Controlling Gel Structure to Modulate Cell Adhesion and Spreading on

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Controlling gel structure to modulate cell adhesion and spreading on the surface of microcapsules Huizhen Zheng, Meng Gao, Ying Ren, Ruyun Lou, Hongguo Xie, Weiting Yu, Xiudong Liu, and Xiaojun Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05778 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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ACS Applied Materials & Interfaces

Controlling gel structure to modulate cell adhesion and spreading on the surface of microcapsules Huizhen Zheng a,c, Meng Gao a,c, Ying Ren a,c, Ruyun Lou a,c, Hongguo Xie a, Weiting Yu a,*, Xiudong Liu b,*, Xiaojun Ma a a

Laboratory of Biomedical Materials Engineering, Dalian Institute of Chemical

Physics, Chinese Academy of Sciences, Dalian 116023, P.R. China b

College of Environment and Chemical Engineering, Dalian University, Dalian

Economic Technological Development Zone, Dalian 116622, P.R. China c

University of the Chinese Academy of Sciences, Beijing 100049, P.R. China

* Corresponding authors Dr. Weiting Yu E-mail: [email protected] Tel: +86-411-84379139 Fax: +86-411-84379096 Dalian Institute of Chemical Physics, CAS 457 Zhongshan Road, Dalian 116023, P. R. China.

Prof. Dr. Xiudong Liu E-mail: [email protected] Tel: +86-411-84379139 Fax: +86-411-84379096 College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, P. R. China

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Abstract The surface properties of implanted materials or devices play critical roles in modulating cell behavior. However, the surface properties usually affect cell behaviors synergetically so that it is still difficult to separately investigate the influence of a single property on cell behavior in practical applications. In this study, alginate-chitosan (AC) microcapsules with a dense or loose gel structure were fabricated to understand the effect of gel structure on cell behavior. Cells preferentially adhered and spread on the loose gel structure microcapsules rather than on the dense ones. The two types of microcapsules exhibited nearly identical surface positive charges, roughness, stiffness and hydrophilicity, thus the result suggested that the gel structure was the principal factor affecting cell behavior. X-ray photoelectron spectroscopy analyses demonstrated that the overall percentage of positively charged amino groups was similar on both microcapsules. Since the different gel structures led to different states and distributions of the positively charged amino groups of chitosan, we conclude that the loose gel structure facilitated greater cell adhesion and spreading mainly because more protonated amino groups remained unbound and exposed on the surface of these microcapsules. Key words: alginate-chitosan microcapsule, gel structure, protonated amino, cell adhesion and spreading, surface property

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1. Introduction Microcapsule has been extensively studied as a carrier for cell transplantation or drug delivery system for the treatment of many diseases.1-2 Alginate-polycation microcapsules, consisting of a three-dimensional network structure and a semi-permeable membrane designed for isolation, have probably become the preferable system for cell transplantation.3-5 However, protein adsorption and acute inflammation (cell adhesion) occur successively on the surface of microcapsules during transplantation.6,7 Cell infiltration and adhesion to the surface of microcapsules significantly promote fibrous overgrowth and fibrosis formation, which could hind the diffusion of nutrients and therapeutic products across the capsule membrane and lead to transplantation failure.4,8 Although the physicochemical properties, including surface energy, roughness, charge, stiffness and wettability, have been demonstrated to have profound effects on cell behavior,9-13 the cell-microcapsule interactions are still unclear. Therefore, it has attracted more and more attentions to clearly understand the correlation between cell adhesion and the surface properties of microcapsules to facilitate the design of well-defined and more biocompatible surfaces. Alginate-polycation microcapsules are often fabricated in a two-steps process: one step for the formation of an inner gel core, and the other for the formation of polyelectrolyte complex (PEC) membrane.14 A variety of parameters can influence the physicochemical properties in both steps, and these can be divided into two categories: properties related to alginate (e.g., molecular weight, G content and purity) and the gelling bath (e.g. ion type, concentration and ionic strength), which affect the 3



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three-dimensional network structure of the inner core; and properties related to the polycation (e.g., molecular weight, charge density and conformation), which directly impact the formation of the PEC membrane.2, 15-16 These parameters usually influence the physicochemical properties in a synergistic way; thus, a change in one parameter can alter more than one property, which may overshadow the effect of the principal factor on biological behaviors and make the comparison and interpretation of results difficult.8 Tam et al. utilized different polycations to form microcapsules and studied the effect of the membrane-forming step on biocompatibility. They demonstrated that the poly-L-ornithine (PLO) capsules have a greater hydrophilicity than poly-L-lysine (PLL) capsules, which resulted in better biocompatibility in vivo.4 However, the use of different polycations may induce not only a change in the surface hydrophilicity/hydrophobicity but also a change in the mechanical stability, making it difficult to solely attribute the improved biocompatibility to surface hydrophilicity. Additionally, de Vos et al. reported that high-guluronic (G) alginate capsules induced more undesired inflammatory reactions than intermediate-G capsules owing to in vivo detachment of PLL from high-G alginate.8 However, this study ignored the fact that G-content differences in alginate can affect not only the PLL binding capacity but also gel structure and stiffness. Hence, distinguishing the specific influence of the physicochemical properties of microcapsules on cell behavior and biocompatibility remains a challenge, which hinders a deeper understanding of the precise mechanisms underlying cell-microcapsule interactions. In the present study, alginate-Ca beads with different gel structures were fabricated by 4


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controlling the ionic strength of the gelling bath. Then, alginate-chitosan (AC) microcapsules were prepared using the classical two-step process and evaluated for membrane formation and fibroblast adhesion. Moreover, two types of AC microcapsules with different gel structures but the same amount of bound chitosan were constructed to decouple the effect of gel structure on cell adhesion from that of other physicochemical properties. The cell behavior on the surface of AC microcapsule was investigated at different pH levels to further elucidate the different cell adhesion affected by the gel structure. This study provides insight into the interaction mechanisms between cell behavior and physicochemical properties of microcapsules and can thus guide the design of microcapsules with better biocompatibility. 2. Materials and methods 2.1 Materials Sodium alginate (molecular weight, 460 kDa; molar ratio of mannuronic acid to guluronic acid, 2/1) was obtained from Qingdao Crystal Salt Bioscience and Technology Corporation (Qingdao, China). Chitosan (molecular weight, 41 kDa; degree of deacetylation, 90%) was degraded from the raw material (Yu Huan Chemical Plant, Zhejiang, China) in our laboratory. Chitosan labeled with fluorescein isothiocyanate (FITC, Sigma) was synthesized as described by Onishi and Machida.17 All other chemical reagents were of analytical grade and were used as received. 2.2 Fabrication of alginate-chitosan microcapsules Alginate-Ca beads were prepared by extruding 1.5% (w/v) sodium alginate, through a 5



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0.5 mm needle into 0.1 M CaCl2 solution (gelling bath) with an electrostatic droplet generator (YD-04, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China). After 30 min of gelling, alginate-Ca beads, which were denoted as A-A beads, were collected. Next, the beads were immersed in 0.5% (w/v) chitosan solution to form AC microcapsules, which are referred to as group A. Another group of alginate-Ca beads was prepared controlling the gelling bath to have a 0.025 M CaCl2 and 0.1155 M NaCl solution (B-A beads). Next, the beads were reacted with chitosan (0.5% w/v) to form AC microcapsules, which are referred to as group B. The amount of bound chitosan was measured by gel permeation chromatography (GPC), using a method established in our previous study.18 Then, to fabricate two types of microcapsules with a similar amount of bound chitosan (Fig. S1), A-A beads were reacted with chitosan for 12 min to form A-AC microcapsules, whereas B-A beads were reacted with chitosan for 3 min to form B-AC microcapsules. 2.3 Characterization of the gel structure by SEM Two types of alginate-Ca hydrogel beads were dehydrated with a graded series of ethanol (30%, 50%, 70%, 90% and 100%, v/v) for 15 min at each concentration. Next, the samples were dried in a critical point dryer (Quorum K850, Quorum Technologies, Lewes, UK). Subsequently, the morphology and gel structure were observed with field emission scanning electron microscopy (FE-SEM) (JSM-7800F, JEOL, Japan) by mounting the gel beads on metal stubs at a high vacuum (10-4 Pa) and using an accelerated voltage of 2.0 kV. 2.4 Cell culture 6


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Mouse fibroblast L929 cells (RCB 0081, Cell Bank, Japan) were cultivated in RPMI-1640 medium with 20 mM NaHCO3 supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (P/S) (all cell culture materials were from Invitrogen, Carlsbad, CA) at 37 °C and 5% CO2. The medium was replaced every other day and cells were sub-cultured when they reached over 90% confluence. 2.5 Cell behavior on the surface of microcapsules L929 cells were harvested after trypsinization using a 0.25% (w/v) trypsin solution (Invitrogen) and resuspended in the culture medium (RPMI-1640 medium with 20 mM HEPES buffer, pH6.9) to achieve cell suspension with a final concentration of 5 × 105 cells/mL. Next, 2 mL of the cell suspension was seeded onto a 12-well plate in which each well was filled with microcapsules. All of the 12-well plates used for this experiment were pre-coated with agarose gel (2% w/v) to prevent cellular adhesion to the bottom. Next, the cells were cultured at 37 °C and 5% CO2. For the initial 4 h of cultivation, gentle shaking (5 min every half an hour) of the plates was employed. After incubation for 24 h, the cell adhesion to the surface of AC microcapsules was evaluated under a phase contrast microscope (Nikon Eclipse TE 2000, Nikon Corp. Japan). The samples were incubated with live/dead stain composed of 2 µM calcein AM (Sigma-Aldrich) and 4 µM ethidiumhomodimer-1 (ED-1, Sigma-Aldrich) at 37 °C for 1 h and then scanned using confocal laser scanning microscopy (CLSM, Leica SP2, Heidelberger, Germany) at excitation wavelengths of 480 nm and 533 nm. 2.6 Quantification of the genomic DNA of cells adhering to the microcapsules 7



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Cell adhesion to the surface of microcapsules was quantitatively determined using DNA content analysis. After harvesting of all the samples, the DNA content was measured using a PicoGreen DNA Assay following the protocol provided by the manufacturer (Molecular Probes, Eugene, OR). The fluorescence was measured with a microplate reader (BioTek, Synergy H1, USA) at excitation and emission wavelengths of 480 nm and 520 nm, respectively. 2.7 Characterization of the cell morphology on microcapsules 2.7.1 SEM After incubation, microcapsules with adhered cells were washed in a saline solution and fixed with paraformaldehyde (4% w/v) for 4 h at room temperature. Afterwards, the samples were rinsed with saline three times and dehydrated with a graded series of ethanol (30%, 50%, 70%, 90% and 100%, v/v). Finally, the samples were dried by vacuum drying overnight. The morphology of the cells adhering to the microcapsules was observed by FE-SEM (JSM-7800F, JEOL, Japan). 2.7.2 Immunofluorescence assay After the fixation of microcapsules with adherent cells, as described in 2.7.1, a permeabilization buffer (0.1% TritonX-100, v/v, Aladdin) was added for 15 min. Next, the samples were washed with saline three times and incubated in 10% goat serum (v/v) at room temperature for 2 h to prevent non-specific binding of antibodies. Then, samples were incubated with a monoclonal mouse antibody against vinculin (1:400 in 10% goat serum; Sigma) and Alexa Fluor 488-conjugated phalloidin (1:100 in 1% goat serum, v/v; Invitrogen) at 4 °C overnight. Rhodamine red-conjugated goat 8


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anti-mouse IgG antibody (1:100 in 1% goat serum, v/v; Invitrogen) was then added at room temperature and kept for 4 h. Finally, the samples were washed with saline three times. Confocal fluorescence imaging studies were performed with an Olympus (Japan) laser-based point scanning FV 1000 confocal microscope. 2.7.3 Quantitative real-time PCR Approximately 106 cells were harvested from the surface of AC microcapsules for RNA extraction. Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Japan). The RNA quantity and quality were determined by NanoPhotometer (Implen, Germany) with the absorbance ratio at 260 nm/280 nm. Reverse transcription was performed using PrimeScriptTM RT Master Mix (TaKaRa, Japan) according to the manufacturer's instructions in a 5333 PCR Mastercycler (Eppendorf, Germany). The qRT-PCR amplifications were performed with at least 3 biological replicates using 2X SYBR Premix Ex TaqTM II (DRR081A, TaKaRa, Japan) and the Stratagene MX3000P (Agilent Technologies, CA, USA). The housekeeping gene β-actin was used as a control for normalization. The primer sequences are shown in Tab. S2. 2.8 Characterization of the physicochemical properties of microcapsules 2.8.1 Surface roughness The surface roughness of microcapsules was measured using a noncontact, three-dimensional white-light optical interferometer (New-View 5020, ZYGO, USA).19 The samples were vertically scanned, and the interference fringes were recorded using a CCD camera. Then, the actual surface roughness of the microcapsules was obtained by spherical aberration correction. Average values were 9



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obtained from at least three microcapsules of each sample. 2.8.2 Contact angle Alginate (500 µL) was first cast onto microscope slides. After gelling in two types of gelling bath, each type of film was reacted with chitosan for 12 min and for 3 min. For removal of excess chitosan molecules, the films were quickly rinsed with sterile water before being allowed to dry overnight. The contact angle was measured using a telescopic goniometer (Powereach JC2000C2, Shanghai, China). One microliter of sterile water was dispensed from a mechanically controlled 100 µL syringe fitted with a 22 G blunt tip needle. The sample was raised to touch the suspended water droplet; then, a photo was taken within 2 s of contact with the water droplet. Finally, the contact angle of the droplet was measured using the software provided with the machine. Average values were obtained from at least three droplets of each sample. 2.8.3 Surface positive charge Microcapsules were first rinsed with sterile water to remove excess salt and then dehydrated with a graded series of ethanol. After the samples were dried, the elemental compositions (except hydrogen) of the samples were analyzed by high-resolution X-ray photoelectron spectrometry (XPS; ESCALAB250, Thermo VG, USA) with an Al Kα source operating at a take-off angle of 45°. The approximate depth of the profile was 2-10 nm. Charge shift correction was conducted by setting the C1s peak value of saturated hydrocarbons (C-C) to 284.6 eV. The surface atomic percentage was calculated using XPS Peak Software 4.1 with the sensitivity factor provided by the manufacturer. The high-resolution N1s spectra were curve-fitted with 10


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an 80% Gaussian - 20% Lorentzian peak shape, and the peak integral was calculated with subtraction of a Shirley background.20 For each sample, three batches were analyzed. 2.8.4 Stiffness measurement The mechanical resistance of the microcapsules was characterized by ElectroForce (ELF3100, BOSE, USA), a high-precision biomechanical test instrument mounted with a 250 g load cell. The tests were performed to compress a single microcapsule at a speed of 0.05 mm/s with the probes set from the position of -1.0 mm to -1.4 mm. Then, a force-displacement curve was obtained. The resistance force of the sample at 50% deformation (in order to avoid any damage to the probe and breakage of microcapsules) was used to determine the stiffness. All average values were calculated from at least three microcapsules of each sample. 2.9 Statistical analysis All reported values were averaged (n = 3 repeats except for the specific experiments where explanations are provided) and expressed as the mean ± standard deviation (SD). Significant differences were determined by Student’s two-tailed test and differences were considered statistically significant at p < 0.05. 3. Results 3.1 The effect of ion strength on gel structure, membrane formation and cell adhesion In this study, two types of alginate-Ca beads with the same diameter were fabricated by dropping the same alginate solution into two gelling baths of different ion 11



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strengths (a 0.1 M CaCl2 solution; a 0.025 M CaCl2 and 0.1155 M NaCl solution); these beads were denoted as A-A and B-A beads, respectively. The gel structure of the beads was characterized by SEM. The A-A beads exhibited a dense gel structure with some small nanopores (ranging in size from 20 to 80 nm), whereas the B-A beads demonstrated a loose gel structure with a fiber-like network and larger pores (ranging from 100 to 300 nm) (Fig. 1). On the one hand, the loose gel structure might be due to the lower Ca2+ concentration in the gelling bath. On the other hand, the presence of Na+ can also reduce both the gelling rate and strength, and both factors might contribute to the looser gel structure of the B-A beads than that of the A-A beads.

Fig. 1. SEM images of alginate-Ca beads crosslinked with calcium ions at 0.1 M (a, A-A beads) and 0.025 M (b, B-A beads), scale bar = 100 nm. Chitosan is a deacetylated derivative of chitin, and it has numerous protonated amino groups (positive charges) when dissolved in acetate buffer (pH 4.3). Chitosan can diffuse into the three-dimensional network and bind to alginate forming the PEC membrane of alginate-chitosan (AC) microcapsules. The membrane thickness of the AC microcapsules formed by complexing FITC-labeled chitosan with the dense gel structure (A-A beads) was thinner than that of the AC microcapsules with the loose 12


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gel structure (B-A beads) (Fig. 2a-b). Although the amount of chitosan bound on both gel structures increased dramatically as the reaction time increased, the reaction rate of chitosan with B-A beads was much faster than that with A-A beads (Fig. 2c). This result is probably due to the loose gel structure of the B-A beads, which better facilitates the inward diffusion of chitosan.

Fig. 2. CLSM images of AC microcapsule formed by complexing chitosan-FITC with two types of alginate-Ca beads for 15 min (a, A-AC microcapsules; b, B-AC microcapsules) (scale bar = 150 µm). Measurement of the rate of binding of chitosan to the two types of alginate-Ca beads (c). After co-incubation of fibroblasts with the two groups of AC microcapsules, the cells exhibited very different behaviors. Cell adhesion occurred on microcapsules with both dense and loose gel structures and increased as the reaction time with chitosan increased (Fig. 3). However, cell adhesion was significantly enhanced on the B-AC microcapsules, suggesting that chitosan complexation might be the primary factor that promotes cell adhesion to the surface of AC microcapsules. The greater cell adhesion to the B-AC microcapsules than on the A-AC microcapsules at the same time points can be ascribed to more binding of chitosan (Fig. 2), which was definitely decided by the gel structure. These results suggest that both the gel structure and subsequent 13



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bound chitosan on the AC microcapsules affected cell adhesion.

Fig. 3. Effect of the chitosan reaction on cell adhesion to AC microcapsules under the membrane formation time of 3 min and 15 min (a1) A-AC-3, (b1) A-AC-15, (c1) B-AC-3 and (d1) B-AC-15 (scale bar = 100 µm). CLSM images using live/dead stain (a2-d2) indicate the viability of the adherent cells on the surface of AC microcapsules, scale bar = 150 µm. 3.2 The effect of gel structure on cell adhesion and spreading To investigate the effect of gel structure alone on cell adhesion and spreading, two types of AC microcapsules were fabricated while maintaining the same amount of bound chitosan. A-A beads were reacted with chitosan for 12 min to form A-AC microcapsules, whereas B-A beads were reacted with chitosan for only 3 min to form B-AC microcapsules. The two types of AC microcapsules had nearly the same amount of bound chitosan (approximately 1.0 µg/mm2), as determined by GPC,18 as well as a similar membrane thickness of 15 µm (Fig. S1). Fibroblasts were incubated with microcapsules for 24 h, and the cells attached to two types of AC microcapsules with significant differences. Fewer cells attached to A-AC 14


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microcapsules and exhibited spherical adhesion, whereas many more cells adhered to B-AC microcapsules and spread in a fusiform shape (Fig. 4a-b). The morphology of the cells adhered to the AC microcapsules was then observed by SEM. The cells clearly adhered in a spherical shape on the A-AC microcapsules with a small contact surface area. On the cell-microcapsule interface, fewer pseudopodia formed and connected with the gel matrix (Fig. 4c-c1). However, the cells that adhered to the surface of B-AC microcapsules exhibited a spindle or branch shape and formed a high density of lamellipodia and filopodia to contact with the microcapsules (Fig. 4d-d1). This finding suggested that the cells had more cross-talk and stronger interactions with the B-AC microcapsules than with the A-AC microcapsules. The number of cells that adhered to the surface of AC microcapsules was analyzed by quantifying the genomic DNA content. As shown in Fig. 4e, the DNA concentration on the surface of B-AC microcapsules was significantly higher than that on A-AC microcapsules, which is consistent with the above morphology analysis.

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Fig. 4. Cell morphology on the surface of A-AC microcapsules (a and c) and B-AC microcapsules (b and d) evaluated using light microscopy (scale bar = 100 µm) and SEM (scale bar = 100 nm). Partially magnified SEM images are shown as inserts c1 and d1. The concentration of genomic DNA isolated from cells that adhered to two types of microcapsules was measured (e) (p < 0.05). Moreover, Immunofluorescence and real-time PCR analysis were studied to explore the mechanism underlying the differences in cell adhesion. Immunofluorescence 16


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studies of the cytoskeleton organization and focal adhesions provided insight into the differences in cell adhesion on the AC microcapsules. The cell adhesion and spreading on microcapsules were monitored by analysis of F-actin (green) and vinculin (red), which associated with cell cytoskeleton and focal adhesions, respectively. On the surface of A-AC microcapsules, the overall fluorescence intensity was lower, and the F-actin was condensed with no signs of fibrillar organization. Additionally, vinculin staining showed a weak fluorescence signal and was mainly located to the cell periphery, which suggested that few focal adhesions formed (Fig. 5a-c). Conversely, cells spread and developed a prominent spindle shape on the surface of B-AC microcapsules. The formation of adhesion plaques was obvious and the actin filaments were organized in bundles in the cells (Fig. 5d-f). Moreover, real-time PCR analysis was used to verify the variation of gene expression related to focal adhesion proteins. It suggested that the relative expressions of genes encoded integrin (Itgα1 and Itgβ1) and paxillin (Pxn) were significant higher on the surface of B-AC than that of A-AC microcapsules (Fig. 5g). The higher gene expressions of cells adhered to the surface of B-AC microcapsules could induce the formation of focal adhesion and finally facilitate the rearrangement of F-actin. Therefore, these findings suggest that the cells adhering to the B-AC microcapsules performed more stable and mature focal points at the cell membrane.

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Fig. 5. Immunofluorescence staining of F-actin structures (green) and vinculin focal adhesion (red) of cells adhered to A-AC microcapsules (a-c) and B-AC microcapsules (d-f), scale bar = 10 µm. The relative expression of key genes deciding adhesive proteins and subsequent cell adhesion on the surface of two AC microcapsules (* p < 0.05) (g). 3.3 The effect of surface properties on cell adhesion 3.3.1 Surface properties of microcapsules 18


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The fact that changing only the gel structure of AC microcapsules induced significantly different cell adhesion and spreading greatly aroused our interest. Numerous studies have reported the profound effects of surface properties on the interaction between cells and materials.13,

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Therefore, to further determine the

principal factor that affects the cell adhesion to microcapsules, the surface properties of two types of microcapsules were further investigated. Surface positive charge is an important property that affects cell adhesion.22 Chitosan, a polysaccharide with positive charges in acetate buffer, was reacted with alginate-Ca beads and contributed to the surface positive charge of the microcapsules. Thus, we utilized XPS to analyze the positively charged amino groups on the surface of microcapsules. The percentage of positively charged amino groups distributed on the two types of microcapsules was similar without significant difference (Fig. 6a). Surface roughness has also been reported to affect cell behaviors.10, 23 According to our previous report,19 the surface roughness of two types of microcapsules was characterized in wet and in situ using an optical interferometer. The results indicated that both the A-AC and B-AC microcapsules had a smooth surface with a surface roughness of approximately 25 nm in wet condition (Fig. 6b). Thus, these microcapsules with different gel structures had nearly the same surface roughness. Hydrophobic surfaces have been reported to improve the adsorption of proteins,2 which may facilitate cellular adhesion and induce immune reactions. In our study, the contact angles were measured to evaluate the surface hydrophilicity/hydrophobicity of these two types of microcapsules. The contact angles were both of approximately 70 ° 19



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(Fig. 6c), which is much larger than that of alginate alone (less than 15 °).4 Although the chitosan coating on alginate-Ca beads reduced the hydrophilicity, which may facilitate cell adhesion, it is worth noting that no significant difference was observed between the two types of microcapsules. Moreover, stiffness can also affect the adhesion force of cells and cytoskeleton constriction.24 To characterize the stiffness of these microcapsules, the compression force was measured by compressing the microcapsules to 50% deformation. The compression force of B-AC microcapsules was 5.1 g, similar to A-AC microcapsules with no statistically significant difference (Fig. 6d). Other properties, such as surface chemical compositions, were characterized by XPS and ATR-FTIR (Tab. S1 and Fig. S3). It indicated that these two types of microcapsules have very similar surface chemical compositions. Therefore, the two types of AC microcapsules with different gel structure but the same amount of bound chitosan exhibited nearly identical surface properties, including surface chemical compositions, positive charge, roughness, hydrophilicity and stiffness. Remarkably, these same surface properties actually resulted in a significant difference in cell adhesion to the two types of AC microcapsules. The principal difference between both microcapsules was the gel structure, which aroused our further interest to explore how the different gel structures affected cellular adhesion.

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Fig. 6. Characterization of the surface properties of A-AC and B-AC microcapsules, including surface positive charges (percentage of protonated amino groups) (a), surface roughness (b), contact angle (c) and stiffness (the compression force for 50% deformation) (d). 3.3.2 Effect of the protonated amino groups of the microcapsules on cell adhesion The two types of AC microcapsules with different gel structure but the same amount of bound chitosan were incubated with fibroblasts, and the pH of the medium was precisely adjusted to 6.9, 7.1, 7.3 and 7.5. On the surface of A-AC microcapsules, cell adhesion occurred at pH 6.9 and 7.1, but was sharply reduced at pH 7.3 and 7.5 (Fig. 7a). On the surface of B-AC microcapsules, more cell adhesion was observed at pH 6.9, 7.1 and 7.3, although few cells adhered at pH 7.5 (Fig. 7b). These results demonstrated that such minor variation in the pH of the medium can strongly affect cell adhesion to AC microcapsules. The quantitative analysis of the genomic DNA 21



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concentration on both types of microcapsules showed decreasing trend as the pH of the medium increased, but the DNA concentration on the B-AC microcapsules was much higher than that on the A-AC microcapsules at each pH value (Fig. 7c). As a whole, the increase in the pH of the medium (that is, a decrease in protonated amino groups) can reduce the extent of cell adhesion to these two types of AC microcapsules, especially the A-AC microcapsules with a dense gel structure. This finding indirectly suggests that the different numbers or distributions of protonated amino groups on both microcapsules were caused by their different gel structures.

Fig. 7. Effect of the pH of the medium (6.9, 7.1, 7.3 and 7.5) on the cell adhesion to the surface of A-AC microcapsules (a) and B-AC microcapsules (b), scale bar = 100 µm. The genomic DNA content of cells adhered to both types of microcapsules at 22


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various pH values was measured (c) (p< 0.05). 4. Discussion Microcapsules have been extensively studied for cell transplantation, the understanding of cell-microcapsules interactions is essential for their applications. Chitosan has positively charged amino groups that can react with alginate-Ca beads to form AC microcapsules with PEC membrane. The amount of chitosan bound on alginate-Ca beads increased along with reaction time, thus resulting in enhanced cell adhesion to the surface of AC microcapsules. This phenomenon indicated that chitosan is a crucial factor in inducing cell adhesion to surface. Interestingly, the amount of bound chitosan was much greater on the loose alginate-Ca beads (B-A beads) than on the dense one (A-A beads), which contributed to the larger number of cells attached to the surface of B-AC microcapsules than to the A-AC microcapsules (Fig. 2 and Fig. 3). These findings suggested that the significantly different gel structure induced the different chitosan binding and the subsequent cell behavior. To further distinguish the influence of gel structure and chitosan binding on cell behavior, two types of microcapsules with the same amount of bound chitosan but different gel structures were formed (Fig. S1-S2). Cells still preferentially attached and spread to the surface of B-AC microcapsules with the loose gel structure. By characterizing the surface properties, we demonstrated that the two types of microcapsules exhibited nearly identical surface properties, including surface chemical compositions, positive charge, roughness, stiffness and hydrophilicity. Thus the principal difference between these two types of microcapsules is the gel structure. 23



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Subsequently, we supposed that whether the gel structure affected cell behaviors on the two types of AC microcapsules indirectly by modulating the conditions to obtain diverse chitosan complexation and distributions. Chitosan has been reported to have a pKa value of 6.1-6.6,25 so the amino groups will be protonated at pH values lower than this pKa. When the pH of the medium was adjusted to values higher than the pKa, the surface amino groups were deprotonated, which resulted in decrease in cell adhesion to the surface. This finding clearly demonstrated that the positively charged amino groups on the surface introduced by chitosan are a major determining factor in cell adhesion. The percentages of surface protonated amino measured by XPS displayed no significant difference between the two types of microcapsules (Fig. 6a). Nevertheless, cell adhesion and spreading were always observed preferentially on B-AC microcapsules with the loose gel structure. Reconsidering the XPS analysis suggested that this measurement provides the overall percentage of positively charged amino groups on both microcapsules. Because the binding force between the protonated amino groups of chitosan and the negatively charged carboxyl of alginate is an electrostatic interaction, there is no electron or charge transfer and no change in binding energy (Fig. S4). Therefore, XPS cannot distinguish the bound protonated amino groups from the unbound ones. Furthermore, the bound protonated amino groups cannot contribute to the adhesion of fibroblasts, which was indicated by the results of almost no cells adhered on the surface of A-ACA and B-ACA microcapsules (coating outer layer of alginate for AC microcapsules) (Fig. S5). Hence, it was further 24


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supposed that the unbound protonated amino groups of the two types of microcapsules were distributed differently on the distinct gel structures. Also, it was calculated that the ratio of Chi/Alg (w/w) for A-AC microcapsules (0.14 ± 0.001) is significant lower than that of B-AC microcapsules (0.21 ± 0.005), because the B-A beads showed looser gel structure and the alginate density was lower. The calculated pore size of the loose gel structure is 4- or 5-fold higher than that of the dense gel structure; the porosity was 92% for the former and 33% for the latter, which were estimated by image J software according to SEM images. These results indicated that the molecular skeleton of alginate perunit area of the loose gel beads was far less than that of the dense gel beads; therefore, the available carboxyl perunit area of the loose gel beads was far less than that of the dense gel beads. When chitosan was used to coat alginate-Ca beads with the dense gel structure, chitosan was able to sufficiently bind to form the PEC membrane. Thus few unbound protonated amino groups would be left and exposed on the surface of A-AC microcapsules. However, in the case of B-A beads, chitosan was easily able to diffuse into the loose gel structure but encountered fewer carboxyl groups on the surface. Thus, a greater number of positively charged amino groups might remain unbound and exposed on the surface of B-AC microcapsules, as depicted in Fig. 8, which was supposed to be the main reason to facilitate the cell adhesion and spreading on these microcapsules. Therefore, the distribution of protonated amino groups, which is dependent on the gel structure, is of paramount importance in determining cell behavior on AC microcapsules.

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Fig. 8. Schematic representation of the binding of chitosan on alginate-Ca beads with different gel structures, and the subsequent effect of inducing different cell adhesion on the surface of microcapsules. 5. Conclusion This study found that both the gel structure and the binding of chitosan affected cell adhesion to the surface of AC microcapsules. Two types of AC microcapsules were constructed, with either a dense or loose gel structure but the same amount of bound chitosan, to determine the effect of gel structure alone on cell behavior. We demonstrated that cell adhesion and spreading preferentially on the surface of AC microcapsules with the loose gel structure. Furthermore, study of the surface properties and analysis of the gel structure revealed that the binding efficiency of protonated amino groups on the microcapsules with the loose gel structure was less than that of the dense ones. Hence, more unbound protonated amino groups were exposed on the loose gel structure microcapsules that facilitated the formation of focal adhesion. This new finding suggests that the states and distributions of protonated amino groups caused by different gel structures played a dominant role in the cell 26


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adhesion and spreading on AC microcapsules. This study has improved the understanding of the interaction mechanisms underlying cell adhesion and spreading on microcapsules, thus providing guidance for designing and optimizing microcapsules to have a better biocompatibility. Supporting Information The characterization of chitosan bound on the surface of alginate-Ca beads; SEM images of A-AC and B-AC microcapsules; the results of ATR-FTIR spectra and XPS for the surface of microcapsules; and the results of cell adhesion on the surface of ACA microcapsules. Acknowledgments The authors greatly acknowledge the financial support from National Basic Research Program of China (grant 2012CB720801), Scientific and Technological projects of Guangdong Province (2013B091100001) and the National Natural Science Foundation of China (21276033). References (1) de Vos, P.; van Hoogmoed, C. G.; van Zanten, J.; Netter, S.; Strubbe, J. H.; Busscher, H. J., Long-term Biocompatibility, Chemistry, and Function of Microencapsulated Pancreatic Islets. Biomaterials 2003, 24, 305–312. (2) Rokstad, A. M.; Lacik, I.; de Vos, P.; Strand, B. L., Advances in Biocompatibility and Physico-chemical Characterization of Microspheres for Cell Encapsulation. Adv. Drug Delivery Rev. 2014, 67-68, 111-130. (3) Zimmermann, H.; Ehrhart, F.; Zimmermann, D.; Müller, K.; Katsen-Globa, A.; Behringer, M.; Feilen, P. J.; Gessner, P.; Zimmermann, G.; Shirley, S. G.; Weber, M. M.; Metze, J.; Zimmermann, U., Hydrogel-based Encapsulation of Biological, Functional Tissue: Fundamentals, Technologies and Applications. Appl. Phys. A 2007, 89, 909-922. (4) Tam, S. K.; Bilodeau, S.; Dusseault, J.; Langlois, G.; Halle, J. P.; Yahia, L. H., Biocompatibility and Physicochemical Characteristics of Alginate-polycation Microcapsules. Acta biomater. 2011, 7, 1683-1692. (5) de Vos, P.; Faas, M. M.; Strand, B.; Calafiore, R., Alginate-based Microcapsules for 27



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Immunoisolation of Pancreatic Islets. Biomaterials 2006, 27, 5603-5617. (6) Bryers, J. D.; Giachelli, C. M.; Ratner, B. D., Engineering Biomaterials to Integrate and Heal: the Biocompatibility Paradigm Shifts. Biotechnol. Bioeng. 2012, 109, 1898-1913. (7) Hernandez, R. M.; Orive, G.; Murua, A.; Pedraz, J. L., Microcapsules and Microcarriers for in situ Cell Delivery. Adv. Drug Delivery Rev. 2010, 62, 711-730. (8) de Vos, P.; Spasojevic, M.; de Haan, B. J.; Faas, M. M., The Association between in vivo Physicochemical Changes and Inflammatory Responses against Alginate based Microcapsules. Biomaterials 2012, 33, 5552-5559. (9) E., S.; Bruinsma, R. F., Cell Adhesion as Wetting Transition? ChemPhysChem 2002, 3, 262-269. (10) Zan, Q.; Wang, C.; Dong, L.; Cheng, P.; Tian, J., Effect of Surface Roughness of Chitosan-based Microspheres on Cell Adhesion. Appl. Surf.Sci. 2008, 255, 401-403. (11) Kim, J.; Kim, D. H.; Lim, K. T.; Seonwoo, H.; Park, S. H.; Kim, Y. R.; Kim, Y.; Choung, Y. H.; Choung, P. H.; Chung, J. H., Charged Nanomatrices as Efficient Platforms for Modulating Cell Adhesion and Shape. Tissue eng. Part C 2012, 18, 913-923. (12) Best, J. P.; Javed, S.; Richardson, J. J.; Cho, K. L.; Kamphuis, M. M. J.; Caruso, F., Stiffness-mediated adhesion of cervical cancer cells to soft hydrogel films. Soft Matter 2013, 9, 4580-4584. (13) Bacakova, L.; Filova, E.; Parizek, M.; Ruml, T.; Svorcik, V., Modulation of Cell Adhesion, Proliferation and Differentiation on Materials Designed for Body Implants. Biotechnol. Adv. 2011, 29, 739-767. (14) Bhatia, S. R.; Khattak, S. F.; Roberts, S. C., Polyelectrolytes for Cell Encapsulation. Curr. Opin. Colloid Interface Sci. 2005, 10, 45-51. (15) Huang, X.; Zhang, X.; Wang, X.; Wang, C.; Tang, B., Microenvironment of Alginate-based Microcapsules for Cell Culture and Tissue Engineering. J. Biosci. Bioeng. 2012, 114, 1-8. (16) Tam, S. K.; Dusseault, J.; Polizu, S.; Ménard, M.; Hallé, J.-P.; Yahia, L. H., Physicochemical Model of Alginate–Poly-l-lysine Microcapsules Defined at the Micrometric/nanometric Scale Using ATR-FTIR, XPS, and ToF-SIMS. Biomaterials 2005, 26, 6950-6961. (17) Onishi, H.; Machida, Y., Biodegradation and Distribution of Water-soluble Chitosan in Mice. Biomaterials 1999, 20, 175-182. (18) Yu, W.; Lin, J.; Liu, X.; Xie, H.; Zhao, W.; Ma, X., Quantitative Characterization of Membrane Formation Process of Alginate–Chitosan Microcapsules by GPC. J. Membrane Sci. 2010, 346, 296-301. (19) Zheng, H.; Xie, H.; Wu, H.; Wang, F.; Liu, X.; Yu, W.; Ma, X., Investigation of Spherical Hydrogel Surface with Optical Interferometer. Colloid Surf. A-Physicochem. Eng. Asp. 2015, 484, 457-462. (20) Yeh, H. Y.; Lin, J. C., Surface Characterization and in vitro Platelet Compatibility Study of Surface Sulfonated Chitosan Membrane with Amino Group Protection-deprotection Strategy. J. Biomater. Sci. Polym. Ed. 2008, 19, 291-310. (21) Yao, X.; Peng, R.; Ding, J., Cell-material Interactions Revealed via Material Techniques of Surface Patterning. Adv. Mater. 2013, 25, 5257-5286. (22) Chen, Y. H.; Chung, Y. C.; Wang, I. J.; Young, T. H., Control of Cell Attachment on pH-responsive Chitosan Surface by Precise Adjustment of Medium pH. Biomaterials 2012, 33, 1336-1342. (23) Zheng, J.; Xie, H.; Yu, W.; Tan, M.; Gong, F.; Liu, X.; Wang, F.; Lv, G.; Liu, W.; Zheng, G.; Yang, 28


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Y.; Xie, W.; Ma, X., Enhancement of Surface Graft Density of MPEG on Alginate/Chitosan Hydrogel Microcapsules for Protein Repellency. Langmuir 2012, 28, 13261-13273. (24) Van Tam, J. K.; Uto, K.; Ebara, M.; Pagliari, S.; Forte, G.; Aoyagi, T., Mesenchymal Stem Cell Adhesion but not Plasticity is Affected by High Substrate Stiffness. Sci. Technol. Adv. Mater. 2012, 13, 064205. (25) Wang, Q. Z.; Chen, X. G.; Liu, N.; Wang, S. X.; Liu, C. S.; Meng, X. H.; Liu, C. G., Protonation Constants of Chitosan with Different Molecular Weight and Degree of Deacetylation. Carbohyd.Polym. 2006, 65, 194-201.

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Controlling Gel Structure to Modulate Cell Adhesion and Spreading on

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