Bioinspired, Calcium-Free Alginate Hydrogels with Tunable Physical

May 2, 2013 - and Technology, Daejeon 305-701, Republic of Korea. ∥ ... a bioinspired approach for preparing divalent ion-free alginate hydrogels th...
1 downloads 0 Views 2MB Size
Article pubs.acs.org/Biomac

Bioinspired, Calcium-Free Alginate Hydrogels with Tunable Physical and Mechanical Properties and Improved Biocompatibility Changhyun Lee,† Jisoo Shin,† Jung Seung Lee,† Eunkyoung Byun,‡ Ji Hyun Ryu,§ Soong Ho Um,∥ Dong-Ik Kim,⊥ Haeshin Lee,*,‡,§ and Seung-Woo Cho*,† †

Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea Department of Chemistry and §The Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea ∥ School of Chemical Engineering and SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, Republic of Korea ⊥ Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea ‡

ABSTRACT: Alginate hydrogels are for various biomedical applications including tissue engineering, cell therapy, and drug delivery. However, it is not easy to control swelling or viscoelastic and biophysical properties of alginate hydrogels prepared by conventional cross-linking methods (ionic interaction using divalent cations). In this study, we describe a bioinspired approach for preparing divalent ion-free alginate hydrogels that exhibit tunable physical and mechanical properties and improved biocompatibility due to the absence of cations in the gel matrices. We conjugated dopamine, a minimalized adhesive motif found in the holdfast pads of mussels, to alginate backbones (alginate-catechol) and the tethered catechols underwent oxidative cross-linking. This resulted in divalent cation-free alginate hydrogels. The swelling ratios and moduli of the alginate-catechol hydrogels are readily tunable, which is difficult to achieve in ionic bond-based alginate hydrogels. Furthermore, alginate-catechol hydrogels enhanced the survival of various human primary cells including stem cells in the three-dimensional gel matrix, indicating that intrinsic cytotoxicity caused by divalent cations becomes negligible when employing catechol oxidation for alginate cross-linking. The inflammatory response in vivo was also significantly attenuated compared to conventional alginate hydrogels with calcium crosslinking. This biomimetic approach for the preparation of alginate hydrogels may provide a novel platform technology to develop tunable, functional, biocompatible, three-dimensional scaffolds for tissue engineering and cell therapy.



cations such as Ca2+, Mg2+, and Ba2+.2,7 Alginate hydrogels have been used to inject cells and drugs; they have also been used as wound dressings and for dental implants because they have minimal toxicity, are low cost, and gel easily due to the divalent cations.2 Despite these advantages, alginate hydrogel biomaterials cross-linked by ionic interactions have intrinsic disadvantages. Specifically, the mechanical and biophysical properties (e.g., elastic modulus, swelling ratio, and degradation rate) of these hydrogels are difficult to control due to uncontrollable cross-linking and unavoidable, rapid loss of ions.7−9 Therefore, intermolecular cross-linking methods such as conjugating various types of cross-linkers to the alginate backbone have been developed.2 Unfortunately, the reagents and reaction conditions for conjugation and cross-linking are

INTRODUCTION

Hydrogels are hydrophilic polymers swollen by large amounts of water and are widely used in biomedical applications for tissue engineering and drug delivery. Physical or chemical crosslinking of these gels creates a three-dimensional (3D) polymeric network. Their high water content and soft, porous 3D structure mimics the in vivo extracellular matrix (ECM) microenvironment making them useful for biomedical applications.1,2 Hydrogels are typically injectable and can deliver cells or drugs into the body in a minimally invasive manner. Applications that hydrogels have been used for include the creation of 3D scaffolds for cell culture and transplantation and as carriers for local release of proteins and drugs.3−6 Alginate is a linear polysaccharide with homopolymeric blocks of (1,4)-linked β-D-mannuronate and α-L-guluronate that is extracted from seaweed and is widely used as a natural polymer for hydrogels.2 In general, alginate forms a hydrogel via ionic interactions between carboxylic acids and divalent © XXXX American Chemical Society

Received: March 8, 2013 Revised: May 1, 2013

A

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 1. Schematic illustration of the preparation of mussel-inspired, calcium-free, alginate hydrogels by conjugation of dopamine to the alginate backbone and subsequent oxidation of catechol moieties for cross-linking.

typically toxic to encapsulated cells and can cause denaturation of growth factors.7,10 Biomimetic approaches can provide alternative and more efficient ways of preparing alginate hydrogels. Marine mussels, Mytilus edulis, secret protein material that initiates robust adhesion and energy dissipation for mitigating the impact of waves.11 Adhesive proteins in the mussel adhesive pads promote adhesion to virtually any type of organic or inorganic material.12−14 Byssal threads connecting the adhesive pads and the mussel’s body contain a catecholic amino acid called 3,4dihydroxy-L-phenylalanine (L-DOPA). This amino acid has redox activity that allows oxidative cross-linking to form protein networks.12 Oxidative transition of the catechol group in the LDOPA to o-quinone triggered by alkaline pH (>7.5) results in chemical cross-linking by forming catechol-catechol adducts such as poly(DOPA).12,15 In fact, this chemical cross-linking principle has been used to generate 3D hydrogel networks by conjugating catechol moieties to polymers such as poly(ethylene glycol) (PEG)16 and chitosan17 followed by oxidative cross-linking of the catechol groups. Interestingly, the catecholcontaining polymers were not toxic in vitro or in vivo when used as tissue adhesives or for surface functionalization;18−20 however, conjugation of the catechol moieties and subsequent use in alginate-based biomaterials has not been reported. Therefore, this cross-linking approach based on oxidation of conjugated catechol groups could be advantageous for preparing alginate hydrogels by replacing the conventional method of using divalent cations. The degree of catechol conjugation can be used to control physicochemical and mechanical properties of the hydrogel while the divalent cationfree environment could provide a highly biocompatible 3D matrix in the hydrogel construct. In this study, we report divalent cation-free alginate hydrogels made of bioinspired adhesive polymer, catecholconjugated alginate. The swelling, rheological, and biophysical properties of the alginate-catechol hydrogels were readily tunable compared to alginate hydrogels prepared by the conventional cross-linking method of using divalent cations.

The swelling ratios of the alginate-catechol hydrogels were greater (180−230%) than those of alginate-calcium hydrogels (40−80%), which may lead to more efficient mass transfer. We were able to vary the alginate-catechol moduli from 300 to 6000 Pa by changing the content of alginate-catechol. The alginate-catechol hydrogels did not exhibit significant cytotoxicity during cell encapsulation and culture, and they induced minimal inflammatory response both in vitro and in vivo. Our study demonstrates that catechol-functionalized alginate polymers inspired by mussel adhesive chemistry can generate highly organized, controllable, biocompatible 3D hydrogels appropriate for biomedical applications such as cell transplantation.



EXPERIMENTAL SECTION

Synthesis of Alginate-Catechol Conjugates. The alginatecatechol conjugate was synthesized by chemical reaction using 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS; Figure 1). To conjugate the dopamine, alginate (Pronatal LF10/60, FMC Biopolymer, Philadelphia, PA, U.S.A.) was dissolved in distilled water at a concentration of 1% (w/v). EDC (Thermo Scientific, Waltham, MA, U.S.A.) and NHS (Sigma, St. Louis, MO, U.S.A.) were then added to the alginate solution at a molar ratio equal to the alginate. Dopamine hydrochloride (Sigma) was added to the solution at a 1:1 molar ratio of dopamine hydrochloride to alginate at pH 4.5−5.5. The reaction was performed at room temperature overnight. The reaction mixture was dialyzed using Dulbecco’s Phosphate Buffered Saline (DPBS, pH = 4) and acidic distilled water (pH 5−6) for 12 h and subsequently freeze-dried. Synthesis of the alginate-catechol conjugate was confirmed by nuclear magnetic resonance (NMR; Bruker 400 MHz, Bruker, Billerica, MA, U.S.A.). The conjugation efficiency of dopamine to alginate backbone was determined by measuring the absorbance at 280 nm using an UV−vis spectrophotometer because the aromatic ring structure of conjugated catechol group exhibits absorption peak at this wavelength.17 Dopamine hydrochloride solution was used to generate a standard curve for catechol concentrations (ranging from 1 to 0.0078 mg/mL by 1/2 serial dilution). Alginate-catechol solution (1 mg/mL) was used as a sample. Alginate Hydrogel Formation. To produce alginate-catechol hydrogels, the catechol moieties conjugated to the alginate backbone B

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Fluorescent signals were observed under a fluorescent microscope (IX71, Olympus, Shinjuku, Tokyo, Japan). Immune Activation. The immune-stimulating effect of alginate hydrogels was examined by measuring secretion levels of the immune cytokine tumor necrosis factor-α (TNF-α) from macrophages (Raw 264.7 cells) cocultured with the alginate hydrogels. Raw 264.7 cells were cocultured with alginate hydrogels in 24-transwell plates (1.5 × 105 cells/well) for 1 day. The collected medium was used to quantify the amount of TNF-α secreted from Raw 264.7 cells. The quantity of TNF-α in the medium was measured using a TNF-α Duo Set enzymelinked immunosorbent assay (ELISA, R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s instructions. In Vivo Implantation. In vivo biocompatibility of alginate hydrogels was evaluated by implanting the hydrogels into mice. Animal experiments for hydrogel implantation have been approved and performed in compliance with the Institutional Animal Care and Use Committee at the Yonsei Laboratory Animal Research Center (IACUC protocol number: 2010−0049). Briefly, Balb/c mice (4 weeks old, Nara Biotech, Pyungtaek, Korea) were anesthetized with xylazine (20 mg/kg) and ketamine (100 mg/kg). The alginate hydrogels (volume: 100 μL) were subcutaneously injected into each side of the backs of mice using 24-gauge syringes (two implants per mouse). The mice were followed up to 4 weeks after implantation of alginate hydrogels. The hydrogel constructs were retrieved with adjacent host tissues at several time points after implantation (7, 14, and 28 days; three mice for each time point) and embedded in an OCT compound (TISSUE-TEK 4583, Sakura Finetek USA Inc., Torrance, CA, U.S.A.) for histological analysis. The specimens were sliced into 10-μm sections and stained with hematoxylin and eosin (H&E) to examine inflammation and fibrosis in the retrieved hydrogel constructs. The sections were immunofluorescently stained against myeloperoxidase (MPO), a neutrophil marker, using mouse monoclonal FITC-conjugated anti-MPO (ab90812, Abcam, Cambridge, U.K.) to check infiltration and migration of inflammatory cells into the implanted alginate hydrogels and surrounding tissues. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Fluorescence signals were observed under a fluorescence microscope (IX71, Olympus). Encapsulation and Release of Growth Factors. We tested the alginate hydrogels for sustained and controlled delivery of growth factors. We prepared the alginate hydrogels under three different conditions to examine their release profiles for encapsulated basic fibroblast growth factor (bFGF, R&D Systems). Basic FGF (11.1 μg/ mL) was encapsulated into the 2% (w/v) alginate-catechol hydrogels cross-linked with NaIO4 or into the 2% (w/v) unmodified alginate hydrogels cross-linked with CaCl2. The alginate-catechol hydrogels prepared by CaCl2 cross-linking were also tested for bFGF encapsulation to investigate the effect of cross-linking methods on the release profiles of growth factors. The alginate hydrogels bearing bFGF were incubated in DPBS at 37 °C for 15 days. The buffer solution was collected for ELISA at several time points during incubation. The amount of bFGF released from the hydrogels was quantified using a human bFGF Duo Set ELISA kit (R&D Systems). Statistical Analysis. Quantitative data are expressed as mean ± standard deviation. Statistical analyses were performed using an unpaired Student’s t test with Sigma-Plot software (Systat Software Inc., Chicago, IL, U.S.A.). Values of p < 0.01 and 0.05 were considered statistically significant.

were cross-linked with an oxidizing agent. Alginate-catechol dissolved in DPBS (at 2% and 4% (w/v) concentration) was mixed with a gelation solution containing 0.4 M sodium hydroxide (NaOH) and an oxidizing agent (sodium periodate (NaIO4): 4.5 mg/mL, equal molar ratio to catechol) to initiate cross-linking of catechol. Alginate without the catechol modification was cross-linked by adding calcium chloride (CaCl2). Unmodified alginate was dissolved in DPBS solution at 2% (w/v) concentration and then mixed with 100 mM CaCl2 solution for cross-linking via ionic interaction. The alginate gels were formed immediately after cross-linking. The formed gel constructs were washed with DPBS solution to remove excess CaCl2. Characterization of the Alginate Hydrogels. The swelling properties of the alginate hydrogels were determined by examining water uptake capacity. Alginate-catechol hydrogels (2 and 4% (w/v) concentrations) were incubated in DPBS (pH = 7.2) at 37 °C. The wet weight of the hydrogels was measured at several time points (days 3, 7, 10, and 14) during the incubation. The swelling ratio (%) was calculated with the following equation: (Ws − Wi)/Wi × 100 (%), where Ws represents the weight of the swollen hydrogel at each time point and Wi represents the weight of the unswollen hydrogel at day 0.21 The moduli of the hydrogels were measured using a rotating rheometer (Bohlin Advanced Rheometer, Malvern Instruments, Worcestershire, U.K.) set at a frequency sweep mode of 0.1−10 Hz to determine the mechanical properties of the alginate hydrogels. The modulus of the hydrogel was also measured at a time sweep mode by recording storage modulus (G′) and loss modulus (G″) with increasing time. In this mode, the frequency and stress were constant at 1 Hz and 100 Pa, respectively. The gelation time was considered as a crossover time of G′ and G″ in the modulus graph produced at a time sweep mode. The microporous structure of the alginate hydrogels was observed by scanning electron microscopy (SEM) in lyophilized hydrogel samples (S-4800, Hitachi, Japan) at a voltage of 10 kV. The pore size of the hydrogel was calculated from the SEM images of hydrogel constructs. The shortest and the longest diameters of each pore in the SEM images were measured by using Image J software (Image J, Bethesda, MD, U.S.A.) and then the average of two diameters was calculated and represented as a pore size. Cell Viability. The alginate hydrogels were used for 3D culture of human and animal cells including Huh-7 cells, Neuro-2a cells, human umbilical vein endothelial cells (HUVECs), human adipose-derived stem cells (hADSCs), and human neural stem cells (hNSCs). The cells were encapsulated (2.0 × 106 cells/mL) into the alginate-catechol hydrogels prepared by NaIO4 cross-linking and into the unmodified alginate hydrogels prepared by CaCl2 cross-linking. In brief, 9 mg of alginate-catechol was dissolved in 288 μL of DPBS buffer and 50 μL of medium containing cells was then added to alginate-catechol solution. Then, 112 μL of a gelation solution containing 0.4 M NaOH and an oxidizing agent (NaIO4: 4.5 mg/mL, equal molar ratio to catechol) was mixed with alginate-catechol solution containing cells to initiate cross-linking of catechol and construct cell-encapsulating hydrogel samples. The formed gels were transferred to multiwell plates, washed with DPBS, and the medium was added to the hydrogel constructs. For cell encapsulation within alginate-calcium hydrogel, 9 mg of unmodified alginate was dissolved in 300 μL of DPBS buffer and 50 μL of medium containing cells was added to the alginate solution. The alginate solution containing cells was mixed with 100 μL of 100 mM CaCl2 solution for cross-linking and the formed gels were transferred to multiwell plates for the culture. The cells were cultured in the following medium conditions. Huh-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, Gaithersburg, MD, U.S.A.) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL), Neuro-2a cells were cultured in DMEM supplemented with 10% (v/v) FBS, HUVECs were cultured in Endothelial Growth Medium-2 (EGM-2, Lonza, Hopkinton, MA, U.S.A.), hADSCs were cultured in DMEM supplemented with 10% (v/v) FBS, and hNSCs were cultured in DMEM/F12 with N-2 supplements (Gibco BRL). The viability of cells cultured in the hydrogels was examined using a LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, U.S.A.). In this kit, calcein stains the cytoplasm of viable cells green and an ethidium homodimer stains the nuclei of nonviable cells red.



RESULTS AND DISCUSSION Synthesis of Alginate-Catechol Conjugates. The alginate-catechol conjugate was synthesized by standard carbodiimide coupling chemistry (EDC/NHS chemistry). In this reaction, the carboxyl group of alginate is activated by EDC/NHS and the amine group of dopamine is then coupled to the activated carboxyl group (Figure 1). The reaction was carried out in an aqueous buffer (pH 4.5−5.5) at an equal molar ratio of alginate to dopamine. The conjugation efficiency C

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 2. Characterization of alginate-catechol conjugate synthesis. (A) NMR analysis confirmed alginate-catechol conjugation. NMR analysis showed that the peaks of the catechol protons (a) appeared at around 7 ppm. The analysis was performed in triplicate. (B) Gelation of the alginate solution is shown (alginate-catechol hydrogel and alginate-calcium hydrogel).

of dopamine to the carboxyl group in the alginate backbone was 5.5 ± 0.1%, as determined by absorbance at 280 nm using UV− vis spectrophotometer. The conjugation of dopamine to polymer backbone depends upon dopamine concentration in the reaction solution. We previously confirmed that the conjugation efficiency of dopamine to hyaluronic acid (HA) backbone was 8.4 ± 0.1 and 14.0 ± 0.1% at 1:1 and 1:3 molar ratios of HA to dopamine, respectively. NMR analysis also confirmed the conjugation of dopamine to alginate by showing that the peaks of the catechol protons appeared at around 7 ppm (Figure 2A). Gelation of Alginate-Catechol Conjugates. The synthesized alginate-catechol conjugate was cross-linked to form a hydrogel under basic conditions in the presence of a chemical oxidizing agent, sodium periodate (NaIO4). The mechanism of alginate-catechol assembly might be explained by oxidative conversion of the catechol moiety to o-quinone.12 Deprotonation of hydroxyl groups in the catechol moiety results in quinone formation, leading to cohesive catechol-catechol crosslinking reactions that produce 3D hydrogels. 22,23 This mechanism was demonstrated in a previous study regarding catechol-functionalized PEG hydrogels and is reminiscent of the mechanism of melanin formation in mammals.22 Because oxidizing agents can accelerate this conversion of catechol to quinone, sodium periodate (NaIO4), used in this study, can facilitate polymerization of the alginate-catechol conjugate and, consequently, hydrogel formation. We found that equal molar equivalents of NaIO4 to catechol in the alginate-catechol conjugate resulted in formation of the hydrogel within 3−4 min after induction of gelation. Upon gelation, the alginate-catechol solution turns into a brown colored hydrogel due to the unique color of cross-linked catechol (Figure 2B).12 Unmodified alginate cross-linked by adding divalent cations (Ca2+) formed a hydrogel with no color change within a few seconds (Figure 2B). Gelation of alginate-catechol conjugate can be modulated by changing the concentration of oxidizing agent and pH condition, which is more controllable compared to formation of alginate-calcium hydrogel. Microporous Structure of the Alginate Hydrogels. We used SEM to analyze the microporous structure of the alginate hydrogel (Figure 3). SEM showed interconnected pores in the alginate hydrogels cross-linked by catechol oxidation (Figure

Figure 3. SEM analysis of the microporous structure of the alginate hydrogels: (A) alginate-catechol hydrogel (2%), (B) alginate-catechol hydrogel (4%), and (C) alginate-calcium hydrogel (2%), scale bars = 100 μm. SEM analysis was performed in duplicate. The representative images were chosen from duplicated experiments. (D) The pore sizes of alginate hydrogels are also shown (p < 0.01; alginate-catechol groups vs alginate-calcium group).

3A,B). The average pore size for the 2 and 4% alginate-catechol hydrogels were 37.7 ± 7.4 and 29.1 ± 1.9 μm, respectively, which were both smaller than that of the 2% alginate-calcium hydrogel (69.4 ± 11.1 μm; Figure 3A−D). SEM analysis demonstrated that alginate-catechol hydrogels have highly porous structures and therefore may provide favorable microenvironments for cell adhesion, proliferation, and migration. Swelling Kinetics of the Alginate-Catechol Hydrogels. The swelling properties of alginate-catechol hydrogels were examined by measuring the change in hydrogel weight during incubation under physiological conditions (in a DPBS solution at 37 °C). Alginate hydrogels cross-linked by catechol oxidation or calcium binding were both completely swollen 3 days after incubation (Figure 4A). The wet weight of the hydrogel in the D

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 4. Swelling properties of the alginate hydrogels. (A) Wet weight increase of the alginate hydrogels was measured at several time points during incubation in DPBS buffer at 37 °C. (B) Swelling ratios of the alginate hydrogels at 14 days after incubation was measured (n = 3, p < 0.01; alginatecatechol groups vs alginate-calcium group). The swelling ratio of the alginate hydrogels was measured in duplicate.

Figure 5. Rheological properties of the alginate-catechol hydrogels. The storage (G′) and loss (G″) moduli of the (A) 2% (w/v) alginate-catechol hydrogel and (B) 4% (w/v) alginate-catechol hydrogel at a frequency sweep mode are provided. (C) Elastic moduli of the alginate-catechol hydrogels were measured at 1 Hz of frequency (n = 4, p < 0.01). (D) Storage (G′) and loss (G″) moduli of the 2% (w/v) alginate-catechol hydrogel at a time sweep mode (frequency: 1 Hz, stress: 100 Pa). The rheological analysis was performed in triplicate.

The swelling property of the hydrogel is important for efficient mass transfer of nutrients, oxygen transfer, and waste exchange. In general, highly porous 3D hydrogels exhibit greater swelling ratios, and therefore, would be expected to mediate more efficient mass transfer.24 This type of hydrogel can enhance cellular functions by facilitating mass transfer to encapsulated cells within the hydrogel construct.24 In fact, Ji et al. reported that chitosan hydrogels with high porosity promote fibroblast proliferation due to improvements in the swelling properties.24 In this regard, alginate-catechol hydrogels with

DPBS solution increased more rapidly in the alginate-catechol hydrogel group than in the alginate-calcium hydrogel group up to the first 3 days of incubation and then remained constant during the rest of the incubation period (∼2 weeks; Figure 4A). We observed no significant weight loss in all tested alginate hydrogels for the 14 days of incubation (Figure 4A). The swelling ratios of the alginate-catechol hydrogels (2% = 229.7 ± 3.8% and 4% = 192.9 ± 14.5%) at 14 days were much greater than that of the alginate-calcium hydrogel (2%: 63.5 ± 27.7%; Figure 4B). E

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 6. In vitro biocompatibility of the alginate hydrogels. (A) Cells were stained by live/dead assay in the alginate-catechol hydrogel (2%) and alginate-calcium hydrogel (2%) immediately after cell encapsulation, scale bar = 50 μm. (B) Live/dead staining was performed on hADSCs encapsulated in the 2% alginate-catechol hydrogel 3 and 7 days after encapsulation, scale bar = 500 μm. The cell viability assay was performed in duplicate. The representative images were chosen from duplicated experiments. (C) Viability of hNSCs in the alginate-catechol hydrogel (2%) and alginate-calcium hydrogel (2%) immediately after cell encapsulation (n = 3; p < 0.05). (D) ELISA was used to quantify the secretion of TNF-α from macrophage cells (Raw 264.7) cocultured with the alginate hydrogels (1 day after culture; n = 3, p < 0.01; alginate-catechol hydrogel vs lipopolysaccharide). Immune stimulation of the alginate hydrogels was tested in triplicate.

cross-linking did not induce uniform or controlled cross-linking throughout the hydrogel construct.17 The average moduli measured (at 1 Hz of frequency) in the 2 and 4% alginatecatechol hydrogels were 378.9 ± 38.9 and 5445.3 ± 666.6 Pa, respectively (Figure 5C). The modulus data of alginate-catechol hydrogel measured at a time-sweep mode showed the G′/G″ crossover point indicating the gelation time of alginate-catechol hydrogel around 3−4 min after gelation induction (Figure 5D). Mechanical properties including elastic modulus and stiffness of substrates and scaffolds regulate cellular behavior. Engler et al. reported the sensitivity of mesenchymal stem cells (MSCs) to elasticity of culture substrates.26 Specifically, soft (0.1−1 kPa), stiffer (8−17 kPa), and more rigid matrices (25−40 kPa) committed the cells to neuronal, myogenic, and osteogenic lineages, respectively.26 Several studies also report lineagespecific differentiation of stem cells modulated by the modulus and stiffness of 3D hydrogels used for stem cell encapsulation.27,28 In a study performed by Huebsch et al., MSC fate was examined by encapsulating MSCs into alginate hydrogels of varying matrix rigidity.27 They demonstrated that hydrogels with an elastic modulus of 10−30 kPa induced osteogenic differentiation of MSCs and that the softer hydrogels with an elastic modulus of 2.5−5 kPa induced adipogenesis. 27 Guvendiren et al. reported similar results regarding human MSC response to hydrogel stiffening from soft (∼3 kPa) to stiff (∼30 kPa) in methacrylate-conjugated hyaluronic acid hydrogels.28 Considering the modulus of alginate-catechol hydrogels (∼6 kPa) measured in this study, these soft hydrogels may be suitable for neuronal or adipocyte differentiation of MSCs derived from bone marrow, cord blood, or adipose tissue.

highly porous structures and greater swelling ratios (Figures 3 and 4) may be more potent biomaterials for cell transplantation compared to conventional alginate hydrogels cross-linked by divalent cations. The alginate-catechol hydrogel can exhibit improved stability in aqueous conditions in vitro and in vivo compared to alginate hydrogel with calcium cross-linking. The stability of alginate hydrogel cross-linked via ionic interaction following calcium addition usually decreases due to loss of calcium ion caused by ion exchange in aqueous conditions. In contrast, alginatecatechol hydrogel cross-linked by oxidative catechol polymerization does not employ ionic interaction for gelation and thus can resist ion exchange, which may render this type of hydrogel more stable in aqueous environment in vitro and in vivo. Rheological Properties of the Alginate Hydrogels. We examined the rheological and viscoelastic properties of the alginate hydrogels using dynamic mechanical analysis. The storage modulus (G′) of the alginate-catechol hydrogel was nearly consistent over the frequency range tested in this study and was higher than the loss modulus (G″) at each frequency (Figure 5A,B). This indicates that cross-linking in the alginatecatechol hydrogel was highly stable and uniform.17,25 This rheological analysis demonstrated that the alginate hydrogel cross-linked by oxidative catechol polymerization exhibited solid-like behavior after gelation. As the content of alginatecatechol in the hydrogel increased from 2 to 4%, the storage modulus (G′) of the hydrogel increased more than 10-fold (Figure 5A,B). The alginate hydrogel cross-linked by calcium chloride did not show such a consistent storage modulus (G′) or loss modulus (G″) over the tested frequency range (data not shown). This may indicate that the use of divalent cations for F

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 7. In vivo implantation of the alginate hydrogels. (A) H&E staining shows the alginate hydrogels retrieved 7 and 14 days after implantation into the subcutaneous space of mice, scale bars = 200 μm. In vivo implantation was performed in duplicate. The representative images were chosen from duplicated experiments. (B) Gross morphology and H&E-stained images were taken of the alginate-catechol hydrogels (2%) retrieved at 14 and 28 days after implantation, scale bars = 200 μm. H&E images showed no sign of inflammatory response or fibrotic capsule formation. (C) Immunofluorescent staining of a neutrophil marker (myeloperoxidase; MPO) in the retrieved alginate hydrogels 14 days after implantation, scale bars = 100 μm.

Biocompatibility of the Alginate Hydrogels In Vitro. We examined the alginate-catechol hydrogel’s biocompatibility in vitro by assaying its effect on cytotoxicity and immunestimulation. Human primary cells and stem cells were encapsulated in the alginate-catechol hydrogels and stained with a Live/Dead assay kit. Immediately after encapsulation, most cells in the alginate hydrogel constructs cross-linked by catechol or calcium were viable (Figure 6A). In particular, hADSCs remained viable in the alginate-catechol hydrogel 1 week after encapsulation (Figure 6B). In this study, pH condition and oxidizing agent (NaIO4) concentration were optimized for efficient and noncytotoxic cell encapsulation using alginate-catechol hydrogel. Highly basic and oxidizing conditions with the increase in pH and NaIO4 concentration resulted in rapid gelation of alginate-catechol, but also increased cytotoxicity (data not shown). Therefore, we usually apply equal molar ratio of NaIO4 to conjugated catechol and pH condition adjusted to around 7.4−8.0 for cell encapsulation. The cell viability was quantified with Live/Dead stained images of encapsulated cells in the alginate hydrogels. The viability of certain types of cells that are relatively robust to environmental changes (e.g., hepatocarcinoma, neuroblastoma cell line) was not different between alginate-catechol and alginate-calcium hydrogel group. Indeed, Huh-7 and Neuro-2a exhibited good viability in both alginate-catechol and alginatecalcium hydrogel. Most cell types were viable in both alginatecatechol (99−100% viability for Huh-7, Neuro-2a, and hADSC) and alginate-calcium hydrogel (95−98% viability for Huh-7, Neuro-2a, and hADSC) immediately after encapsulation. However, cell types that are vulnerable and sensitive to environmental changes occurring during hydrogel encapsula-

tion (e.g., primary cells, stem cells) showed higher viability in alginate-catechol hydrogel than in alginate-calcium hydrogel. For example, the viability of encapsulated hNSCs was higher (p < 0.05) in alginate-catechol hydrogel compared to alginatecalcium hydrogel (Figure 6C). The viability of HUVECs was also higher (p < 0.01) in alginate-catechol hydrogel (99−100%) than in alginate-calcium hydrogel (86.1 ± 2.3%). These data may demonstrate the general applicability of alginate-catechol hydrogel for culture and transplantation of various cell types in tissue engineering. In this study, FBS-containing media were used for other cell types except for hNSCs. The presence of serum can induce spontaneous differentiation of hNSCs and thus it was removed from the medium for undifferentiated hNSC culture in the alginate-catechol hydrogel. It seems that serum influences the viability of cells requiring serum for growth and survival during and after oxidative catechol crosslinking for cell encapsulation. We also investigated the immune-stimulating effect of the alginate hydrogel by measuring TNF-α secretion from macrophage cells (Raw 264.7) incubated with the alginate hydrogels. Several reports have demonstrated that TNF-α secretion from Raw 264.7 cells is an established method to determine immune-stimulating effect of exogenous materials.29,30 ELISA results showed that TNF-α secretion from macrophages cocultured with the alginate-catechol hydrogel was much lower than that of cells treated with an immune stimulant, lipopolysaccharide (Figure 6D). These ELISA results may indicate that soluble components eluted from the catechol cross-linked alginate hydrogels marginally activate immune lineage cells but we need to further investigate immunestimulating effect of alginate-catechol hydrogel in a future G

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

considered in the design of scaffolds. Spatial distribution of cells/drugs, regulation of cellular behavior, and tissue formation are affected by hydrogel degradation.2 However, degradation of alginate hydrogels cross-linked by divalent cations is difficult to precisely control because loss and dissolution of cations are typically uncontrollable and unpredictable.2 For these reasons, research groups have tried to develop alginate hydrogels with tunable biodegradation properties.7,33 In our study, we measured degradation of the alginate-catechol hydrogels for 2−4 weeks in vitro and in vivo (Figures 4 and 7) and did not observe significant mass loss or degradation; however, this should be measured for a longer period of time. The specific mechanism by which degradation occurs in alginate-catechol hydrogels will also need to be elucidated. Release Profiles of Growth Factors from Alginate Hydrogels. The potential of catechol-functionalized alginate hydrogels to be used as a drug delivery system was assessed by examining the release profile of bFGF from the hydrogels. Basic FGF was chosen because it is a potent growth factor that can promote cell proliferation and angiogenesis.34 Surprisingly, bFGF loaded into the alginate-catechol hydrogel that was crosslinked with an oxidizing agent (NaIO4) was not released at all from the hydrogel construct during 2 weeks of incubation in DPBS buffer at 37 °C (Figure 8). Some functional groups in

study. TNF-α secretion by the alginate-catechol hydrogel group was not lower than that of the alginate-calcium hydrogel group because the difference was not statistically significant (p > 0.05; Figure 6D). Biomimetic hydrogels modified by mussel-inspired adhesive chemistry are expected to further improve the therapeutic efficacy of cell transplantation by facilitating in vivo localization of transplanted cells. In fact, Brubaker and colleagues developed a catechol-modified PEG hydrogel that can be cross-linked under oxidizing conditions; they then used it for islet transplantation to treat type I diabetes.16 The catechol moiety not only participates in nontoxic cross-linking of PEG hydrogels, but also forms strong covalent interactions with nucleophiles including amines, thiols, and imidazoles in proteins and peptides.3,12,31 Therefore, this catechol-PEG hydrogel containing islet cells was efficiently grafted onto the surface of the liver in diabetic animals via catechol binding to ECM proteins. As a result, glucose levels were effectively managed by islet transplantation using the catechol-PEG hydrogel.16 The alginate-catechol hydrogel developed in our study could also be a useful scaffold for transplantation of diverse cell types including stem/progenitor cells with accessibility to regions that are not feasible with conventional transplantation scaffolds. Biocompatibility of the Alginate Hydrogels In Vivo. To test the toxicity and immunogenicity of the alginatecatechol hydrogels in vivo, we injected the hydrogels subcutaneously in mice. The body weight of mice increased normally after alginate-catechol hydrogel injection (data not shown), indicating that there was no sign of toxicity. When the alginate hydrogels were retrieved 7 days after injection, the highly organized internal microstructures were maintained in the alginate-catechol hydrogel, but the alginate-calcium hydrogel did not show such organized structure (Figure 7A). H&E staining revealed that inflammatory cells were recruited to the edges of the alginate-calcium hydrogel at 7 days in vivo (Figure 7A), but infiltration of inflammatory cells was not detected in the alginate-catechol hydrogel for up to 28 days in vivo (Figure 7B). Dramatic degradation or structural deformations were not observed at retrieval of the alginate-catechol hydrogel constructs 14 days or 28 days after injection (Figure 7B). Alginate-catechol hydrogel may lack unfavorable tissue interaction to hydrogel function compared to alginate hydrogel with calcium cross-linking. H&E-stained images of host tissues adjacent to implanted alginate hydrogels (14 days after implantation) indicated that a larger number of migrating and infiltrating cells were observed in the surrounding tissue around implanted alginate-calcium hydrogel compared to tissue adjacent to implanted alginate-catechol hydrogel (Figure 7A). A previous study demonstrated that neutrophils migrate and infiltrate into the implanted alginate hydrogels early time point after implantation.32 Immunofluorescent staining of a neutrophil marker (MPO) revealed neutrophil migration and infiltration into the alginate-calcium hydrogels 14 days after implantation into subcutaneous space of the mice (Figure 7C). In contrast, neutrophil infiltration was less extensive in the alginate-catechol hydrogels (Figure 7C). Overall these data demonstrate the improved biocompatibility of catecholfunctionalized, calcium-free alginate hydrogels. The biodegradation of this biomimetic alginate-catechol hydrogel needs to be examined further in future studies. Degradation properties of biomaterials are a critical feature that can control the efficiency of tissue regeneration and should be

Figure 8. bFGF release profiles from the alginate hydrogels. ELISA was used to quantify the amount of bFGF released from the three types of alginate hydrogels: (1) the alginate-catechol hydrogel that was NaIO4 cross-linked, (2) the alginate-catechol hydrogel that was CaCl2 cross-linked, and (3) the unmodified alginate hydrogel that was CaCl2 cross-linked (n = 4 per each group).

proteins (e.g., ε-amino groups in the lysine residue and thiol groups in the cysteine residue) are known to have strong binding affinity to the oxidized catechols.31,35 Therefore, bFGF not released from the alginate-catechol hydrogel cross-linked with NaIO4 is likely due to strong covalent conjugation of the growth factor to the catechol moieties in the alginate-catechol hydrogel. In contrast, bFGF showed a sustained-release from the alginate hydrogel that was cross-linked with a divalent cation (calcium) over a period of 2 weeks (∼60% of loaded bFGF; Figure 8). Interestingly, when the alginate-catechol hydrogel was prepared by cross-linking using divalent cations (calcium), about 10% of the loaded bFGF was released from the alginate-catechol hydrogel (Figure 8), indicating that the cross-linking method may alter the release profile of growth factors from the hydrogel. Introduction of catechol moieties with high affinity to proteins and peptides can improve the therapeutic efficacy of cell transplantation using alginate hydrogels. Despite several H

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(2) Lee, K. Y.; Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 2001, 101, 1869−1879. (3) Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 2012, 336, 1124−1128. (4) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 2009, 324, 59−63. (5) Lin, C. C.; Anseth, K. S. PEG hydrogels for the controlled release of biomolecules in regenerative medicine. Pharm. Res. 2009, 26, 631− 643. (6) Cho, S. W.; Kim, I. K.; Bhang, S. H.; Joung, B.; Kim, Y. J.; Yoo, K. J.; Yang, Y. S.; Choi, C. Y.; Kim, B. S. Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction. Eur. J. Heart Fail. 2007, 9, 974−985. (7) Jeon, O.; Bouhadir, K. H.; Mansour, J. M.; Alsberg, E. Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties. Biomaterials 2009, 30, 2724−2734. (8) Lee, K. Y.; Alsberg, E.; Mooney, D. J. Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering. J. Biomed. Mater. Res. 2001, 56, 228−233. (9) Jeon, O.; Powell, C.; Ahmed, S. M.; Alsberg, E. Biodegradable, photocrosslinked alginate hydrogels with independently tailorable physical properties and cell adhesivity. Tissue Eng., Part A 2010, 16, 2915−2925. (10) Fedorovich, N. E.; Oudshoorn, M. H.; van Geemen, D.; Hennink, W. E.; Alblas, J.; Dhert, W. J. The effect of photopolymerization on stem cells embedded in hydrogels. Biomaterials 2009, 30, 344−353. (11) Lee, B. P.; Messersmith, P. B.; Israelachvili, J. N.; Waite, J. H. Mussel-inspired adhesives and coatings. Annu. Rev. Mater. Res. 2011, 41, 99−132. (12) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-inspired surface chemistry for multifunctional coatings. Science 2007, 318, 426−430. (13) Kang, S. M.; You, I.; Cho, W. K.; Shon, H. K.; Lee, T. G.; Choi, I. S.; Karp, J. M.; Lee, H. One-step modification of superhydrophobic surfaces by a mussel-inspired polymer coating. Angew. Chem., Int. Ed. 2010, 49, 9401−9404. (14) Cui, J.; Yan, Y.; Such, G. K.; Liang, K.; Ochs, C. J.; Postma, A.; Caruso, F. Immobilization and intracellular delivery of an anticancer drug using mussel-inspired polydopamine capsules. Biomacromolecules 2012, 13, 2225−2228. (15) Zhu, L. P.; Yu, J. Z.; Xu, Y. Y.; Xi, Z. Y.; Zhu, B. K. Surface modification of PVDF porous membranes via poly(DOPA) coating and heparin immobilization. Colloids Surf., B 2009, 69, 152−155. (16) Brubaker, C. E.; Kissler, H.; Wang, L. J.; Kaufman, D. B.; Messersmith, P. B. Biological performance of mussel-inspired adhesive in extrahepatic islet transplantation. Biomaterials 2010, 31, 420−427. (17) Ryu, J. H.; Lee, Y.; Kong, W. H.; Kim, T. G.; Park, T. G.; Lee, H. Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic materials. Biomacromolecules 2011, 12, 2653− 2659. (18) Hong, S.; Kim, K. Y.; Wook, H. J.; Park, S. Y.; Lee, K. D.; Lee, D. Y.; Lee, H. Attenuation of the in vivo toxicity of biomaterials by polydopamine surface modification. Nanomedicine 2011, 6, 793−801. (19) Bilic, G.; Brubaker, C.; Messersmith, P. B.; Mallik, A. S.; Quinn, T. M.; Haller, C.; Done, E.; Gucciardo, L.; Zeisberger, S. M.; Zimmermann, R.; Deprest, J.; Zisch, A. H. Injectable candidate sealants for fetal membrane repair: bonding and toxicity in vitro. Am. J. Obstet. Gynecol. 2010, 202, 85.e1−e9. (20) Ku, S. H.; Ryu, J.; Hong, S. K.; Lee, H.; Park, C. B. General functionalization route for cell adhesion on non-wetting surfaces. Biomaterials 2010, 31, 2535−2541. (21) Ferreira, L. S.; Gerecht, S.; Fuller, J.; Shieh, H. F.; VunjakNovakovic, G.; Langer, R. Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells. Biomaterials 2007, 28, 2706−2717.

advantages of alginate hydrogels, the lack of cellular interaction is considered one of the critical limitations to using this material for tissue engineering applications. It is known that alginate usually discourages protein adsorption due to its hydrophilic character and that it is unable to specifically interact with mammalian cells.2 For this reason, alginate has been engineered with cell adhesion peptides such as Arg-Gly-Asp (RGD) to promote cell adhesion. These modified alginate hydrogels could facilitate adhesion, proliferation, and differentiation of primary cells.2,9,36,37 However, typical chemical conjugation methods that immobilize peptides, ligands, and growth factors require multistep, complicated procedures such as activation or functionalization steps that require additional chemical treatments.38,39 Considering the capability of catechols to covalently conjugate nucleophiles (amine, thiol, and imidazole groups), diverse types of peptides, proteins, and molecules with these functional groups may be easily immobilized to the alginatecatechol hydrogel.31,35,40 Therefore, simply mixing alginatecatechol with peptides or proteins would create biologically functional hydrogel scaffolds conjugated with bioactive molecules for improved therapeutic efficacy of cell transplantation.



CONCLUSIONS In this study, alginate hydrogels engineered by mussel-inspired adhesive chemistry were created by calcium-free cross-linking. The alginate-catechol hydrogel exhibited not only tunable physical and mechanical properties, but also low immunogenicity and no cytotoxicity. Therefore, mussel-inspired nontoxic cross-linking via oxidative catechol polymerization may overcome several drawbacks of currently available alginate crosslinking methods that use ionic interaction of divalent cations (i.e., cytotoxicity, inflammatory response, and limited control of physical and mechanical properties). This biomimetic catecholmediated cross-linking strategy may also be applicable to other types of natural polymers (e.g., hyaluronic acid) for hydrogel formation. This novel class of biomimetic alginate hydrogels can provide tunable, functional, biocompatible 3D scaffolds for tissue engineering and cell transplantation.



AUTHOR INFORMATION

Corresponding Author

*Tel.: +82-2-2123-5662 (S.-W.C.); +82-42-350-2849 (H.L.). Fax: +82-2-362-7265 (S.-W.C.); +82-42-350-2810 (H.L.). Email: [email protected] (S.-W.C.); [email protected]. kr (H.L.). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by a grant from the Korea Health Technology R&D Project of the Ministry of Health & Welfare of the Republic of Korea (Grant No. A110552). This work was also supported by grants (2010-0025982 and R31-10071 (WCU)) funded by the National Research Foundation of Korea. We thank Jin Kim for her assistance in preparing the schematic illustration in Figure 1.



REFERENCES

(1) Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review. Biomacromolecules 2011, 12, 1387−1408. I

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(22) Lee, B. P.; Dalsin, J. L.; Messersmith, P. B. Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels. Biomacromolecules 2002, 3, 1038−1047. (23) Liu, B.; Burdine, L.; Kodadek, T. Chemistry of periodatemediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins. J. Am. Chem. Soc. 2006, 128, 15228−15235. (24) Ji, C.; Khademhosseini, A.; Dehghani, F. Enhancing cell penetration and proliferation in chitosan hydrogels for tissue engineering applications. Biomaterials 2011, 32, 9719−9729. (25) Ghosh, K.; Shu, X. Z.; Mou, R.; Lombardi, J.; Prestwich, G. D.; Rafailovich, M. H.; Clark, R. A. Rheological characterization of in situ cross-linkable hyaluronan hydrogels. Biomacromolecules 2005, 6, 2857−2865. (26) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677− 689. (27) Huebsch, N.; Arany, P. R.; Mao, A. S.; Shvartsman, D.; Ali, O. A.; Bencherif, S. A.; Rivera-Feliciano, J.; Mooney, D. J. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 2010, 9, 518−526. (28) Guvendiren, M.; Burdick, J. A. Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics. Nat. Commun. 2012, 3, 792. (29) Hasko, G.; Szabo, C.; Nemeth, Z. H.; Kvetan, V.; Pastores, S. M.; Vizi, E. S. Adenosine receptor agonists differentially regulate IL-10, TNF-α, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol. 1996, 157, 4634−4640. (30) El-Sherbiny, I. M.; Smyth, H. D. Controlled release pulmonary administration of curcumin using swellable biocompatible microparticles. Mol. Pharmaceutics 2012, 9, 269−280. (31) Lee, H.; Rho, J.; Messersmith, P. B. Facile conjugation of biomolecules onto surfaces via mussel adhesive protein inspired coatings. Adv. Mater. 2009, 21, 431−434. (32) Spargo, B. J.; Rudolph, A. S.; Rollwagen, F. M. Recruitment of tissue resident cells to hydrogel composites: in vivo response to implant materials. Biomaterials 1994, 15, 853−858. (33) Jeon, O.; Alt, D. S.; Ahmed, S. M.; Alsberg, E. The effect of oxidation on the degradation of photocrosslinkable alginate hydrogels. Biomaterials 2012, 33, 3503−3514. (34) Bhang, S. H.; Cho, S. W.; Lim, J. M.; Kang, J. M.; Lee, T. J.; Yang, H. S.; Song, Y. S.; Park, M. H.; Kim, H. S.; Yoo, K. J.; Jang, Y.; Langer, R.; Anderson, D. G.; Kim, B. S. Locally delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs. Stem Cells 2009, 27, 1976−1986. (35) Yang, K.; Lee, J. S.; Kim, J.; Lee, Y. B.; Shin, H.; Um, S. H.; Kim, J. B.; Park, K. I.; Lee, H.; Cho, S. W. Polydopamine-mediated surface modification of scaffold materials for human neural stem cell engineering. Biomaterials 2012, 33, 6952−6964. (36) Alsberg, E.; Anderson, K. W.; Albeiruti, A.; Franceschi, R. T.; Mooney, D. J. Cell-interactive alginate hydrogels for bone tissue engineering. J. Dent. Res. 2001, 80, 2025−2029. (37) Drury, J. L.; Boontheekul, T.; Mooney, D. J. Cellular crosslinking of peptide modified hydrogels. J. Biomech. Eng. 2005, 127, 220−228. (38) Shen, H.; Hu, X.; Yang, F.; Bei, J.; Wang, S. The bioactivity of rhBMP-2 immobilized poly(lactide-co-glycolide) scaffolds. Biomaterials 2009, 30, 3150−3157. (39) Nakaji-Hirabayashi, T.; Kato, K.; Arima, Y.; Iwata, H. Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells. Biomaterials 2007, 28, 3517−3529. (40) Shin, Y. M.; Lee, Y. B.; Kim, S. J.; Kang, J. K.; Park, J. C.; Jang, W.; Shin, H. Mussel-inspired immobilization of vascular endothelial growth factor (VEGF) for enhanced endothelialization of vascular grafts. Biomacromolecules 2012, 13, 2020−2028.

J

dx.doi.org/10.1021/bm400352d | Biomacromolecules XXXX, XXX, XXX−XXX