Using glucosamine to improve the properties of photocrosslinked gelatin scaffolds.

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Using glucosamine to improve the properties of photocrosslinked gelatin scaffolds Hairui Suo, Kedi Xu and Xiaoxiang Zheng J Biomater Appl published online 23 September 2014 DOI: 10.1177/0885328214551009 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/09/23/0885328214551009

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Article

Using glucosamine to improve the properties of photocrosslinked gelatin scaffolds

Journal of Biomaterials Applications 0(0) 1–11 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214551009 jba.sagepub.com

Hairui Suo1,3, Kedi Xu1,2,3 and Xiaoxiang Zheng1,2,3

Abstract The use of hydrogel-based cell transport scaffolds holds great promise in regenerative medicine, such as treating osteoarthritis. Gelatin and glucosamine are the ideal materials to be used in the hydrogel scaffolds for cartilage regeneration for they could act as compositions of cartilage. To overcome the weak strength of traditional gelatin hydrogels and down-regulate cell toxicity of glucosamine, gelatin and glucosamine molecules were grafted with acrylate groups and covalently crosslinked under photo-radiation to form hydrogels. Hydrogels with tuning physiochemical properties were produced according to different proportions of methacrylate gelatin (GelMA) and N-acryloyl glucosamine (AGA). The process of photocrosslinking was elaborated, and the hypothesis of increasing AGA concentration leading to higher strength of hydrogels was corroborated by testing rheological property and scanning micro-morphological features. A serial of properties, including smaller swelling ratio, lower gelatin dissolution and slower degradation of GelMA/AGA hydrogels with higher AGA concentration further proved our hypothesis. Moreover, AGA molecules showed less cytotoxicity than unmodified glucosamine molecules and the incorporation of AGA molecules in GelMA/AGA hydrogels upregulated cell adhesion and spreading on the hydrogel surface. All of these results indicated that addition of AGA molecules could significantly alter the physiochemical properties of GelMA/AGA hydrogels, which may have broad application prospects in the future. Keywords Hydrogel, photocrosslinking, gelatin, glucosamine, acrylate, scaffold

Introduction Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases in human, which could cause joint pain and significantly reduce the life quality of patients.1 OA often causes focal cartilage lesions which would seldom heal and frequently lead to degenerative change in the joint.2 So far, there is no effective therapeutic treatment to cure this disease. Currently available medical therapies primarily target palliation of joint pain by using analgesics, which often have suboptimal effectiveness and may increase risk of cardiovascular diseases.3 If the drug treatment does not work, surgical joint replacement might be the last opinion, which is highly invasive and expensive.4 A potential treatment for OA is employing tissue engineering technology. By using biocompatible materials to transport bone mesenchymal stem cells (BMSCs) to lesion area, the biomatrix could act as a temporary substitute for extracellular matrix (ECM), which can be replaced by

neo-tissue during tissue regeneration.5 Recently, using hydrogels as cell scaffolds attracts considerable attention of biomaterial scientists, since hydrogels have adjustable physiochemical properties close to human tissues and could provide an ideal aqueous environment for cell growth. Another great advantage of using hydrogels as cell transportation scaffolds is that they can be injected in situ with a minimally 1 Department of Biomedical Engineering, Key Laboratory of Biomedical Engineering of Education Ministry, Zhejiang University, Hangzhou, China 2 Qiushi Academy for Advanced Studies (QAAS), Zhejiang University, Hangzhou, China 3 Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou, China

Corresponding author: Kedi Xu, Qiushi Academy for Advanced Studies (QAAS), Zhejiang University, Hangzhou, 310027 China. Email: [email protected]



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invasive manner.6 Several methods have been developed for preparing injectable hydrogels. One widely accepted way is using chemical-crosslinking agents such as carbodiimide, glutaraldehyde, genipin and adipic dihydrazide.7–9 Although those chemical agents are very effective, they are also well known for high cytotoxicity, which limited their usage in human disease treatment.10,11 Photoinitiated chain polymerization is another simple, clean and tailorable way to fabricate hydrogels. A lot of materials have been employed in different kinds of hydrogels with this method, such as polyethylene glycol, gelatin, alginate, dextran, hyaluronic acid (HA), chitosan and so on.12–16 Compared with chemical crosslinking methods, photo-sensitive solutions could be fully mixed before injection, which is very desirable in regenerative therapies.5 Furthermore, the photoinitiated reaction provided a capable way to control the formation of hydrogel structures spatially and temporally.17 The components of hydrogels could be divided into two major categories, synthetic materials and natural macromolecules. The advantage to apply natural macromolecules, such as collagen, HA and elastin, is that they will not cause severe tissue rejection in vivo since they are also the components of ECM. Compared with most of the synthetic materials, natural macromolecules contain many different kinds of bioactive motifs, which can influence cellular functions, such as attachment, differentiation and proliferation. Thus, more and more natural macromolecules are recruited to fabricate novel hydrogels. The major components of human cartilaginous ECM are collagen and glycosaminoglycan (GAG).18 Thus, it is rational to think of using collagen as the substrate of hydrogel for OA treatment. However, collagen molecule is hard to produce and modify, made it not an ideal material for hydrogel fabrication. A possible alternative to replace collagen molecule is gelatin. Gelatin is the product of collagen hydrolysis that can be derived from a variety of sources, while retaining bioactive motifs such as cell binding sites and matrix metalloproteinase-sensitive degradation sites.19 Gelatin also possesses advantageous features as a building block for tissue regeneration, that it is nonimmunogenic and can be crosslinked or functionalized with side chains to form tailorable biomaterials. Previous studies have reported various methods for preparing gelatin hydrogels.15,19,20 Ali Khademhosseini’s group introduced a method to modify gelatin molecule with methacrylic anhydride (MA),19 demonstrated as an easy manipulation to fabricate gelatin hydrogels by photocrosslink. Thus, gelatin molecule was chosen in our study to form the main structure of hydrogels for potential OA treatment. The other components of human cartilaginous ECM are mainly GAGs. As one of the constituents of GAGs, glucosamine (GlcN) is

widely used in OA therapeutics.21,22 It has been reported that GlcN is proposed to be a chondroprotective agent that may stop the degradation of cartilage and stimulate production of new cartilage.23 GlcN has also been introduced in several hydrogel systems. In Wang’s research,17 GlcN was incorporated in poly(ethylene glycol) hydrogel to form injectable biomatrix. Therefore, it is of great value to contain GlcN molecule in a gelatin hydrogel network for cartilage tissue engineering, especially for the purpose of curing OA. In our study, gelatin and GlcN were modified with MA and acryloyl chloride, respectively, to form a photo-sensitive hydrogel. Photocrosslinking of gelatin molecules produced much stronger network than physical crosslinking, which could overcome the weak strength of unmodified gelatin hydrogel. Differently with other previous reports, GlcN molecule was modified into N-acryloyl glucosamine (AGA) and conveniently crosslinked into the gelatin hydrogel to form a hybrid methacrylate gelatin (GelMA)/AGA hydrogel. We hypothesized that covalently crosslinking GlcN molecule into the gelatin network will influence the bulk structure of the hydrogel and retain the GlcN molecule in the hydrogel for a long time. Thus, gelatin hydrogels with different concentration of AGA molecules were investigated for their physiochemical characteristics, including the mechanical strength, swelling and degradation properties and micro-morphological features. Besides these physiochemical property tests, BMSCs were also applied on the GelMA/AGA hydrogels to examine the cytotoxicity and biocompatibility. Our results showed that hydrogels formed by photocrosslinking of GelMA and AGA would have tunable physiochemical properties and might be further used in OA treatment in the future.

Materials and methods Materials Type A gelatin (250 g bloom from porcine skin, G108395), D-GlcN hydrochloride, MA and acryloyl chloride were purchased from Aladdin Industrial Inc. (Shanghai, China). The photoinitiator, 2-hydroxy-1[4-(hydroxyethoxy) phenyl]-2-methy-1-propanone (Irgacure 2959), was obtained from Sigma-Aldrich Corp. (St. Louis, USA). Low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM) and fetal bovine serum (FBS) were bought from Gibco. Millipore water was used throughout the study.

Synthesis of GelMA and AGA GelMA was synthesized as literature described.19,24 Briefly, 10% (w/v) gelatin was dissolved in



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phosphate-buffered saline (PBS) at 60 C. MA was added at a rate of 0.5 mL/min under stirring condition at 50 C. After reaction for 3 h, the reacted solution was diluted and dialyzed against distilled water using 12– 14 kDa cutoff dialysis tubes for seven days at 40 C. The dialyzed GelMA solution was then filtered through a 0.22-mm filter membrane and finally lyophilized and stored at 80 C for further use. The degree of methacrylation of gelatin was quantified by the Habeeb method.25,26 GelMA was finally examined by 1H NMR at a frequency of 400 MHz using a BRUKER ADVANCE2B NMR spectrometer (Switzerland) and deuterium oxiden as the solvent. AGA was synthesized according to literature method with minor modification.17 Briefly, 20 mL of 1 mol/L K2CO3 solution containing 0.02 mol D-GlcN hydrochloride was cooled in ice bath. 0.024 mol acryloyl chloride was subsequently added dropwise to the solution under vigorous stirring. The reaction was maintained at 0–4 C for 4 h, and the mixture solution was then slowly warmed to room temperature in fuming cupboard for 24 h to evaporate the unreacted acryloyl chloride. The crude product was further dried by lyophilization and purified by column chromatography on silica gel with a methanol/ethyl acetate mixture as eluent (20/80 v/v). AGA molecule was finally lyophilized and stored at 80 C for further use. Electrospray ionization-mass spectrometry (ESI-MS) was used to identify its structure. The acrylation of GlcN was quantified using 1H NMR with deuterium oxiden as the solvent at a frequency of 400 MHz.

Photocrosslinking of GelMA/AGA hydrogels To form GelMA/AGA hydrogels, AGA solutions were firstly prepared to final concentration of 0, 5, 10, 20 and 40 mM in PBS with 0.1% (w/v) photoinitiator. Then the polymer precursors (GelMA) were dissolved in different AGA solutions at a final concentration of 5% (w/v) under stirring at 37 C. Once the GelMA was homogeneously dissolved, the solutions were exposed to a 9 mW/cm2 UV light (max ¼ 365 nm) and photocrosslinked for 3 min.

Bulk rheological characterization Bulk rheological characterization of the hydrogels was performed with a RS6000 rheometer (HAAKE, Germany) equipped with 20 mm parallel disk geometry. Hydrogel disks with 20 mm diameter were evaluated in small amplitude oscillatory shear at 37 1 C, over a frequency range of 0.1–10 Hz. The complex shear modulus, G* ¼ G0 þ iG00 , was investigated by measuring the shear storage modulus (G0 ) and the shear loss modulus (G00 ).27 The magnitude ofpthe complex shear ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi modulus is, thus, given by jGj ¼ G02 þ G002 .

Micro-structure of GelMA/AGA hydrogels The micro-morphology of the photocrosslinked GelMA/AGA hydrogels (5% GelMA, separately with 0, 5, 10, 20 and 40 mM AGA) was analyzed using a scanning electron microscope (SEM, HITACHI S-4800) with an accelerating voltage of 3.0 KV. Freshly prepared GelMA/AGA hydrogel samples were frozen in 80 C for 12 h and freeze-dried for 48 h. The samples were sputter coated twice using palladium–platinum alloy target materials with 40 mA current for 40 s before SEM observation. Three replicates were used for each hydrogel.

Hydrogel characterization: Swelling, gelatin dissolution and degradation To prepare samples for swelling analysis, 300 mL mixed solution including 0.1% photoinitiator was placed in a glass cylinder (the diameter is 12 mm) and photocrosslinked. Once the photo-polymerization complete, the hydrogel disks were placed in measuring cups containing 2 mL PBS for 24 h to reach equilibrium swelling. The wet swollen hydrogel disks were then weighed after gently blotting the excess liquid by Kim-wipes. This was followed by freezing and lyophilization steps to measure the dry weight of the disks. The swelling ratio (Qs) is defined as Qs ¼ ðWs Wd Þ=Wd where Ws is the gel weight after swelling and Wd is the weight of dried hydrogel. Four replicates were used for each hydrogel formulation. The hydrogels for evaluating gelatin dissolution profiles were produced same as previously described. Once removed from the glass cylinder, the hydrogel disks were rinsed with PBS and incubated in measuring cups containing 5 mL PBS at 37 C on a shaker of 130 rpm. At each time point (0.5, 2, 4, 8, 16, 24, 32, 40 and 48 h), the entire volume of supernatant was collected and replaced with fresh PBS. The gelatin concentration in the supernatant was determined using a Bradford protein assay kit (Beyotime, China). The gelatin dissolution percentage (Qd) was calculated as the cumulative gelatin weight in the supernatant (Ws) compared with the gelatin weight in the initial hydrogel (Wh) Qd ¼ ðWs =Wh Þ 100% Degradation of the hydrogels was studied as follow. After crosslinking, the hydrogel disks were rinsed with PBS and deposited in 2 mL eppendorf tubes. Then the hydrogels were lyophilized, and the initial weights (Wi) were recorded. Dried hydrogels were then rehydrated in



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PBS for 24 h. A total of 1 mL of 2.5 U/mL of collagenase type II solution in PBS was added to the hydrogels. They were then incubated at 37 C on a shaker at 130 rpm. At different time points (4, 8, 12, 18 and 24 h), the enzyme solution was removed, and the hydrogels were rinsed with PBS. Then the hydrogels were lyophilized to determine the dry weight of remaining polymer (Wr). Four replicates were used for each hydrogel formulation at each time point. The mass remaining percent after enzymatic degradation (Qr) was calculated as

30 min. The hydrogels were then imaged under a florescence microscope. Three replicates were used for each hydrogel composition.

Statistical analysis All the data were subjected to statistical analysis and were reported as mean standard deviation of samples. Statistical significance (*p < 0.05, **p < 0.01) was determined using unpaired Student t test. At least three independent experiments were performed for each study.

Qr ¼ ðWr =Wi Þ 100%

Results and discussion Cell culture and viability assay The committee on animal experimentation of Zhejiang University approved all the animal experiments. BMSCs were isolated from femora of four- to sixweek-old S-D rats as literature described with minor modification.28,29 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 the media of L-DMEM supplemented with 10% FBS (v/v). The marrow was collected and cultured for three days in a humidified atmosphere of 95% air and 5% CO2. The first medium change was done after four days and twice a week, thereafter. BMSCs were passaged once they reached 80–90% confluence. BMSCs between the passages four and nine were used for all experiments. The viability of BMSCs treated with GlcN or AGA was estimated with 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenytetrazolium bromide (MTT).30 After reaching 80% confluence, culture medium of the cells in the 96well plate was changed separately with the medium containing GlcN at the concentrations of 1, 2, 5, 10 and 20 mM or AGA at the counterpart concentrations. After 48 h, the medium was removed, and the plate was gently rinsed with PBS. 150 mL MTT solutions (0.5 mg/ mL) were added to each well and incubated for another 4 h. The amount of viable cells in each well was determined by the absorbance of solubilized Formosan. The optical density was measured at 490 nm with a microplate reader (Versa Max, Molecular Devices).

Cell attachment and spreading assay GelMA/AGA hydrogels were prepared for cell attachment and spreading assay same as described above. The crosslinked hydrogels were seeded with 1 105 cells/cm2 on the top surface and cultured in a 24-well plate. The culture medium was half changed every two days. At day seven, the cell-seeded disks were rinsed with PBS and incubated with 5 mM Calcein-AM for

Gelatin and GlcN molecule modification and GelMA/AGA hydrogel preparation A widely used macromolecule, gelatin, was chosen as the backbone of our hydrogel system. One gram of type A gelatin contains about 0.35 mmol reactive amino groups, resulting in a great potential of chemical modification by amino based reaction.24,31 In our system, the amino groups on gelatin macromolecules were methacrylated with photo-reactive vinyl double bonds (GelMA, Figure 1(a)). The methacrylation degree of gelatin molecules was approximately linear to the molar ratio of the two reactants (Figure 1(b)). According to the methacrylation ratio, the final reaction system was confirmed as 1.0 mL (about 4.88 mmol) MA reacted with 2 g gelatin to ensure the overreaction of amino groups on the gelatin molecules in all batches of GelMA. In order to keep the consistency of the experiments, all batches of synthesized GelMA were examined for their methacrylation degree and only those highest modified batches were used in the following researches. GlcN molecules were also modified to introduce photo-reactivity (Figure 1(c)). The molecular weight of AGA was identified by ESI-MS with the result of 256.1 g/mol, which is equivalent to the calculated result of one AGA molecule plus a sodium ion. Figure 1(d) showed the proton NMR of our synthesized AGA molecule. Two distinctive peaks presented at 5.72–5.76 ppm and 6.13–6.32 ppm (the square in Figure 1(d)) were attributed to the acrylate protons (the circle in Figure 1(b)), thus confirming the successful grafting of the acrylate groups on GlcN molecules. With the presence of photoinitiator, the vinyl double bonds on GelMA and AGA molecules could react with each other during photo-polymerization and form a hybrid network from seconds to minutes, depending either on the concentration of the reactants or the mole ratio between them. Both the large GelMA macromolecules and the small AGA molecules could act as crosslinkers in this photocrosslinking reaction,




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(a) GelMA synthsis NH

NH2

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CH2OH H H OH

NH OH NH

H

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HN

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H OH

OH

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NH

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NH O

O

Figure 1. Gelatin and glucosamine molecule modification and photocrosslinking of GelMA/AGA hydrogels. (a) Modification of gelatin molecule with methacrylic anhydride. (b) The varying degree of modification measured by Habeeb method. (c) Modification of glucosamine molecule with acryloyl chloride. (d) The 1H NMR spectra of AGA. (e) Photocrosslinking of GelMA/AGA hydrogel.

since the surplus electrons will exist on the either end of the vinyl bond (illustrated as the dash lines in Figure 1(e)) once the vinyl addition reaction occurs. As drawn in Figure 1(e), four kinds of possible linkages could exist in this system: the intra-chain linkage, the AGA-chain linkage, the AGA–AGA linkage and the chain–chain linkage. Except those four covalent linkages, physical entanglement between the long chains of gelatin macromolecules could also exist in this hybrid system. Thus, the whole network of GelMA and AGA photocrosslinking hydrogel could be the combination of all of linkages mentioned above. Since the small AGA molecules could be used as active crosslinkers in this hybrid system, we hypothesized that the addition of AGA in the GelMA solution could enhance the photocrosslinking reaction and have deep influence on

whole structure of the hydrogel as well as physiochemical characteristics.

Bulk characterization of GelMA/AGA hydrogels When using hydrogels as cell scaffolds, it is imperative that the hydrogels could maintain their shape under specific loads and provide necessary mechanical support to surrounding cells and tissues.31,32 To test the influence of small AGA molecules on the structure and physical property of GelMA/AGA hydrogels, viscoelastic and rheological tests were then performed at the frequency of 0.1–10 Hz in our study. The shear storage (also named real or elastic) modulus G0 and the shear loss (also named imaginary or viscous) modulus G00 of each sample with different AGA concentration was shown



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in Figure 2(a). Compared with storage modulus, loss modulus values were much smaller, exhibiting predominantly and elastic character,33 and without significant difference between each hydrogel formulation. Meanwhile, with fixed GelMA concentration (5%), the G0 values of GelMA/AGA hydrogels increased obviously as the AGA concentration increased from 0 to 40 mM. Thus, the defined mechanical loss angle (tan ¼ G00 /G0 ) became smaller in the hydrogels with higher AGA concentration, which indicated the incorporation of AGA molecules upregulated elasticity and compressive strength of the hydrogels. Generally speaking, our GelMA/AGA hydrogels had similar bulk physical characteristic as natural human articular cartilage. Both the values of G0 and G00 were a little higher in the high frequency of all the five groups. This feature was consistent with the frequency-dependent responses of articular cartilage.34,35 The complex shear modulus pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (jG j ¼ G02 þ G002 ), which provided the gel stiffness property under dynamic conditions, also increased significantly with higher AGA concentration in GelMA/ AGA hydrogels. As illustrated in Figure 2(c), the G* values increased from 1636.59 31.14 Pa (10 mM AGA) to 2060.57 57.02 Pa (20 mM AGA) and

(a) 3500 3000

G’

0

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2758.78 26.39 Pa (40 mM AGA), in accordance with an analogous positive linear correlation between G* values and the AGA concentration in the system. This result indicated that the compressive strength and stiffness of the GelMA/AGA hydrogels could be tuned by adjusting AGA concentrations, which could be explained by the forming of dense chemical bonds between GelMA molecules and small AGA molecules. Since the scaffolds may be exposed to mechanical forces, the improved mechanical properties of hydrogels may contribute to improve their stability when used in vivo.12

Micro-morphological features of GelMA/AGA hydrogels The rheology property of hydrogels is highly related to their physical structures. We further demonstrated the micro-morphological features of GelMA/AGA hydrogels by SEM. Figure 3 showed detailed surface structure of different GelMA/AGA hydrogels and illustrated that the micro-morphology was greatly affected by incorporation different concentration of AGA. Consistent with rheology measurement, the lyophilized hydrogels with lower concentration of AGA molecule showed a sponge-like structure with lots of cave-like structures inside the bulk hydrogel, indicating lower crosslink density of hydrogel network. However, GelMA/AGA hydrogels with higher AGA concentration showed denser structures with a smooth leaf-like surface, indicating the network formation under higher AGA concentration condition is relative compact. Compared with other gelatin hydrogel system, the application of AGA photocrosslink with gelatin network proved a novel way to control the detailed structure of the hydrogels to some extent. Furthermore, it is worthwhile to note that the SEM analysis may not reflect real situation of the hydrogels, since the lyophilization procedure before SEM analysis may influence the bulk structure. Thus, it is necessary to further estimate the influence of AGA molecule by other experiments, such as the measurement of swelling and degradation characteristics.

Swelling and degradation characteristics of GelMA/AGA hydrogels

2000 1500 1000 500 0 0

20 5 10 AGA concentration (mM)

40

Figure 2. Bulk rheology (a) and complex shear moduli (b) of GelMA-AGA composite hydrogels. The complex shear modulus shows significant differences (**p < 0.01) between two adjacent concentrations.

Adequate mechanical performance of a scaffold depends on the material mechanical properties including elasticity, compressibility, viscoelastic behavior, tensile strength and failure strain.36 For hydrogels, these properties are affected by polymer and crosslinker characteristics, gelling conditions (e.g. temperature and pH), swelling and degradation.37 Hydrogels are normally hydrophilic and could maintain over 90% water in their 3D structures due to their porous structure. Therefore, the swelling behavior of hydrogels is greatly related to the pore size and the



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Figure 3. Morphology of freeze-dried GelMA/AGA hydrogels with different concentrations of AGA: (a) 0 mM AGA, (b) 5 mM AGA, (c) 10 mM AGA, (d) 20 mM AGA and (e) 40 mM AGA. Scale bar is 100 mm.

mechanical properties.15 In this study, the swelling behavior of GelMA/AGA hybrid hydrogels was found to be tunable by varying the composition of AGA (Figure 4(a)). The swelling ratio decreased from 32.72 1.67 of 5% GelMA without AGA to 23.69 1.40 of hydrogels containing 5 mM AGA. Correspondently, significant decrease was observed upon comparison of hydrogels containing 20 mM AGA and 40 mM AGA (p < 0.01). The swelling result further approved our hypothesis that the addition of small AGA molecules into the hybrid GelMA/AGA system could greatly increase the chance of crosslinking between double bonds on GelMA chains. Accompanied with the incorporation of AGA molecules, the photocrosslinking reaction became more active and a much denser hydrogel network with smaller pore size was produced. Degradation is another important property when using hydrogels in tissue engineering application. In our GelMA/AGA hydrogel system, release of gelatin from the hydrogel is an obvious characteristic of degradation. Gelatin molecules could release out from the hydrogel by two ways: diffusion of noncovalently crosslinked Gelatin molecules and degradation of the bulk structure. For the former, the unmodified or uncrosslinked gelatin will diffuse into the surrounding water on the effect of osmotic pressure. This kind of release mainly occurs in the first few hours and will reach equilibrium quickly.27 As shown in Figure 4(b), GelMA/AGA hydrogels were quite stable in aqueous buffer, since less than 3% of the hydrogel mass was released after 48 h embedding. The incorporation of AGA molecules decreased gelatin release, indicating less uncrosslinked gelatin molecules or tight structure of the GelMA/AGA hydrogels with higher AGA concentration. This result again validated that the incorporation of AGA molecules strengthened the photocrosslinking

reaction and generated tighter polymeric network in the GelMA/AGA hydrogel system. As mentioned above, the other kind of gelatin release may be due to the hydrolysis, enzymatically catalyzed degradation or mechanical degradation of the bulk structure. This kind of degradation was widely applied to design degradable tissue engineering scaffolds.5,12,13 In this study, we tested GelMA/AGA hydrogel degradation with 2.5 U/ml collagenase under 37 C to mimic in vivo environment.31 Figure 4(c) showed hydrogels with higher concentration of AGA have less mass loss and slower degradation speed. Without AGA addition, the hydrogel remained only less than one-third of the original weight (29.68 4.03%) after 24 h enzymatic degradation, while more than half bulk mass of the hydrogels with 40 mM AGA (59.50 3.27%) remained. The results of swelling and degradation experiments are in good agreement with the results of rheology test and morphology properties analysis. All these experiments lead up to the conclusion that incorporation of AGA molecules could regulate the intensity and stiffness of the hydrogels by adjusting the extent of covalent crosslinking reaction.

BMSCs viability One of the potential applications of GelMA/AGA hydrogels is manufacturing tissue scaffold for cartilage repair. Several groups have studied the potential function of GlcN molecules on the biosynthesis of cartilagespecific proteoglycans.38,39 However, de Mattei et al have mentioned that pharmacological doses of GlcNinduced a broad impairment in the metabolic activity and led to cell death.23 The toxicity of GlcN molecule may come from the free amino group, which was found




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Figure 5. Cytotoxicity test of GlcN and AGA. BMSCs were treated by different concentrations of GlcN and AGA. *p < 0.05 and **p < 0.01 compared with control.

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Similar as previous report, our results validated the cytotoxicity of GlcN molecules (Figure 5). The cell viability decreased obviously when GlcN concentration was larger than 1 mM. After modifying the GlcN molecules with acrylate groups, the AGA molecules showed less cytotoxicity. No significant decrease of BMSCs viability was observed unless treated with higher concentration (10 mM AGA vs. control, p < 0.05), indicating that GlcN modified with acrylate is less cytotoxic at any concentration than its counterpart.

Reamaining mass (%)

0 5 10 20 40 mM AGA 80

BMSCs adhesion on GelMA/AGA hydrogels

60

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0 0

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Figure 4. (a) Swelling profiles of 5% GelMA/AGA hydrogels with varying concentrations of AGA (0, 5, 10, 20 and 40 mM). The swelling ratios show statistically significant differences (*p < 0.05, **p < 0.01). (b) Gelatin dissolution behavior of GelMA/AGA hydrogels (0, 5, 10, 20 and 40 mM AGA) in DPBS water. (c) Degradation of GelMA/AGA hydrogels by 2.5 U/mL collagenase.

to have inhibitory action on chondrocyte proliferation and PG synthesis. In our GelMA/AGA hydrogel system, the GlcN molecules were modified and covalently crosslinked with gelatin network, which may decrease the cytotoxicity but maintain its function on cartilage repair. BMSCs were recruited in the following experiments to identify the cytotoxicity of AGA molecules and cell adhesion on GelMA/AGA hydrogel.

The above cytotoxicity test identified the less toxicity of free AGA molecules, and further study is needed to identify the cell viability after the AGA molecules have been crosslinked in the hydrogel network. BMSCs were then planted on different GelMA/AGA hydrogel surfaces to evaluate the cell functions, such as attachment, spreading and proliferation. We adopted the method of half change of media to ensure that the released substances from hydrogels, such as noncrosslinked gelatin, unmodified GlcN and intercrosslinked AGA, have full effect on the BMSCs adhering on the top surface of the hydrogels. As shown in Figure 6, BMSCs could attach on all the five different hydrogel surfaces with high cell viability, mainly due to the gelatin component in GelMA/AGA hydrogels. Besides the cell binding motifs on the hydrogel surface, it has been demonstrated by previous researches that cell adhesion and spreading are also greatly related with hydrogel stiffness.19,31 Scaffold properties at the microscale are also known to influence cell–ECM interaction behavior.40 In our GelMA/AGA hydrogels, the amount of cells on the surface increased according to AGA concentration (from 0 mM to 20 mM) and BMSCs spread out more on hydrogels with higher AGA concentration, mainly because higher AGA concentration obviously



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Figure 6. BMSCs adhesion on different GelMA/AGA hydrogel surfaces. (a) 0 mM AGA, (b) 5 mM AGA, (c) 10 mM AGA, (d) 20 mM AGA and (e) 40 mM AGA. Scale bar is 100 mm.

increase the elastic modulus of GelMA/AGA hydrogels. Normally, cells would prone to attach on stiff surface and show much flatter morphology on stiff surface than on soft surface.19 It is worth to notice that BMSCs showed more spreading ability but less attachment on the surface of hydrogels with 40 mM AGA. This decrease of cell attachment on 40 mM AGA hydrogels might be explained by the cytotoxicity of released superfluous AGA from hydrogels.

Acknowledgements The authors thank Dr Mingen Xu and Dr Qian Zhao for teaching and guiding during this investigation. We also acknowledge Dr Youliang Li, Dr Chang Li and Dr Shufang Zhang for invaluable technical assistance. This research project was funded by (1) Zhejiang Provincial Natural Science Foundation of China [grant number LQ13H180001]; (2) Fundamental Research Funds for the Central Universities.

Declaration of conflicting interests

Conclusions

None declared.

In recent years, GelMA hydrogel has attracted great attention in tissue engineering and stem cell bioengineering, mainly due to the large amount of cell binding motifs on gelatin molecule and the tunable mechanical robustness of hydrogel.40,41 In this study, AGA and GelMA were successfully used to generate composite hydrogels. We have investigated the material properties and assessed the cellular response to the addition of AGA. Through the investigation of hydrogel properties, we can draw a conclusion that the degree of photocrosslinking can be tuned by varying AGA in a dosedependent manner. Compared to unmodified GlcN, AGA has less cytotoxicity, especially in high dose. Moreover, incorporation of AGA molecules increased the BMSCs attachment on hydrogel surface. Therefore, with excellent biocompatibility, AGA can be used as a regulator to tune the properties of gelatin hydrogels. The ability to precisely control physical and biological properties of engineered constructs may enable widely use of GelMA/AGA hydrogels in the future.

Funding This research project was funded by Zhejiang Provincial Natural Science Foundation of China (grant number LQ13H180001) and Fundamental Research Funds for the Central Universities.

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