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Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering
Accepted Manuscript Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering
Hairui Suo, Deming Zhang, Jun Yin, Jin Qian, Zi Liang Wu, Jianzhong Fu PII: DOI: Reference:
S0928-4931(17)34088-2 doi:10.1016/j.msec.2018.07.016 MSC 8728
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
12 October 2017 19 June 2018 6 July 2018
Please cite this article as: Hairui Suo, Deming Zhang, Jun Yin, Jin Qian, Zi Liang Wu, Jianzhong Fu , Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering. Msc (2018), doi:10.1016/j.msec.2018.07.016
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ACCEPTED MANUSCRIPT Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering
Hairui Suo1,2,†, Deming Zhang1,2,†, Jun Yin1,2,*, Jin Qian3,*, Zi Liang Wu4,*,
The State Key Laboratory of Fluid Power and Mechatronic Systems, School of
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1
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Jianzhong Fu1,2
Mechanical Engineering, Zhejiang University, Hangzhou 310027, China Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School
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2
of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China Key Laboratory of Soft Machines and Smart Devices of Zhejiang Province,
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3
Department of Engineering Mechanics, Zhejiang University, Hangzhou 310027,
4
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China
MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
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Department of Polymer Science and Engineering, Zhejiang University, Hangzhou
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310027, China
† These authors contributed equally to this work.
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* Corresponding authors. E- mail: [email protected], [email protected], and [email protected]
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ACCEPTED MANUSCRIPT Abstract: Gelatin and chitosan (CS) are widely used natural biomaterials for tissue engineering scaffolds, but the poor mechanical properties of pure gelatin or CS hydrogels become a big obstacle that limits their use as scaffolds, especially in load-bearing tissues. This study provided a novel mechanism of forming interpenetrating network (IPN) of
interactions
through
photocrosslinking
and
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gelatin methacryloyl (GelMA) and CS hydrogels by covalent bonds and hydrophobic basification,
respectively.
By
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characterization of the compressive and tensile moduli, ultimate te nsile stress and strain, it was found that semi-IPN and IPN structure can greatly enhance the
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mechanical properties of GelMA-CS hydrogels compared to the single network CS or GelMA. Moreover, the increase of either GelMA or CS concentration can strengthen
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the hydrogel network. Then, the swelling, enzymatic degradation, and morphology of GelMA-CS hydrogels were also systematically
investigated.
The excellent
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biocompatibility of GelMA-CS hydrogels was demonstrated by large spreading area of bone mesenchymal stem cells on hydrogel surfaces when CS concentration was
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less than 2% (w/v). According to this study, the multiple requirements of properties can be fulfilled by carefully selecting the GelMA and CS compositions for IPN
Keywords:
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hydrogels.
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Interpenetrating network, gelatin, chitosan, photocrosslinking, mechanical property
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ACCEPTED MANUSCRIPT 1. Introduction Tissue engineering aims to support and promote regeneration of damaged or diseased tissues using a scaffold seeded with cells and growth factors [1]. The scaffold is expected to substitute extracellular matrix (ECM) and provides a temporary place for cell growth [2]. Therefore, the materials for scaffolds should be
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rigorously selected according to their biocompatibility, degradation, mechanical properties, etc. Gelatin and chitosan (CS) are the most preferred natural biomaterials
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for tissue engineering scaffolds since they are close ly related to ECM. Gelatin is the product of collagen hydrolysis, the main component of ECM, and has been widely
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used in tissue engineering because of its antigenicity and biocompatibility. CS is a partially deacetylated derivative of chitin and its structure is similar to that of
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glycosaminoglycans (GAGs), another important component of ECM, which makes CS an ideal scaffold material for tissue engineering [3]. Moreover, CS exhibits
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considerable antimicrobial and hemostatic properties due to its cationic nature of amino group [4, 5]. Due to their excellent biological properties, the mixture of gelatin
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and CS have been used to fabricate various scaffolds, which have been applied in numerous areas of tissue engineering, such as skin [6], cartilage [7], bone [8, 9],
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osteochondral [10], nerve [11], liver [12, 13], and so on. Although gelatin- and CS-based hydrogels have their advantages, poor mechanical property becomes a big obstacle that limits their applications as scaffolds,
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especially in load-bearing tissues [11, 14]. Several studies have been reported to improve the mechanical properties of hydrogels [15, 16], but very few investigations on improving the mechanical properties of gelatin-CS hydrogels have been carried out. The compressive modulus of gelatin-CS hydrogels neutralized with alcohol is about 1.15 to 3.4 kPa in wet condition [17]. Combining gelatin-CS hydrogels with other synthetic polymers was found to be a promising approach to enhance their mechanical properties, but it either was time-consuming or would weaken the biocompatibility and degradability of natural materials [18]. Chemical crosslinkers also have been introduced to improve the mechanical properties of gelatin-CS hydrogels. For 3
ACCEPTED MANUSCRIPT example, Kathuria et al. [19] synthesized the gelatin-CS hydrogels crosslinked by glutaraldehyde with the compressive modulus varied between 36-39 kPa. However, the remaining unreacted chemical crosslinkers and the toxic by-products may make these hydrogels unfit for tissue engineered applications in vitro and in vivo. Recently, increasing interests and more efforts were devoted to genipin [13, 20, 21], a natural
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biocompatible crosslinker derived from gardenia fruit. However, genipin was reported to possess genotoxicity and showed detrimental effects on chondrocytes above 220
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µM in vitro for a long-time culture [22, 23]. Therefore, how to effectively enhance the mechanical properties of gelatin-CS hydrogels with preserved biocompatibility and
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degradability remains a big challenge for further applications of gelatin-CS hydrogels. Recently, the crosslinking approaches on gelatin-CS hydrogels without organic
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solvents have gained increasing attention, such as solvent exchange, UV irradiation, pH change, and temperature modulation [24]. Since the photocrosslinking leads to [25,
26],
photocrosslinkable
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strong covalent bonds
gelatins obtained
by
methacrylation have been manufactured as tissue engineering scaffolds for cardiac
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tissues [27], blood vessels [28], and skins [29]. Moreover, CS can also be crosslinked by neutralization in either dilute NaOH or ethanol solution to form a hydrophobic
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network before its application in tissue engineering as scaffolds [30]. Therefore, we hypothesized that the interpenetrating of the photocrosslinked gelatin network and the hydrophobic CS network would be an effective way to enhance their mechanical
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properties.
When one hydrophilic polymer chain permeates another polymeric network without any chemical bonds between them, the (semi- )interpenetrating network (IPN) forms. In semi-IPN, only one crosslinked network forms physically or chemically, while in IPN, two or more crosslinked networks form, permeating with each other. The formation of (semi-)IPN would not only preserve the characteristics of each network structure, but also improve the stability and mechanical properties of the crosslinked
networks
[20].
Li et al.
[31]
fabricated
IPN
hydrogels of
gelatin- graft-polyaniline, carboxymethyl-CS and oxided dextran via Schiff base 4
ACCEPTED MANUSCRIPT reaction, which had the storage modulus as high as 924 kPa, good cytocompatibility and enhanced cell proliferation. Thus, the (semi-)IPN leads to the new systems with the combination of favorable properties of each constituent polymer, which quite often are substantially different from those of the individual polymer s [32]. The objective of this work is to develop IPN gelatin methacryloyl (GelMA)-CS hydrogels
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through photocrosslinking and basification, which not only have enhanced mechanical properties than either single network GelMA or CS hydrogel; but also possess the
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advantages of the two biomaterials for biomedical applications. The obtained hydrogels were well characterized by Fourier transform infrared (FTIR) spectra and
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scanning electronic microscope (SEM). Their mechanical properties were evaluated by compressive and tensile tests. The swelling ratio of the materials at different pH
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values and the enzymatic degradation rate were also investigated. Finally, the biocompatibility of the semi- IPN and IPN GelMA-CS hydrogels were evaluated in
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vitro by growing bone mesenchymal stem cells (BMSCs) on the hydrogels. The IPN approach proposed in this study is found to effectively improve the mechanical
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properties of GelMA-CS hydrogels without toxic or chemical crosslinkers, which may
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significantly extend the application of GelMA-CS hydrogels.
2. Materials and Methods 2.1. Synthesis of GelMA
Briefly,
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Gelatin methacryloyl (GelMA) was synthesized as previously described [33, 34]. 15%
(w/v)
gelatin
(MA,
Aladdin)
was dissolved
in
0.25
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carbonate-bicarbonate buffer (7.95 g sodium carbonate and 14.65 g sodium bicarbonate in 1 L distilled water) at 50 o C. Methacrylic anhydride (MA, Aladdin) was added at a rate of 0.5 mL/min under a stirring condition at 50 o C. After reaction for 3 hours, the reacted solution was diluted and dialyzed against distilled water using 8-14 kDa cutoff dialysis tubes for 7 days at 40 o C. The dialyzed GelMA solution was then filtered through 0.22 μm filter membrane, lyophilized and stored at -80 o C for further use. 5
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2.2. Photocrosslinking and centralization of GelMA-CS hydrogels To form GelMA-CS hydrogels, 30% (w/v) GelMA and 3% (w/v) CS (low viscosity: < 200 mPa∙s, Aladdin) was separately dissolved in 1% (v/v) acetyl acid with
0.1%
(w/v)
photoinitiator
2-hydroxy-1-(4-(hydroxyethoxy)
pyenyl)-2-methyl-1-propanone (Irgacure 2959, Sigma). Then the two polymer
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precursors were blended homogeneously under 37 o C in different ratios as listed in
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Table 1, where 10% GelMA and 2% CS with single network were prepared as a control. Then, the precursor solutions were exposed to a 1.5 W/cm2 UV light
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(wavelength: 365 nm) and photocrosslinked for 30 seconds. Finally, the precursor
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solutions were basified in 0.1 mol/L NaOH for 30 minutes to form IPNs.
Table 1. Samples for experiment
CS2 G10 G10CS0.5 G10CS1 G10CS2 G5CS1 G15CS1 G20CS1 G10CS0.5-IPN G10CS1-IPN G10CS2-IPN G5CS1-IPN G15CS1-IPN G20CS1-IPN
Single network
GelMA (w/v %) 0 10 10 10 10 5 15 20 10 10 10 5 15 20
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Network
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Groups
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Semi-IPN
IPN
Chitosan (w/v %) 2 0 0.5 1 2 1 1 1 0.5 1 2 1 1 1
Crosslinking method Basification
Photocrosslink
Photocrosslink and basification
2.3. FT-IR study FT-IR spectra were performed using a Shimadzu FT-IR spectrometer with a resolution of 2 cm-1 at wavenumber ranging of 4000-400 cm-1 . Samples were lyophilized and grinded in liquid nitrogen. The dried sample powder was blended 6
ACCEPTED MANUSCRIPT homogeneously with KBr pellet and scanned against a blank KBr pellet background.
2.4. Morphology of the hydrogels Freshly prepared samples were frozen in -80 o C for 12 hours and lyophilized for 48 hours. The samples were fractured and sputtered with gold vapor for 40 seconds,
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and then the morphology of the fractured surface of the hydrogels was observed using
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a SEM (TM1000, Hitachi) with an acceleration voltage of 15 kV.
2.5. Mechanical tests
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The tensile and compressive properties of the hydrogels were determined using a testing machine ElectroForce (TA Instruments, America) at room temperature.
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Cylindrical samples with the diameter of 12 mm and the thickness of 3 mm were prepared for the compressive test. The samples were placed between two parallel
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plates (diameter of 25 mm) and the compressive force was applied parallel to the longitudinal axis of the samples with the rate of 1.0 mm/min. The compressive
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modulus was determined as the slope of the stress-strain curve within 0-10% strain. At least 3 samples were used for each testing groups.
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For the tensile test, rectangular samples were prepared with the width of 8 mm and thickness of 2.5 mm. Samples were tested between two clamps with initial distance of 5 mm. The uniaxial stretch was performed with the rate of 1 mm/min until
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the samples broke. The tensile properties were represented by Young’s modulus (the tangent slope within 0-10% strain of the stress-strain curve), ultimate stress (stress at failure), and maximum strain (strain at failure) of the GelMA-CS hydrogels.
2.6. Swelling properties of the hydrogels The measurement of equilibrium water uptake can reflect the swelling property of the hydrogel. Briefly, 400 μL of each GelMA-CS precursor solution was placed in a glass vial (diameter: 12 mm) and exposed to UV light to obtain a solid cylindrical hydrogel disc with the thickness of 3 mm (Fig. S1). Once the photopolymerization 7
ACCEPTED MANUSCRIPT was complete, the hydrogel discs were placed in measuring cups, followed by freezing and lyophilization to measure the dry weight (Wd ) of the discs. Then, the dried hydrogels were rehydrated in phosphate buffered solution (PBS, pH = 7) for 24 hours to reach equilibrium swelling. The PBS was pipetted out and the wet swollen hydrogel discs were then weighed (recorded as Ws ) after gently blotting the excess
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liquid by filter paper. The equilibrium water uptake per unit weight of the hydrogel was defined as:
(1)
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Qs = (Ws -Wd )/Wd .
basic (pH = 10) environment as done in PBS.
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2.7. Degradation property
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Equilibrium water uptake of the hydrogels was also tested in the acid ic (pH = 4) and
For degradation analysis, 5 mL PBS with 2.5 U/mL collagenase type Ι was added
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to the swelling hydrogels in the measuring cups. Then, the hydrogels were incubated at 37 o C on a shaker with 130 rpm. At different time points (4, 12, 24, 48 and 96
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hours), the enzyme solution was removed and the hydrogels were frozen and lyophilized to determine the dry weight of the remaining hydrogels (W r). Four
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replicates were used for each hydrogel at each time point. The percentage of remaining mass after enzymatic degradation was calculated as: (2)
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Qd = (Wr/Wd )×100%.
2.8. Cell attachment and spreading assay Since BMSCs can differentiate into chondrocytes that are beneficial for cartilage regeneration, BMSCs were utilized for in vitro cell outgrowth on hydrogels. BMSCs were isolated from femora of 4- to 6-week old S-D rats as previously described with minor modifications [35, 36]. Briefly, both ends of the femora were cut away from the epiphysis, and the bone marrow was flushed out of the diaphysis using a syringe with Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). The marrow was collected and cultured for 3 days in a 8
ACCEPTED MANUSCRIPT humidified atmosphere of 95% air and 5% CO 2 . The first medium change was done after 4 days and twice a week thereafter. BMSCs were passaged once they reached 80-90% confluence. BMSCs between the passages 4 and 9 were used for all experiments. For cell attachment and spreading assay, all samples were prepared as listed in
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Table 1 and lyophilized. Then, the samples were sterilized by ethylene oxide vapor. Before seeding cells, the samples were washed and neutralized with PBS for several
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times until the pH value reached about 7. The hydrogels were seeded with 1×105 cells/cm2 on the top surface and cultured in a 24-well plate. The culture medium was
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half changed every two days. At day 5, the cell-seeded disks were rinsed with PBS and incubated with 1 µg/mL Calcein-AM and 5 µg/mL propidium iodide (PI) for 30
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minutes to stain living and dead cells. The hydrogels were then imaged using a fluorescence microscope (Ti-S, Nikon, Japan). Three replicates were used for each
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sample. The spreading area of cells was analyzed by Image J software.
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2.9. Statistical analysis
All the data were subjected to statistical analysis and reported as the mean ±
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standard deviation of the samples. Statistical significance (*p < 0.05, **p < 0.01) were determined using a Bonferroni’s multiple comparison one-way ANOVA. At
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least three independent experiments were performed for each condition of the study.
3. Results
3.1. Formation of semi-IPN and IPN structure The semi-IPN and IPN GelMA-CS hydrogels were prepared through photocrosslinking and basification. In the first step, UV irradiation covalently crosslinks GelMA network, in which CS is only entrapped but not crosslinked, forming semi-IPN structures. Since CS is a pH-dependent cationic polymer, it is water-soluble when the pH value is below 6.2 [24]. In an acidic environment, the free amino groups (-NH2 ) are protonated as -NH3 +, which causes electrostatic repulsion 9
ACCEPTED MANUSCRIPT between CS molecules. The basification reduced the electrostatic repulsion of protonated -NH3 + and subsequently induced extensive hydrophobic interactions as well as hydrogen bonding between the adjacent chains [24, 37], which eventually led to the formation of another network. The hydrogen-bonded CS network entangles and interpenetrates with the covalently crosslinked GelMA network, forming the hybrid
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IPN structure (Fig. 1).
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Fig. 1. Schematic of the formation of semi-IPN and IPN GelMA-CS hydrogels
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3.2. FTIR spectra analysis
In Fig. 2, FTIR spectra of CS (CS2) show adsorption bands at 3300-3500 cm-1
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corresponding to the partially overlapped amine and hydroxyl stretching vibrations, 1595 cm-1 corresponding to the amide and amine bending vibrations, 1417 cm-1 corresponding to O-H bending vibrations, 1649 cm-1 due to amide C=O stretching
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vibrations of amide I, 1151 and 1027 cm-1 corresponding to C-O-C asymmetric stretching and C-O stretching vibrations, respectively; which are typical CS saccharide structure [21, 38, 39]. FTIR spectrum of GelMA (G10 group) shows the characteristic bands of amide I, amide II and amide III, at 1649 cm-1 , 1542 cm-1 , and 1238 cm-1 , respectively; caused by C-N stretching vibrations, amide N-H in-plane bending vibrations and CH2 wagging vibrations. Bands corresponding to N-H and O-H stretching vibrations overlap in the adsorption peak at 3308 cm-1 .
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Fig. 2. FTIR spectra of semi-IPN and IPN GelMA-CS hydrogels
FTIR spectra of GelMA-CS are very similar to those of pure GelMA due to
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higher proportion of GelMA in the hybrid hydrogels. The increase of CS proportion from 0.5% to 2% could not be shown obviously in the spectra of Fig. 2. Compared to
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semi-IPN samples, the bonds of IPN samples show an increase of amide I band, from 1649 to 1681 cm-1 , indicating the formation of hydrogen bonds among –NH2 and –OH
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groups after the deprotonation of CS [40].
3.3. Hydrogel morphology Figure 3 shows the typical morphology of semi-IPN and IPN structure for GelMA-CS hydrogels. Fig. 3 (A,B) is the morphology of pure CS and GelMA hydrogels with loose structure, respectively. When they are blended together and cured under UV irradiation, the semi-IPN structure shows a much denser structure with smaller pores than those of both pure CS and GelMA hydrogels, as shown in Fig. 3 (C,E,G). The increase of CS concentration doesn’t significantly change the morphology of the semi- IPN groups. In contrast, as the concentration of CS increases, 11
ACCEPTED MANUSCRIPT bigger pores with thicker pore-wall formed in the IPN groups, indicating more
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macromolecular chains are entangled and bonded together.
Fig. 3. Morphology of semi-IPN and IPN GelMA-CS hydrogels (500×, scale bar: 50 μm)
3.4. Mechanical properties
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Fig. 4. (A) Compressive and (B) tensile curves of GelMA-CS hydrogels
Figure 4 (A,B) presents the typical compressive and tensile curves of the GelMA-CS hydrogels. The values of tensile/compressive modulus, strength, and ultimate tensile strain are listed in Table 2. The pure CS hydrogel is fragile with the compressive modulus of only 3.43 kPa, and it is even difficult to make a regular band bulk for tensile test. The pure GelMA hydrogel is much tougher than the pure CS hydrogel, with the compressive modulus of 31.08 kPa and Young’s modulus of 2.05 13
ACCEPTED MANUSCRIPT kPa. The GelMA-CS hydrogels are greatly strengthened by forming a semi-IPN or IPN structure compared to pure GelMA or CS hydrogels. It is shown in Fig. 4 that the IPN structure is much stronger than the semi-IPN structure for all three groups with different GelMA-CS compositions. Both the compressive modulus and Young’s modulus of semi-IPN and IPN increased with the CS concentration, and the modulus
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values of IPN groups are much larger than those of semi- IPN groups, indicating that the formation of CS network significantly enhances the mechanical properties of
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GelMA-CS hydrogels. G10CS2-IPN group had the highest compressive modulus with the value of 116.08 kPa, which is 33 times higher than that of pure CS and 3 times
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higher than that of pure GelMA. G10CS2-IPN also shows the highest Young’s modulus with the value of 59.43 kPa, 28 times higher than that of pure GelMA. Table
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2 shows that both the modulus and strength of GelMA-CS IPN hydrogels increase linearly with the composition of CS in both the tensile and compressive tests. The
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highest tensile strength of G10CS2-IPN reaches 37.75 kPa, about 11 times of the value of pure GelMA hydrogels. Whereas, pure GelMA hydrogel has the largest
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ultimate tensile strain, about 161% of its initial length. The ultimate tensile strain of semi-IPN group increases with CS concentration, while the IPN group exhibits
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opposite tendency.
Table 2. Compressive and tensile properties of the semi-IPN and IPN hydrogels Young’s modulus (kPa)
Tensile strength (kPa)
Ultimate tensile strain (%)
CS2
3.43±0.87
G10 G10CS0.5 G10CS1 G10CS2
31.08±3.61 37.48±8.58 49.24±4.50 56.59±9.15
2.05±0.49 4.87±0.63 9.17±1.97 11.95±2.12
3.42±0.61 3.67±0.63 6.21±3.56 8.85±3.28
161.03±37.65 85.01±7.91 95.79±15.31 100.87±5.45
G10CS0.5-IPN G10CS1-IPN
55.60±7.08 98.67±6.46
13.38±1.34 24.62±4.63
10.45±2.05 19.25±1.77
97.54±4.61 96.06±13.14
G10CS2-IPN
116.08±9.62
59.43±4.51
37.75±5.35
87.2±15.83
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Groups
Compressive modulus (kPa)
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3.5. Swelling property
Fig. 5. (A) Equilibrium water uptake of semi-IPN and IPN GelMA-CS hydrogels in PBS and (B) equilibrium water uptake of IPN GelMA-CS hydrogels in different pH values
In Fig. 5 (A), the equilibrium water uptake of pure CS (CS2 group) reaches 37.4 and that of pure GelMA (G10 group) is about 15.2. After blending of GelMA and CS, 15
ACCEPTED MANUSCRIPT the equilibrium water uptake is significantly decreased for all groups, compared to the value of pure GelMA or CS hydrogels. As the concentration of CS increases, the equilibrium water uptake shows a monotonically decreasing trend, but the variation is not obvious between adjacent groups. The equilibrium water uptake of IPN groups doesn’t decrease significantly compared to the semi-IPN groups with the same ratio of
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GelMA and CS in PBS, and the CS concentration only has a slight influence. However, when the pH value of environment changes, the equilibrium water uptake
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of pure GelMA hydrogels increases with pH value, while that of other groups
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decreases with pH value once CS is added, as shown in Fig. 5 (B).
3.6. Degradation property
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GelMA and CS exhibit reverse degradation properties in the presence of collagenase, as shown in Fig. 6. The weight loss of CS is less than 18% after 96-hour
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degradation; while only 13.8% weight of GelMA hydrogels remains after 24 hours and disappeared after 48 hours degradation. The addition of CS slows down the
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degradation of GelMA hydrogels enormously and the IPN groups are more stable than the semi-IPN groups. The degradation rate of semi-IPN and IPN groups both
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decreases with CS concentration, indicating the formation of a stronger network as the
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increase of CS concentration.
16
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60
CS2 G10 G10CS0.5 G10CS0.5-IPN G10CS1 G10CS1-IPN G10CS2 G10CS2-IPN
40
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Remaining mass (%)
80
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20
0
8
16 24 32 40 48 56 64 72 80 88 96 Time (hour)
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0
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3.7. Cell attachment and spreading assay
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Fig. 6. Enzymatic degradation of semi-IPN and IPN GelMA-CS hydrogels
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Fig. 7. BMSCs adhesion on the surfaces of semi-IPN and IPN GelMA-CS hydrogels (100×, scale bar: 100 μm)
It is obvious in Fig. 7 (A) that the surface of pure CS hydrogel is not suitable for cell adhesion and spreading with only a few cells on the surface. In contrast, BMSCs exhibit vigorous spreading on the GelMA hydrogel surface, and the percentage of spreading area reaches 30.7% (Fig. 7B). These cells exhibit better spreading on the IPN groups than on the semi-IPN GelMA-CS hydrogels. The spreading area of 18
ACCEPTED MANUSCRIPT G10CS1-IPN is not significantly different from that of G10CS0.5-IPN, about 16% of the whole area. However, when the concentration of CS reaches 2%, both the number and spreading area of the attached cells reduce significantly, regardless of the semi-IPN or IPN surface, with the percent of spreading area of 3.6% and 6.9%,
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respectively.
4. Discussion
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The improvement and regulation of the mechanica l properties of hydrogels are crucially important in biomedical applications because of the profound effects of
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materials stiffness on cell adhesion and adhesion-related behaviors. The formation of IPN structures in this work is an effective method to improve the mechanical
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properties as well as incorporating the advantages of two or more hydrogels. In most of previous studies, semi-IPN hydrogels were reported to have better
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flexibility but lower stiffness due to the dissipation of strain energy during deformation [1, 41]. While in this study, the stiffness of semi- IPN GelMA-CS
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hydrogels is found to be dramatically enhanced in comparison with pure GelMA hydrogels. One possible reason is that the moving space of photocrosslinked GelMA
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network in semi-IPN hydrogels is heavily restricted by embedded CS chains. When the hydrogels are basified with NaOH solution, hydrophobic interactions begin to play a dominant role in CS aggregation because CS is not soluble in water above pH = 6.2.
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Hydrogen bonds among –NH2 and –OH are also produced (demonstrated by FTIR in Fig. 1), which further strengthened the IPN hydrogels in a large scale. As the CS concentration increases, more hydrogen bonds are formed in the full IPN structure, so there are more CS chains entangling with GelMA chains. This is the reason of the morphology of G10CS2-IPN hydrogels has larger pores with thicker walls, which lead to a strong enhancement of Young’s modulus (59.43 kPa) and compressive modulus (116.08 kPa) compared to those of pure GelMA and CS hydrogels (Table 2).
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Fig. 8. Young’s modulus of semi-IPN and IPN hydrogels containing 1% CS and
MA
different concentrations of GelMA
Previous studies have reported that the mechanical properties of photocrosslinked
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GelMA hydrogels are influenced by factors such as the duration and intensity of UV curing, the concentration of GelMA, and so on [42, 43]. In this work, we also
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investigated the influence of GelMA concentration on the mechanical properties of GelMA-CS hydrogels. It is shown in Fig. 8 that Young’s modulus of both semi- IPN
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and IPN GelMA-CS hydrogels increases with GelMA concentration from 5% to 20%. It should be noticed that the compressive modulus of G20CS1-IPN hydrogels (GelMA concentration: 20%) reaches 233.25 kPa, which are much stronger than those gelatin-CS hydrogels crosslinked by chemical crosslinkers in previous studies [6, 19]. Such performance can even meet the requirements of some cartilaginous biological tissues, such as immature articular cartilage in compression (100-300 kPa) [44, 45]. The mechanical properties of GelMA-CS hydrogels are effectively regulated by adjusting the GelMA concentration as well. Generally, the hydrogels with higher mechanical properties have a higher crosslinking degree and lower swelling ratio. Here, the semi-IPN and IPN GelMA-CS 20
ACCEPTED MANUSCRIPT hydrogels exhibit similar swelling behavior in PBS. This is probably caused by the fact that the neutral environment of PBS helped the semi- IPN hydrogels crosslink and transform into IPN hydrogels. Also, the formation of semi-IPN and IPN structure was found to greatly slow down the degradation of GelMA. In particular, the IPN groups showed lower degradation rate than the semi- IPN groups due to the more stable
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network of IPN groups. Even though the mechanical properties can be improved by increasing GelMA
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and CS concentration, the higher concentration usually leads to higher crosslinking density in the hydrogels, which is not suitable for further biomedical applications.
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Here, we find when the concentration of CS reaches 2%, the morphology of BMSCs becomes round and their adhesion and spreading area decrease s (Fig. 7G,H). Similar
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observations were reported previously [17], with the finding that CS reduced cell-spreading area, and disrupted F-actin and focal adhesion kinase (FAK)
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distribution in the cells, leading to the lack of specific binding domains for cell adhesion on CS surface. GelMA also plays a significant role in regulating cell
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spreading and size on hydrogel membranes since gelatin contains Arg-Gly-Asp (RGD)- like sequence, which promotes cell adhesion and migration. Thus, the GelMA
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concentration should be increased if cell adhesion and spreading is the predominant requirement in certain applications, such as the surface of tissues. For the regeneration of load-bearing tissues, such as cartilage, the material toughness is a more critical
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factor, so the concentrations of both GelMA and CS should be increased to strengthen the hydrogels. Therefore, in practical biomedical applications, the mechanical properties and cell spreading should be compromised and optimized by tuning the concentration and ratio of GelMA and CS. Interestingly, BMSCs exhibit a more spread morphology on the IPN hydrogels at the same concentration of GelMA and CS, mainly due to the more compact and flat surface of the IPN hydrogels.
5. Conclusions In this study, we employed photocrosslinking and basification to fabricate 21
ACCEPTED MANUSCRIPT semi-IPN and IPN GelMA-CS hydrogels by the formation of covalent bonds and hydrophobic interactions. The formation of IPN network was demonstrated to be an effective approach to tailor the mechanical properties of the resultant GelMA-CS hydrogels. The IPN GelMA-CS hydrogels were confirmed to inherit the excellent biocompatibility as pure GelMA. Therefore, the present approach and the composite
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IPN GelMA-CS hydrogels may provide a promising candidate to fulfill the diversified
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demands in a variety of biomedical applications.
Acknowledgement
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This study was supported by the National Natural Science Foundation of China (NSFC, Grant No. 11402056, 51605426, 11672268, 11621062), the Key Research
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and Development Program of Zhejiang Province (Grant No. 2017C01063).
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Graphical abstract
28
ACCEPTED MANUSCRIPT Highlights
Design and synthesis hydrogel composed of GelMA and chitosan with interpenetrating network using photocrosslinking and basification
Effectively enhancing the mechanical properties of GelMA-chitosan hydrogels
Retaining
excellent
biocompatibility
and
enzymatic
degradability
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GelMA-chitosan hydrogels
29
of
Hairui Suo, Deming Zhang, Jun Yin, Jin Qian, Zi Liang Wu, Jianzhong Fu PII: DOI: Reference:
S0928-4931(17)34088-2 doi:10.1016/j.msec.2018.07.016 MSC 8728
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
12 October 2017 19 June 2018 6 July 2018
Please cite this article as: Hairui Suo, Deming Zhang, Jun Yin, Jin Qian, Zi Liang Wu, Jianzhong Fu , Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering. Msc (2018), doi:10.1016/j.msec.2018.07.016
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ACCEPTED MANUSCRIPT Interpenetrating polymer network hydrogels composed of chitosan and photocrosslinkable gelatin with enhanced mechanical properties for tissue engineering
Hairui Suo1,2,†, Deming Zhang1,2,†, Jun Yin1,2,*, Jin Qian3,*, Zi Liang Wu4,*,
The State Key Laboratory of Fluid Power and Mechatronic Systems, School of
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1
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Jianzhong Fu1,2
Mechanical Engineering, Zhejiang University, Hangzhou 310027, China Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School
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2
of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China Key Laboratory of Soft Machines and Smart Devices of Zhejiang Province,
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3
Department of Engineering Mechanics, Zhejiang University, Hangzhou 310027,
4
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China
MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
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Department of Polymer Science and Engineering, Zhejiang University, Hangzhou
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310027, China
† These authors contributed equally to this work.
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* Corresponding authors. E- mail: [email protected], [email protected], and [email protected]
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ACCEPTED MANUSCRIPT Abstract: Gelatin and chitosan (CS) are widely used natural biomaterials for tissue engineering scaffolds, but the poor mechanical properties of pure gelatin or CS hydrogels become a big obstacle that limits their use as scaffolds, especially in load-bearing tissues. This study provided a novel mechanism of forming interpenetrating network (IPN) of
interactions
through
photocrosslinking
and
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gelatin methacryloyl (GelMA) and CS hydrogels by covalent bonds and hydrophobic basification,
respectively.
By
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characterization of the compressive and tensile moduli, ultimate te nsile stress and strain, it was found that semi-IPN and IPN structure can greatly enhance the
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mechanical properties of GelMA-CS hydrogels compared to the single network CS or GelMA. Moreover, the increase of either GelMA or CS concentration can strengthen
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the hydrogel network. Then, the swelling, enzymatic degradation, and morphology of GelMA-CS hydrogels were also systematically
investigated.
The excellent
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biocompatibility of GelMA-CS hydrogels was demonstrated by large spreading area of bone mesenchymal stem cells on hydrogel surfaces when CS concentration was
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less than 2% (w/v). According to this study, the multiple requirements of properties can be fulfilled by carefully selecting the GelMA and CS compositions for IPN
Keywords:
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hydrogels.
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Interpenetrating network, gelatin, chitosan, photocrosslinking, mechanical property
2
ACCEPTED MANUSCRIPT 1. Introduction Tissue engineering aims to support and promote regeneration of damaged or diseased tissues using a scaffold seeded with cells and growth factors [1]. The scaffold is expected to substitute extracellular matrix (ECM) and provides a temporary place for cell growth [2]. Therefore, the materials for scaffolds should be
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rigorously selected according to their biocompatibility, degradation, mechanical properties, etc. Gelatin and chitosan (CS) are the most preferred natural biomaterials
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for tissue engineering scaffolds since they are close ly related to ECM. Gelatin is the product of collagen hydrolysis, the main component of ECM, and has been widely
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used in tissue engineering because of its antigenicity and biocompatibility. CS is a partially deacetylated derivative of chitin and its structure is similar to that of
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glycosaminoglycans (GAGs), another important component of ECM, which makes CS an ideal scaffold material for tissue engineering [3]. Moreover, CS exhibits
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considerable antimicrobial and hemostatic properties due to its cationic nature of amino group [4, 5]. Due to their excellent biological properties, the mixture of gelatin
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and CS have been used to fabricate various scaffolds, which have been applied in numerous areas of tissue engineering, such as skin [6], cartilage [7], bone [8, 9],
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osteochondral [10], nerve [11], liver [12, 13], and so on. Although gelatin- and CS-based hydrogels have their advantages, poor mechanical property becomes a big obstacle that limits their applications as scaffolds,
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especially in load-bearing tissues [11, 14]. Several studies have been reported to improve the mechanical properties of hydrogels [15, 16], but very few investigations on improving the mechanical properties of gelatin-CS hydrogels have been carried out. The compressive modulus of gelatin-CS hydrogels neutralized with alcohol is about 1.15 to 3.4 kPa in wet condition [17]. Combining gelatin-CS hydrogels with other synthetic polymers was found to be a promising approach to enhance their mechanical properties, but it either was time-consuming or would weaken the biocompatibility and degradability of natural materials [18]. Chemical crosslinkers also have been introduced to improve the mechanical properties of gelatin-CS hydrogels. For 3
ACCEPTED MANUSCRIPT example, Kathuria et al. [19] synthesized the gelatin-CS hydrogels crosslinked by glutaraldehyde with the compressive modulus varied between 36-39 kPa. However, the remaining unreacted chemical crosslinkers and the toxic by-products may make these hydrogels unfit for tissue engineered applications in vitro and in vivo. Recently, increasing interests and more efforts were devoted to genipin [13, 20, 21], a natural
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biocompatible crosslinker derived from gardenia fruit. However, genipin was reported to possess genotoxicity and showed detrimental effects on chondrocytes above 220
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µM in vitro for a long-time culture [22, 23]. Therefore, how to effectively enhance the mechanical properties of gelatin-CS hydrogels with preserved biocompatibility and
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degradability remains a big challenge for further applications of gelatin-CS hydrogels. Recently, the crosslinking approaches on gelatin-CS hydrogels without organic
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solvents have gained increasing attention, such as solvent exchange, UV irradiation, pH change, and temperature modulation [24]. Since the photocrosslinking leads to [25,
26],
photocrosslinkable
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strong covalent bonds
gelatins obtained
by
methacrylation have been manufactured as tissue engineering scaffolds for cardiac
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tissues [27], blood vessels [28], and skins [29]. Moreover, CS can also be crosslinked by neutralization in either dilute NaOH or ethanol solution to form a hydrophobic
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network before its application in tissue engineering as scaffolds [30]. Therefore, we hypothesized that the interpenetrating of the photocrosslinked gelatin network and the hydrophobic CS network would be an effective way to enhance their mechanical
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properties.
When one hydrophilic polymer chain permeates another polymeric network without any chemical bonds between them, the (semi- )interpenetrating network (IPN) forms. In semi-IPN, only one crosslinked network forms physically or chemically, while in IPN, two or more crosslinked networks form, permeating with each other. The formation of (semi-)IPN would not only preserve the characteristics of each network structure, but also improve the stability and mechanical properties of the crosslinked
networks
[20].
Li et al.
[31]
fabricated
IPN
hydrogels of
gelatin- graft-polyaniline, carboxymethyl-CS and oxided dextran via Schiff base 4
ACCEPTED MANUSCRIPT reaction, which had the storage modulus as high as 924 kPa, good cytocompatibility and enhanced cell proliferation. Thus, the (semi-)IPN leads to the new systems with the combination of favorable properties of each constituent polymer, which quite often are substantially different from those of the individual polymer s [32]. The objective of this work is to develop IPN gelatin methacryloyl (GelMA)-CS hydrogels
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through photocrosslinking and basification, which not only have enhanced mechanical properties than either single network GelMA or CS hydrogel; but also possess the
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advantages of the two biomaterials for biomedical applications. The obtained hydrogels were well characterized by Fourier transform infrared (FTIR) spectra and
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scanning electronic microscope (SEM). Their mechanical properties were evaluated by compressive and tensile tests. The swelling ratio of the materials at different pH
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values and the enzymatic degradation rate were also investigated. Finally, the biocompatibility of the semi- IPN and IPN GelMA-CS hydrogels were evaluated in
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vitro by growing bone mesenchymal stem cells (BMSCs) on the hydrogels. The IPN approach proposed in this study is found to effectively improve the mechanical
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properties of GelMA-CS hydrogels without toxic or chemical crosslinkers, which may
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significantly extend the application of GelMA-CS hydrogels.
2. Materials and Methods 2.1. Synthesis of GelMA
Briefly,
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Gelatin methacryloyl (GelMA) was synthesized as previously described [33, 34]. 15%
(w/v)
gelatin
(MA,
Aladdin)
was dissolved
in
0.25
M
carbonate-bicarbonate buffer (7.95 g sodium carbonate and 14.65 g sodium bicarbonate in 1 L distilled water) at 50 o C. Methacrylic anhydride (MA, Aladdin) was added at a rate of 0.5 mL/min under a stirring condition at 50 o C. After reaction for 3 hours, the reacted solution was diluted and dialyzed against distilled water using 8-14 kDa cutoff dialysis tubes for 7 days at 40 o C. The dialyzed GelMA solution was then filtered through 0.22 μm filter membrane, lyophilized and stored at -80 o C for further use. 5
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2.2. Photocrosslinking and centralization of GelMA-CS hydrogels To form GelMA-CS hydrogels, 30% (w/v) GelMA and 3% (w/v) CS (low viscosity: < 200 mPa∙s, Aladdin) was separately dissolved in 1% (v/v) acetyl acid with
0.1%
(w/v)
photoinitiator
2-hydroxy-1-(4-(hydroxyethoxy)
pyenyl)-2-methyl-1-propanone (Irgacure 2959, Sigma). Then the two polymer
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precursors were blended homogeneously under 37 o C in different ratios as listed in
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Table 1, where 10% GelMA and 2% CS with single network were prepared as a control. Then, the precursor solutions were exposed to a 1.5 W/cm2 UV light
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(wavelength: 365 nm) and photocrosslinked for 30 seconds. Finally, the precursor
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solutions were basified in 0.1 mol/L NaOH for 30 minutes to form IPNs.
Table 1. Samples for experiment
CS2 G10 G10CS0.5 G10CS1 G10CS2 G5CS1 G15CS1 G20CS1 G10CS0.5-IPN G10CS1-IPN G10CS2-IPN G5CS1-IPN G15CS1-IPN G20CS1-IPN
Single network
GelMA (w/v %) 0 10 10 10 10 5 15 20 10 10 10 5 15 20
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Network
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Groups
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Semi-IPN
IPN
Chitosan (w/v %) 2 0 0.5 1 2 1 1 1 0.5 1 2 1 1 1
Crosslinking method Basification
Photocrosslink
Photocrosslink and basification
2.3. FT-IR study FT-IR spectra were performed using a Shimadzu FT-IR spectrometer with a resolution of 2 cm-1 at wavenumber ranging of 4000-400 cm-1 . Samples were lyophilized and grinded in liquid nitrogen. The dried sample powder was blended 6
ACCEPTED MANUSCRIPT homogeneously with KBr pellet and scanned against a blank KBr pellet background.
2.4. Morphology of the hydrogels Freshly prepared samples were frozen in -80 o C for 12 hours and lyophilized for 48 hours. The samples were fractured and sputtered with gold vapor for 40 seconds,
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and then the morphology of the fractured surface of the hydrogels was observed using
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a SEM (TM1000, Hitachi) with an acceleration voltage of 15 kV.
2.5. Mechanical tests
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The tensile and compressive properties of the hydrogels were determined using a testing machine ElectroForce (TA Instruments, America) at room temperature.
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Cylindrical samples with the diameter of 12 mm and the thickness of 3 mm were prepared for the compressive test. The samples were placed between two parallel
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plates (diameter of 25 mm) and the compressive force was applied parallel to the longitudinal axis of the samples with the rate of 1.0 mm/min. The compressive
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modulus was determined as the slope of the stress-strain curve within 0-10% strain. At least 3 samples were used for each testing groups.
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For the tensile test, rectangular samples were prepared with the width of 8 mm and thickness of 2.5 mm. Samples were tested between two clamps with initial distance of 5 mm. The uniaxial stretch was performed with the rate of 1 mm/min until
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the samples broke. The tensile properties were represented by Young’s modulus (the tangent slope within 0-10% strain of the stress-strain curve), ultimate stress (stress at failure), and maximum strain (strain at failure) of the GelMA-CS hydrogels.
2.6. Swelling properties of the hydrogels The measurement of equilibrium water uptake can reflect the swelling property of the hydrogel. Briefly, 400 μL of each GelMA-CS precursor solution was placed in a glass vial (diameter: 12 mm) and exposed to UV light to obtain a solid cylindrical hydrogel disc with the thickness of 3 mm (Fig. S1). Once the photopolymerization 7
ACCEPTED MANUSCRIPT was complete, the hydrogel discs were placed in measuring cups, followed by freezing and lyophilization to measure the dry weight (Wd ) of the discs. Then, the dried hydrogels were rehydrated in phosphate buffered solution (PBS, pH = 7) for 24 hours to reach equilibrium swelling. The PBS was pipetted out and the wet swollen hydrogel discs were then weighed (recorded as Ws ) after gently blotting the excess
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liquid by filter paper. The equilibrium water uptake per unit weight of the hydrogel was defined as:
(1)
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Qs = (Ws -Wd )/Wd .
basic (pH = 10) environment as done in PBS.
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2.7. Degradation property
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Equilibrium water uptake of the hydrogels was also tested in the acid ic (pH = 4) and
For degradation analysis, 5 mL PBS with 2.5 U/mL collagenase type Ι was added
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to the swelling hydrogels in the measuring cups. Then, the hydrogels were incubated at 37 o C on a shaker with 130 rpm. At different time points (4, 12, 24, 48 and 96
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hours), the enzyme solution was removed and the hydrogels were frozen and lyophilized to determine the dry weight of the remaining hydrogels (W r). Four
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replicates were used for each hydrogel at each time point. The percentage of remaining mass after enzymatic degradation was calculated as: (2)
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2.8. Cell attachment and spreading assay Since BMSCs can differentiate into chondrocytes that are beneficial for cartilage regeneration, BMSCs were utilized for in vitro cell outgrowth on hydrogels. BMSCs were isolated from femora of 4- to 6-week old S-D rats as previously described with minor modifications [35, 36]. Briefly, both ends of the femora were cut away from the epiphysis, and the bone marrow was flushed out of the diaphysis using a syringe with Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). The marrow was collected and cultured for 3 days in a 8
ACCEPTED MANUSCRIPT humidified atmosphere of 95% air and 5% CO 2 . The first medium change was done after 4 days and twice a week thereafter. BMSCs were passaged once they reached 80-90% confluence. BMSCs between the passages 4 and 9 were used for all experiments. For cell attachment and spreading assay, all samples were prepared as listed in
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Table 1 and lyophilized. Then, the samples were sterilized by ethylene oxide vapor. Before seeding cells, the samples were washed and neutralized with PBS for several
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times until the pH value reached about 7. The hydrogels were seeded with 1×105 cells/cm2 on the top surface and cultured in a 24-well plate. The culture medium was
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half changed every two days. At day 5, the cell-seeded disks were rinsed with PBS and incubated with 1 µg/mL Calcein-AM and 5 µg/mL propidium iodide (PI) for 30
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minutes to stain living and dead cells. The hydrogels were then imaged using a fluorescence microscope (Ti-S, Nikon, Japan). Three replicates were used for each
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sample. The spreading area of cells was analyzed by Image J software.
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2.9. Statistical analysis
All the data were subjected to statistical analysis and reported as the mean ±
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3. Results
3.1. Formation of semi-IPN and IPN structure The semi-IPN and IPN GelMA-CS hydrogels were prepared through photocrosslinking and basification. In the first step, UV irradiation covalently crosslinks GelMA network, in which CS is only entrapped but not crosslinked, forming semi-IPN structures. Since CS is a pH-dependent cationic polymer, it is water-soluble when the pH value is below 6.2 [24]. In an acidic environment, the free amino groups (-NH2 ) are protonated as -NH3 +, which causes electrostatic repulsion 9
ACCEPTED MANUSCRIPT between CS molecules. The basification reduced the electrostatic repulsion of protonated -NH3 + and subsequently induced extensive hydrophobic interactions as well as hydrogen bonding between the adjacent chains [24, 37], which eventually led to the formation of another network. The hydrogen-bonded CS network entangles and interpenetrates with the covalently crosslinked GelMA network, forming the hybrid
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IPN structure (Fig. 1).
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Fig. 1. Schematic of the formation of semi-IPN and IPN GelMA-CS hydrogels
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3.2. FTIR spectra analysis
In Fig. 2, FTIR spectra of CS (CS2) show adsorption bands at 3300-3500 cm-1
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corresponding to the partially overlapped amine and hydroxyl stretching vibrations, 1595 cm-1 corresponding to the amide and amine bending vibrations, 1417 cm-1 corresponding to O-H bending vibrations, 1649 cm-1 due to amide C=O stretching
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vibrations of amide I, 1151 and 1027 cm-1 corresponding to C-O-C asymmetric stretching and C-O stretching vibrations, respectively; which are typical CS saccharide structure [21, 38, 39]. FTIR spectrum of GelMA (G10 group) shows the characteristic bands of amide I, amide II and amide III, at 1649 cm-1 , 1542 cm-1 , and 1238 cm-1 , respectively; caused by C-N stretching vibrations, amide N-H in-plane bending vibrations and CH2 wagging vibrations. Bands corresponding to N-H and O-H stretching vibrations overlap in the adsorption peak at 3308 cm-1 .
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Fig. 2. FTIR spectra of semi-IPN and IPN GelMA-CS hydrogels
FTIR spectra of GelMA-CS are very similar to those of pure GelMA due to
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higher proportion of GelMA in the hybrid hydrogels. The increase of CS proportion from 0.5% to 2% could not be shown obviously in the spectra of Fig. 2. Compared to
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semi-IPN samples, the bonds of IPN samples show an increase of amide I band, from 1649 to 1681 cm-1 , indicating the formation of hydrogen bonds among –NH2 and –OH
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3.3. Hydrogel morphology Figure 3 shows the typical morphology of semi-IPN and IPN structure for GelMA-CS hydrogels. Fig. 3 (A,B) is the morphology of pure CS and GelMA hydrogels with loose structure, respectively. When they are blended together and cured under UV irradiation, the semi-IPN structure shows a much denser structure with smaller pores than those of both pure CS and GelMA hydrogels, as shown in Fig. 3 (C,E,G). The increase of CS concentration doesn’t significantly change the morphology of the semi- IPN groups. In contrast, as the concentration of CS increases, 11
ACCEPTED MANUSCRIPT bigger pores with thicker pore-wall formed in the IPN groups, indicating more
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macromolecular chains are entangled and bonded together.
Fig. 3. Morphology of semi-IPN and IPN GelMA-CS hydrogels (500×, scale bar: 50 μm)
3.4. Mechanical properties
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Fig. 4. (A) Compressive and (B) tensile curves of GelMA-CS hydrogels
Figure 4 (A,B) presents the typical compressive and tensile curves of the GelMA-CS hydrogels. The values of tensile/compressive modulus, strength, and ultimate tensile strain are listed in Table 2. The pure CS hydrogel is fragile with the compressive modulus of only 3.43 kPa, and it is even difficult to make a regular band bulk for tensile test. The pure GelMA hydrogel is much tougher than the pure CS hydrogel, with the compressive modulus of 31.08 kPa and Young’s modulus of 2.05 13
ACCEPTED MANUSCRIPT kPa. The GelMA-CS hydrogels are greatly strengthened by forming a semi-IPN or IPN structure compared to pure GelMA or CS hydrogels. It is shown in Fig. 4 that the IPN structure is much stronger than the semi-IPN structure for all three groups with different GelMA-CS compositions. Both the compressive modulus and Young’s modulus of semi-IPN and IPN increased with the CS concentration, and the modulus
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values of IPN groups are much larger than those of semi- IPN groups, indicating that the formation of CS network significantly enhances the mechanical properties of
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GelMA-CS hydrogels. G10CS2-IPN group had the highest compressive modulus with the value of 116.08 kPa, which is 33 times higher than that of pure CS and 3 times
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higher than that of pure GelMA. G10CS2-IPN also shows the highest Young’s modulus with the value of 59.43 kPa, 28 times higher than that of pure GelMA. Table
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2 shows that both the modulus and strength of GelMA-CS IPN hydrogels increase linearly with the composition of CS in both the tensile and compressive tests. The
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highest tensile strength of G10CS2-IPN reaches 37.75 kPa, about 11 times of the value of pure GelMA hydrogels. Whereas, pure GelMA hydrogel has the largest
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ultimate tensile strain, about 161% of its initial length. The ultimate tensile strain of semi-IPN group increases with CS concentration, while the IPN group exhibits
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Table 2. Compressive and tensile properties of the semi-IPN and IPN hydrogels Young’s modulus (kPa)
Tensile strength (kPa)
Ultimate tensile strain (%)
CS2
3.43±0.87
G10 G10CS0.5 G10CS1 G10CS2
31.08±3.61 37.48±8.58 49.24±4.50 56.59±9.15
2.05±0.49 4.87±0.63 9.17±1.97 11.95±2.12
3.42±0.61 3.67±0.63 6.21±3.56 8.85±3.28
161.03±37.65 85.01±7.91 95.79±15.31 100.87±5.45
G10CS0.5-IPN G10CS1-IPN
55.60±7.08 98.67±6.46
13.38±1.34 24.62±4.63
10.45±2.05 19.25±1.77
97.54±4.61 96.06±13.14
G10CS2-IPN
116.08±9.62
59.43±4.51
37.75±5.35
87.2±15.83
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Groups
Compressive modulus (kPa)
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3.5. Swelling property
Fig. 5. (A) Equilibrium water uptake of semi-IPN and IPN GelMA-CS hydrogels in PBS and (B) equilibrium water uptake of IPN GelMA-CS hydrogels in different pH values
In Fig. 5 (A), the equilibrium water uptake of pure CS (CS2 group) reaches 37.4 and that of pure GelMA (G10 group) is about 15.2. After blending of GelMA and CS, 15
ACCEPTED MANUSCRIPT the equilibrium water uptake is significantly decreased for all groups, compared to the value of pure GelMA or CS hydrogels. As the concentration of CS increases, the equilibrium water uptake shows a monotonically decreasing trend, but the variation is not obvious between adjacent groups. The equilibrium water uptake of IPN groups doesn’t decrease significantly compared to the semi-IPN groups with the same ratio of
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GelMA and CS in PBS, and the CS concentration only has a slight influence. However, when the pH value of environment changes, the equilibrium water uptake
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decreases with pH value once CS is added, as shown in Fig. 5 (B).
3.6. Degradation property
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GelMA and CS exhibit reverse degradation properties in the presence of collagenase, as shown in Fig. 6. The weight loss of CS is less than 18% after 96-hour
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degradation; while only 13.8% weight of GelMA hydrogels remains after 24 hours and disappeared after 48 hours degradation. The addition of CS slows down the
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degradation of GelMA hydrogels enormously and the IPN groups are more stable than the semi-IPN groups. The degradation rate of semi-IPN and IPN groups both
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60
CS2 G10 G10CS0.5 G10CS0.5-IPN G10CS1 G10CS1-IPN G10CS2 G10CS2-IPN
40
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80
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0
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16 24 32 40 48 56 64 72 80 88 96 Time (hour)
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Fig. 6. Enzymatic degradation of semi-IPN and IPN GelMA-CS hydrogels
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Fig. 7. BMSCs adhesion on the surfaces of semi-IPN and IPN GelMA-CS hydrogels (100×, scale bar: 100 μm)
It is obvious in Fig. 7 (A) that the surface of pure CS hydrogel is not suitable for cell adhesion and spreading with only a few cells on the surface. In contrast, BMSCs exhibit vigorous spreading on the GelMA hydrogel surface, and the percentage of spreading area reaches 30.7% (Fig. 7B). These cells exhibit better spreading on the IPN groups than on the semi-IPN GelMA-CS hydrogels. The spreading area of 18
ACCEPTED MANUSCRIPT G10CS1-IPN is not significantly different from that of G10CS0.5-IPN, about 16% of the whole area. However, when the concentration of CS reaches 2%, both the number and spreading area of the attached cells reduce significantly, regardless of the semi-IPN or IPN surface, with the percent of spreading area of 3.6% and 6.9%,
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4. Discussion
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The improvement and regulation of the mechanica l properties of hydrogels are crucially important in biomedical applications because of the profound effects of
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materials stiffness on cell adhesion and adhesion-related behaviors. The formation of IPN structures in this work is an effective method to improve the mechanical
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properties as well as incorporating the advantages of two or more hydrogels. In most of previous studies, semi-IPN hydrogels were reported to have better
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flexibility but lower stiffness due to the dissipation of strain energy during deformation [1, 41]. While in this study, the stiffness of semi- IPN GelMA-CS
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hydrogels is found to be dramatically enhanced in comparison with pure GelMA hydrogels. One possible reason is that the moving space of photocrosslinked GelMA
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network in semi-IPN hydrogels is heavily restricted by embedded CS chains. When the hydrogels are basified with NaOH solution, hydrophobic interactions begin to play a dominant role in CS aggregation because CS is not soluble in water above pH = 6.2.
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Hydrogen bonds among –NH2 and –OH are also produced (demonstrated by FTIR in Fig. 1), which further strengthened the IPN hydrogels in a large scale. As the CS concentration increases, more hydrogen bonds are formed in the full IPN structure, so there are more CS chains entangling with GelMA chains. This is the reason of the morphology of G10CS2-IPN hydrogels has larger pores with thicker walls, which lead to a strong enhancement of Young’s modulus (59.43 kPa) and compressive modulus (116.08 kPa) compared to those of pure GelMA and CS hydrogels (Table 2).
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Fig. 8. Young’s modulus of semi-IPN and IPN hydrogels containing 1% CS and
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different concentrations of GelMA
Previous studies have reported that the mechanical properties of photocrosslinked
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GelMA hydrogels are influenced by factors such as the duration and intensity of UV curing, the concentration of GelMA, and so on [42, 43]. In this work, we also
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investigated the influence of GelMA concentration on the mechanical properties of GelMA-CS hydrogels. It is shown in Fig. 8 that Young’s modulus of both semi- IPN
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and IPN GelMA-CS hydrogels increases with GelMA concentration from 5% to 20%. It should be noticed that the compressive modulus of G20CS1-IPN hydrogels (GelMA concentration: 20%) reaches 233.25 kPa, which are much stronger than those gelatin-CS hydrogels crosslinked by chemical crosslinkers in previous studies [6, 19]. Such performance can even meet the requirements of some cartilaginous biological tissues, such as immature articular cartilage in compression (100-300 kPa) [44, 45]. The mechanical properties of GelMA-CS hydrogels are effectively regulated by adjusting the GelMA concentration as well. Generally, the hydrogels with higher mechanical properties have a higher crosslinking degree and lower swelling ratio. Here, the semi-IPN and IPN GelMA-CS 20
ACCEPTED MANUSCRIPT hydrogels exhibit similar swelling behavior in PBS. This is probably caused by the fact that the neutral environment of PBS helped the semi- IPN hydrogels crosslink and transform into IPN hydrogels. Also, the formation of semi-IPN and IPN structure was found to greatly slow down the degradation of GelMA. In particular, the IPN groups showed lower degradation rate than the semi- IPN groups due to the more stable
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network of IPN groups. Even though the mechanical properties can be improved by increasing GelMA
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and CS concentration, the higher concentration usually leads to higher crosslinking density in the hydrogels, which is not suitable for further biomedical applications.
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Here, we find when the concentration of CS reaches 2%, the morphology of BMSCs becomes round and their adhesion and spreading area decrease s (Fig. 7G,H). Similar
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observations were reported previously [17], with the finding that CS reduced cell-spreading area, and disrupted F-actin and focal adhesion kinase (FAK)
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distribution in the cells, leading to the lack of specific binding domains for cell adhesion on CS surface. GelMA also plays a significant role in regulating cell
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spreading and size on hydrogel membranes since gelatin contains Arg-Gly-Asp (RGD)- like sequence, which promotes cell adhesion and migration. Thus, the GelMA
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concentration should be increased if cell adhesion and spreading is the predominant requirement in certain applications, such as the surface of tissues. For the regeneration of load-bearing tissues, such as cartilage, the material toughness is a more critical
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factor, so the concentrations of both GelMA and CS should be increased to strengthen the hydrogels. Therefore, in practical biomedical applications, the mechanical properties and cell spreading should be compromised and optimized by tuning the concentration and ratio of GelMA and CS. Interestingly, BMSCs exhibit a more spread morphology on the IPN hydrogels at the same concentration of GelMA and CS, mainly due to the more compact and flat surface of the IPN hydrogels.
5. Conclusions In this study, we employed photocrosslinking and basification to fabricate 21
ACCEPTED MANUSCRIPT semi-IPN and IPN GelMA-CS hydrogels by the formation of covalent bonds and hydrophobic interactions. The formation of IPN network was demonstrated to be an effective approach to tailor the mechanical properties of the resultant GelMA-CS hydrogels. The IPN GelMA-CS hydrogels were confirmed to inherit the excellent biocompatibility as pure GelMA. Therefore, the present approach and the composite
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IPN GelMA-CS hydrogels may provide a promising candidate to fulfill the diversified
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Acknowledgement
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This study was supported by the National Natural Science Foundation of China (NSFC, Grant No. 11402056, 51605426, 11672268, 11621062), the Key Research
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and Development Program of Zhejiang Province (Grant No. 2017C01063).
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Cell- laden microengineered gelatin methacrylate hydrogels, Biomaterials, 31 (2010)
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concentration cell- laden gelatin methacrylate (GelMA) bioinks with two-step crosslinking strategy, Acs Appl. Mater. Inter., 10 (2018) 6849-6857. [44] Q.T. Nguyen, Y. Hwang, A.C. Chen, S. Varghese, R.L. Sah, Cartilage-like mechanical properties of poly (ethylene glycol)-diacrylate hydrogels, Biomaterials, 33 (2012) 6682-6690. [45] A.K. Williamson, A.C. Chen, R.L. Sah, Compressive properties and function-composition relationships of developing bovine articular cartilage, J. Orthop. Res., 19 (2001) 1113-1121.
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ACCEPTED MANUSCRIPT
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights
Design and synthesis hydrogel composed of GelMA and chitosan with interpenetrating network using photocrosslinking and basification
Effectively enhancing the mechanical properties of GelMA-chitosan hydrogels
Retaining
excellent
biocompatibility
and
enzymatic
degradability
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GelMA-chitosan hydrogels
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