Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation

Accepted Manuscript Title: Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation Author: Do Yoon Kim Honghyun Park Sang Woo Kim Jae Won Lee Kuen Yong Lee PII: DOI: Reference:

S0144-8617(16)31258-9 http://dx.doi.org/doi:10.1016/j.carbpol.2016.11.002 CARP 11712

To appear in: Received date: Revised date: Accepted date:

15-7-2016 6-10-2016 2-11-2016

Please cite this article as: Kim, Do Yoon., Park, Honghyun., Kim, Sang Woo., Lee, Jae Won., & Lee, Kuen Yong., Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Submitted to Carbohydrate Polymers (revised manuscript)

Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation

Do Yoon Kima, Honghyun Parka, Sang Woo Kima, Jae Won Leea, Kuen Yong Leea,b,*

a Department b Institute

of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea

of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of Korea

*To whom correspondence should be addressed: Kuen Yong Lee, Ph.D., Professor Department of Bioengineering, Hanyang University 222 Wangsimni-ro, Seongdong-gu Seoul 04763, Republic of Korea Phone: +82-2-2220-0482 Fax: +82-2-2293-2642 E-mail: [email protected] 1

Highlights  Partially oxidized hyaluronate can form gels in the presence of glycol chitosan.  Oxidation degree and polymer composition & concentration control gel properties.  These gels can be useful as an injectable in tissue engineering applications.

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ABSTRACT Hyaluronate has attracted great attention in tissue engineering as a scaffolding material. However, hyaluronate typically requires chemical cross-linking molecules to form hydrogels, which may induce undesirable side effects in the body. In this study, hyaluronate was partially oxidized with sodium periodate to generate aldehyde groups in the backbone, and simply mixed with glycol chitosan to form gels via Schiff base formation. The effects of the degree of oxidization, polymer concentration, and polymer composition on the mechanical properties of oxidized hyaluronate/glycol chitosan hydrogels were investigated in vitro. Degradation behavior and biocompatibility of oxidized hyaluronate/glycol chitosan gels were also evaluated in vitro. This system may be potentially useful as an injectable system in many tissue engineering applications, including cartilage regeneration.

KEYWORDS: hyaluronate, glycol chitosan, hydrogel, cartilage, tissue engineering

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1. Introduction There have been many efforts to replace damaged tissues or organs of patients. Artificial substitutes, non-living tissue from animals, or organ transplantation typically have been exploited to treat patients. However, these may not provide fundamental solutions to patients due to their disparate composition when compared with original host tissues or organs. Recently, tissue engineering has emerged as an alternative to traditional surgical procedures (Vacanti & Langer, 1999). In tissue engineering approaches, a scaffold prepared from biocompatible materials is typically required (Kim & Mooney, 1998). A scaffold functions as an extracellular matrix (ECM) of natural tissue, which regulates cell attachment, migration, proliferation, and differentiation (Jones, Sepulveda, & Hench, 2001; Yang, Leong, Du, & Chua, 2001). Scaffold design and fabrication requires control of physical, chemical, and biological properties (Collins & Birkinshaw, 2013; Sikavitsas, Temenoff, & Mikos, 2001; Wang, He, Wang, & Cui, 2012). Hydrogel is a very useful scaffold in many tissue engineering applications, as it can be injected into the body using a syringe and can maintain its hydrated structure, providing suitable microenvironments for delivered cells after injection (Ruel-Gariepy & Leroux, 2004). Hydrogels have been widely used as space filling agents, as well as delivery carriers of various bioactive agents, including genes, proteins, and cells (Drury & Mooney, 2003). Hydrogels can be designed and fabricated to enhance interaction with cells simply by modification with various ligands, such as adhesion peptides (Mann, Gobin, Tsai, Schmedlen, & West, 2001). In addition, degradation of hydrogels by hydrolysis and/or enzymatic cleavage can be modulated depending on the rate of tissue formation (Metters, Anseth, & Bowman, 2000). Injured articular cartilage has a poor regenerative capability. Therefore, tissue engineering may provide a fundamental therapeutic solution to repair cartilage injury. Articular cartilage is 4

composed of dense ECMs and chondrocytes, and hyaluronic acid is the major component of glycosaminoglycans in the ECM (Buckwalter, Rosenberg, & Hunziker, 1990). Hyaluronic acid is an anionic, linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and D-N-acetylglucosamine (Fig. 1a) (Necas, Bartosikova, Brauner, & Kolar, 2008). Hyaluronic acid comprises liquid connective tissues, such as joint synovial and eye vitreous fluid, and it is one of the most hydrophilic molecules in nature (Balazs, Watson, Duff, & Roseman, 1967; Fagerholm, Koul, & Trocmé, 1987; Scott, 1992). Hyaluronic acid can bind to proteins and cells through cell surface receptors, such as CD44 and intercellular adhesion molecule 1 (ICAM-1) (Knudson, 2003). In particular, CD44 receptors in cells interact with hyaluronic acid and express chondrogenic markers such as collagen type 2, SOX-9, and aggrecan (Jakobsen, Shahdadfar, Reinholt, & Brinchmann, 2010; Knudson, 2003). Hyaluronic acid is degradable by the action of hyaluronidase under physiological conditions (Csoka, Frost, Stern, & Csóka, 1996). Hyaluronic acid can form hydrogels by chemical cross-linking with hydrazide, polyfunctional epoxide, glutaraldehyde, and carbodiimide (Collins & Birkinshaw, 2013; Prestwich, Marecak, Marecek, Vercruysse, & Ziebell, 1998; Tomihata & Ikada, 1997). However, limitations still remain when applying these methods to deliver cells and engineer tissues, due to severe cross-linking conditions and the generation of toxic by-products (Caravaggi et al., 2003; Guida et al., 2004; Zhang, Wu, Huang, Peng, Chen, & Tang, 2012). In this study, we report the preparation and characterization of hyaluronate-based hydrogels without excipient chemical cross-linkers, which are potentially useful for cartilage regeneration. Aldehyde groups were generated in the hyaluronate backbone by partial oxidation (Fig. 1a) and allowed to react with amino groups of glycol chitosan (Fig. 1b) to form hydrogels via Schiff base formation (Fig. 1c). Oxidized polysaccharides have been used to prepare hydrogels in the presence of chitosan. 5

However, chitosan is hardly soluble in physiological conditions; chitosan derivatives, such as carboxymethyl chitosan (Cheng et al., 2014; Li et al., 2014), N-carboxyethyl chitosan (Wei et al., 2015), and N-succinyl-chitosan (Sun, Xiao, Tan, & Hu, 2013), were used instead. Glycol chitosan, a cationic polysaccharide, has good solubility in water compared with chitosan, which has been frequently used in many biomedical applications (Amsden, Sukarto, Knight, & Shapka, 2007; Park, Saravanakumar, Kim, & Kwon, 2010) and can be considered useful for tissue regeneration in combination with partially oxidized hyaluronate, allowing for promising potential in tissue engineering (Yu et al., 2011). Various characteristics of hydrogels, such as physicochemical properties and degradation behavior, were investigated. In addition, cytotoxicity as well as viability of chondrocytes encapsulated within hydrogels were tested in vitro to evaluate the potential for cartilage regeneration.

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Figure 1. Chemical structure of (a) oxidized hyaluronate and (b) glycol chitosan. (c) Schematic description of Schiff base formation between oxidized hyaluronate and glycol chitosan, resulting in the gel formation.

2. Materials and methods 2.1. Materials Sodium hyaluronate (MW 1,000,000) was purchased from Humedix (Seoul, Korea). 7

Glycol chitosan (MW 50,000) was provided from Wako (Osaka, Japan). Sodium periodate and tbutyl carbazate were supplied from Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle medium and Ham’s F-12 nutrient mixture (DMEM/F-12), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and penicillin-streptomycin (PS) were purchased from Gibco (Carlsbad, CA, USA). Formaldehyde and 2,4,6-trinitrobenzene sulfonic acid were purchased from Junsei (Tokyo, Japan) and Thermo Scientific (Waltham, MA, USA), respectively, and used without further purification.

2.2. Preparation of oxidized hyaluronate Hyaluronate (1 g) was dissolved in 90 ml of distilled water, and a solution of sodium periodate (Sigma; St. Louis, MO, USA) dissolved in distilled water (10 ml) was added to the hyaluronate solution, dropwise, in a dark room. The mixture was then stirred for 24 h, and an equimolar amount of ethylene glycol was added to stop the oxidation reaction. The resultant solution was purified by dialysis against distilled water (molecular weight cut-off = 3500) for 3 days. After dialysis, the solution was treated with charcoal, filtered through a 0.22-μm filter and lyophilized (Lee, Bouhadir, & Mooney, 2000).

2.3. Characterization of oxidized hyaluronate A 1H-NMR spectrometer (Avance-500, Bruker; Karlsruhe, Germany) was used to confirm the formation of aldehyde groups in oxidized hyaluronate (D2O, 10 mg/ml). The amount of aldehyde groups in oxidized hyaluronate (OHA) was quantified using 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Lee, Bouhadir, & Mooney, 2000). In brief, an excess of t-butyl carbazate was reacted with OHA solution overnight ([OHA] = 0.16 mM), and the amount of unreacted t8

butyl carbazate was quantified using a TNBS solution. Each sample (0.5 ml) was treated with TNBS (0.01%, 0.25 ml) at 37oC for 2 h, followed by addition of SDS (10%, 0.25 ml) and HCl (1 N, 0.125 ml). Absorbance was measured at 335 nm using a spectrophotometer (SpectraMax M2, Molecular Devices; Sunnyvale, CA, USA). Formaldehyde was used as a standard sample. The degree of oxidation (%) was defined as the number of oxidized units per 100 repeating saccharide units. The number behind OHA indicates the theoretical degree of oxidation.

2.4. Hydrogel formation and characterization Oxidized hyaluronate (OHA) and glycol chitosan (GC) dissolved in DPBS were mixed at various ratios using two syringes and a female connector (Park & Lee, 2011). The glycol chitosan solution was also treated with charcoal, filtered, and lyophilized before use. Fourier transform infrared spectroscopy (Nicolet IS50; Thermo Scientific; Waltham, MA, USA) was used to confirm the formation of imine bonds in hydrogels. Briefly, the sample was mixed with dry potassium bromide and the mixture was ground into a fine powder using an agate mortar before being compressed into disks. Each disk was scanned at a resolution of 4 cm-1 (scan rate, 4 mm/s; wavenumber region, 600–2000 cm-1). Viscoelastic properties of OHA/GC hydrogels were measured using a rotational rheometer (Bohlin Gemini 150, Malvern; Malvern, Worcestershire, UK) equipped with a cone and plate fixture (20 mm diameter plate, 4° cone angle) at 25oC (5 Pa, 1 Hz).

2.5. Degradation test OHA/GC gel disks were prepared using a punch (15 mm diameter, 1 mm height) and incubated in DPBS at 37oC for 46 days. The DPBS was changed every 3 days. At the 9

predetermined time points, gel disks were collected, and changes in their mechanical stiffness and weight were measured. A rotational rheometer equipped with a parallel plate fixture (20 mm diameter) at 25oC was used to determine the mechanical stiffness of gel disks over time.

2.6. In vitro cell culture and cytotoxicity test ATDC5 cells were seeded in a 96-well tissue culture plate ([cell] = 1 x 105 cells/well), and cultured with DMEM/F-12 cell culture media containing 5% FBS and 1% PS at 37oC in a humidified 5% CO2 atmosphere. Cells were then treated with a solution of HA, OHA, or GC at various polymer concentrations ([polymer] = 250, 500, and 1000 µg/ml). After 24 h or 48 h, each well was treated with EZ-CYTOX (Daeil Lab Service; Seoul, Korea) for 2 h according to the manufacturer’s instructions, and absorbance was measured at 450 nm using a spectrophotometer (SpectraMax M2, Molecular Devices; Sunnyvale, CA, USA).

2.7. Viability of cells encapsulated within hydrogels ATDC5 cells were encapsulated into hydrogels using syringes, and cut into disks. In brief, OHA solution was premixed with a cell suspension ([cell] = 1 x 106 cells/ml) using two syringes and a female connector, and the mixture was then cross-linked with GC solution to form hydrogels. The gels were placed between two glass plates with spacers for 10 min and cut into disks using a punch (15 mm diameter, 1 mm height). Gel disks were incubated in DMEM/F-12 media containing 5% FBS and 1% PS at 37oC for 24 h. A Live/Dead Viability/Cytotoxicity Kit (Invitrogen; Grand Island, NY, USA) was used to evaluate the cell viability according to the manufacturer’s instructions. Briefly, gel disks were washed twice with PBS. A working solution (2 mM calcein AM and 4 mM EthD-1 in PBS) was added to each gel disk, followed by 10

incubation for 30 min at 37oC in a humidified 5% CO2 atmosphere. Images were taken by fluorescence microscopy (ECLIPSE TE2000-E, Nikon; Japan). Image analysis was carried out using Image J software (NIH; Bethesda, MD, USA). The numbers of live and dead cells were counted from images to evaluate the cell viability. ATDC5 cells cultured in OHA/GC hydrogels were treated with mouse anti-human vinculin monoclonal antibody, followed by treatment with rhodamine-conjugated affinity donkey anti-mouse IgG to visualize vinculin as previously reported (Lee et al., 2010). Images of the cells were then taken using a FLUOVIEW laser scanning confocal microscope (Olympus, Tokyo, Japan).

2.8. RT-PCR Hydrogels encapsulating ATDC5 cells were frozen in liquid nitrogen and were homogenized in lysis buffer (Takara; Otsu, Japan). The amount of isolated RNA was determined using an UV-VIS spectrometer at 260 nm, and the concentration and quality were checked on agarose gel. The isolated RNA was reverse transcribed to cDNA using a Maxime RT PreMix kit (iNtRON Biotechnology; Gyeonggi-do, Korea). Expression of chondrogenic marker genes (SOX-9 and aggrecan) and housekeeping gene (GAPDH) was evaluated by RT-PCR (Bioline; London, UK). PCR products were analyzed by conventional agarose gel electrophoresis. The sequences of primers used are as follows (Integrated DNA Technologies; Coralville, IA, USA): SOX-9, 5'-ACCTCAAGAAGGAGAGCGAAGA-3', 5'-CGGGTGGTCTTTCTTGTGCT-3' (354 bp); aggrecan, 5'-GAGGTCGTGGTGAAAGGTGT-3', 5'-GTGTGGATGGGGTACCTGAC-3' (206 bp); GAPDH, 5'-TCACCATCTTCCAGGAGCGA-3', 5'-CACAATGCCGAAGTGGTCGT3' (293 bp).

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2.9. Statistical analysis All data are presented as the mean ± standard deviation (n = 4). Statistical analyses were performed using analysis of variance (ANOVA).

3. Results and discussion 3.1. Preparation of OHA/GC hydrogels Partially oxidized hyaluronate containing aldehyde groups was first prepared using sodium periodate. Sodium periodate is often used to generate two aldehyde groups in the repeating unit of hyaluronate (Jia et al, 2004; Su et al, 2011) and alginate (Bouhadir, Hausman, & Mooney, 1999). New peaks appeared at 4.9 ppm and 5.0 ppm on the 1H-NMR spectrum of oxidized hyaluronate (OHA) (Fig. S1 in Supplementary data), which may indicate the existence of the aldehyde groups in OHA (Li et al., 2014). To quantify the aldehyde groups in OHA, the TNBS assay was used. Since aldehyde groups are not able to react with TNBS directly, an excess amount of t-butyl carbazate was added to the aldehyde groups in advance, and the amount of unreacted t-butyl carbazate was determined to calculate the degree of oxidation. Actual value for the degree of oxidation was slightly lower than the theoretical value (Table 1). An absorption peak of aldehyde groups (C=O) in OHA appeared at 1690 cm−1 on the FTIR spectrum, which reacted with amino groups of glycol chitosan (GC) via Schiff base formation, resulting in the formation of hydrogels. Formation of cross-links in hydrogels was also investigated by FT-IR spectroscopy. Imine bonds (C=N) in OHA/GC gels were observed at 1456 cm-1 (Fig. S2 in Supplementary data).

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Table 1. Degree of oxidation of partially oxidized hyaluronate used (n = 4).

3.2. Viscoelastic properties of OHA/GC hydrogels The degree of oxidation and polymer concentration of OHA were fixed ([OHA50] = 2 wt%), and the optimum OHA/GC ratio for gel formation was first determined. An OHA50 solution was mixed with a GC solution at various mixing ratios (wt/wt). The mechanical stiffness of OHA50/GC gels increased as the GC content increased, due to the increased number of crosslinks in the gels. Addition of GC improved stiffness of OHA50/GC hydrogels up to G = 8 kPa at [OHA50]:[GC] = 1:1 (Fig. 2). However, the gels were hardly injectable through a syringe with an 18G needle due to the high mechanical stiffness. In contrast, gels were very weak at [OHA50]:[GC] = 4:1. For this reason, hydrogels prepared at [OHA50]:[GC] = 2:1 were considered suitable for cell delivery as an injectable.

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10

(a)

G¢ (kPa)

8

6

4

(b) 2

0 0.0

0.5

1.0

1.5

2.0

2.5

[GC] (wt%) Figure 2. (a) Changes in the storage shear moduli (G') of hydrogels prepared from oxidized hyaluronate (OHA50) and glycol chitosan (GC) depending on the GC concentration. The OHA concentration was kept constant ([OHA50] = 2 wt%, n =4). (b) Photograph of OHA50/GC hydrogel ([polymer] = 3 wt%, [OHA]:[GC] = 2:1).

Changes in the moduli of OHA/GC hydrogels prepared from OHA with different degrees of oxidation were next investigated ([OHA]:[GC] = 2:1). Highly oxidized OHA (e.g. [OHA] > 50%) improved the mechanical properties of OHA/GC hydrogels, but gelation was instant, and inhomogeneous gels were formed. This finding may be attributed to the fact that a fast Schiff base reaction induces partial gelation and disturbs the formation of homogeneous gel structures (Cheng et al., 2014). Hydrogels prepared from OHA with the oxidation degrees in the range of 10% to 50% were thus used for further experiments (Fig. 3). Hydrogels prepared at [polymer] = 1 wt% and 2 wt% ([OHA]:[GC] = 2:1) had low mechanical stiffness, irrespective of the oxidation degree, which may not be suitable for cartilage regeneration. The mechanical 14

properties of hydrogels prepared at [polymer] = 3 wt% ([OHA]:[GC] = 2:1) were dependent on the oxidation degree ranging from 10% to 50% (Fig. 3 & Table 2), consistent with previous reports that used oxidized hyaluronate and chitosan to fabricate gels (Li et al., 2014; Sun, Xiao, Tan, & Hu, 2013). All G values of OHA/GC hydrogels were higher than G values measured at various frequencies, which clearly indicates the hydrogel formation (Fig. S3 in Supplementary data).

4

G' (kPa)

3

2

1

0 0

10

20

30

40

50

60

Degree of oxidation (%) Figure 3. Changes in the storage shear moduli (G) of hydrogels prepared from oxidized hyaluronate (OHA) and glycol chitosan (GC) depending either on the total polymer concentration (squares, 1 wt%; triangles, 2 wt%; circles, 3 wt%; n = 4) or on the degree of oxidation ([OHA]:[GC] = 2:1, n = 4).

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Table 2. Storage shear modulus (G) and complete gelation time of OHA/GC gels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n =4).

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G' G¢,(kPa) G¢¢ (kPa)

3

2

1

0 0

150

300

450

600

Time (s) Figure 4. Changes in the storage shear moduli (G) and loss shear moduli (G) of various OHA/GC gels as a function time (squares, OHA10/GC; triangles, OHA25/GC; circles, OHA50/GC) (filled symbols, G'; empty symbols, G''). Total polymer concentration was kept constant for all hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n = 3).

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Time-dependent changes in the viscoelastic properties of OHA/GC hydrogels were next investigated (Fig. 4). Gelation time is normally the time when the storage shear modulus (G′) value becomes larger than the loss shear modulus (G″) value. However, the Gvalues were greater than G values at all time points measured in this study, as hydrogels were instantly formed when OHA and GC solutions were mixed. Thus, complete gelation time was determined when the equilibrium G values were obtained. Complete gelation time decreased as the oxidation degree increased at a constant polymer concentration and polymer composition ([polymer] = 3 wt%, [OHA]:[GC] = 2:1) (Table 2). This may be explained by the increasing number of cross-linking sites in the polymers with increasing the oxidation degree of hyaluronate.

3.3. Degradation behavior of OHA/GC hydrogels In vitro hydrolytic degradation of OHA/GC hydrogels was next investigated. Hydrogel disks prepared from OHA and GC lost their weight during degradation, as imine bonds were hydrolyzed. OHA25/GC and OHA50/GC gels retained more than 50% of their original weight for 46 days. In contrast, OHA10/GC gels were completely disintegrated after 30 days of incubation (Fig. 5a). A gradual decrease in the mechanical stiffness of OHA25/GC and OHA50/GC gels during degradation was also observed (Fig. 5b). However, the G values of OHA10/GC gels could not be measured after 19 days of incubation. This can be attributed to the low cross-linking density of OHA10/GC gels, compared with those of OHA25/GC and OHA50/GC gels. From these results, OHA10/GC gels may not be useful for long-term applications, and OHA25/GC and OHA50/GC gels are considered promising as a delivery vehicle for chondrocytes, as cartilage tissue formation normally takes several months in vivo. 17

(a)

125

W/W (%) W/W (%) 0 0

100

75

50

25

0 0

10

20

30

40

50

40

50

Time Time (days) (days)

(b)

2500

G' G¢(Pa) (kPa)

2000

1500

1000

500

0 0

10

20

30

Time (days) (days) Time Figure 5. Changes in the (a) weight and (b) storage shear modulus (G') of OHA/GC hydrogels (squares, OHA10/GC; circles, OHA25/GC; triangles, OHA50/GC) incubated in DPBS at 37oC ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n =4). The weight of each gel disk (W) was measured at the predetermined time point and normalized to the initial value before degradation (W0).

3.4. In vitro cell viability 18

Cytotoxicity of the polysaccharides used to form hydrogels was first investigated in vitro using various cell types such as chondrocytes (ATDC5), osteoblasts (MC3T3), and fibroblasts (NIH3T3). Cells were seeded on 96-well tissue culture plates ([cell] = 1×105 cells/ml) and cultured for 24 h. The cells were then treated with a solution of HA, OHA, or GC at various polymer concentrations. Cell viability was evaluated by a WST assay. No significant toxicity of HA, OHA, and GC at various concentrations was observed compared to the control group (nontreated cells) (Fig. S4 in Supplementary data). It has been previously reported that oxidized hyaluronate and chitosan derivatives did not show critical toxicity at concentrations below 1 mg/ml (Li et al., 2014). ATDC5 cells were next encapsulated into OHA/GC hydrogels ([cell] = 1×106 cells/ml), and their viability was investigated by the Live/Dead assay after 7 days of culture. Both OHA25/GC and OHA50/GC hydrogels showed good biocompatibility (Fig. 6a); approximately 80% of cells were viable after 7 days of culture within the gels (Fig. 6b). Vinculin was also clearly observed for ATDC5 cells encapsulated within OHA25/GC or OHA50/GC hydrogels and cultured for 7 days in vitro (Fig. 6c). In contrast, OHA10/GC hydrogels quickly disintegrated within 24 h of incubation when encapsulating cells. Cell encapsulation caused a decrease in the mechanical strength of these hydrogels (Table 2), due to the inherent volume of cells in the gels. Chondrogenic differentiation of ATDC5 cells encapsulated and cultured in OHA/GC hydrogels was next investigated by RT-PCR analysis. Expression of typical chondrogenic marker genes such as SOX-9 and aggrecan was observed for ATDC5 cells in OHA/GC hydrogels (Fig. 6d). SOX-9 is known to be critical for chondrocyte differentiation and function (Akiyama, 2008), and aggrecan is an important ECM component of cartilage tissues (Barry, Boynton, Liu, & Murphy, 2001). This finding may indicate potential of OHA/GC hydrogels as an injectable 19

delivery vehicle of chondrocytes for cartilage regeneration.

(a)

(b)

OHA25/GC

200 mm

OHA50/GC

Cell viability Cell viability(%) (%)

100

n.s.

80

60

40

20

0

200 mm

(c)

OHA25/GC

OHA25/GC

OHA50/GC

OHA25/GC OHA50/GC

(d)

OHA50/GC

C C 5/G 0/G 2 5 A A OH OH

GAPDH SOX-9 Vinculin/DAPI

100 mm

Vinculin/DAPI

100 mm

Aggrecan

Figure 6. (a) Images from the Live/Dead assay of ATDC5 cells encapsulated in OHA25/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 7 days). (b) The number of viable cells per total cells was determined by image analysis (n.s., no statistical significance; n =4). (c) Confocal laser scanning microscopic images of ATDC5 cells cultured within OHA/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 7 days). (d) Chondrogenic gene expression of ATDC5 cells encapsulated in OHA/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 5 days). GAPDH was used as a housekeeping gene. 20

4. Conclusions A simple mixing of oxidized hyaluronate (OHA) and glycol chitosan (GC) solutions formed hydrogels in the absence of excipient chemical cross-linkers, via Schiff base formation between aldehyde groups of oxidized hyaluronate and amino groups of glycol chitosan. Viscoelastic properties of OHA/GC hydrogels were dependent on the degree of oxidation of OHA, the mixing ratio between OHA and GC, and the total polymer concentration. OHA25/GC and OHA50/GC hydrogels prepared at [polymer] = 3 wt% and [OHA]:[GC] = 2:1 were considered useful for cell delivery as an injectable. OHA/GC hydrogels were degradable by hydrolysis of imine bonds in physiological conditions over 6 weeks. ATDC5 cells showed good viability within OHA/GC hydrogels in vitro. These hydrogels display good biocompatibility and durability under physiological conditions, and may have potential as an injectable cell delivery system in tissue engineering applications, including cartilage tissue engineering.

Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (MSIP) (NRF-2016R1A2A2A10005086).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at 21

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Figure captions

Figure 1. Chemical structure of (a) oxidized hyaluronate and (b) glycol chitosan. (c) Schematic description of Schiff base formation between oxidized hyaluronate and glycol chitosan, resulting in the gel formation.

Figure 2. (a) Changes in the storage shear moduli (G') of hydrogels prepared from oxidized hyaluronate (OHA50) and glycol chitosan (GC) depending on the GC concentration. The OHA concentration was kept constant ([OHA50] = 2 wt%, n =4). (b) Photograph of OHA50/GC hydrogel ([polymer] = 3 wt%, [OHA]:[GC] = 2:1).

Figure 3. Changes in the storage shear moduli (G) of hydrogels prepared from oxidized hyaluronate (OHA) and glycol chitosan (GC) depending either on the total polymer concentration (squares, 1 wt%; triangles, 2 wt%; circles, 3 wt%; n = 4) or on the degree of oxidation ([OHA]:[GC] = 2:1, n = 4).

Figure 4. Changes in the storage shear moduli (G) and loss shear moduli (G) of various OHA/GC gels as a function time (squares, OHA10/GC; triangles, OHA25/GC; circles, OHA50/GC) (filled symbols, G'; empty symbols, G''). Total polymer concentration was kept constant for all hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n = 3).

Figure 5. Changes in the (a) weight and (b) storage shear modulus (G') of OHA/GC hydrogels (squares, OHA10/GC; circles, OHA25/GC; triangles, OHA50/GC) incubated in DPBS at 37oC 28

([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n =4). The weight of each gel disk (W) was measured at the predetermined time point and normalized to the initial value before degradation (W0).

Figure 6. (a) Images from the Live/Dead assay of ATDC5 cells encapsulated in OHA25/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 7 days). (b) The number of viable cells per total cells was determined by image analysis (n.s., no statistical significance; n =4). (c) Confocal laser scanning microscopic images of ATDC5 cells cultured within OHA/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 7 days). (d) Chondrogenic gene expression of ATDC5 cells encapsulated in OHA/GC hydrogels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, [ATDC5] = 1×106 cells/ml, 5 days). GAPDH was used as a housekeeping gene.

29

Table 1. Degree of oxidation of partially oxidized hyaluronate used (n = 4).

Table 1

Sample

Sodium periodate used (mg/g hyaluronate)

Theoretical degree of oxidization (%)

Actual degree of oxidization (%)

OHA10

53.5

10

6.8 ± 3.7

OHA25

OHA50

133.7

25

267.4

50

p-value

<

0.05

<

0.05

18.0 ± 3.5

33.8 ± 3.7

Table 2. Storage shear modulus (G) and complete gelation time of OHA/GC gels ([polymer] = 3 wt%, [OHA]:[GC] = 2:1, n =4).

Table 2

Sample

G (kPa)

OHA10/GC

1.8 ± 0.4

OHA25/GC

p-value

2.4 ± 0.3

3.2 ± 0.7

G (kPa) with ATDC5 ([cell] = 1×107/ml)

121 ± 12

0.7 ± 0.1

105 ± 19 <

OHA50/GC

Complete gelation time (s)

30

<

0.01

<

0.01

1.2 ± 0.1

0.05 97 ± 17

p-value

1.6 ± 0.1