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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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This article is protected by copyright. All rights reserved.

Thi s article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jbm.a.36678

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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.

REFERENCES

(1) Lu, H.; Kawazoe, N.; Kitajima, T.; Myoken, Y.; Tomita, M.; Umezawa, A.; Chen, G.; Ito, Y. Spatial

immobilization of bone morphogenetic protein-4 in a collagen-PLGA hybrid scaffold for enhanced

osteoinductivity. Biomaterials 2012, 33(26), 6140-6146.

(2) Manoukian, S.; Aravamudhan, A.; Lee, P.; Arul, M.; Yu, X. J.; Rudraiah, S.; Kumbar, S. Spiral

Acc

epte

d A

rticl

e


23

Layer-by-Layer Micro-Nanostructured Scaffolds for Bone Tissue Engineering. ACS Biomater. Sci. Eng.

2018, 4, 2181-2192.

(3) Higuchi, A.; Ling, Q. D.; Chang, Y.; Hsu, S. T.; Umezawa, A. hysical Cues of Biomaterials Guide Stem

Cell Differentiation Fate. Chem. Rev. 2013, 113(5), 3297-3328.

(4) Fernandez-Yague, M. A.; Abbah, S. A.; McNamara, L.; Zeugolis, D. I.; Pandit, A.; Biggs, M. J.

Biomimetic approaches in bone tissue engineering: Integrating biological and physicomechanical

strategies. Adv. Drug Deliv. Rev. 2015, 84, 1-29.

(5) Zang, S.; Sun, Z.; Liu, K.; Wang, G.; Zhang, R.; Liu, B.; Yang, G. Ordered manufactured bacterial

cellulose as biomaterial of tissue engineering. Mater. Lett. 2014, 128, 314-318.

(6) Sharma, C.; Dinda, A. K.; Potdar, P. D.; Chou, C. F.; Mishra, N. C. Fabrication and characterization of

novel nano-biocomposite scaffold of chitosan-gelatin-alginate-hydroxyapatite for bone tissue

engineering. Mater. Sci. Eng. C Mater. Biol. Appl. 2016, 64, 416-427.

(7) Pawar, S. N.; Edgar, K. J. Alginate derivatization: A review of chemistry, properties and applications.

Biomaterials 2012, 33(11), 3279-3305.

(8) Lee, K. Y.; Mooney, D. J. Alginate: Properties and biomedical applications. Prog. Polym. Sci. 2012,

37(1), 106-126.

(9) Sarker, B.; Li, W.; Zheng, K.; Detsch, R.; Boccaccini, A. R. Designing porous bone tissue engineering

scaffolds with enhanced mechanical properties from composite hydrogels composed of modified

alginate, gelatin, and bioactive glass. ACS Biomater. Sci. Eng. 2016, 2, 2240-2254.

(10) Valente, J.; Valente, T.; Alves, P.; Ferreira, P.; Silva, A.; Correia, I. Alginate based scaffolds for bone

tissue engineering. Mater. Sci. Eng. C Mater. Biol. Appl. 2012, 32(8), 2596-2603.

(11) Venkatesan, J.; Bhatnagar, I.; Manivasagan, P.; Kang, K. H.; Kim, S. K. Alginate composites for bone

tissue engineering: A review. Int. J. Biol. Macromol. 2015, 72, 269-281.

Acc

epte

d A

rticl

e


24

(12) Tonnesen, H. H.; Karlsen, J. Alginate in drug delivery systems. Drug Dev. Ind. Pharm. 2002, 28(6),

621-630.

(13) Kuo, C. K.; Ma, P. X. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1.

Structure, gelation rate and mechanical properties. Biomaterials 2001, 22(6), 511-521.

(14) Chan, L. W.; Jin, Y.; Heng, P. Cross-linking mechanisms of calcium and zinc in production of alginate

microspheres. Int. J. Pharm. 2002, 242(1-2), 255-258.

(15) Braccini, I.; Perez, S. Molecular basis of Ca2+

-induced gelation in alginates and pectins: The egg-box

model revisited. Biomacromolecules 2001, 2(4), 1089-1096.

(16) Luo, Y.; Lode, A.; Wu, C.; Chang, J.; Gelinsky, M. Alginate/nanohydroxyapatite scaffolds with

designed core/shell structures fabricated by 3D plotting and in situ mineralization for bone tissue

engineering. ACS Appl. Mater. Interfaces 2015, 7(12), 6541-6549.

(17) Han, Y.; Zeng, Q.; Li, H.; Chang, J. The calcium silicate/alginate composite: Preparation and evaluation

of its behavior as bioactive injectable hydrogels. Acta Biomater. 2013, 9(11), 9107-9117.

(18) Kolanthai, E.; Sindu, P. A.; Khajuria, D. K.; Veerla, S. C.; Kuppuswarny, D.; Catalani, L. H.; Mahapatra,

D. R. Graphene oxide-a tool for the preparation of chemically crosslinking free

alginate-chitosan-collagen scaffolds for bone tissue engineering. ACS Appl. Mater. Interfaces 2018,

10(15), 12441-12452.

(19) Levengood, S.; Zhang, M. Chitosan-based scaffolds for bone tissue engineering. J. Mater. Chem. B

2014, 2(21), 3161-3184.

(20) Shen, J. L.; Shi, D. J.; Dong, L. L.; Zhang, Z. Y.; Li, X. J., Chen, M. Q. Fabrication of polydopamine

nanoparticles knotted alginate scaffolds and their properties. J. Biomed. Mater. Res., Part A 2018, 106A,

3255-3266,.

(21) Bhattacharjee, M.; Chameettachal, S.; Pahwa, S.; Ray, A. R.; Ghosh, S. Strategies for replicating

Acc

epte

d A

rticl

e


25

anatomical cartilaginous tissue gradient in engineered intervertebral disc. ACS Appl. Mater. Interfaces

2014, 6(1), 183-193.

(22) Du, Y.; Liu, H.; Yang, Q.; Wang, S.; Wang, J.; Ma, J.; Noh, I.; Mikos, A. G.; Zhang, S. Selective laser

sintering scaffold with hierarchical architecture and gradient composition for osteochondral repair in

rabbits. Biomaterials 2017, 137, 37-48.

(23) Tang, G.; Zhang, H.; Zhao, Y.; Zhang, Y.; Li, X.; Yuan, X. Preparation of PLGA scaffolds with graded

pores by using a gelatin-microsphere template as porogen. J. Biomaterials Sci., Polym. Ed. 2012, 23(17),

2241-57.

(24) Zhang, Q.; Lu, H.; Kawazoe, N.; Chen, G. Preparation of collagen porous scaffolds with a gradient pore

size structure using ice particulates. Mater. Lett. 2013, 107, 280-283.

(25) Ma, H.; Xue, L.; Nie, T. Fabrication of PLLA scaffold with gradient macro/micro/nano structure by

electrophoretic deposition of carbon nanotube. Mater. Lett. 2015, 159, 185-188.

(26) Suchanek, W.; Yoshimura, M. Processing and properties of hydroxyapatite-based biomaterials for use as

hard tissue replacement implants. J. Mater. Res. 1998, 13(1), 94-117.

(27) Zhou, S.; Bismarck, A.; Steinke, J. H. G. Interconnected macroporous glycidyl methacrylate-grafted

dextran hydrogels synthesised from hydroxyapatite nanoparticle stabilised high internal phase emulsion

templates. J. Mater. Chem. 2012, 22(36), 18824-18829.

(28) Boehler, R. M.; Shin, S.; Fast, A. G.; Gower, R. M.; Shea, L. D. A PLG/HAp composite scaffold for

lentivirus delivery. Biomaterials 2013, 34(21), 5431-5438.

(29) Liu, X.; Wei, D.; Zhong, J.; Ma, M.; Zhou, J.; Peng, X.; Ye, Y.; Sun, G.; He, D. Electrospun nanofibrous

P(DLLA-CL) balloons as calcium phosphate cement filled containers for bone repair: in Vitro and in

Vivo studies. ACS Appl. Mater. Interfaces 2015, 7(33), 18540-18552.

(30) Turco, G.; Marsich, E.; Bellomo, F.; Semeraro, S.; Donati, I.; Brun, F.; Grandolfo, M.; Accardo, A.;

Acc

epte

d A

rticl

e


26

Paoletti, S. Alginate/Hydroxyapatite biocomposite for bone ingrowth: a trabecular structure with high

and isotropic connectivity. Biomacromolecules 2009, 10(6), 1575-1583.

(31) Dittrich, R.; Tomandl, G.; Despang, F.; Bernhardt, A.; Hanke, T.; Pompe, W.; Gelinsky, M. Scaffolds for

hard tissue engineering by ionotropic gelation of alginate-influence of selected preparation parameters.

J. Am. Ceramic Soc. 2007, 90(6), 1703-1708.

(32) Sanosh, K. P.; Chu, M. C.; Balakrishnan, A.; Lee, Y. J.; Kim, T. N.; Cho, S. J. Synthesis of nano

hydroxyapatite powder that simulate teeth particle morphology and composition. Curr. Appl. Phys. 2009,

9(6), 1459-1462.

(33) Giardina, M. A.; Fanovich, M. A. Synthesis of nanocrystalline hydroxyapatite from Ca(OH)2 and

H3PO4 assisted by ultrasonic irradiation. Ceram. Int. 2010, 36(6), 1961-1969.

(34) Zhu, A. P.; Lu, Y.; Si, Y. F.; Dai, S. Frabicating hydroxyapatite nanorods using a biomacromolecule

template. Appl. Surface Sci. 2011, 257, 3174-3179.

(35) Lee, H.; Dellatore, S.M.; Miller, W.M.; Messersmith, P.B. Mussel-inspired surface chemistry for

multifunctional coatings. Science 2007, 318, 426-430.

(36) Moulay, S. Dopa/catechol-tethered polymers: bioadhesives and biomimetic adhesive materials. Polym.

Rev. 2014, 54, 436–513.

(37) Levingstone, T. J.; Matsiko, A.; Dickson, G. R.; O'Brien, F. J.; Gleeson, J. P. A biomimetic

multi-layered collagen-based scaffold for osteochondral repair. Acta Biomater. 2014, 10(5), 1996-2004.

(38) Karageorgiou, V.; Kaplan, D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005,

26(27), 5474-5491.

(39) Huang, W.; Shi, X.; Ren, L.; Du, C.; Wang, Y. PHBV microspheres-PLGA matrix composite scaffold

for bone tissue engineering, Biomaterials 2010, 31(15), 4278-4285.

(40) Hu, Y.; Ma, S.; Yang, Z.; Zhou, W.; Du, Z.; Huang, J.; Yi, H.; Wang, C. Facile fabrication of

Acc

epte

d A

rticl

e


27

poly(L-lactic acid) microsphere-incorporated calcium alginate/hydroxyapatite porous scaffolds based on

Pickering emulsion templates. Colloids Surf. B-Biointerfaces 2016, 140, 382-391.

(41) Nonoyama, T.; Wada, S.; Kiyama, R.; Kitamura, N.; Mredha, M. T. I.; Zhang, X.; Kurokawa, T.;

Nakajima, T.; Takagi, Y.; Yasuda, K.; Gong, J. P. Double-network hydrogels strongly bondable to bones

by spontaneous osteogenesis penetration. Adv. Mater. 2016, 28(31), 6740.

(42) Song, Y.; Wang, H.; Zeng, X.; Sun, Y.; Zhang, X.; Zhou, J.; Zhang, L. Effect of molecular weight and

degree of substitution of quaternized cellulose on the efficiency of gene transfection. Bioconjugate

Chem. 2010, 21(7), 1271-1279.

(43) Ran, J.; Jiang, P.; Liu, S.; Sun, G.; Yan, P.; Shen, X.; Tong, H. Constructing multi-component

organic/inorganic composite bacterial cellulose-gelatin/hydroxyapatite double-network scaffold

platform for stem cell-mediated bone tissue engineering. Mater. Sci. Eng. C, Mater. Biol. Appl. 2017, 78,

130-140.

(44) Wu, J.; Zhang, L.; Wang, Y.; Long, Y.; Gao, H.; Zhang, X.; Zhao, N.; Cai, Y.; Xu, J. Mussel-inspired

chemistry for robust and surface-modifiable multilayer films. Langmuir 2011, 27(22), 13684-13691.

(45) Kim, S.; Park, C. B. Mussel-inspired transformation of CaCO3 to bone minerals. Biomaterials 2010,

31(25), 6628-6634.

(46) Shen, J. L.; Shi, D. J.; Shi, C.; Li, X. J.; Chen, M. Q. Fabrication of dopamine modified

polylactide-poly(ethylene glycol) scaffolds with adjustable properties. J. Biomaterials Sci., Polym. Ed.

2017, 28(17), 2006-2020.

(47) Gao, F.; Xu, Z. Y.; Liang, Q. F.; Liu, B.; Li, H.; Wu, Y. H.; Zhang, Y. Y.; Lin, Z. F.; Wu, M. M.; Ruan, C.

S.; Liu, W. G. Direct 3D printing of high strength biohybrid gradient hydrogel scaffolds for efficient

repair of osteochondral defect. Adv. Funct. Mater. 2018, 28, 1706644.

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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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