Multifunctional Collagen and Hyaluronic Acid Multilayer Films on Live

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Multifunctional collagen and hyaluronic acid multilayer films on live mesenchymal stem cells Daheui Choi, Jaeseong Park, Jiwoong Heo, Tong In Oh, Eun Ah Lee, and Jinkee Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00365 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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ACS Applied Materials & Interfaces

Multifunctional Collagen and Hyaluronic Acid Multilayer Films on Live Mesenchymal Stem Cells Daheui Choi1, Jaesung Park2, Jiwoong Heo1, Tong In Oh2,3, EunAh Lee2,* and Jinkee Hong1,* 1

School of Chemical Engineering & Materials Science, College of Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea 2

Impedance Imaging Research Center, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea

3

School of Medicine, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea

Keywords: Mesenchymal stem cell, Collagen, Hyaluronic acid, Multilayer nanofilm, Openness of film, Mutlifunctional nanofilm

Abstract Cell encapsulation has been reported to convey cytoprotective effects and to better maintain cell survival. In contrast to other studies, our report shows that the deposition of two major biomacromolecules, collagen type I (Col) and hyaluronic acid (HA), on mesenchymal stem cells (MSCs) does not entirely block the cell plasma membrane surface. Instead, a considerable amount of the surface remained uncovered ofr only slightly covered, as confirmed by TEM observation and by FACS analysis based on quantitative surface labeling. Despite this structure showing openness and flexibility, the multilayer Col/HA films 1

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significantly increased cell survival in the attachment-deprived culture condition. In terms of stem cell characteristics, the MSCs still showed functional cell activity after film deposition, as evidenced by their colony-forming activity and in vitro osteogenic differentiation. The Col/HA multilayer films could provide cytoprotective effect and induce osteogenic differentiation without deteriorating effect or inhibition of cellular attachment, showing that this technique can be a valuable tool for modulating stem cell activities.

Introduction Mesenchymal stem cell (MSC) transplantation has been used extensively in medicine not only for tissue regeneration purposes (e.g., for stroke, bone damage, tissue defects, and injury)1, but also to deal with immune disorders (e.g., sepsis, graft-versus-host disease, and autoimmune disease). Administration of the MSCs can be done intravenously, intra-arterially, intracerebrally, or intra-articularly, depending on the type of disease to be treated2-5. When MSCs enter the circulation by injection, the cells migrate to the damaged tissue and execute wound

healing

through

their

immunomodulatory

or

trophic

effects,

or

via

transdifferentiation6. MSC therapy is highly advantageous compared with pluripotent stem cells (PSCs) such as embryonic stem cells or induced PSCs, in that they are free from ethical controversy and the risk of teratoma formation1, 7. However, there are still challenges facing the use of MSC therapy for future clinical trials since the limited in vivo cell viability prevents the injected cells from exerting high effectiveness. For intravenous administration of MSCs, cells expanded in vitro are detached from substrata to make a single-cell suspension. After subsequent injection, such anchoragedetached MSCs face shear stress in the circulation without structural support coming from the 2


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extracellular matrix (ECM) or nearby cells. An extended duration of the single-cell suspension state in circulation can lead to cellular anoikis, a type of apoptosis caused by an attachment-deprived state8. In studies conducted to increase cell protection efficiency, some researchers have demonstrated that the cytoprotective effect could be attained via functionalized hydrogel or shell formation. For example, tannic acid and metal ion complexes coated mammalian cell line like HeLa cell showed higher viability under UV-C irradiation9. Another report revealed that a silica hydrogel coating on cells provides cytoprotection against oxidative and mechanical stress10. However, embedding cells within thick shells or macroscopic hydrogels could lead to poor nutrient exchange and reduced cell division9, 11. Therefore, although encapsulated cells are endowed with cytoprotective effects, the purpose of such treatments are for maximizing cell survival during administration rather than for fully exerting cell activity. Besides the reduced cell division, cell migration and membrane protein functions could also be affected depending on stiffness or pore size of surrounding environments12. To solve these limitations of cell encapsulation, we have tried to use a layer-by-layer (LbL) assembly technique to deposit nanofilms on the plasma membrane of live MSCs13. The LbL assembly system involves the absorption of building blocks selected from a broad range of materials, such as polymers, DNA, graphene, nanoparticles, and proteins14-21. Using the LbL assembly, the desired structure and morphology can be precisely tuned by modulating the pH, salt condition, and type of building materials used22-23. Here, we used collagen type I (Col) and hyaluronic acid (HA) as the LbL building blocks on live cellular membranes. Being a fibrous matrix protein, Col is characterized by its high mechanical strength and structural support, and can be found abundantly in skin and bone. HA is a negatively charged polysaccharide that fills in extracellular space and endows resistant power against 3



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compression by absorbing a significant amount of water24. The main driving forces for building the Col/HA multilayer film are electrostatic and Van der Waals interactions at physiological condition25. Prior to this, however, the two matrix molecules bind first to their corresponding receptors integrin and CD44 (with binding affinities to Col and HA, respectively) on the cell membrane26-27. Deposition of the Col/HA LbL assembly onto MSCs supported cell survival under the attachment-deprived state, possibly by activating survival signals, as was confirmed in our previous study28. In this study, we have focused on the deposition characteristics of the Col/HA LbL multilayer film. Contrary to other previous cell encapsulation studies9, 11, 29, our Col/HA LbL films did not block the entire surface of the cell plasma membrane in their multilayered structure, but instead left a vast extent of the plasma membrane in a naked state, as confirmed by transmission electron microscopy (TEM) observation and fluorescence-activated cell sorting (FACS) analysis based on quantitative surface labeling. However, even in its mesh-like form, the Col/HA film supported cell survival under the attachment-deprived state. This characteristic of “openness” helped the cells to keep up with their normal activities, such as attachment, survival in clonogenic density, and osteogenic differentiation, as shown in this study. By LbL assembly, we could stabilize the survival of stem cells in an attachmentdeprived condition without any cytotoxicity effect or functional blocking of cell activities. The Col/HA multilayer film proposed in this study can be a potential approach to potentiate MSC stability for biomedical applications.

Materials and Methods 1. Materials 4


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The solution of collagen type I (100 mg/30 mL conc.) extracted from rat tail was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Hyaluronic acid (Mw 10K) was purchased from PolyScience Inc. (Niles, IL, USA). Phosphate-buffered saline (PBS; 10×, without Mg2+ and Ca2+) was purchased from Gibco (Grand Island, NY, USA). 2. Film preparation on flat substrates The Col/HA LbL film was prepared by a dipping method. The substrate (silicon wafer and Quartz Crystal Microbalance (QCM) electrode) was cleaned by piranha treatment (1:3 = H2O2:H2SO4) for 5 min and then treated with O2 plasma (CUTE; Femto Science, Yongin, Korea) for 2 min to provide the negative charge. The charged substrate was immersed in Col solution for 10 min and then rinsed twice with 1× PBS for 1 min each time. Subsequently, the substrate was dipped into HA solution for 10 min and washed twice as described above. The multilayer film was prepared by sequentially repeating the Col and HA layering cycles. The concentration of each polymer solution was 1 mg/mL, dissolved with 1× Phosphate Buffered Saline (PBS). 3. Film characterization The film thicknesses prepared on silicon wafer were measured with a profilometer (Dektak 150; Veeco, Plainview, NY, USA). For quantitative analysis of the polymer adsorption, QCM (QCM 200; Stanford Research Systems, Inc., Sunnyvale, CA, USA) measurement was carried out. The surface morphologies and roughness of the multilayer films were measured by Atomic Force Microscopy (AFM) (NX-10; Park Systems, Suwon, Korea). The films on the MSCs were confirmed by TEM (JEM1010; JEOL, Tokyo, Japan). For the statistical analysis of film thicknesses, we have measured the thickness at 10 different sites of film area and averaged. Thickness of the nanofilm was statistically analyzed by F test and was found to 5



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be significantly different (P=0.0245).

4. Cell culture Bone marrow stromal cells were isolated from the bone marrow of female New Zealand white rabbits (average weight 3.5 kg), as described previously30. In brief, the femur, tibia, or humerus was collected, and the adjoining cartilages with epiphysis and soft connective tissues were completely removed before breaking the bone to collect the cells in the bone marrow. The bone fragments were vigorously shaken with alpha-minimal essential medium (α-MEM; Gibco) to flush out the bone marrow mononuclear cells. These bone marrow cells were pipetted several times to obtain a single-cell suspension and then passed through a 100µm cell strainer before plating at a density of 1 × 107 cells per 100-mm tissue culture dish with standard medium, consisting of α-MEM supplemented with 20% fetal bovine serum (JR Scientific Inc., Woodland, CA, USA), 1% penicillin/streptomycin (Lonza, Walkersville, MD, USA), 2 mM L-glutamine (Gibco), 10-8 M dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 10-4 M ascorbic acid, for establishing the primary culture. Upon reaching confluency, the adhesive mononuclear bone marrow cells were subcultured at a density of 5 × 105 cells per 100-mm tissue culture dish. All the animal study procedures were performed in accordance with animal study protocols approved by the institutional animal care and use committee of Kyung Hee University (KHUASP-10-002 and KHUASP-10-014). 5. Film preparation on MSCs MSCs were grown on culture dishes until enough cells were obtained. In order to assemble the multilayer film on the cells, the MSCs were detached by trypsin treatment and collected via centrifugation (1500 rpm, 5 min). Col solution was added to the MSC pellet, and mild 6


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pipetting (50–60 times) was carried out for 30 s. After Col deposition, the cells were collected by centrifugation and the supernatant was removed. For removal of unbound and remained polymer, growth medium was added and the mixture pipetted for 10 s. HA adsorption was carried out in the same way as done for the Col layer. The Col/HA deposition cycle was repeated 2, 3, and 4 times (4, 6, and 8 layers). For preparation of Col solution, the Col was dissolved in Dulbecco’s modified Eagle’s Medium (DMEM) and sterilized reconstitution buffer (2.2 g NaHCO3 in 100 mL of 0.05 N NaOH and 200 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) for adjusting the pH. HA was dissolved in the cell growth medium. The polymer concentrations were set at 1 mg/mL. The entire film preparation process, including mild pipetting and centrifugation, was carried out at 4°C to maintain constant cell conditions and thus avoid a reduction of cell viability, as well as to prevent collagen self-assembly during the deposition process. 6. Biotinylation EZ-Link Sulfo-N-hydroxysuccinimide (NHS)-biotin and fluorescein isothiocyanate (FITC)labeled streptavidin were purchased from ThermoFisher Scientific (Waltham, MA, USA). Biotinylation on MSCs was carried out according to the manufacturer’s manual. In brief, MSCs were harvested by trypsin treatment and collected into 15 mL conical tubes (105 cells each). The single-cell suspension was washed three times with ice-cold PBS before 2 mM biotin in PBS was added and mixed well by gentle pipetting. The mixtures were kept at 4°C for 1 h. After the biotinylation reaction, unbound biotin molecules were washed out twice with 100 mM of glycine solution. The biotinylated cells were subjected to Col/HA multilayer film deposition, as indicated in the Results section (4 and 8 layers), using the same method described above. After the deposition, FITC-labeled streptavidin (dissolved in 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid in PBS) was added to the cell 7



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suspension, and the mixture was kept at 4°C for 30 min to allow the streptavidin to interact with biotin before washing three times with PBS. The intensity of the FITC-labeled streptavidin, which is reduced by the extent of the masking effect caused by the Col/HA multilayer, was measured by FACS (FACSort Flow Cytometer; BD Biosciences, Franklin Lakes, NJ, USA). 7. Test of cell viability and cytoprotective effect of the nanofilms by MTT assay Cell viability during the film deposition process and in the attachment-deprived state was confirmed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To test the cell viability after the film deposition process, MSCs with or without Col/HA films were collected into a 15 mL conical tube (1 × 105 cells; n = 3) and further incubated with 10 mg/mL MTT in medium for 2 h. The relative amounts of viable cells were compared by reading the optical density at 540 nm wavelength. To test the cytoprotective effect of the nanofilm in the attachment-deprived state, MSCs with or without Col/HA films were seeded at a density of 5 × 104 cells per poly 2-hydroxyethyl methacrylate (poly-HEMA)-coated 6well culture plate in order not to allow cell thickness during culture31, 32. At each time point, (1, 2, and 3 days), the cultured MSCs were divided into two portions. Half of the cells (n = 3) were seeded on a normal 6-well plate, fixed with 4% paraformaldehyde (PFA), and stained with methyl violet after 1 day of incubation in order to count viable attached cells by image analysis using ImageJ software. The rest of the cells (n = 3) were subjected to MTT assay to measure viable cells. Values obtained with the MTT assay for each day were normalized to the proportion of viable attached cells at each day obtained from image analysis. 8. Cell attachment test Col/HA multilayer films were deposited on MSCs as indicated in the Results section (4, 6, 8


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and 8 layers) and seeded into 24-well plates at a density of 3 × 104 cells per well (n = 3). At a certain period of time (10 min, 30 min, 1 h, and 16 h), the cells were fixed with 4% PFA for 1 h and then stained with methyl violet for visualization. The area occupied with attached cells was calculated using Image J software. 9. Colony-forming assay Single-cell suspensions of MSCs were coated with 4 and 8 layers of Col/HA film. MSCs with or without Col/HA film were seeded at two different cell densities of 1 × 103 and 3 × 103 cells per T-25 flask (Corning, Corning, NY, USA) for each coating condition (n = 5) and cultured at 37°C in a CO2 incubator for 10 days without medium change. Colonies formed by MSCs were visualized by methyl violet staining before counting. 10. Evaluation of osteogenic differentiation The osteogenic differentiation potential of the MSCs was confirmed by culturing the cells in differentiation induction medium as described previously33. MSCs with or without nanofilm (4 and 8 layers) were seeded at a density of 2 × 104 cells per well in 6-well plates (n = 3). Osteogenic differentiation of the cells was induced with osteogenic differentiation induction medium, consisting of α-MEM supplemented with antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), 20% fetal bovine serum, 10-8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate. The osteogenic medium was changed three times a week, and the culture was maintained for 3 and 5 weeks. To visualize calcium-rich granule accumulation, cells were fixed with 4% PFA for 2 h and then stained with alizarin red S solution. The stained area was numerically calculated using Image J software. 11. Statistical analysis

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Numerical data obtained by FACS analysis, MTT assay, and image analysis was represented in a graph with mean value ± error bar. Differences between two groups were compared by two-tailed, paired t-test. Differences between several groups were analyzed by 2-way ANOVA. P values greater than 0.05 was marked as “NS” indicating non-significance. All of the experiments were repeated at least three times.

Results and Discussion Figure 1 depicts the formation of Col/HA multilayer films as confirmed by the adsorption amount, film thickness, and surface morphology. For a model film, we had carried out preparation of the Col/HA films on flat substrates (QCM electrode and silicon wafer) and provided strong negative charges on the surfaces through oxygen plasma treatment. The main driving force in building the Col/HA film layers is electrostatic interaction, due to the pI of Col (around 8.2–8.4)34 and the pKa of HA (= 2.9). At the film preparation condition of pH 7.4, Col has a slightly positive charge and HA has a strong negative charge. The charges of Col and HA were screened using salts and charged molecules in PBS or standard medium that we used as solvents. Therefore, Van der Waals interaction between Col and HA also participates to form the multilayers. At high salt concentration in the solvent, Col and HA could lose their charge density, resulting in Van der Waals interaction being the main contributor to multilayer film formation25. Figure 1A depicts the quantitative analysis of the adsorbed amounts of Col and HA according to the number of layers. To examine the effect of reaction temperature on the deposition profile, we compared the amount of film deposited at 4°C with that at room temperature. At the first several layers, the attached amount was unstable owing to the lower interaction between the QCM electrode and the biomolecules; however, the steady decrease 10


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of frequency was overcome after stabilization of the bottom layers. Furthermore, a higher amount of material adsorption onto the electrode was confirmed at the higher temperature35. This phenomenon was due to the increased amount of self-assembly exhibited by Col, the swelling effect of the films, and the trapping of materials within the multilayer at the higher temperature. The thickness of 10 bilayers was 46.16 nm at room temperature and 40.11 nm at 4°C measured by profilometer, indicating that a higher temperature increases the polymer conformational dynamics (Figure 1B). At 4°C, there was an even deposition of the matrix molecules, and the surface roughness of the film was significantly lower than that deposited at room temperature. In case of previous studies on preparation of Col/HA multilayer LbL film36-37, different from in this study, the films were constructed at lower pH condition to obtain positively charged Col molecules. In addition, the main driving force to make Col/HA film is electrostatic interaction, indicating that thicker film was prepared which thickness was around 300 nm for 5 bilayers. For the AFM images, Col formed fibrils during deposition at room temperature (size and length of 500 nm and >5 µm, respectively), creating fibril segments within the films38. Although Col can self-assemble at neutral pH, little or no Col fibril or fiber formation occurred at the lower temperature (Figure 1C). The fibrillogenesis of Col is highly affected by the salt condition, pH, and temperature39. A higher salt concentration at neutral pH induces the formation of Col fibers, whereas a low temperature prevents hydrogel formation. In the previous study, Col/HA film topography was observed that Col fibers were tightly entangled into network even though the films were prepared at lower pH condition (pH 4)36. In this study, Col/HA multilayer films were prepared at lower temperature and the AFM image of Col/HA multilayer films shows no entangled Col fibers were detected, indicating that film deposition temperature also impacts on the entanglement of Col molecules. 11



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Figure 2A depicts the Col/HA film formation based on LbL assembly for deposition onto MSCs. Aside from electrostatic and Van der Waals interactions, additional interactions between the Col/HA and cell membrane proteins are responsible for forming the LbL films. The α1β1 and α2β1 subunits of integrin, a transmembrane protein that is the focal contact between cells and the ECM environment, can recognize and bind the tripeptide RGD (ArgGly-Asp) motif that exists in matrix molecules such as Col and fibronectin27, 40. In addition, HA is a well-known natural polymer that bind to cells through its membrane protein receptor, CD4426. The simplified procedure for preparation of LbL films for deposition onto MSCs is shown in Figure 2B. Because the cell plasma membranes have a negative charge due to the phosphate group facing outward in the lipid bilayer, the Col/HA film can be deposited directly onto the cells without any pretreatment. Repetitive adsorption of the Col and HA molecules onto the MSC membrane is carried out by a centrifugation method (Figure 2B). During deposition, the temperature was kept at 4°C in order to halt cell metabolism, thereby maintaining cell viability and preventing the excessive Col gel formation that occurs at higher temperature (as indicated in Figure 1C). In our previous study28, it was suggested that the deposited LbL assembly does not entirely cover the cell plasma membrane. Therefore, the extent of plasma membrane coverage by the LbL assembly was determined by quantitative detection of biotin and avidin interaction and by TEM analysis. Biotin is a small molecular vitamin (Mw 244.31 g/mol) that has a strong binding affinity for avidin protein. In this study, we used sulfo-NHS-biotin, which can react with primary amine groups to form a stable covalent bond. Therefore, cellular membrane proteins containing several amine groups were labeled with biotin, and these labeled MSCs were then subjected to Col/HA deposition by LbL assembly (4 and 8 layers), following which FITC-labeled streptavidin was added to bind with the biotin. Because streptavidin has a high 12


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molecular mass (around 66–69 kDa), we considered that it cannot penetrate the films and instead binds only with exposed biotin molecules that have not or only slightly been covered with the film layer. To quantify the percentage of biotin molecules exposed on the cell surface after film deposition, we measured the fluorescence intensity of FITC-labeled streptavidin conjugated with biotin on the MSCs. In Figure 2D, the negative control ((-) control) indicates non-biotin-treated cells that were treated with FITC-labeled streptavidin only, with a normalized fluorescence intensity of 0.0018 in comparison with the positive control ((+) control). The biotinlyation intensity showed a slightly decreasing tendency depending on the number of layers, indicating an increase in the LbL coverage of biotin molecules according to the number of layers. However, there were still areas of exposed plasma membrane surface after the film preparation (Figure 2 C, D). The Col/HA film-coated MSCs were subjected to TEM for direct observation of the film structure on the plasma membrane (Figure 2E and Figure S2). Films were clearly observed on the plasma membrane, and their thicknesses increased according to the number of layers; that are, 21.24 ± 6.11 nm for 4 layers and 29.74 ± 13.71 nm for 8 layers. On the other hand, a sharp and smooth boundary was seen on the clearly noticeable plasma membrane surface of the bare MSCs (Control). The film thickness difference together with the profilometry data inferred that more Col and HA molecules could be deposited on the cell membrane by both additional biological interactions and electrostatic interaction. However, a considerable portion of the plasma membrane surface was still naked. In fact, the cell plasma membrane is composed of various types of molecules; namely, lipid bilayer and proteins in glycosylated and non-glycosylated forms. The cell plasma membrane surface is completely different from the flat substrate on which the film preparation was performed (Figure 1); it contains many membrane ruffles in order to exert various cell activities. Therefore, it is not surprising that the films did not show an even distribution and 13



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regular thickness on the plasma membrane surface (black and red arrows in Figure 2E). This “openness” of the Col/HA LbL film can confer flexibility to the structure, allowing it to conform to the cell morphology and permitting ligand-receptor interaction and channel activity for the cells to sense the microenvironment. TEM images showing MSCs in their entirety after Col/HA LbL film deposition revealed that the film coating had not eliminated the membrane ruffles (Figure S1). Even though the building blocks used for LbL assembly (i.e., Col and HA) are cell-friendly matrix molecules, the accumulation of such molecules on the plasma membrane might cause undesirable effects to the cells. Therefore, the cytotoxicity of the building blocks and the film preparation method was examined by MTT assay immediately after film preparation. Figure 3A shows that the survival fraction of MSCs was not significantly changed after the film deposition. Being an adherent stem cell, the viability of the MSC can be maintained by adhesion culture on a plastic culture vessel. When MSCs are maintained in a non-attached condition, the stress resulting from the attachment-deprived state activates the apoptotic signaling pathway and leads to cell death, a process called anoikis. MSCs with or without Col/HA LbL film deposition were subjected to the attachment-deprived state by culturing on poly-HEMA-coated dishes, which do not allow cell attachment31. Whereas the viable cells with metabolic activity in control MSCs kept decreasing, that of the film-coated MSCs remained significantly high at all the time points examined (Figure 3B). Being natural matrix molecules, the Col/HA multilayer films could exert a cytoprotective effect in the attachmentdeprived state by tentatively mimicking cell-ECM interaction and creating focal adhesion stimuli, thereby prolonging the viability of the MSCs41. In our previous study28, multilayer deposition of poly-L-lysine/HA also showed a cytoprotective effect, as evidenced by the activation of Akt, a key component in the cell survival signal transduction pathway. MSCs 14


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with or without LbL film deposition still showed adhesive property when seeded on plastic culture vessels, as shown in Figure 4A. The film-coated MSCs showed slower rate of attachment compared with bare MSCs at the early time points although there were no statistical significance. However, after overnight incubation, the film-coated MSCs showed increased adhesion in a dose-dependent manner according to the number of LbL layers, and there was no significant difference in adhesion rate between the MSCs with 8 film layers and the control group. For verification of the “stemness” of MSCs after film deposition, their clonogenicity and differentiation potentials were compared with those of bare MSCs42-43. As confirmed by colony-forming unit fibroblast assay, there was no significant difference in clonogenicity between the bare and 4- and 8-layered film-coated MSCs (Figure 5 and Figure S3). The osteogenic potential of the MSCs was examined by in vitro incubation in osteogenesis induction medium for 3 and 5 weeks. Calcium-rich granule formation is the first step of mineralized matrix formation, which can be visualized by alizarin red S staining and analyzed using ImageJ software. Based on the average values, there was a tendency toward increased calcium-rich granule accumulation in the film-coated MSCs compared with the bare MSCs, being 1.3 and 1.5 times higher at 3 and 5 weeks, respectively (Figure 6A). However, statistical analysis resulted in P values greater than 0.05 indicating non significance between experimental groups. Considering the fact that Col is the most abundant ECM molecule present in bone matrix and its polymeric characteristics direct the mineralization process during bone mineralized matrix formation44-45, it is probable that its presence on the plasma membrane surface upregulates the pace of osteogenesis. The Col-coated surface of culture dishes were reported to promote MSC proliferation and osteogenesis through the Erk and Akt signaling pathways46, and the hydrogels could control the differentiation fate of MSCs depending on the specific functional groups in the hydrogels47. These previous reports 15



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and our results support the idea that Col/HA LbL film deposition onto the plasma membrane surface can enhance the differentiation tendency of MSCs.

Conclusion In this study, we have prepared Col/HA multilayer films on MSCs by using the LbL assembly technique. The driving force for building the Col and HA layers is mainly electrostatic and Van der Waals interactions, and the biomacromolecules were successfully incorporated on the flat substrates. The stem cell membrane contains receptors for extracellular molecules, such as integrin and CD44 protein, that can interact and bind with Col and HA deposited on the plasma membrane surface. The Col/HA multilayer film was deposited on the MSC membrane induced cellular metabolic activity and osteogenic differentiation without any cytotoxicity. The open structure and flexible characteristics of the multilayer film were confirmed by quantitative binding analysis using biotin-avidin binding, cell adhesion testing, and TEM observation. The Col/HA LbL film increased the survival of the MSCs under anoikisinducing conditions. Moreover, film deposition upregulated osteogenic differentiation of the MSCs. These results show that the LbL film assembly technique on stem cells can be a valuable tool for modulating the survival and differentiation activity of cell therapeutic agents.

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Figures

Figure 1. (A) Quantitative analysis of adsorbed Col and HA layers measured by QCM at different deposition temperatures (black line: room temperature; gray line: 4°C). (B) Thickness and roughness of 10 bilayer films analyzed by profilometry and AFM, respectively. (C) AFM images of (Col/HA)10 films at different deposition temperatures.

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Figure 2. (A) Schematic illustration of the Col/HA LbL film on MSCs. Col and HA can bind with integrin and CD44, respectively, forming a stable multilayer on the MSC. (B) Schematic diagram of the film preparation method. (C) Number of FITC-streptavidin-conjugated MSCs counted for (+) control and film-covered MSCs, according to FACS analysis. (D) Normalized biotinylated index of MSCs after 4- and 8-layer deposition based on the (+) control index. (-) control and (+) control indicate FITC-streptavidin-conjugated MSCs only (nonfluorescent) and FITC-streptavidin coupled with biotin on MSCs (fully fluorescent), respectively. (E) TEM images of the MSC membrane with and without Col/HA films. Red and black arrows indicate parts of the plasma membrane covered and non-covered with Col/HA films, respectively.

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Figure 3. (A) Comparison of proportion of viable cells with metabolic activity immediately after film preparation, as determined by MTT assay. (B) Normalized proportion of viable cells with metabolic activity cultured for 3 days on HEMA-coated dishes. Statistical significance (***p < 0.05) determined by t-test.

Figure 4. (A) Cell attachment capacity depending on the number of film layers. Cells were fixed and then stained with methyl violet at indicated time points. (B) Areas occupied by MSCs attached onto the plastic surface of culture plates were calculated using ImageJ software.

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Figure 5. Number of colonies formed by bare and film-coated MSCs.

Figure 6. Osteogenic differentiation assay. (A) Alizarin-red-stained areas at 3 and 5 weeks, calculated using ImageJ software. (B) Representative images of the alizarin red staining of MSCs.

Author information Corresponding authors *E-mail: [email protected], [email protected] Notes 20


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The authors declare no competing financial interest.

Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Korean Government (Grant 2012M3A9C6050104 and 2016M3A9C6917405). Additionally, this research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI14C-3266, HI15C-1653). This research was also supported by the Basic Science Research Program (2015R1D1A1A01059702) and Mid-career Research Program (NRF-2015R1A2A2A04006172) through the National Research Foundation of Korea funded by MEST, and by a grant from the Korea Health Technology R&D Project (HI16C1010) through the Korea Health Industry Development Institute (KHIDI) funded by the Korea Ministry of Health & Welfare.

Supporting Information TEM images and Colony forming units-fibroblast (CFU-F) results are displayed on Supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Multifunctional Collagen and Hyaluronic Acid Multilayer Films on Live

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