaffold with
the homogenous structure. The novel
multi-layered scaffold with gradient structure provided an
interesting option for bone tissue engineering.
Keywords: Gradient scaffold, Dopamine, Biopolymer, Bone tissue
engineering
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INTRODUCTION
Three-dimensional (3D) porous scaffolds have been widely used in
bone tissue engineering to provide
3D space for cell growth and guide new tissue formation1-3
. Numerous different natural polymer-based
porous scaffolds have attracted considerable attention due to
their significant potentials of fulfilling these
features simultaneously4-6
. Alginate (Alg), an anionic linear polysaccharide consisting of
1,4-linked
β-D-mannuronic acid (M) and α-L-guluronic acid (G)
residues7-9
, has been considered as a promising porous
scaffold material because of its good biocompatibility,
biodegradability, non-toxicity and
non-immunogenicity10-13
. Alg is easily to form stable 3D gel networks with the
so-called “egg-box’’
structure through ionotropic crosslinking14-16
. However, Alg-based scaffolds are still limited in bone
tissue
engineering because of poor mechanical properties and biological
properties17
. To overcome these
shortcomings, various Alg-based 3D porous composite scaffolds
such as Alg/gelation, Alg/chitosan,
Alg/graphene and Alg/synthetic polymers were developed for the
bone tissue applications9,11,18,19
. In our
work, we had demonstrated an Alg/polydopamine scaffold with
enhanced mechanical and biological
properties for bone repairing materials20
. Unfortunately, in these previous reports, the mechanical
properties
as well as other properties are still dissatisfied with the
clinical application.
An ideal scaffold for the bone tissue engineering must fulfill
the following requirements: (1) similar
structure and components with natural bone tissue; (2) good
mechanical properties to support the bone repair,
maintain the stable structure and provide suitable micro-stress
environment for osteoblasts; (3) suitable
biodegradation cycles to match the cell and tissue growth rate
without toxic degradation products; (4) good
affinity for the scaffolds to the surrounding tissues; and (5)
good bone conductivity and osteoinductivity as a
template for osteoblasts to grow, proliferate, differentiate,
and speed up the repair of defective bone tissue.
Accordingly, it is a major challenge in fabricating the 3D
scaffolds with functional structure as well as high
mechanical properties, controllable biodegradation behavior, and
cell viability.
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The natural bone is found to be not homogeneous, but shows dense
cortical tissue and highly porous
cancellous tissue, that is gradient structure from dense to
sponge. This gradient structure of bone tissue is of
critical importance to ensure the biological tissue work
properly21
. Therefore, it is of significance in the
development of biomaterials for osteochondral application to
adequately mimic the gradient structure of
natural osteochondral tissue22
. Some methods have been developed to prepare the gradient
scaffolds,
including thermally-induced phase separation and porogen
leaching, electrospinning, rapid prototyping
technology, freeze-drying, and so on23
. Zhang et al. prepared collagen porous scaffolds with pore size
using
ice particulates as a porogen material. These gradient scaffolds
possessed spherical pore structures with good
interconnectivity and showed beneficial effect on cartilage
regeneration24
. Ma et al. fabricated poly (L-lactic
acid) (PLLA) scaffolds with a functionally gradient structure by
a thermally induced phase separation
method. The gradient scales in the scaffolds were favorable to
control cellular environment and cell-matrix
interactions25
. Thus, adequately mimic the gradient structure of the natural
bone tissue is a promising
approach in the development of the scaffolds for bone
repair.
On the other hand, incorporation of inorganic biomaterials into
scaffolds is an effective approach to
improve the mechanical properties. Hydroxyapatite (HA) has
recently drawn more interests, due to its
chemical structure similarity to the natural bone tissue,
outstanding biocompatibility, and excellent
mechanical strength26-29
. In comparison with the Alg scaffolds, HA/alginate composite
scaffolds exhibited
improved mechanical properties30-31
. However, the easy aggregation of HA resulting in its low
bioactivity
limits the applications in the fabrication of individual HA
scaffolds. Therefore, a suitable decoration is
necessary for HA to enable the incorporation of HA into the
Alg-based 3D porous composite scaffolds.
Regretfully, the traditional methods would produce the HA with
uncontrolled bioactivity due to the
differences in size, morphology, crystallinity, and surface
properties of HA with the natural HA in bone
tissue32, 33
. In this regard, a suitable synthesis method mimicking the
formation process of natural HA is
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highly desirable to prepare HA with excellent mechanical
properties and biological activity. During the
biomimetic synthesis process of HA, the biomacromolecules
enabling interactions with Ca2+
or PO43-
plays a
vital role of controlling HA nucleation as an effective
template34
. Quaternized chitosan (QCS) with
tetrahedral structured ammonium group can bind with PO43-
by electrostatic interaction and stereochemical
effect. Through these two interactions, a biomimetic nucleation
and growth process of HA could be readily
achieved.
The implanted scaffolds might easily cause inflammation,
possibly due to the less affinity to the
surrounding tissues. Moreover, the scaffolds have the
possibility of falling off the defect in vivo because of
the movement of the organism during the repair process. This
fault might lead to loss the bone repair
property of the scaffolds. Thus, it is very important to design
the scaffolds with good affinity to the original
tissues for improving the osteoblasts to grow, proliferate,
differentiate, and speed up the repair of defective
bone tissue. Dopamine (DA), an amino acid, is reported to have
biocompatibility and show high adhesive
property on various materials, due to its strong interactions
with other materials via chemical and physical
interactions35
. Moreover, DA was also confirmed to have the reactivity with
tissues via hydrogen bonds and
covalent bonds36
. Therefore, the scaffolds with DA functional group would have
good affinity and help
themselves to be stable in the defected tissues.
Herein, in order to mimic the natural bone structure, improve
the mechanical properties, control the
degradation behavior and increase good affinity, a novel
biomimetic porous 3D scaffold with gradient and
layered micro-structure was designed and fabricated by
compositing pre-prepared DA modified Alg
(Alg-DA) and quaternized chitosan-templated hydroxyapatite
(QCHA) by ‘‘iterative layering’’ freeze-drying
technique and further crosslinking by calcium ions (Ca2+
), Then, microstructures and porosity were
confirmed, and mechanical properties, biodegradation behavior,
biomineralization capacity,
anti-inflammatory adsorption properties, cell viability and in
vivo osteogenetic activity of the obtained
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gradient scaffold were investigated in detail.
MATERIALS & METHODS
Materials
Sodium alginate (Alg), dopamine hydrochloride (dopamine, DA),
chitosan (CS),
(3-chloro-2-hydroxypropyl) trimethyl-ammonium chloride
(CHPTAC),
1-(3-dmethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC·HCl), N-hydroxysuccinimide (NHS)
and levofloxacin (LVFX) were obtained from Aladdin Reagent Co.,
Ltd. (Shanghai, China), and used
without purification. Lithium hydroxide (LiOH), potassium
hydroxide (KOH), urea, calcium chloride
(CaCl2), dipotassium phosphate (K2HPO4), sodium dihydrogen
phosphate (NaH2PO4), disodium hydrogen
phosphate (Na2HPO4), glutaraldehyde and ethanol (C2H5OH) were
purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle
medium (DMEM), fetal bovine serum
(FBS), and penicillin-streptomycin (P/S) were purchased from
Gibco (Thermo Fisher, Shanghai, China).
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) and dimethylsulfoxide (DMSO) were
purchased from Amersco (Beijing, China). Fibroblasts (L929) and
Phosphate buffer (PBS) were purchased
from Optimization biological Co., Ltd (Shanghai, China). Human
chondrocytes cells (CC-H107) were
purchased from Liaoding Biological Co. Ltd (Shanghai,
China).
Synthesis of Quaternized Chitosan-Templated Hydroxyapatite
(QCHA).
Quaternized chitosan (QCS) and QCHA were prepared as report
reference34
. Firstly, chitosan (CS, 4 g)
was dispersed into a mixed solution of KOH/LiOH·H2O/urea/H2O
with weight ratio to 7:8:8:77, and then
the above CS suspension was frozen at -20 °C overnight and
thawed at 5 °C with stirring to get a transparent
CS solution. Sequentially, (3-chloro-2-hydroxypropyl) trimethyl
ammonium chloride (CHPTAC) (60 wt%,
60 mL) was added dropwise into the CS solution to further stir
vigorously at 30 °C for 24 h (Scheme S1).
The mixture was neutralized with excess HCl aqueous solution and
dialyzed in distilled water for 1 week
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while the dialysis solvent was changed for each 8 h. The final
product QCS was lyophilized and kept in a
moisture-free desiccator. The reaction scheme was shown in
Scheme S1.
Then, QCS (0.5 g) was dissolved in 0.3 M Na2HPO4 solution with
stirring at room temperature for 1 h.
0.5 M CaCl2 solution (atom ratio of Ca/P to 1.67) was
subsequently added dropwise to the CS solution under
magnetic stirring and titrated with 1 M NaOH solution at 90 ℃ to
keep pH at 10 for 2 h. After the
hydrothermal reaction, the precipitates were harvested by
centrifugation and washed with deionized water
until pH closed to neutral. QCHA was then obtained by
freeze-drying (Figure 1A). Structures of QCS and
QCHA were recorded on Nicolet iS50 Fourier transform infrared
spectroscopy (FTIR, Thermo Fisher
Scientific, Madison, WI, USA) and Proton nuclear magnetic
resonance (1H-NMR, Bruker, Fällanden,
Switzerland). The identification of QCHA crystal phases and the
component of the mineral layers were
carried out by X-ray powder diffraction (XRD) technique using a
D8 X-ray diffractometer (Bruker-Axs,
Germany) equipped with Cu-K incident radiation at room
temperature over the range of 20-60◦.
Microstructures and morphological features of QCHA were observed
by scanning electron microscope
(S-4800, SEM, Hitachi Limited, Tokyo, Japan) and transmission
electron microscopy (JEM-2100, TEM,
Hitachi Limited, Tokyo, Japan).
Fabrication of Alg-DA/QCHA Gradient Scaffolds
DA modified Alg (Alg-DA) was prepared according to our previous
research20
and described detail in
supporting information. 0.6 g Alg-DA was dissolved in PBS buffer
(pH = 6.0) with a concentration of 3%
(w/v). 1.2 g, 0.6 g and 0.3 g QCHA were subsequently added to
the above Alg-DA solution, respectively,
and the obtained mixtures were abbreviated as Alg-DA/QCHA2.0,
Alg-DA/QCHA1.0 and Alg-DA/QCHA0.5.
Alg-DA/QCHA gradient scaffold was fabricated through a
‘‘iterative layering’’ freeze-drying technique37
. As
shown in Scheme 1, 0.5 mL Alg-DA/QCHA2.0 was added into a
cylindrical mold and frozen quickly by
liquid nitrogen. Then, the second layer was added by pipetting
0.5 mL Alg-DA/QCHA1.0 on top of the
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Alg-DA/QCHA2.0 layer and frozen again. Subsequently,
Alg-DA/QCHA0.5 (0.5 mL) as the third layer was
injected on the surface of the two-layer scaffold using a
similar protocol as before. Following freeze-drying,
the scaffold was immersed in CaCl2 solution (5 wt%) for 5 h at
room temperature for further cross-linking
(Scheme 1), and subsequently in ultrapure water for 48 h to
remove residual CaCl2. The Alg-DA/QCHA
gradient scaffold was acquired by freeze-drying finally. The
whole preparation procedure was shown in
scheme 1.
For comparison, three homogeneous scaffolds with one layer
composed of pure Alg-DA/QCHA2.0,
Alg-DA/QCHA1.0 and Alg-DA/QCHA0.5 scaffolds were also prepared
as the same procedure with the above
method (Scheme S2).
Morphologies of the scaffolds were observed by SEM. Mechanical
properties of the scaffolds were
assessed through unconfined compression testing using a
universal testing machine (5967, INSTRON, UK)
with a crosshead speed of 0.2 mm/min under ambient conditions.
Compressive modulus was defined as the
strain was 10%.
Porosity of Gradient Scaffolds
Porosity that defined as the percentage of void space in a solid
is necessary for bone tissue formation
because pores allow migration and proliferation of cells, as
well as vascularization38
. The porosity of the
Alg-DA/QCHA gradient scaffolds was determined by liquid
displacement method using ultrapure water as
replacement fluid39, 40
. Firstly, the scaffold was weighted to be signed as m1, and
weight of a pycnometer
with full of ultrapure water was as m2. The scaffold was putted
into the dried pycnometer and then sucked
out the air in the scaffold with a vacuum apparatus. Afterwards,
enough water was added into the
pycnometer for water uptake of scaffold. m3 was then got by
measured the total quality of the pycnometer
together with the scaffold and water. Finally, after removed the
wet scaffold, the final weight of the
remaining liquid together with the pycnometer was registered as
m4. The percentage porosity was calculated
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by equation 1.
(1)
Where Vp and Vs are the volumes of pore structure and solid
structure, respectively, and ε is the
porosity of scaffold.
Degradation of Gradient Scaffolds in vitro
Degradability of the Alg-DA/QCHA gradient scaffolds was
determined by mass changes of the
scaffolds after incubation in a simulated body fluid (SBF), in
which ion concentrations nearly equal to those
of human blood plasma of pH 7.4 at 37 ℃. A dried weighted
gradient scaffold was incubated in SBF at
37 °C. The gradient scaffold was removed from SBF after 7, 14,
21 and 35 days of incubation, thoroughly
washed with distilled water and weighed after lyophilization.
Degradation rate (%) of the scaffold was
calculated in terms of equation 2.
Degradation rates (%) = [(W0-W1)/W0] ×100% (2)
Where W0 and W1 are the weights of the dried scaffolds before
and after degradation, respectively.
Biomineralization of Gradient Scaffolds in vitro
Biomineralization performance of the Alg-DA/QCHA scaffolds was
measured by alternating
immersion method41
. Briefly, gradient scaffolds were firstly immersed in 0.1 M
Tris buffer (pH 9.0). Then,
the Alg-DA/QCHA scaffolds were alternatively dipped in 0.3 M
K2HPO4 solution for 100 s and washed by
ultrapure water, and then transferred to 0.5 M CaCl2 solution
for continue dipping 100 s and washed by
ultrapure water. This alternative dipping process was repeated
for five times to mineralize hydroxyapatite
(HA). After alternative dipping, the gradient scaffold was
further dipped in 0.5 M CaCl2 aqueous solution at
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37 ℃ for 24 h to ripen the mineralized amorphous calcium
phosphate (ACP) to form HA. Ca/P ratio of the
mineral layers on the gradient scaffolds was determined by
energy dispersive X-ray (EDX) spectrometry.
Loading Efficacy and Release of Levofloxacin (LVFX)
LVFX was selected as a model drug to be dissolved in a phosphate
buffered solution (PBS, pH 7.4, 10
mg/L) with 2.5 mM CaCl2. After incubation of the Alg-DA/QCHA
gradient scaffold into the LVFX solution
at 37 °C for 12 h, the scaffold was washed with ultrapure water
and then freeze-dried. The adsorbed LVFX
in the gradient scaffold was calculated by subtracting the
amount of LVFX which remained in the PBS
solution from the initial amount. Loading efficacy (LE) value
was calculated according to following
equation 3.
LE (%) = ([Total LVFX]-[Free LVFX])/[Total LVFX]×100 (3)
Release characteristics of the Alg-DA/QCHA gradient scaffold
were carried out in PBS buffer solution
with 2.5 mM CaCl2 (pH 7.4). LVFX-loaded scaffolds were immersed
in PBS solution (10 mL) at 37 °C with
shaking speed of 100 rpm. At predetermined intervals, the
release solution (2 mL) was withdrawn for
characterization, and equal amount of fresh PBS was added to the
release medium. Amount of the released
LVFX was measured at 288 nm using an Ultraviolet-visible
spectrophotometer (UV-Vis, TU-1901, UV-vis,
Purkinje General Co., LTD, Beijing). All the tests were repeated
three times.
Cell Viability
Cell culture studies were conducted using CC-H107 and L929 cells
as model cells. The Cells were
cultured in DMEM with 10% FBS and 1% PS at 37 ℃ and then placed
under standard cell culture
conditions. The culture medium was changed every 2-3 days. When
the cells were permitted to confluence,
they were trypsinized using 0.25% trypsin-EDTA solution and were
used to investigate the cells proliferation
and morphology of the Alg-DA/QCHA gradient scaffolds. Prior to
cell seeding, the gradient scaffold was
sterilized with ultraviolet (UV) light and soaking in 100%
ethanol for 6 h, followed by rehydrating in sterile
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PBS overnight.
Cell proliferation on the gradient scaffolds was investigated by
using the MTT assay. CC-H107 and
L929 cells were seeded on the scaffolds and cultured at a
density of 1 × 104 cells/mL in 24 well culture
plates. At predetermined time,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
solution (20
μL, 2 mg/mL in PBS) was added to each well in culture plates and
incubated in darkness for 4 h. After
removed supernatant, the formed formazan crystals were dissolved
in 150 μL dimethylsulfoxide (DMSO).
Afterward, optical density (OD) of the obtained solutions was
determined at 570 nm using an Infinite
M200Pro microplate reader. The results were expressed as means
of five parallel replicates.
The morphologies of CC-H107 and L929 cells on the scaffolds were
observed by scanning electron
microscope (SEM). After 12 h of incubation, the scaffolds with
the adherent cells were transferred into
another dish, rinsed three times with PBS, and fixed in 2.5%
glutaraldehyde solution for another 24 h at
room temperature. The fixed scaffolds were dehydrated by ethanol
in an increasing concentration gradient
(30%, 50%, 70%, 90%, and 100%) and then lyophilized for SEM
observation.
Bone Regeneration of Gradient Scaffolds in vivo
In this study, animal experiment was carried out according to
the Rules and Regulations of the Jiangnan
University’s Animal Care and Use Committee [JN. No.
20170930R0120130[32]]. Twelve white New
Zealand rabbits (2.5–3 kg) with an age of 6 months were employed
to evaluate bone regeneration of the
Alg-DA/QCHA gradient scaffolds. Operation for each animal was
performed under intravenous anaesthesia
(10% chloral hydrate) and sterile conditions. After disinfection
and incision, femoral defect (4 mm in
diameter and 5 mm deep) was drilled through the femur using a
Kirschner wire, and then the Alg-DA/QCHA
gradient scaffolds were implanted into the femoral defects
(right leg) while the defects without any scaffolds
were prepared as controls (left leg). Bone regeneration of the
Alg-DA/QCHA gradient scaffolds was
observed after 4, 8 and 12 weeks (n = 4). After implantation at
designated time points, CT was used to
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determine new bone formation in the femoral defect.
Three-dimensional images from CT scanning were
analyzed with RadiAnt DICOM Viewer to measure regenerated bone
areas. Then, implantation segments
were extracted for histological examination, which was fixed
with 4% paraformaldehyde for 24 h and
washed with water for 12 h. Tissue sections (5 μm in thickness)
were cut by mucosa and stained with
hematoxylin and eosin (H&E). The stained samples were
examined under microscope at 20× magnification.
RESULTS
Synthesis and Characterization of QCHA
Quaternization of chitosan (QCS) was carried out in a mixed
solution of KOH/LiOH·H2O/urea/H2O
with CHPTAC as etherifying agent42
. Structure of QCS was characterized by FTIR and 1H-NMR spectra,
as
shown in Figure S1. The results demonstrated the successful
synthesis of QCS with high degree of
substitution (DS) of quaternary ammonium salt to around 96.8%.
Quaternized chitosan-templated
hydroxyapatite (QCHA) was synthesized by hydrothermal method in
the presence of QCS (Figure 1A).
Morphology of QCHA was needle-like structure with an aspect
ratio of 10.9 (diameter and length ~22 nm ×
240 nm), which was observed by TEM as shown in Figure 1B.
Identification of QCHA crystal phases were characterized by
WXRD. As shown in Figure 1C,
diffraction patterns of QCHA matched well with the
characteristic pattern of HA without any additional
peaks. (002), (211), (112), (300), (202), (222), (213) and (004)
corresponding to different crystal planes of
HA could be also observed in QCHA, indicating formation of an
apatite crystal structure of QCHA.
However, the diffraction peaks of QCHA became wider and wider
compared to pure HA.
The structure of QCHA was also characterized by FTIR spectrum.
As shown in Figure 1D,
characteristic bands of QCHA were in good agreement with HA
signals. Peak at 958 cm-1
was assigned to
the symmetric P-O stretching vibration, while the bands at 1091
and 1018 cm-1
were related to the
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asymmetric stretching of the P-O group. Peak range of 3800-2400
cm-1
was amplified as an inset Figure in
Figure 1D. Compared with HA, new absorption peaks were appeared
at 2929 and 2878 cm-1
in the spectrum
of QCHA, which were corresponded to the stretching vibration of
-CH3 and -CH2, respectively. These
results further verified the formation of QCHA.
Architecture and Microstructure of Alg-DA/QCHA Gradient
Scaffolds
The Alg-DA/QCHA gradient scaffold was fabricated through
‘‘iterative layering’’ freeze-drying
technique by changing the QCHA compositions in each layer
(Scheme 1). For well understanding
microstructures of the scaffolds, cross sections of the
homogeneous scaffolds including the
Alg-DA/QCHA0.5, Alg-DA/QCHA1.0 and Alg-DA/QCHA2.0 homogenous
scaffolds and the Alg-DA/QCHA
gradient scaffold were investigated by SEM measurements, showing
in Figure 2A. All the scaffolds showed
a high degree of pore interconnectivity throughout the
architecture and clearly different pore sizes with the
various layers. The pore sizes of the Alg-DA/QCHA0.5,
Alg-DA/QCHA1.0 and Alg-DA/QCHA2.0 scaffolds
were calculated about 20~30 pores from SEM images, and averaged
about 75, 65 and 50 μm (Figure
2A(a)-(c)), respectively, which decreased with increasing the
QCHA compositions. For the gradient scaffold,
the microstructure of the cross section was showed a gradient
pore size change (Figure 2A(d)). The pore size
was gradually decreased from the top to bottom layer, well
corresponding to the homogenous scaffolds.
Moreover, structural continuity at the interfaces was evident,
and the individual layers were tightly bonded
with one another.
Porosity plays an important role on the biological properties of
the bone repair scaffolds. A suitable
porosity is beneficial for the adhesion, propagation and
migration of the cells on the scaffold, as well as
effectively promote the transport of nutrients and the discharge
of metabolic wastes. Thus, the porosities of
the scaffolds were tested, and the results were shown in Figure
2B. The top layer (Alg-DA/QCHA0.5) was
found to have the higher porosity of 77.5±2.7 %, while the
porosities of the other two layers decreased to
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73.7±2.5% and 70.5±1.8%. By combined these three layers into a
gradient scaffold, the total porosity was
about 72.2±2.3%, while the obtained scaffold had a gradient
porosity from dense to relatively loose, similar
with the human natural bone.
Mechanical Property of Alg-DA/QCHA Gradient Scaffolds
Mechanical property is a vital parameter in the fabrication of
scaffolds to meet the requirements for
tissue engineering applications. Excellent mechanical property
is a key factor to maintain the pore structure
and ensure successful implantation of scaffolds. Herein, the
compression tests were performed to assess the
mechanical properties of Alg-DA/QCHA gradient scaffolds. Figure
3 showed the stress-strain curve of the
gradient scaffolds (A) and compression modulus of the scaffolds
at 10% strain (B). For the individual layers,
at the same strain, the bottom layer (Alg-DA/QCHA2.0) showed
highest compression modulus (about 2.0
MPa), and it decreased with the lower QCHA composition. In the
scaffold with higher QCHA, the formed
HA crystal was higher, resulting good compressive strength. The
compression modulus of the gradient
scaffold was 1.7±0.1 MPa, even in a wet state with a low polymer
concentration. Moreover, the gradient of
the pressure distribution for scaffolds was great significance.
When scaffolds implanted in the human body,
the dense part of the scaffolds bore larger stress, while the
loose part was smallest. Thus, the compression
performance of gradient scaffolds could match well with the
requirements.
In vitro Biodegradation Behavior of Alg-DA/QCHA Gradient
Scaffolds
Biodegradability is an important index to evaluate whether the
bone repair material could be applied to
clinical treatment, which provides time and space for tissue
growth and matrix deposition. In this study,
biodegradation behavior of the Alg-DA/QCHA gradient scaffolds
was detected in a simulated body fluid
(SBF) for five weeks, as shown in Figure 4. The degradation of
the individual layers enhanced with
increasing the QCHA composition. The bottom layer with high QCHA
composition displayed a quicker
degradation, compared to the other layers with lower QCHA
compositions. Higher QCHA might contain a
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certain proportion of amorphous calcium phosphate, resulting in
a low crystallinity, and thus leading a
relatively higher degradation rate. After 35 days of in vitro
biodegradation, the degraded rates of the bottom,
middle and top layers were degraded by 33%, 28% and 20 %,
respectively (Figure 4). The degradation rate
of the gradient scaffold was 30 % after 35 days of degradation,
enough for the promotion of bone tissue
production. Meanwhile, the scaffolds after degradation provided
a sufficiently space for bone regeneration.
These results also indicated that the degradation of the
scaffolds could be controlled by adjusting the QCHA
composition to satisfy with the bone regeneration22, 29
.
In vitro Biomineralization Behavior of Alg-DA/QCHA Gradient
Scaffolds
Natural bone is an inorganic-organic complex composed of
hydroxyapatite (HA) and macromolecular
collagen fibers. Since HA has excellent bone conduction
properties, the bone repair scaffold enabling
promote the formation and deposition of HA will be beneficial
for the osteogenic properties of scaffold. In
this study, biomineralization behavior of the Alg-DA/QCHA
gradient scaffolds was investigated in vitro by
alternating immersion in aqueous solutions of calcium chloride
and dipotassium phosphate. Contents of Ca
and P atoms and Ca/P ratio of Alg-DA/QCHA gradient scaffolds
before and after mineralization were
measured by EDX, as shown in Table 1. The contents of Ca and P
atoms on the surface of the
non-mineralized scaffolds were low, regardless of the gradient
and homogenous scaffolds. With the reducing
QCHA composition, the contents also decreased. It would be noted
that the atomic contents of Ca and P on
all the scaffold’s surfaces were significantly increased to
around 10 times than those non-mineralized
scaffolds after mineralization. This phenomenon indicated that
the mineral layers were successfully
deposited on the surface of the scaffolds. In addition, the Ca/P
ratios of the non-mineralized and mineralized
gradient scaffolds were above 1.85, higher than the theoretical
ratio of HA (Ca/P ratio of HA is1.65). The
enriched Ca2+
was endowed to the capture of Ca2+
by the catechol groups, to form stronger chelating
interaction, as reported44-46
. During mineralization, the scaffolds thus possessed the
ability to capture Ca2+
,
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resulting in more Ca2+
enriched on the scaffold surface. Therefore, the obtained
Alg-DA/QCHA gradient
scaffolds had high biomineralization property.
XRD was used to further characterize the composition of the
mineralized layers on the gradient
scaffolds, and the results were shown in Figure 5. XRD patterns
(Figure 5a-d) of mineral layers were very
similar to the standard pattern of HA (Figure 5e).
Characteristic diffraction peaks of HA at (002), (211),
(112), (300), (202), (222), (213) and (004) were observed in the
mineral layers, indicating that the main
component of the mineralized matter was HA. The results showed
that Alg-DA/QCHA gradient scaffolds
was favorable for the calcium phosphate depositing on their
surface.
Microstructures of the mineralized layers on the gradient
scaffolds were observed by SEM, as shown in
Figure S2. The mineralized matter was uniformly deposited on the
surface of the scaffolds with regular size.
This excellent mineralization properties of the Alg-DA/QCHA
gradient scaffolds could promote the
deposition of calcium phosphate on the surface and accelerate
new bone formation.
In vitro Load and Release of Alg-DA/QCHA Gradient Scaffolds
Once the bone repair scaffolds are infected in vivo, induced
osteomyelitis easily cause limb dysfunction.
Loading a certain amount of anti-inflammatory or antimicrobial
drugs in the scaffolds is a better way to
prevent orthopedic implants infection. Thus, Levofloxacin (LVFX)
was selected as a model drug to dissolve
in a phosphate buffered solution (PBS, pH 7.4) with 2.5 mM CaCl2
to 10 mg/L. The adsorption ability of the
anti-inflammatory drugs was studied by immersing the Alg-DA/QCHA
gradient scaffold into the LVFX
solution. After 12 h of incubation at 37 °C, the LVFX-loaded
scaffold was washed with ultrapure water for
three times and then freeze-dried. The loading amount of LVFX
was detected and calculated after
determining the free LVFX remaining in PBS solution by UV-vis
spectra. As shown in Figure S3, after 12 h
of incubation, the drug loading of the gradient scaffolds was
very high and reached around 958.5 ± 21.3
ng/mg, possibly due to the high porosity of the scaffolds and
the π-π stacking and hydrogen interactions
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between catechol groups in DA and LVFX.
LVFX release curves and cumulative release rates in PBS were
shown in Figure 6. The drug release
curves of the scaffolds showed a gradual upward trend over time
(Figure 6A). Within the first period of 12 h,
the drug released quickly, and the release rate was about
30~40%. In additional 36 h, the release became a
little slowly. The cumulative release rate of the drugs within
48 h was shown in Figure 10B. The cumulative
release rate from the gradient scaffolds was 57.0 ± 4.7%.
Amongst each individual layer, the drug release
rate at the same release time was not significantly different.
This slow-release of the drug in the scaffolds
was mainly due to the interactions between the catechol groups
and the drugs, preventing the burst release of
the drugs. Thus, catechol was deemed to endow the well
slow-release property of the Alg-DA/QCHA
gradient scaffold.
Cell behavior of Alg-DA/QCHA Gradient Scaffolds
Cytotoxicity is an important property to evaluate the
biocompatibility of bone repair materials. MTT
assay was performed for 1 and 3 days to evaluate the cell
viability and proliferation of CC-H107 and L929
cells over the Alg-DA/QCHA scaffolds. Optical density (OD) was
measured for the cell-seeded scaffold
which was proportional to cell viability. As shown in Figure 7A,
the gradient scaffolds displayed an increase
in CC-H107 cell viability and proliferation with time. The cell
growth rate of the individual layers improved
with the increase of the QCHA, which may be due to QCHA with the
similarity inorganic constituents of the
natural bone. It endued good biocompatibility, adsorption
properties and biological activity for the gradient
scaffolds, and promoted the adhesion and growth of cells.
Morphologies of the CC-H107 cells cultured on
the Alg-DA/QCHA gradient scaffolds in each layer were also
observed by SEM (Figure 7B). After 12 h of
culture, cells with spherical morphology spread out and tightly
attached onto the scaffolds. In addition, the
cells attached well on each layer, and the cell numbers were
different with the pore size, indicating that cell
adhesion and growth could be slightly adjusted by the gradient
structure of the scaffolds.
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The Alg-DA/QCHA gradient scaffolds showed good biocompatibility
not only to CC-H107 cells, but
also to L929 cells, as shown in Figure S4 and Figure S5. These
results suggested that the pore size and inner
gradient structure of the scaffold were suitable for cell
attachment and proliferation.
In vivo Bone Regeneration of Alg-DA/QCHA Gradient Scaffolds
To evaluate bone repairing property, the Alg-DA/QCHA gradient
scaffold was implanted in a femoral
defect model and analyzed the property of bone formation in
vivo. At 4, 8 and 12 weeks after surgery, CT
observation was carried out to assess the in vivo bone
regeneration of the Alg-DA/QCHA gradient scaffold
in the rabbit right femoral. CT images and three-dimensional
(3D) images from CT scanning were showed in
Figure 8. Figures 8a1~a3 were the CT images of defects, which
were supported with the Alg-DA/QCHA
gradient scaffolds, respectively. In the white iris, the
gradient scaffolds and the surrounding bone tissue
showed different colors due to different densities, and the area
indicated by the arrow were the gradient
scaffolds. The boundary of the scaffold was still relatively
clear after 4 weeks of implantation, indicating
that there was no new bone regeneration at that time. With the
increment of the implantation time, the
boundaries of the gradient scaffolds gradually blurred and began
to fuse with the surrounding bone tissue
after 8 and 12 weeks. With prolonging time, the scaffolds became
smaller and smaller, suggesting the
generation of bone tissue after longer time. For well
understanding, 3D images from CT scanning were also
reconstructed to measure regenerated bone areas, as shown Figure
8b-c. In the control groups (Figure
8b1~b3), there was no significant change in the femoral defect
at 4 and 8 weeks. Until to 12 weeks, the bone
defect area was slightly reduced, indicating that a small amount
of bone tissue was produced by host bone
self-repair. However, large part of the defect area still did
not achieve repairing. For the Alg-DA/QCHA
gradient scaffolds group (c1~c3), the 3D images revealed that a
certain amount of new bone tissue formed at
the bone defect after the gradient scaffolds implanted for 8
weeks. Moreover, the area of the defect was
significantly reduced in the Alg-DA/QCHA gradient scaffolds
group at 12 weeks after surgery.
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To further study the osteogenic activity of the Alg-DA/QCHA
gradient scaffold, histological analyses
were employed at designated time points and shown in Figure 9.
Figures 9a~c were the histological images
of the control group at 4, 8 and 12 weeks after surgery. At 4
weeks, a small amount of fibrous was formed at
the edge of the host bone, but there was no obvious new bone
tissue growth. At 8 and 12 weeks, the fibrous
increased gradually and a small amount of new bone tissue was
formed around the host bone. Histological
analyses were also used to assess the in vivo bone promotion
behavior of the Alg-DA/QCHA gradient
scaffold, as shown in Figure 9d~f. At 4 weeks after operation,
small gaps became apparent between the
scaffolds and the host bone, indicating that new bone could not
be regenerated in such short time. In addition,
a few new bone tissues were found at the junction of the
scaffolds and the host bone after 8 weeks after
implantation. The gap between the scaffolds and the host bone
became gradually unclear, and a few formed
bone trabeculae appeared inside the scaffolds. At 12 weeks, the
gradient scaffolds were fully fused with the
host bone, and a large amount of new bone tissue was generated
inside the scaffolds. All the bone trabecula
surrounding the scaffolds was mutual connection with each other.
Importantly, there is no any inflammation
induced by the scaffolds to be found.
Effect of the gradient structure of the scaffolds on the bone
regeneration was also detected in vivo. As
shown in Figure 10a, the homogeneous scaffold (Alg-DA/QCHA1.0 as
an example) showed no clear gap
with the host bone, and a large amount of new bone tissues were
regenerated around the scaffolds after 12
weeks of surgery. For the gradient scaffolds, there were lots of
bone trabeculaes appeared not only around
the scaffolds, but also on the surface of the scaffolds and
inside the scaffolds (Figure 10b), indicating the
new bone was fully fused with the scaffolds.
DISCUSSION
HA is an important ingredient in the natural bone tissue and
often used to complex with polymers for
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preparation of the bone scaffolds, because of its excellent
biocompatibility and bioactivity. However, HA is
easy to aggregate during compositing process. Using quaternized
chitosan as template to in situ
mineralization of HA during fabrication process could increase
the compatibility of HA and polymers. When
dissolving CS in Na2HPO4 solution, PO43-
ions were apt to react with QCS, because of the
electrostatic
interactions between PO43-
groups and positively charged quaternary ammonium salt groups.
What’s more,
the quaternary ammonium salt groups of QCS have a similar
tetrahedral structure with PO43-
, forming their
combination by the spatial stereo-chemical effect and thus
providing a template for the nucleation and
growth of HA crystals34
. Then, the needle-like HA crystals could be facilitated by the
long and rigid
backbone of QCS. The characteristic pattern of HA in WXRD
patterns of the obtained QCHA suggested the
successfully synthesized needle-like HA. Moreover, the
diffraction peaks of QCHA became wider, revealing
that the low crystallinity of QCHA. Thus, the higher QCHA
composition showed better degradation
performance.
For preparation of the scaffolds, one of the major challenges is
to adequately mimic the gradient
structure of natural bone tissue with seamlessly integrated
layers. Freeze-drying technique is confirmed as a
simple and clear method without any additional compound. By
mixing the Alg-DA and QCHA solutions
with a design concentration and composition, the first layer of
the Alg-DA/QCHA was firstly frozen to fix
the scaffold structure and then added the second and subsequent
the third layer. Thanking to the adhesive
property induced by the strong interactions of the DA-DA groups
and DA-polymers and crosslinking of
Ca2+
, there was no significant boundary among the layers from the
optical image and SEM images. The
individual layers were tightly bonded with one another. By
varying the QCHA compositions, the interactions
between the Alg-DA and QCHA were changed, leading the difference
of the crosslinking degree of the
Alg-DA/QCHA hydrogel. Thus the porosity was changed depending on
the QCHA compositions. The
seamless integration and porosity of the gradient scaffold are
of vital importance for drug loading and
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release and promoting attachment, migration and reproduction of
the human chondrocytes CC-H107 cells.
The mechanical property of scaffolds is one essential criteria
for clinical repair. The obtained gradient
scaffolds showed high compression modulus, which were
contributed to the existing of HA component and
the stronger hydrogen-bond and electrostatic interactions among
the DA groups and the Alg and QCS chains.
This compression modulus was much higher than many CS-based and
other biopolymer-based scaffolds,
which were reported to have only several or hundred KPa, even
with a high HA composition in their
examples9,43
.
By implanted the Alg-DA/QCHA gradient scaffold in rabbit femoral
defect in vivo, the area of the
defect was significantly reduced and lots of the new bone
tissues grew in the Alg-DA/QCHA gradient
scaffolds group at 12 weeks after surgery, compared with the
blank group and homogenous scaffold.
Moreover, the gradient scaffold was degraded after implanting
into the animal body, which was satisfied
with the growth of bone in the first degradation period. These
results further indicated that the gradient
scaffolds had good histocompatibility and osteogenic activity,
due to well binding with the host bone tissue,
effectively promoting bone tissue regeneration and then
accelerating the repair of bone defect. Although the
reason why the gradient scaffold had better osteogenic activity
than the homogeneous scaffold was unclear
now, we believe that the gradient pore size might be easy for
bone cell fibrous to penetrate and promote the
biological tissue to work well, as other researches reported
47
.
CONCLUSION
In summary, a novel catechol-modified alginate/quaternized
chitosan-templated hydroxyapatite scaffold
(Alg-DA/QCHA) with gradient pore structure was rationally
designed and fabricated by ‘‘iterative layering’’
freeze-drying technique. The scaffold with a seamlessly
integrated layer structure delivered high levels of
porosity at around 77.5% with high compression modules of 1.7
MPa. The gradient scaffolds showed
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appropriate degradation rate to satisfy with the bone
regeneration. Moreover, model drugs could be loaded
and slowly released from the scaffolds. Both human chondrocytes
and fibroblasts could adhesive and growth
well on the scaffolds in vitro. Furthermore, the gradient
scaffold had been demonstrated to be highly
effective for the proliferation of cells and new bone growth due
to the catechol group (promoting cell
adhesion and growth) and hydroxyapatite (enhanced biomechanical
and biological properties) in the
scaffolds. This novel gradient scaffold would be a good
candidate for bone tissue engineering.
CONFLICT OF INTEREST. No potential conflict of interest was
reported by the authors.
ACKNOWLEDGEMENTS. This study was supported by the National
Nature Science Foundation of
China (No.21571084), the Natural Science Foundation of Jiangsu
Province (Grants No. BK20181349),
MOE & SAFEA for the 111 Project (B13025), National
First-Class Discipline Program of Light Industry
Technology and Engineering (LIFE2018-19) and the Innovation of
Graduate Student Training Project of
Jiangsu (KYCX18_1813).
SUPPORTING INFORMATION. Synthesis of dopamine-modified alginate
and quaternized chitosan,
fabrication of the Alg-DA/QCHA homogenous scaffolds, and In
vitro biomineralization behavior, drug
loading and cell hehavior of L929 of the Alg-DA/QCHA gradient
scaffolds. Additional Supporting
Information may be found in the online version of this
article.
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Figure caption:
Scheme1. Schematic illustration of fabrication of Alg-DA/QCHA
gradient scaffold
Figure 1. (A) Schematic illustration of the synthesis of QCHA,
(B) TEM image of QCHA, and (C) XRD
patterns and (D) FTIR spectra of HA and QCHA.
Figure 2. (A) SEM micrographs of (a) Alg-DA/QCHA0.5, (b)
Alg-DA/QCHA1.0 and (c) Alg-DA/QCHA2.0
homogenous scaffolds and (d) Alg-DA/QCHA gradient scaffold. (B)
Porosity of Alg-DA/QCHA scaffolds
(n=3).
Figure 3. Stress-strain behavior (A) and compression modulus (B)
of Alg-DA/QCHA scaffolds at 10%
strain during compression (n=3)
Figure 4. In vitro biodegradation curves of Alg-DA/QCHA gradient
scaffolds
Figure 5. XRD patterns of Alg-DA/QCHA2.0 (a), Alg-DA/QCHA1.0 (b)
and Alg-DA/QCHA0.5 (c)
homogeneous scaffolds, (d) Alg-DA/QCHA gradient scaffold after
mineralization, and (e) HA
Figure 6. Cumulative release curves of Levofloxacin in
Alg-DA/QCHA gradient scaffold (A) and
cumulative release rate after 48 h (B). (n = 3)
Figure 7. (A) Cytotoxicity of Alg-DA/QCHA gradient scaffold and
(B) distribution and proliferation of
seeded cells on gradient scaffolds after 12 h of culture
Figure 8. CT images (a1~a3) and three-dimensionally
reconstructed CT images (b1~b3: control, c1~c3:
Alg-DA/QCHA scaffolds) of defects at 4, 8 and 12 weeks after
surgery
Figure 9. HE staining sections of defects supported by blank
(a-c) and Alg-DA/QCHA gradient scaffold (d-f)
at 4, 8 and 12 weeks after surgery (M: Gradient scaffolds; N:
New bone; H: Host bone; 20×)
Figure 10. HE staining sections of defects supported by
Alg-DA/QCHA homogeneous (a) and gradient
scaffolds (b) at 12 weeks after surgery (M: Gradient scaffolds;
N: New bone; H: Host bone; 20×)
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Table 1. Atomic content of Ca and P atoms and Ca/P ratio of
Alg-DA/QCHA scaffolds before and after
mineralization.
Sample aCa %
bCa %
aP %
bP %
aCa/P
bCa/P
Top Layer 2.63 22.4 1.24 11.9 2.12 1.88
Middle Layer 2.94 22.7 1.54 12.2 1.91 1.86
Bottom Layer 3.55 26.6 1.91 14.5 1.86 1.83
Gradient Scaffold 2.89 24.5 1.53 13.2 1.89 1.85
a: before mineralization;
b: after mineralization
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