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Development of Flexible Cell-Loaded Ultrathin Ribbons for Minimally Invasive Delivery of Skeletal Muscle Cells Sahar Salehi, Serge Ostrovidov, Majid Ebrahimi, Ramin Banan Sadeghian, Xiaobin Liang, Ken Nakajima, Hojae Bae, Toshinori Fujie, and Ali Khademhosseini ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00696 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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ACS Biomaterials Science & Engineering

Development of Flexible Cell-Loaded Ultrathin Ribbons for Minimally Invasive Delivery of Skeletal Muscle Cells Sahar Salehi§, Serge Ostrovidov§, Majid Ebrahimi§, Ramin Banan Sadeghian§, Xiaobin Liang$, Ken Nakajima$, Hojae Bae&, Toshinori FujieΦ,@, Ali Khademhosseini*,§,&,#,ß,†,‡ §WPI-Advanced

Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan.

$School

of Materials and Chemical Technology, Tokyo Institute of Technology, Tokyo 1528550, Japan. &Department

of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Korea.

ΦWaseda @Japan

Institute for Advanced Study, Waseda University, Shinjuku, Tokyo 162-8480, Japan.

Science and Technology Agency, PRESTO, Kawaguchi, Saitama 332-0012, Japan.

#Biomaterials Innovation Research Center, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02139, USA. ßHarvard-MIT

Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

†Wyss

Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115,

USA. ‡Department

of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia

* Corresponding author, Email: [email protected]

ABSTRACT 1

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Cell transplantation therapy provides a potential solution for treating skeletal muscle disorders, but cell survival after transplantation is poor. This limitation could be addressed by grafting donor cells onto biomaterials to protect them against harsh environments and processing, consequently improving cell viability in situ. Thus, we present here the fabrication of poly (lactic-co-glycolic acid) (PLGA) ultrathin ribbons with “canal-like” structures using a microfabrication technique to generate ribbons of aligned murine skeletal myoblasts (C2C12). We found that the ribbons functionalized with a solution of 3,4-dihydroxy-L-phenylalanine (DOPA) and then coated with poly-L-lysine (PLL) and fibronectin (FN) improve cell attachment and support the growth of C2C12. The viability of cells on the ribbons is evaluated following the syringe-handling steps of injection with different needle sizes. C2C12 cells readily adhere to the ribbon surface, proliferate over time, align (over 74%), maintain high viability (over 80%), and differentiate to myotubes longer than 400 μm. DNA content quantification carried out before and after injection and myogenesis evaluation confirm that cell-loaded ribbons can safely retain cells with high functionality after injection and are suitable for minimally invasive cell transplantation.

KEYWORDS: immobilization, injectable materials, microfabrication, skeletal muscle cells, tissue engineering, ultrathin ribbons. 1. INTRODUCTION Skeletal muscles have a high capacity of self-repair but are not able to regenerate when a significant loss of tissue occurs, resulting in severe loss of function.1 In diseased muscles, such as those degraded by muscular dystrophy, repeated cycles of degeneration and regeneration cannot be sustained by the muscle tissue, which gradually results in the formation of adipose and fibrotic tissue instead of muscle tissue.2,3 The treatment of skeletal muscle tissues by the delivery of cells or drugs remains a challenge, as regenerative strategies using intramuscular injection of cells provide poor therapeutic outcomes.4 To improve the outcome, localized 2


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delivery of cultured cells on soft carriers may provide an alternative to increase the efficiency of cell delivery at the injury site. 5-8 Much effort has been made to deliver cells locally via engineered and injectable polymeric carriers.8,9 Such injectable carriers avoid invasive surgery, whereas intramuscular injection via a syringe and a needle decreases the cost of treatment, the risk of infection and scar formation, and patient discomfort.10,11 Various polymeric scaffolds composed of synthetic or natural materials that solidify or stiffen in vivo have been used as injectable scaffolds for the regeneration of different tissues such as cartilage,12 bone,11 nerve,13 and cardiac14 tissue and even as angiogenic growth factor delivery carriers.15 However, when scaffolds are chemically crosslinked in situ, the crosslinking reaction may adversely affect the cells, the scaffolds and potentially the encapsulated biomolecules.11,16 Other scaffold types, such as microparticles, microspheres, microbeads and microgels, face challenges involving size, thickness, or the necessary flexibility to be aspirated and injected through a conventional syringe needle, or otherwise do not allow the cells to form an organized cellular structure.17-21 To overcome these issues, the development of biocompatible cell micro/nanocarriers such as micropatterned polymeric ultrathin films (referred to as “nanosheets”) for effective injection has great significance.9,22 Such micro/nano cell carriers not only support local cell attachment and proliferation before injection but also localize the cells at the target site of injection and can enhance cell retention and viability at the injection sites compared to cells transplanted without a biomaterial substrate, thereby improving the repair of damaged tissue.20 Thus, Yoon et al.23 fabricated miniaturized microcylinders using electrohydrodynamic cojetting and automated microsectioning. They coated one side of the cylinders with poly (ethylene glycol) to prevent cell attachment and the other side with FN to promote the attachment of cells. These microcylinders were used as cardiomyocytes-driven actuators. In another study, Han et al.24 fabricated microribbon structure fibers using wet spinning. These microribbons had irregular shapes because the diameters of the fibers were not adjustable. Moreover, changing the rate of spinning and the temperature also changed the shape and the thickness of the microribbons. Patil et al.25 and Szymanski 3



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et al.26 also fabricated freestanding alginate microfibers that were micropatterned by extracellular matrix (ECM) protein such as FN to enhance the adhesion of C2C12 cells. Various approaches such as physical adsorption and covalent chemical conjugation have been used to modify the performance of materials and to regulate cell adhesion and function by immobilizing biomolecules such as peptides or proteins via covalent bonds onto surfaces.27 However, most of these methods require multiple treatments or have low surface conjugation efficiency. One of the immobilization approaches, inspired by a mussel adhesive protein, is surface modification by DOPA precursor residues (a critical adhesive motif) which promotes cell attachment and proliferation and enhances the immobilization of bioactive molecules such as growth factors and ECM proteins. These bioactive molecules will covalently conjugate to the oxidized catechol group of DOPA coating layer via their amine or thiol groups.28,29 This molecular conjugation allows a better immobilization of the bioactive molecules than a simple adsorption of them on the material surface and therefore a DOPA coating followed by the immobilization of these molecules is more efficient to promote the cell adhesion and proliferation.28,30 In this study, we delivered C2C12 cells adhering to microfabricated PLGA carriers via a conventional syringe needle. We previously developed biodegradable PLGA nanosheets composed of nanoribbons.22 We showed that these nanoribbon sheets, with their high aspect ratio and flexibility, are efficient substrates for cell culture, facilitating cell proliferation and also regulation of cell orientation, which is beneficial in skeletal muscle cell therapy and tissue engineering.22 Furthermore, we have also shown that polymeric nanosheets can be used to transplant cells in narrow tissue space (subretinal space) by injection9. Herein, we extended our previous studies by fabrication of individual freestanding ultrathin ribbons that can be used to deliver cells by injection. These ribbons exhibit a high aspect ratio and a unique morphology of “canallike” structures because of the presence of edges, which are useful for retaining cells on the ribbons. The canal-like PLGA ribbons were functionalized with DOPA to immobilize PLL and FN in the goal to improve cell adhesion and proliferation. Moreover, topographical cues of the ribbons induce cytoskeletal alignment and as a result of their high flexibility, these ribbons can 4


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mechanically support the cells to withstand the pressure experienced during injection. To our knowledge, this is the first report of the development of flexible carriers loaded with cells that allow for minimally invasive delivery of an elongated cellular organization.

2. EXPERIMENTAL SECTION 2.1. Fabrication and functionalization of freestanding and dispersed PLGA ultrathin ribbons Freestanding PLGA (75/25; MW: 66,000-107,000, Sigma, USA) ribbon sheets were produced as previously described22 by a combination of micropatterning and spin-coating techniques. First, PLGA ribbon sheets were prepared by spin-coating (1000 rpm, 10 s; 2000 rpm, 10 s; and 4000 rpm, 40 s) of 5, 7, and 10 mg mL-1 PLGA solutions in dichloromethane (Wako, Japan) on a micro-grooved polydimethylsiloxane replica (PDMS, 1 × 1 cm2) with a width and ridge size of 50 μm, followed by baking on a hot plate (80 °C, 90 s). Next, 10 wt.% poly(vinyl alcohol) solution in water (PVA, 300 μL, MW: 13,000-23,000, Kanto Chemical Inc., Japan) was cast on the PLGA sheet as a supporting/sacrificial layer. Then, the residual water in the PVA layer was removed by drying in a desiccator overnight. The PLGA/PVA bilayer was then carefully removed from the PDMS replica with tweezers. Dispersed PLGA ultrathin ribbons were obtained after cutting the flat edges of the PLGA/PVA bilayer sheet and depositing it in water to dissolve the PVA layer. To obtain shorter pieces, the length of the ribbons was also cut to 1 mm length with scissors before PVA dissolution in water. After 4 days (4 d) and every 2 d subsequently, the water was carefully replaced to avoid aspirating the soaked ribbons and to complete the removal of the residual PVA. After dissolving PVA, we removed the water gently by syringe needle and dried the dish containing ribbons in desiccator overnight. In the next step, freestanding PLGA ribbons were coated with a solution of 3,4-dihydroxy-Lphenylalanine (DOPA, 2 mg mL-1, Sigma, China) in Tris buffer solution (10 mM, pH 8.5, Sigma-

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Aldrich, USA) at room temperature for 4 h. The ribbons were washed once in Dulbecco’s phosphate-buffered saline (DPBS, pH = 7.4, Gibco, UK) and freeze-dried for 4 h. To obtain high cell attachment, the DOPA-mediated ribbons were coated with PLL (20 μg mL-1, PLL Hydrobromide, Sigma, USA) for 1 h. After one wash with DPBS, the ribbons were incubated for another hour with FN (10 μg mL-1, Sigma, USA). After washing with DPBS, we transferred the ribbons to nonadherent culture dishes (HydroCell, CellSeed Inc., Japan) by simply pouring the whole suspension and following aspiration of DPBS using a syringe needle. Ribbons at last were sterilized with UV light for 20 min before cell seeding. For better visualization of the ribbons during injection experiments, we mixed 0.2 wt.% Nile red (Sigma, Germany) with PLGA solution in dichloromethane. To show the efficiency of PLL immobilization on PLGA ribbons surface with DOPA coating, we coated the DOPA-mediated ribbons with fluorescent-labeled PLL, fluorescein isothiocyanate-PLL (FITC-PLL, Sigma, USA), and took pictures of their surface using fluorescence microscopy. FITCPLL powder was diluted at 100 μg mL-1 in phosphate-buffered saline (PBS, 10X, 0.1 M, pH 7.4) and filtered through 0.22-μm filter to remove debris from the fluid. After incubation for 2 h at 37 °C, the FITC-PLL solution was removed and samples were rinsed with deionized water. Fluorescent images were acquired using a fluorescence microscope (Zeiss, Germany). Ribbons soaked in PBS were used as a negative control. 2.2. Morphology, chemical properties and Young’s modulus of the ribbons The surface morphology of the dispersed PLGA ribbons was observed by field emission scanning electron microscopy (FE-SEM, JEOL JIB-4600F, Japan). The samples were first coated with a platinum layer (20 mA, 40 s, 5 nm thickness) using a JEOL ion-sputtering coater SM7826004 (JEOL Ltd., Japan) and then images were taken with the FE-SEM operating at 15 kV. Elemental composition of PLGA film spin-coated on round glass coverslips was also determined using energy dispersive X-ray spectroscopy (EDS, Oxford Instruments, UK) operated at 5 kV and scanning X-ray photoelectron spectroscopy (XPS, PHI 5000 VersaProbe II, ULVAC PHI Inc., 6


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Japan) to measure the atomic percentages of C, O, and N after functionalization with DOPA, PLL and FN. Infrared reflection spectra were obtained using an fourier transform infrared spectroscopy (FTIR, JASCO FTIR-6600, Japan). The spectra were recorded with 32 scans per sample, ranging from 400 to 4000 cm-1. The thickness of the dispersed PLGA ribbons was analyzed by a surface profiler (Dektak150, Veeco Instruments Inc., USA). Analysis was made on 5 different ribbons from each concentration and we repeated the measurements 2 times. The Young’s modulus of the dispersed ribbons from different concentrations of PLGA (5, 7, and 10 mg mL-1) was characterized by nanomechanical mapping. Ribbons were collected on round glass coverslips (12 mm, Matsunami glass IND., Japan) from water and were characterized after drying for 24 h. The nanomechanical mapping was recorded in fast force-volume (FV) mode, in which force curves were collected over selected surface areas at a resolution of 128 × 128 pixels. All of the measurements were taken on a Bruker MultiMode AFM with a NanoScope V controller at room temperature. OMCL-TR800PSA-1 cantilevers were purchased from Olympus (Japan). The nominal spring constant of the Si3N4 cantilevers was 0.76 N m-1 and the actual spring constant was measured by the thermal method. The obtained force-distance curves were analyzed using a previously developed procedure.31 By this method, the Young’s moduli can be calculated and a modulus map together with a modulus distribution histogram can be constructed. 2.3. Cell cultures Ribbons were sterilized by UV light for 20 min. C2C12 cells (1 × 105 cells mL-1, 50 μL) (less than 6 passages from the American Type Culture Collection) were then seeded on 5000 ribbons mL-1 and incubated for 1 h under a 5% CO2 atmosphere at 37 °C for cell attachment before the culture dish was filled with growth medium. In cell culture experiments before injection, in accordance with Yoon et al. study,23 we used longer ribbons for better attachment of ribbons to the nonadherent culture dishes and easy handling of them. The growth medium was composed of Dulbecco’s modified eagle’s medium supplemented with GlutaMAX (DMEM, Gibco, USA), 10% 7



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(v/v) fetal bovine serum (Biowest, Chile), 20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (Sigma, Germany), and 1% (v/v) penicillin streptomycin (Sigma, USA). 2.4. Cell viability measurements before and after injection After 2 d of culture, the cell viability was determined by a Live/Dead assay (Invitrogen, USA) according to the manufacturer instructions. Cells on ribbons produced from different concentrations of PLGA (5, 7, and 10 mg mL-1) were washed with DPBS (3 times) and incubated for 20 min in 2 mL of DPBS containing 0.5 μl mL-1 calcein AM and 2 μl mL-1 ethidium homodimer 1. After staining, the cells were washed with DPBS (3 times) and fluorescent images were acquired using a fluorescence microscope. The images were analyzed using ImageJ software (v.1.46r, NIH) and the live cell percentage was calculated by dividing the number of viable cells by the total number of counted cells. One day after cell seeding on ribbons produced from 7 mg mL-1 PLGA, we collected the cellloaded ribbons along with culture medium with a syringe and injected them into an empty nonadherent culture dish using different gauges of needles (23G × 1 inch (NN-2325R, 400 μm inner diameter), 25G × 1 inch (NN-2525R, 320 μm inner diameter), and 27G× 3/4 inch (NN2719s, 220 μm inner diameter), Terumo Co., Japan) connected to a 1 mL syringe (SS-01T, Terumo Co., Japan). One day after injection we measured the cell viability as described above. Ribbon injection was also tested using an intravenous catheter (IV) needle (24G × 3/4 inch: SRFS2419, 470 μm inner diameter and 700 μm outer diameter, Terumo Co., Japan). 2.5. Fluorescent staining and myoblast alignment before injection After 2 d of culture, cells adhered on ribbons fabricated from different concentrations of PLGA (5, 7, and 10 mg mL-1) were washed with DPBS and fixed in a 3.7% (v/v) formaldehyde (SigmaAldrich, USA) solution in DPBS for 15 min. The cells were then permeabilized in 0.1% (v/v) Triton X-100 (Sigma-Aldrich, USA) solution in DPBS for 5 min and stained with Alexa-Fluor 488conjugated phalloidin (Invitrogen, USA) and 4’,6-diamidino-2-phenylindole (DAPI, Sigma8


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Aldrich, USA) to visualize F-actin and cell nuclei, respectively. The F-actin (green) and cell nuclei (blue) were imaged using fluorescence microscopy. The nuclear alignment on ribbons was quantitatively analyzed based on the alignment angle, meaning the angle between the long axis of a nucleus (as elliptical) and the wall of ribbon, and was measured using the Carl Zeiss imaging system (Axiovision software (version 4.8.1, Germany)). Cells with alignment angles less than 10° were considered aligned. Cell elongation was also quantified based on the ratio of the length of the nuclei to its width.32 To quantify the aligned cell nuclei and nuclear aspect ratio on ribbons fabricated form different concentrations, we captured 5 images of DAPI-stained samples per each concentrations of PLGA from 3 independent cultures. 2.6. Morphology of cells by FE-SEM The cell morphology on cell-loaded ribbons after 2 d of culture was analyzed by FE-SEM. The samples in cell culture dishes were fixed with 3.7% (v/v) formaldehyde solution in DPBS for 15 min at room temperature and washed 2 times with DPBS. Briefly, the samples were dehydrated in graded concentrations of ethanol (50, 70, 80, 90, 100 and 100% (v/v) ethanol) for 5 min on ice and then with a mixture (1:1 (v/v)) of ethanol and t-butyl alcohol (Kanto Chemical Inc., Japan) for 5 min at room temperature followed by treatment twice with 100% t-butyl alcohol for 5 min. The cell-loaded ribbons were freeze-dried overnight and imaged using the FE-SEM following the procedure described before. 2.7. DNA content quantification C2C12 cells were cultured on ribbons and then divided into 2 groups. The first group was incubated for 2 d in an incubator and the second group was injected using a 27G needle in a culture dish 1 d after cell seeding and then incubated for another day in the incubator. After 2 d, both groups were rinsed with DPBS and the total DNA in each group was isolated following TRIzol reagent protocol (Life Technologies, Ambion, USA). DNA concentrations were quantified using a spectrophotometer (NanoDrop Lite, Thermo Fisher Scientific Inc., USA). Three samples

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from each condition were considered per experiment, and experiments were repeated two times. 2.8. Myoblast differentiation before and after injection To induce the formation of myotubes on the ribbons, the growth culture medium was replaced after 2 d of culture by a differentiation medium composed of DMEM supplemented with GlutaMAX, 2% horse serum (Gibco, New Zealand), 20 mM HEPES, and 1% penicillin/streptomycin. Seven days after inducing differentiation, the cell-loaded ribbons (7 mg mL-1 PLGA) were fixed in a 3.7% (v/v) formaldehyde solution in DPBS for 15 min at room temperature washed 3 times with DPBS (10 min each) and then permeabilized with a 0.3% Triton X-100 solution in DPBS for 5 min followed by blocking with 5% bovine serum albumin (Sigma, USA) solution in DPBS for 30 min at room temperature. The cells were then incubated with primary antibody mouse monoclonal anti-fast skeletal myosin MY32 (1:1000 dilution; Abcam, UK) overnight at 4 °C. The cells adhering to the ribbons were washed 3 times with DPBS (10 min each) and then incubated with Alexa-Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:1000 dilution; Invitrogen, USA) and DAPI (1:1000 dilution) for 1 h at room temperature. The samples were washed with DPBS (3 times, 10 min each) and fluorescent images were acquired using fluorescence microscopy. The same procedure was performed on cells after injection. Cells adhering to ribbons produced from 7 mg mL-1 PLGA were maintained after injection in growth medium for 2 d and then in differentiation medium for 7 d. The length of the myotubes (containing more than 3 nuclei) was measured using fluorescence microscopy images and Axiovision software. 2.9. Statistical analysis The data were expressed as the mean ± standard deviation (SD) (3 replicates were conducted). Student’s t-test and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests were performed to analyze the differences between 2 and more than 2 experimental groups, respectively. A value of p < 0.05 was considered statistically significant. 10


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3. RESULTS AND DISCUSSION 3.1. Fabrication and morphology of ribbons Micropatterned sheets of ribbons were generated on PDMS molds by a combination of spincoating and microcontact printing techniques.9,22 We used 3 PLGA concentrations (5, 7, and 10 mg mL-1) to fabricate 3 different types of ribbons (Figure 1A). After cutting the edges of the strips and the rest of the sheet into small pieces, the dissolution of PVA sacrificial/supporting layer allowed for the release of freestanding dispersed ribbons in water (Figure 1B). We measured the thickness of individual ribbons using surface profilometry technique at their center region for each PLGA concentration. As indicated in the graph of Figure 1C the average thickness of the ribbons for all 3 PLGA concentrations ranged between 175 and 280 nm. As shown in Figure 1D-F, the PLGA ribbons fabricated using 3 different concentrations had a uniform morphology with a width of 50 μm, which is in accordance with our previous study.22 This size is imposed by the PDMS mold. Figure S1 shows the nanomechanical mapping of ribbons with different PLGA concentrations (5, 7, and 10 mg mL-1) together with histograms of the Young’s elastic modulus. The ribbons present a relative uniform modulus mapping for each composition tested as shown in the modulus histograms. The average values of the calculated Young’s modulus for PLGA concentrations of 5, 7, and 10 mg mL-1 were 750 ± 170, 600 ± 110, and 645 ± 95 MPa, respectively. These values are not significantly different (p = 0.21). However, these Young’s modulus values are much lower than the Young’s modulus of bulk PLGA measured previously (2 GPa), because of the ultrathin thickness of the ribbons,33,34 while they are comparable to values obtained from aligned electrospun PLGA fibers.35 It is known that Young’s modulus of materials at low dimensions is reduced from those of the bulk values.34

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Figure 1. Generation of the micropatterned PLGA ultrathin ribbon sheet and dispersed ribbons. (A) Schematics showing the fabrication of canal-like ribbons. (B) Optical images of the ribbon sheet and schematic of the process for preparing dispersed ribbons in water. (C) Thickness measured in the center region of individual PLGA ribbons fabricated from different PLGA concentrations. (D-F) FE-SEM images of ribbons with a width of 50 μm produced from different concentrations of PLGA (scale bars: 100 μm). 3.2. DOPA functionalization and PLL and FN immobilization Figure 2A shows pre-coating of freestanding ribbons with DOPA and immobilization of PLL and FN on their surfaces before cell seeding. These two biomolecules are conjugated to the DOPA 12


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layer via covalent bonds formed between their amine or thiol groups and the oxidized catechol group of DOPA.28, 36-38 We have applied PLL as a well known positively charged synthetic

homopolymer of amino acid L-lysine to modify the surface charge of the ribbons to allow a better dispersion of them in culture medium by electrostatic interactions. We also have applied FN as an ECM protein to promote the cell adhesion on the ribbons. Advantageously, the presence of both PLL and FN together can act synergistically on the cell attachment.39 Furthermore, the surface morphology of dispersed PLGA ribbons was studied using FE-SEM both before and after DOPA coating as shown in Figure 1D-F and Figures 2B and S2, respectively. After DOPA functionalization, we observed DOPA aggregations covering the surface of the ribbons (Figure 2B, S2) in agreement with Shi et al.40 Table 1 shows the quantification of the chemical composition of the PLGA film after different steps of functionalization (atom percentage of C, O, and N are extracted from EDS spectroscopy). After fabrication of the PLGA film, only carbon and oxygen were detected, whereas nitrogen appeared after coating with DOPA and further immobilization of PLL and FN. As shown in Table 1, the ratio of N to C in DOPA-coated PLGA was close to the theoretical value of dopamine (0.125),41 confirming the modification of the surface with DOPA. This ratio was further increased by immobilization of PLL and FN. Higher ratio of N to O was also detected after immobilization of PLL. This shows successful immobilization of PLL on DOPA layer because PLL has higher ratio of N/O comparing to DOPA.42 For further characterization of the PLGA substrates, the chemical composition was analyzed by XPS and the results showed efficient immobilization of PLL and FN on DOPA mediated surface of PLGA (Figure 2C and Table S1). As shown in Figure 2C, only C1s and O1s peaks were detected on bare surface of PLGA; however after coating with DOPA the N1s peak originated from nitrogen components of DOPA layer appeared. Notably, the intensity of this peak increased after immobilization of PLL and FN indicating the efficiency of DOPA layer to assist immobilization of PLL and FN (Table S1).

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Table 1. Quantification of atomic compositions (C, O, and N) in PLGA film surfaces after different functionalization steps using EDS method Atom %

C

O

N

Pt

PLGA

75.41

23.47

0

1.12

PLGA-DOPA

78.07

9.86

10.24

1.83

PLGA-DOPA-PLL

80.49

6.82

11.07

1.62

PLGA-DOPA-PLL-FN

82.37

3.73

12.22

1.61

Figure 2D further confirms functionalization of PLGA surface. FTIR spectra shows a broad band at 3700-3300 cm-1 that is labeled to N-H and O-H stretching modes; peaks at 2990 and 2900 cm-1 are assigned to an aliphatic C-H stretching modes; peaks at 1600 and 1580 cm-1 to C=C stretching modes in aromatic rings; peaks between 1460 and 1390 cm-1 to benzene ring stretching vibration of DOPA layer and peak at 1530 cm-1 is assigned to the amide N-H.43-45 These features are in accordance with those observed in spectra of DOPA powder (Figure S3).46 Typical PLGA absorption bands at around 1070 C-O, 1395 C-H, 2900 and 2990 C-H cm-1 can be observed clearly in the spectrums.44

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Figure 2. (A) Schematic showing the surface functionalization steps of PLGA ultrathin ribbons with DOPA coating and PLL and FN immobilization before cell seeding. (B) FE-SEM micrographs showing the surface morphology of PLGA ribbon covered by DOPA aggregation after DOPA functionalization at different magnifications. (C) XPS and (D) FTIR analysis of PLGA substrate 15



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after different modifications (unmodified, DOPA coated, DOPA-PLL, and DOPA-PLL-FN).

Visualization analysis of FITC-PLL immobilized on the surface of PLGA ribbons with and without DOPA coating was also performed and shown in Figure 3A. We observed weak green autofluorescence of ribbons soaked in PBS used as a control. Similarly, PLL coated on ribbons with or without DOPA did not show more fluorescence. When FITC-PLL was added on ribbons without DOPA coating, only few spots of fluorescence was observed showing that the protein did not adsorb well on the ribbons without DOPA coating. In contrary, when FITC-PLL was added on ribbons coated with DOPA a green fluorescence was observed homogenously on the whole surface of the ribbons showing that the PLL immobilized well on DOPA, in agreement with other studies (Ko et al.36). The effect of this functionalization was observed after immobilization of PLL and FN via catechol chemistry30,40 by a dramatic improvement of the cell affinity for the ribbons compared to cell affinity and adhesion to ribbons without DOPA or simply coated with PLL and FN. Figure 3B-F shows the fluorescence microscopy images of stained cells with calcein AM on ribbons with and without DOPA-PLL-FN coating. Figure 3B shows that after 2 d of culture, only a few cells were attached to the ribbons without any coating or functionalization, even after coating by PLL or FN cell attachment did not improve (Figure 3C,D). Cells adhered on DOPA coated ribbons (Figure 3E)

compared to ribbons without coating or ribbons coated with only PLL or FN. However, as shown in Figure 3F, DOPA coated ribbons with immobilized bioactive molecules showed synergistic effect on cell attachment and provided a chemically defined and functional substrate for cell culture. This is in accordance with Figure 3A regarding the efficiency of PLL immobilization on the surface of PLGA ribbon with and without DOPA coating.

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Figure 3. (A) Efficiency of PLL immobilization on DOPA-coated ultrathin ribbon visualized by FITC-PLL. Ribbons soaked in PBS were used as a negative control. (scale bars: 50 μm) (B-F) Fluorescence microscopy images of cells adhered on ribbons and stained with calcein AM after 2 d of culture. (B) No coating, (C) PLL coating, (D) FN coating, (E) only DOPA and (F) DOPA-PLL-FN functionalization (scale bars: 200 μm). 17



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3.3. Myoblast viability and alignment before injection C2C12 cells were cultured on covalently conjugated FN and PLL to DOPA-mediated ribbons with different PLGA concentrations (5, 7, and 10 mg mL-1). Cell adhesion on ribbon surfaces was evaluated inside a nonadherent culture dish to promote the attachment of cells on the ribbons. The schematic picture in Figure 4A describes the change in cell morphology observed by phase contrast microscopy (Figures 4B-D). After 1 h of contact with ribbons functionalized with DOPAPLL-FN, immobilized biomolecules promoted fast attachment of cells and the anisotropic morphology of the ribbon promoted cell alignment by contact guidance (Figure 4B-D). However, after 1 day of culture, the cells had spread, covering the entire surface of the ribbons and exhibiting an elongated morphology in the direction of the ribbons (Figure 4D). Next, we determined the morphology and alignment of cells adhered on the surface of PLGA ribbons using FE-SEM. Figure 4E shows that cells covered the surface and elongated in the direction of the ribbon. The viability after 2 d of culture was also evaluated with a Live/Dead assay. Figure 4F and Figure S4 show fluorescence microscopy images of live (green) and dead (red) cells cultured on ribbons. It can be observed that cells are localized on the ribbons and spread across their entire surface; furthermore, cell viability is excellent with only rare dead cells (red) visible. Because PLGA ribbons exhibit red autofluorescence, ribbons are generally visualized in red in fluorescent images. The cell viability quantification (Figure 4G) indicates a cell viability of 80% when cells were cultured on ribbons, with no detectable influence of PLGA concentration (p = 0.39). Figure 4H and Figure S5 show F-actin and nuclei staining by AlexaFluor 488-conjugated phalloidin and DAPI, respectively, after 2 d of culture. The fluorescence micrograph illustrates the cell elongation and alignment with the ribbons. It can be observed that the cells grew not only on the ribbons but also on the edges and that the canal shaped morphology (presence of edges) of the ribbons helped the cells to remain in the central part of the ribbons. A similar observation was reported in previous studies from our group by Fujie et 18


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al. and Shi et al.22,33 Thus, after 2 d of culture, cells grown on PLGA nanoribbons showed excellent spatial organization and high alignment with the direction of the nanoribbons. On the contrary, Han et al.24 did not observe any cell organization when cells (human adipose-derived stromal cells) grew on gelatin flat microribbons, even after 16 d of culture. Therefore, our PLGA ribbons displayed strong cell supportive properties with tremendous effects on cellular organization due to their nanofeature scale and their unique canal shape, which condenses cells in the middle of the ribbons favoring cell-cell contacts and cross-talk. Developing a muscle tissue with high

functionality requires mimicking the muscle structure, which is comprised of multiple bundles of muscle fibers that are formed by the fusion of undifferentiated myoblasts into long cylindrical, multinucleated structures called myotubes. Therefore, importance of delivering aligned cells on nanocarriers to the lesion site will facilitate and enhance the myogenesis of the delivered cells. In addition, if autologous stem cells are used for transplantation, it is requested to differentiate them before transplantation to avoid teratogenic effect. Therefore, the use of nanocarriers allowing cell alignment by contact guidance is crucial.2,3 Quantification of cell nuclei alignment in myoblasts cultured on ribbons made from different PLGA concentrations showed approximately 74% aligned cells within a 10° angle of the direction of the ribbons (Figure S6 A). No significant differences were detected between the cell cultures on ribbons using different PLGA concentrations (p = 0.47). It has been well established that the cellular alignment is improved on groove/ridge micropatterns having a groove width less than 100 μm.32,47,48 Thus, it is not unexpected that our micropatterned substrate with a groove spacing of 50 μm promotes cell alignment. Furthermore, the quantification of the nuclear aspect ratio also indicated that the cells are highly elongated with a nuclear aspect ratio greater than 2.7 (Figure S6 B). Since we did not observe a significant difference in the Young’s modulus of the ribbons or in the cell viability and alignment when cells were cultured on ribbons produced from different PLGA concentrations, we continued our experiments with the ribbons composed of 7 mg mL-1 of PLGA. We evaluated the functionality of the adherent C2C12 cells on ribbons in terms of 19



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myotube formation. Figure 4I shows the formation of several long myotubes on ribbons. The myotubes show an average length of 400 μm (Figure 4I). Furthermore, we observed aligned myotubes on the direction of ribbons with sharp A-bands (Figure 4I, enlargement), which shows the generation of matured myotubes with considerable sarcomere development and intracellular organization.49,50

Figure 4. (A) Schematic illustration of cell attachment and elongation on the surface and edges of canal-like ribbons (FN and PLL immobilized on DOPA-mediated ribbons) after 1 h and 1 d of culture. (B) FE-SEM image of a single ribbon (scale bar: 50 μm) and (C, D) phase contrast microscopic images of cells on a ribbon after 1 h and 1 d of cell seeding, respectively (scale bar: 20


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50 μm). (E) FE-SEM image of cells adhered and spread on a ribbon after 2 d of culture (scale bar: 10 μm). (F) Cell viability of C2C12 cells on ribbons, stained with calcein AM (live cells, green) and ethidium homodimer 1 (dead cells, red) after 2 d of culture (ribbons produced from 7 mg mL-1 PLGA; scale bar: 20 μm). (G) Cell viability quantification after 2 d of culture on individual ribbons produced from different concentrations of PLGA (5, 7, and 10 mg mL-1). (H) Fluorescence image showing Alexa-Fluor 488-conjugated phalloidin (F-actin, green) and DAPI (nuclei, blue) stained C2C12 myoblasts cultured on ribbons after 2 d of culture showing the cell alignment on individual ribbons (scale bar: 100 μm). (I) Fluorescence microscopy image of myotubes showing myotube formation on freestanding ribbons and exhibiting sharp A-bands (enlargement (scale bar: 5 μm)) (7 mg mL-1 PLGA). The images show myosin (stained with mouse-anti myosin and revealed with Alexa-Fluor 488-conjugated goat anti-mouse antibody, green) and nuclei (stained with DAPI, blue) of myotubes on ribbons (red autofluorescent) formed after 7 d of differentiation (scale bar: 50 μm).

3.4. Cell viability, alignment and myogenesis after injection Next, we evaluated the injectability of the ribbons and the state of ribbons and cells after injection. As shown in Figure 5A, as a result of their highly flexible structure, ribbons stained with Nile red (light red color) can be aspirated and injected through an IV needle. This process is shown in Movie S1. The ribbons were capable of flexibly deforming inside a 24-gauge IV needle (24G, 470 μm inner diameter) and bending along the inner wall of the needle without damage, as shown by fluorescent imaging (Figure 5A, magnified fluorescent images). Such flexibility is a significant advantage for minimally invasive cell delivery because it can mechanically protect the cells from shear forces during injection and decreases the damage incurred to the targeted and surrounding tissue during the transplantation procedure. Furthermore, this injectability via an IV needle is advantageous for quicker clinical translation.51 Figure 5B-D show that our flexible 21



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ribbons allow the cellular organization to be retained under the mechanical stress applied by injection. Thus, we injected the cell-loaded ribbons using a 27G needle in a culture dish and quantified the DNA content collected from the remaining cells on the ribbons after 1 d. The amounts of DNA collected from cells before and after injection (3.25 and 3.11 μg, respectively) were not significantly different (p = 0.94), indicating that the flexible ribbons could successfully support cells during and after injection. Moreover, we evaluated the cell viability following syringe manipulation with different syringe needle gauges 1 d after injection. We used nonadherent culture dishes to facilitate ribbon harvesting. We did not observe any significant difference in cell viabilities when different needle gauges (23, 25 and 27G) were used compared to the control, which was the cell viability before the injection of cells cultured on ribbons fabricated from 7 mg mL-1 PLGA. Thus, approximately 80% of cells adhered on micropatterned ribbons with thicknesses of a few hundreds nm (175- 280 nm), withstood deformation in different sizes of syringe needles and remained viable on their surfaces (Figure 5C,D and Figure S7). Our results show a marked technical improvement compared to other studies. For example, Song et al.52 used bigger size of needles, including a 20G needle, to inject cells adhered on PLGA fibrous micro-scaffolds and reported 87% cell viability and the loss of cells during injection as a result of direct contact between the cells and the needle walls. We did not observe dramatic changes in the shape of cell-loaded ribbons after injection, and only some of the ribbons twisted, bended or waved, as shown in the fluorescent images in Figure 5D and Figure S7. We also evaluated the injectability of the ribbons via a 30G needle (159 μm inner diameter). However, the injections were impaired because the ribbons with adhered cells clogged in the needle or the cells detached from the ribbons. Myotube formation on the ribbons was evaluated after injection via a 27G needle. Figure 5E shows the fluorescent image of myotubes formed on ribbons after injection and stained for myosin (green) and nuclei (blue) at 7 d after differentiation. Because multiple myotubes with an average length of 400 μm formed on ribbons, the myoblasts remained functional after injection. Figure S8 shows F-actin and nuclei staining after 7 d of differentiation. The fluorescence 22


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microscopy images show the formation of myotubes elongated on ribbons among undifferentiated myoblasts at two different magnifications. The elongation and alignment of both differentiated and undifferentiated myoblast cells on the surface of the ribbons is clearly visible. Together, our results indicate that ultrathin flexible ribbons are excellent carriers for cells even under stressful conditions such as aspiration into the narrow space of a syringe needle, providing protection to cells and reducing the effects of mechanical stress. Cell-loaded ribbon constructs have several advantages over the microgels and microcapsules that are currently used as injectable cell carriers.53 First, PLGA ribbons have a much greater surface-tovolume ratio, allowing more cells to be cultured on the substrate. Second, their high aspect ratio induces cell alignment after cell seeding (over 74% aligned nuclei) as well as myotube alignment. Third, they can mechanically support the cells during injection and keep the cell organization intact due to their flexibility, which is not achieved by loading cells in microgels. This cell alignment by contact guidance favors myotube formation.

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Figure 5. (A) Nile red stained ribbons were aspirated into a 24G IV needle (470 μm inner diameter and 700 μm outer diameter) (scale bar: 5 mm). Magnified fluorescence microscopy images of ribbons show that they are flexible and easily deformed along the inner wall of the needle (scale bars: 100 μm middle panel and 20 μm right panel). (B) Quantification of DNA content differences collected from the myoblast cells cultured on ribbons before and after injection. (C) Quantification of cell viability for C2C12 cell-loaded ribbons 1 d after injection with different needle gauges (23, 25 and 27G). The control is the cell viability value observed for cells grown on 7 mg mL-1 PLGA before injection (extracted from Figure 3G). (D) Fluorescence 24


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microscopy image showing the cell viability of injected cell-loaded ribbons (7 mg mL-1 PLGA), live (green) and dead (red) cells, 1 d after injection using a conventional 27G syringe needle (scale bar: 100 μm). (E) Fluorescence microscopy image of myotube formation on freestanding ribbons (7 mg mL-1 PLGA) showing the myosin (green) and nuclei (blue) of myotubes on ribbons (red) formed after injection and 7 d of differentiation (scale bar: 50 μm). (The results are expressed as the mean ± SD).

4. CONCLUSION In summary, we have described the development of surface functionalized biodegradable PLGA ultrathin ribbons with DOPA-PLL-FN for the local delivery of C2C12 cells for the treatment of muscle injury. The functionalization of the ribbons by DOPA improved the cell attachment and allowed the formation of stable, adhered, organized and aligned cells that could differentiate into long and matured myotubes. They could mechanically support the cells during injection and keep the cell organization intact. As a result of their high flexibility, the cell-loaded ribbons could be injected through a clinical syringe with a needle as thin as 27G (220 μm inner diameter) without a significant loss of cell viability. This non-invasive method of cell delivery by injection is advantageous because in transplantation it will reduce the incision size in the muscle tissue and the subsequent inflammatory response, enabling this method to be easily translated into clinical applications. Therefore, cell-seeded PLGA ribbons hold great promise for the localized transplantation of organized cellular structures in a lesion site with a higher survival rate and engraftment of cells and constitute an advanced development in non-invasive local cell delivery systems.

ASSOCIATED CONTENT

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Supporting Information Available: The following files are available free of charge. Quantification of atomic percentage of C, O, and N in unmodified and functionalized PLGA. Young’s modulus nanomechanical mapping and histogram of Young’s modulus distribution for ribbons produced from different concentrations of PLGA. FE-SEM micrographs of surface morphology of PLGA ribbon after DOPA functionalization; FTIR spectrum of DOPA powder; Fluorescence microscopy images showing viability and F-actin/DAPI staining of cells adhered on ribbons after 2 d of culture; Quantification of cell nucleus alignment and nuclear aspect ratio; Fluorescence microscopy images of live and dead cells after injection; and a Movie showing aspiration and injection of Nile red stained ribbons through a 24G IV needle.

AUTHUR INFORMATION Corresponding Author *Email: [email protected]

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Author Contributions S. S. designed and performed the experiments, and analyzed the results. S. S. fabricated the ribbons. S. O. and M. E. helped in performing and analyzing the cell culture experiments and data. X. L. collected the AFM measurements under the supervision of K. N. S. S. and S. O. wrote the manuscript. H. B. and R. B. S aided in editing the manuscript. T. F. and A. K supervised the project. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the World Premier International Research Center Initiative (WPI) and JSPS KAKENHI: Grant Number 15H05355 (T. F.) from MEXT, Japan.

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