Gelatin-based micro-hydrogel carrying genetically engineered human endothelial cells for neovascularization

Acta Biomaterialia 95 (2019) 285–296

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Gelatin-based micro-hydrogel carrying genetically engineered human endothelial cells for neovascularization q Young Hwan Choi a,1, Su-Hwan Kim a,b,1, In-Seon Kim a, KyungMin Kim a,c, Seong Keun Kwon d,e,⇑, Nathaniel S. Hwang a,b,f,⇑ a

School of Chemical and Biological Engineering, Institute for Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea Institute of Engineering Research, Seoul National University, Seoul 08826, Republic of Korea Interdisciplinary Program in Bioengineering, Seoul National University, Seoul 08826, Republic of Korea d Department of Otorhinolaryngology-Head and Neck, Seoul National University Hospital, Seoul 03080, Republic of Korea e Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea f Bio-MAX/N-Bio Institute, Seoul National University, Seoul 08826, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 2 October 2018 Received in revised form 16 January 2019 Accepted 28 January 2019 Available online 31 January 2019 Keywords: Gelatin methacrylate (GelMA) Micro-hydrogel Electrospray hVEGF-secreting HUVEC Hindlimb ischemia

a b s t r a c t Cell delivery systems based on micro-hydrogels may facilitate the long-term survival of cells upon transplantation. Micro-hydrogels may effectively support cell proliferation, attachment, and migration in ischemic environments. In this study, we report the fabrication of a gelatin methacrylate (GelMA)based micro-hydrogel for efficient in vivo delivery of genetically engineered endothelial cells. Microhydrogels were initially processed via electrospraying of GelMA and alginate (ALG) mixtures (at different ratios) on to calcium chloride (CaCl2) solution. Electrospraying of the GelMA/ALG mixture resulted in the formation of a micro-hydrogel, owing to ALG crosslinking. Secondary crosslinking of GelMA with UV light and ALG hydrogel chelation using sodium citrate solution resulted in GelMA-based micro-hydrogel formation. We observed the angiogenic response of human umbilical vein endothelial cells (HUVECs) in GelMA concentration-dependent manner. The seeding of HUVECs engineered to express human vascular endothelial growth factor on to the GelMA micro-hydrogel and the subsequent transplantation of the micro-hydrogel into a hindlimb ischemia model effectively attenuated the ischemia condition. This facile and simple micro-hydrogel fabrication strategy may serve as a robust method to fabricate efficient cell carriers for various ischemic diseases. Statement of Significance For the therapeutic angiogenesis, it is important to provide the therapeutic cells with a carrier that could stabilize therapeutic cells and facilitate long-term survival of cells. Furthermore, it is also important to administer as many therapeutic cells as possible in a fixed volume. From these cues, we fabricated ECM-based micro-hydrogel produced by the high through-put system. And we intended to facilitate activation of therapeutic cells by coating the therapeutic cells onto the micro-hydrogel. In this manuscript, we fabricated methacrylate gelatin (GelMA) based micro-hydrogels using the electro-spraying method and coated HUVECs engineered to express hVEGF onto the micro-hydrogels. Then, we identified that GelMA concentration-dependent angiogenic response of HUVECs. Furthermore, we demonstrated that the VEGF secreting HUVEC-GelMA micro-hydrogels induced the restoration of blood flow and neovascularization in a hind-limb ischemia mouse model. These findings demonstrate that the high-throughput fabrication of ECM micro-hydrogels could be a novel platform to apply in neovascularization and tissue engineering. Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

q

Part of the Cell and Tissue Biofabrication Special Issue, edited by Professors Guohao Dai and Kaiming Ye.

⇑ Corresponding authors at: Department of Otorhinolaryngology-Head and Neck, Seoul National University Hospital, Seoul 03080, Republic of Korea (S.K. Kwon). School of Chemical and Biological Engineering, Institute for Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea (N.S. Hwang). E-mail addresses: [email protected] (S.K. Kwon), [email protected] (N.S. Hwang). 1 Y.H Choi and S.H Kim contributed equally to this work. https://doi.org/10.1016/j.actbio.2019.01.057 1742-7061/Ó 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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1. Introduction

2. Materials and methods

Peripheral arterial disease (PAD) is commonly defined as the narrowing of the blood vessels of the lower part of arteries or arterioles. One promising strategy to inhibit the progression of PAD and facilitate disease treatment is through the stimulation of the growth of the pre-existing vessel and induction of neovascularization. For therapeutic angiogenesis, administration of growth factors or therapeutic cells has been proposed in clinical trials [1–4]. However, the administration of therapeutic cells has several limitations such as clearance by the circulatory system, low survival rate of cells, and inflammation at the injected site [5]. To overcome these limitations and improve the therapeutic potency, microhydrogel-based cell delivery systems have been developed [6–8]. Micro-hydrogels could provide mechanical stability and durability, induce cell-responsive moieties, and exhibit biocompatibility and biodegradable properties [9–11]. There are several methods to produce micro-hydrogels, such as the use of microfluidic chip [12], electrospray [13], and emulsion techniques [14]. In this report, we applied electrospray techniques to fabricate micro-hydrogels in a rapid process. This technique allows the user to control over electrospray setups such as applied voltage and nozzle size, resulting in the formation of micro-hydrogels with uniform size and mono-dispersity. In addition, the high-throughput generation of micro-hydrogels could be achieved without the use of any organic solvent. The combined strategy using therapeutic cells and growth factors with hydrogels was recently attempted to improve therapeutic effects [13]. Loading of therapeutic cells and growth factors within functional hydrogels allow sustained release of growth factors and the paracrine factors produced by therapeutic cells [15]. In addition, previous studies have demonstrated increased survival rate upon transplantation of cells in the presence of growth factor supplementation [15–18]. Alternatively, one may engineer cells to express therapeutic factors in a sustained manner. Several studies have reported that the stem cells overexpressing vascular endothelial growth factor (VEGF) stimulated angiogenesis and attenuated myocardial infarction [19,20]. With safety concerns in recent years, researchers have genetically engineered stem or endothelial cells to overexpress VEGF using non-viral gene delivery methods and reported their effects on angiogenesis in hindlimb ischemia or wound repair [21–23]. In this study, we fabricated a gelatin methacrylate (GelMA)based micro-hydrogel as a carrier for engineered cells. Previous studies have reported that the endothelial cells actively attached, proliferated, and migrated on or within the GelMA hydrogel [24,25]. To fabricate the GelMA-based micro-hydrogel, alginate (ALG) was used as a sacrificial material. ALG was rapidly crosslinked in the presence of calcium chloride (CaCl2) solution and the ALG hydrogel freely dissolved upon treatment with sodium citrate. Using this system, we prepared a GelMA/ALG-based micro-hydrogel (entrapment of GelMA in ALG hydrogel) with electrospraying method. The GelMA/ALG micro-hydrogel was subsequently exposed to UV light (cross linking of GelMA hydrogel) and ALG was dissolved using sodium citrate solution, resulting in the fabrication of the GelMA micro-hydrogel. We investigated the properties of GelMA micro-hydrogel at different concentrations of GelMA and evaluated the optimal concentration required for the activation of endothelial cells. Furthermore, the in vivo angiogenic tissue regeneration ability of the GelMA micro-hydrogel coated with VEGF-secreting human umbilical vein endothelial cells (HUVECs) was investigated using a murine hindlimb ischemia model.

2.1. Materials Phosphate-buffered saline (PBS), gelatin (Porcine skin, type A, 300 Bloom), methacrylic anhydride, sodium citrate, rhodamine B isothiocyanate (RITC), fluorescein isothiocyanate (FITC), 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC
HCl), N-hydroxysuccinimide (NHS), and PKH26 red fluorescent cell linker kit were all purchased from Sigma-Aldrich (Yongin, Korea). Sodium ALG was obtained from FMC biopolymer (Drammen, Norway). 2.2. Cell culture A tissue culture plate was coated with 0.1% (w/v) gelatin (Millipore, EmbryomaxÒ, USA) dissolved in ultrapure water for 1 h. HUVECs were seeded on the tissue culture plate and cultured with endothelial cell basal medium-2 (EBM-2; Lonza, Walkersville, MD, USA) supplemented with endothelial growth medium (EGMTM-2) singleQuotsÒ. The medium was replaced every day. HUVECs were used at passages 6–8 for all experiments. 2.3. Synthesis of GelMA We dissolved 10% (w/v) gelatin in PBS at 60 °C for 1 h and 8% (v/v) methacrylic anhydride was slowly added to the gelatin solution; the reaction was proceeded at 60 °C for 3 h. The final solution was diluted with 10 PBS to stop the reaction and dialyzed against distilled water for 5 days and lyophilized for 1 week. The lyophilized GelMA was stored at 20 °C until further experiment. GelMA and the degree of methacrylation were identified using a 1H NMR (Bruker AVANCE III 400 MHZ, Bruker TopSpin Software 4.0.2, Supplementary Fig. 1). 2.4. Synthesis of RITC-GelMA and FITC-ALG To synthesize the RITC-GelMA and FITC-ALG, we referred to the previous studies [26–28]. A total of 5 mg of RITC was reacted with 1% (w/v) of GelMA in PBS and the pH of the solution was raised to 9.5 by adding 1 M sodium hydroxide (NaOH) solution. The solution was stirred for 1 h at 60 °C and the RITC-conjugated GelMA solution was dialyzed at 60 °C against water to remove the excess of unbound RITC. The mixture was lyophilized. Sodium ALG (120 mg) was mixed with EDC/NHS (50 mg/30 mg) in 0.1 M sodium acetic buffer (pH 5.0) for 30 min. Hexamethylene diamine (60 mg) was added and reacted for 4 h, followed by precipitation in isopropanol to remove unreacted diamine. The ALG-amine solution was stirred with FITC (0.5 mg) in pH 8.5 sodium bicarbonate solution for 4 h. FITC-ALG was precipitated from acetone by removing the unbounded FITC. The final solution was lyophilized. 2.5. Fabrication of GelMA micro-hydrogels GelMA and ALG mixtures (final concentration; GelMA = 2.5%, 3%, 3.5%, 4% [w/v] respectively, ALG = 0.5% [w/v]) containing a photo-initiator (Irgacure 2959, 0.5% [w/v]) were exposed to UV light (360–480 nm, 3.5 mW/cm2) for 30 s. After pre-gelation, GelMA/ALG mixtures were electrosprayed on 20 mL of 100 mM CaCl2 solution through a 28-gauge needle (NNC-ESP 200, Seoul, South Korea) using a syringe pump (flow rate: 3 mL/h) at a high voltage of 10 kV [13]. GelMA/ALG micro-hydrogels were then cross-linked using UV light (360–480 nm, 3.5 mW/cm2) for 5 min.

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After the production of micro-hydrogels, CaCl2 was removed and the micro-hydrogels were washed several times with PBS. ALG was removed using a chelation reagent (5% [w/v] sodium citrate) and the GelMA micro-hydrogels were washed several times with PBS and overnight stabilized with EGM. In the same manner, trace of elements in micro-hydrogels was proceeded using RITC-GelMA and FITC-ALG. The micro-hydrogels were then observed with a confocal microscope (LSM 780, Carl Zeiss). Five serial images were taken including inner core of micro-hydrogel (term = 50 mm).

analyzed with PCR by measuring the expression level of angiogenesis-related genes (angiogenin, angiopoietin-1, and vascular cell adhesion protein-1 [VCAM-1]); b-actin was used as a reference gene. The primers used were as follows: Angiogenin (forward: 50 -CATCATGAGGAGACGGGG-30 and reverse: 50 -TCCAAG TGGACAGGTAAGCC-30 ), angiopoietin-1 (forward: 50 -AGCTGT GATCTTGTCTTGGC-30 and reverse: 50 -GTTCAAGTCTCGTGGTCTGA30 ), VCAM-1 (forward: 50 -TAAAATGCCTGGGAAGATGG-30 and reverse: 50 -GGTGCTGCAAGTCAATGAGA-30 ).

2.6. Physicochemical characterization of hydrogels

2.8. Genetically engineered HUVECs

A total of 200 mL of GelMA/ALG mixture (final concentration; GelMA = 2.5%, 3%, 3.5%, 4% [w/v] respectively, ALG = 0.5% [w/v]) containing photo-initiator (Irgacure 2959, 0.5% w/v) was poured into the cap of a 1.2-mL Eppendorf tube and exposed to UV light (360–480 nm, 3.5 mW/cm2) for 30 s, followed by the addition of 200 mL of 100 mM CaCl2 solution. The hydrogels were gelated with UV light treatment for 5 min. CaCl2 was removed, and the hydrogel was washed several times with PBS. ALG was removed using 5% (w/v) sodium citrate, which was eventually decanted. The hydrogel was washed several times with PBS. To investigate surface morphology, scanning electron microscopy (SEM) analysis was performed. Unchelated GelMA/ALG hydrogels and chelated GelMA hydrogels were lyophilized for 48 h. Then, the hydrogels were mounted on a sample holder with carbon tape and sputtercoated with platinum. The images were captured at an acceleration voltage of 10 kV. To analyze swelling property, the weights of the unchelated GelMA/ALG hydrogel and chelated GelMA hydrogel were measured. In addition, the weight of the lyophilized hydrogel was measured and the swelling ratio (volume of swollen gel in water/volume of dry gel) of the hydrogel was derived. Hydrogels were swollen in PBS for a day before the compression test. Young’s modulus of each hydrogel was measured using Instron Model 5966 (Instron Corporation, MA, USA). Stress and strain were calculated from the pressure applied on the hydrogels and the distance between the hydrogel and the compressor. Young’s modulus was obtained through the stress-strain curve (Supplementary Fig. 2). To verify the success of the synthesis of the micro-hydrogels (GelMA), we recorded X-ray diffraction (XRD) patterns using Cu Ka radiation source (D8 Advance, Bruker, Germany). All mechanical properties of hydrogel were analyzed based on the standardized testing methods from the American Society for Testing and Materials (ASTM) F2900-11, F2214-16 and F2150-13.

Cells were washed with PBS and re-suspended in a resuspension buffer (included with NeonTM Kits, 100 mL per 1 million cells). GFP-tagged human VEGF-A plasmid (7.5 mg per 2 million cells) was added to the re-suspension mixture and the mixture was aspirated using NeonTM Pipette. The NeonTM Pipette with sample was vertically inserted into the NeonTM Tube filled with 3 mL of an electrolytic buffer. Electric pulse was delivered to the sample, as per the electroporation protocol (pulse voltage: 1,350 V, pulse width: 30 ms, pulse number: 1).

2.7. HUVEC coating and angiogenic response of HUVECs A petri dish was coated with 1% (w/v) bovine serum albumin (BSA, Koma Biotech, Seoul, Korea) dissolved in distilled water for 1 h. BSA solution was then removed and the petri dish was washed several times with PBS. HUVECs, EGM, and chelated GelMA microhydrogels were shaken using a rotating shaker for a day. The day after, to identify the cytotoxicity, HUVEC-coated GelMA microhydrogels were stained by LIVE/DEADTM Kit (Invitrogen, L3224, 0.5 mL of calcein-AM/1 mL of PBS, 2 mL of ethidium homodimer1/1 mL of PBS). For quantifying the expression of mRNA, the HUVEC-coated GelMA micro-hydrogels were incubated with EGM for 7 days and each group of hydrogel construct was transferred into a 1.8-mL E-tube. Trizol reagent was added to each tube for the extraction of RNA. Micro-hydrogels were manually broken using pestle tip. Reverse transcription of RNA into cDNA was performed using the SuperScriptÒ First-Strand Synthesis System for reverse-transcription polymerase chain reaction (RT-PCR; InvitrogenTM). Real Time-PCR was performed using the SYBR Green PCR Master Mix and the ABI StepOnePlusTM Real-Time PCR System (Applied Biosystems). Angiogenic response of HUVECs was

2.9. Quantification of VEGF released from the genetically engineered HUVECs A total of 5  104 VEGF-A plasmid-transfected HUVECs and 5  104 untransfected HUVECs were cultured with 1 mL of EBM supplemented with 2% fetal bovine serum (FBS) in 12-well tissue culture plates for 7 days. At pre-determined time points, the culture media were collected and replaced with fresh EBM with 2% FBS. The collected media were immediately stored at 80 °C before analysis. The amount of released VEGF from the collected medium was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems). The cumulative release data were presented with each replicate as standard deviation was too small to visualize. 2.10. Evaluation of degradation profiles To test hydrogel degradation profiles, we fabricated GelMA micro-hydrogels using RITC conjugated GelMA. Then, they were incubated with GelMA micro-hydrogels (200 mL of microhydrogels in PBS) or GelMA micro-hydrogels (200 mL of microhydrogels in collagenase 1 U/ml) or GelMA micro-hydrogels coated with unmodified HUVECs (200 mL of GelMA micro-hydrogels coated with 5  105 of HUVECs) or GelMA micro-hydrogels coated with genetically engineered HUVECs (200 mL of GelMA microhydrogels coated with 5  105 of genetically engineered HUVECs). The supernatants of each hydrogel were collected at each time points, and the fluorescence intensity was evaluated by Infinite 200 PRO TECAN microplate reader (Tecan, Durham USA). All data were presented in Box-and-Whisker plots as standard deviation was too small to visualize. 2.11. Tube formation assay 200 mL of MatrigelTM (BD Biosciences, BD Cat No. 354234) were added to each well of 24-well culture plates and plates were incubated at 37 °C for 30 min. 5  104 of HUVECs were seeded onto the layer of Matrigel. Then, they were incubated with GelMA microhydrogels (50 mL of micro-hydrogels re-suspended in 50 mL of PBS, total volume: 100 mL) or GelMA micro-hydrogels coated with unmodified HUVECs (50 mL of GelMA micro-hydrogels coated with 10  104 of HUVECs re-suspended in 50 mL of PBS, total volume: 100 mL) or GelMA micro-hydrogels coated with genetically engineered HUVECs (50 mL of GelMA micro-hydrogels coated with 10  104 of genetically engineered HUVECs re-suspended in 50 mL

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of PBS, total volume: 100 mL) using TranswellTM (Corning, Lowell, MA, USA). After 6 h, cells were stained by calcein-AM, and tube formation was analyzed by quantifying the area and loops of tube networks.

3. Results 3.1. Fabrication of GelMA micro-hydrogel with electrospray method and its characterization

2.12. Hindlimb ischemia model and in vivo neovascularization Animal experiments were performed using protocols (protocol number: SNU-170728-3) approved by the Seoul National University Institutional Animal Care and Use Committees. BALB/c-nu mice (female, age 6 weeks, weighing 20–25 g) were purchased from Orient Bio (Orient Bio Inc, Seongnam, Korea). All mice were anesthetized with an intraperitoneal injection of Zoletil 50 (Tiletamine + Zolazepam, 0.006 mL/10 g) and rompun (Xylazine, 0.004 mL/10 g). Similar to our previous study and other, hindlimb ischemia was induced in the murine [29–31]. The inguinal skin was longitudinally incised along the femoral artery visible through the skin. The femoral artery was excised from its proximal origin as a branch of the external iliac artery to the distal point where it bifurcates into the saphenous and popliteal arteries. In other words, the common femoral artery and superficial femoral artery were occluded by surgical knots, and the blood vessel between them was excised. After arterial excision, the mice were treated with PBS (100 mL), GelMA micro-hydrogels (50 mL of microhydrogels re-suspended in 50 mL of PBS, total volume: 100 mL), genetically engineered HUVECs (1 million cells re-suspended in 100 mL of PBS), and GelMA micro-hydrogels coated with genetically engineered HUVECs (50 mL of GelMA micro-hydrogels coated with 1 million HUVECs re-suspended in 50 mL of PBS, total volume: 100 mL). The extent of necrosis in the ischemic hindlimb was analyzed on day 28 after surgery. Blood flow of the ischemic right limb and normal left limb was measured using a laser Doppler perfusion imaging (LDPI) analyzer (Moor Instruments, Devon, UK) on day 0, 7, 14, 21, and 28 after sample injection. The degree of blood perfusion was calculated on the basis of colored histogram pixels. Blood perfusion was expressed as LDPI index, indicating the ratio of ischemic versus non-ischemic limb blood flow. 2.13. Histological evaluation Tissue samples were collected and fixed with 4% (w/v) formaldehyde, followed by dehydration with serial concentrations of ethanol (50%, 75%, 90%, 95%, and 100%) and paraffin embedment. Samples were then sectioned into 10-lm thickness, rehydrated and stained with hematoxylin and eosin (H&E).

In the present study, we focused on the facile formation of GelMA micro-hydrogels which could be a carrier for engineered HUVECs. In our study, we utilized methacrylated gelatin and the degree of methacrylation was confirmed to have 75 as determined by 1H NMR (Bruker AVANCE III 400 MHz, Bruker TopSpin Software 4.0.2, Supplementary Fig. 1). Methacrylated gelatin provides mechanical robustness that facilitates the fabrication of micro-engineered hydrogel as described in the previous studies [24,32]. To investigate the geometry and size of the micro-hydrogel, the GelMA/ALG solution was electrosprayed (Fig. 1). A precursor solution (Table 1) was electrosprayed at a flow rate of 3 mL/h and a voltage of 10 kV. The final concentration of GelMA varied from 2.5% to 4% (w/v), and ALG was fixed at 0.5% (w/v). At first, the GelMA/ALG mixture with photo-initiator was pre-cured for 30 s in the presence of UV light. The partially cross-linked solution was electrosprayed on CaCl2. At this point, ALG showed spontaneous cross-linking; further cross-linking of GelMA hydrogel was performed with UV light treatment for 5 min, resulting in the fabrication of the GelMA/ALG micro-hydrogel. We removed ALG from the GelMA/ALG micro-hydrogel using the chelating agent sodium citrate. As sodium citrate could trap divalent cations, only GelMA micro-hydrogel was fabricated after chelation. The average diameter of the unchelated and chelated micro-hydrogels was approximately 450–500 mm (Fig. 2A). The micro-hydrogels exhibited a clear circular geometry. After chelation of ALG using sodium citrate, the overall geometry was retained regardless of the precursor ratios (Fig. 2B). To evaluate the removal of ALG, we conjugated fluorescent dyes to hydrogel (FITC to ALG and RITC to GelMA). We detected the fluorescence intensity using same exposure time and gain of laser. Surface and center of micro-hydrogels were investigated with some serial images (Fig. 2C, D). After chelation, the FITC-Alginate was fully removed from the surface of microhydrogel as well as from the inner-core of the micro-hydrogel while the RITC-GelMA still remained. No residual ALG was observed in the fluorescent image of micro-hydrogels both surface and inner-core.

3.2. Physicochemical characterization of hydrogel 2.14. Immunostaining analysis To evaluate the angiogenic response in vivo, the sectioned tissue samples were prepared as mentioned above. After antigen retrieval step, the specimens were treated with a blocking solution containing 1% BSA (w/v), Triton X-100 (0.1% w/v), and normal goat serum (10% w/v) for 45 min. After blocking and washing, the samples were incubated with primary antibodies (rabbit anti-CD-31 or rabbit anti-a-smooth muscle actin [SMA] antibody, Abcam, Cambridge, UK) for overnight at 4 °C. The samples were incubated with secondary antibodies (DyLightTM 594 or 488-conjugated AffiniPure Goat Anti-Rabbit IgG, Jackson Laboratory, USA) and counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). To quantify capillaries and a-SMA-positive cells, the CD-31- and a-SMA-stained samples were imaged and the number of cells in five random fields was counted. 2.15. Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical significance between groups was determined by Student’s t-test with *p < 0.05, **p < 0.01, and ***p < 0.005.

Surface morphology of hydrogels was analyzed by SEM (Fig. 3A). Chelated samples showed more uneven surface than the unchelated samples. In the stress-strain curve, the compressive stress smoothly increased upon increasing GelMA amount. Similar trend was observed with unchelated and chelated samples (Supplementary Fig. 2). However, the unchelated 3.5% group had higher resistance to compressive stress than the 4% group, which comprised higher GelMA amount than the 3.5% group. The Young’s modulus decreased after chelation in all groups and increased with an increase in GelMA concentration (Fig. 3B). In 2.5% group, Young’s modulus was 9.77 ± 1.53 kPa before chelation, while this value decreased to 6.53 ± 0.41 kPa after chelation. In 3% group, Young’s modulus was 10.48 ± 0.66 kPa before chelation, while this value decreased to 7.72 ± 0.52 kPa after chelation. In 3.5% group, Young’s modulus was 13.87 ± 2.38 kPa before chelation, while this value decreased to 8.05 ± 0.59 kPa after chelation. In 4% group, Young’s modulus was 12.83 ± 0.74 kPa before chelation, while this value decreased to 9.98 ± 0.98 kPa after chelation. The Young’s modulus increased from 6.53 ± 0.41 kPa in the chelated 2.5% group to 9.98 ± 0.98 kPa in the chelated 4% group. The swelling properties

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Fig. 1. (A) Schematic illustration of the process of the delivery of GelMA micro-hydrogel in a hindlimb ischemia model. After producing GelMA micro-hydrogels through electrospraying method, HUVECs transfected with plasmids carrying VEGF gene were coated on the surface of GelMA micro-hydrogels. The cell-coated micro-hydrogels were injected into the hindlimb ischemia model to evaluate the angiogenic response. (B) Illustration of the fabrication process of GelMA micro-hydrogels. i) GelMA and ALG mixtures were electrosprayed on CaCl2 solution to crosslink ALG. ii) The mixture was further crosslinked with UV irradiation. iii) After the construction of the interpenetrating network (IPN) structures of GelMA and ALG, ALG was removed using sodium citrate solution. iv) GelMA micro-hydrogels were produced in a high-throughput manner.

Table 1 GelMA/ALG solutions prepared at different concentrations of GelMA and alginate with electrospraying and the final concentration of GelMA in the micro-hydrogel. Microsphere composition

Concentration (w/v)

Unchelated

Alginate GelMA

0.5% 2.5%

3%

3.5%

4%

Chelated

GelMA

2.5%

3%

3.5%

4%

were inversely related to mechanical properties (Fig. 3C). After chelation, all groups showed an increase in the swelling ratio, but no significant difference was observed between the unchelated and chelated samples. Additionally, we evaluated X-ray diffraction (XRD) analysis of hydrogel (Fig. 3D). The diffraction pattern of alginate and GelMA consists of crystalline peaks at 2h = 13.4° and 20.4°, respectively. When the alginate and GelMA mixture was exposed to UV radiation, the acrylate radical in GelMA partially oxidized glycosidic bonds of alginate, following the formation of oxidized alginate [33,34]. Thus, the unchelated hydrogel presents two additional peaks at 2h = 31.6° and 45.3°. After treating sodium citrate solution, the chelated hydrogel exhibited only broad peak at 2h = 20.4° which indicates the GelMA compositions. It was demonstrated that alginate composition was clearly removed by chelation process and the crystal structure of GelMA was retained during fabrication processes. We also investigated the degradation profiles of GelMA microhydrogels (Fig. 3E). In collagenase group, the GelMA microhydrogels were fully degraded in 3 days by enzymatic forces. In HUVECs and transfected HUVECs coated groups, however, the degradation was only affected by metalloproteinase (MMP) from endothelial cells. The results indicated that the endogenous MMP can degrade hydrogels partially about 10% of weight at 2 weeks.

Even though the transfected HUVECs exhibited enhanced angiogenic activity compared to HUVECs, the degradation rate of GelMA has no difference up to 2 weeks. It was demonstrated that the degradation forces of GelMA micro-hydrogels dominantly depended on external enzymatic activity, not the endogenic MMP from HUVECs. Additionally, it was indicated that the GelMA micro-hydrogels might be stayed in vivo environments for a few months as the MMP was not vigorously secreted in static states from native tissues. 3.3. Characteristics of HUVECs coated on the GelMA micro-hydrogels GelMA micro-hydrogels were fabricated at different concentrations, followed by their coating with the genetically engineered HUVECs. On the following day, cell viability was confirmed by the Live/Dead assay. The viability of the HUVECs coated on GelMA micro-hydrogels was up to 99% in all groups, and these cells were homogeneously coated on micro-hydrogels (Supplementary Fig. 3). The angiogenic response of HUVECs was analyzed after 7 days (Fig. 4A-C). In PCR analysis, the expression level of all genes increased with an increase in the concentration of GelMA from 2.5% to 3.5%, while the gene expression of angiopoietin-1 and VCAM-1 in 4% group was lower than that in 3.5% group. Therefore, we chose the 3.5% group for further experiments, as it showed the

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Fig. 2. Fabrication of GelMA micro-hydrogel with electrospraying method and its characterization. (A) Electrospraying of alginate and GelMA mixture to fabricate GelMA micro-hydrogels and size of micro-hydrogels (length of all scale bars = 500 mm). (B) The shape of GelMA hydrogels after chelation at different concentrations of GelMA (scale bar = 1 mm) was investigated. The GelMA hydrogels were fabricated by droplet manner using micropipette (volume 20–200 mL). Significant differences of shape were not found in varying concentrations of GelMA after chelation. (C, D) To evaluate the removal of alginate, RITC-conjugated GelMA and FITC-conjugated alginate were used. After chelation, the FITC-Alginate was fully removed from the surface of micro-hydrogel as well as from the inner-core of the micro-hydrogel while the RITC-GelMA still remained (scale bar = 500 mm).

highest level of angiogenesis-related gene expressions. To enhance the angiogenic potential to HUVECs, we transfected HUVECs with plasmids carrying VEGF-A (Supplementary Fig. 4). The level of VEGF secreted from the genetically engineered HUVECs was analyzed by ELISA (Fig. 4D, E). A total of 5  104 cells transfected with VEGF-A plasmids or unmodified HUVECs were cultured in EBMTM for 7 days, and the basal medium was collected and replaced every day. Until day 2, the secreted VEGF level substantially increased in the transfected cells than in unmodified HUVECs (mean of cumulative VEGF release: transfected HUVECs, 1244.3 pg and unmodified HUVECs, 38.1 pg). After day 2, the transfected HUVECs showed saturated VEGF release up to 1 week. The genetically engineered HUVECs coated on the GelMA micro-hydrogels were observed with the optical microscope (Fig. 4F). To evaluate whether the GelMA micro-hydrogels coated with genetically engineered HUVECs can activate the endothelial cells, we performed tube formation assay (Fig. 4G). Bead + modified cell group (GelMA micro-hydrogels coated with genetically engineered HUVECs) induced formation of the largest tubule area and the highest number of tube loops (Fig. 4H, I). In addition, we also identified that the Bead + cell group (GelMA micro-hydrogels coated with unmodified HUVECs) significantly induced tube formation of endothelial cells compared with Control bead group (GelMA micro-hydrogels). 3.4. Genetically engineered HUVEC-coated GelMA micro-hydrogels improve blood perfusion and ischemic limb salvage in a hindlimb ischemia model To evaluate whether the genetically engineered HUVEC-coated GelMA micro-hydrogels could improve neovascularization and tissue regeneration in vivo, ischemic hindlimb was induced in BALB/c nude mice. After femoral arterial excision, animals were directly

injected into the muscle with PBS (control), only GelMA microhydrogels (bead), genetically engineered HUVECs (cell), or genetically engineered HUVEC-coated GelMA micro-hydrogels (bead + cell), and blood perfusion was measured up to 4 weeks. The blood flow was almost completely blocked after arterial excision (Fig. 5A). After 4 weeks, all groups except bead + cell group displayed necrotic himdlimb due to lack of blood flow. Blood flow was observed only in bead + cell group (Fig. 5A). In control and cell-treated groups, blood flow decreased up to 4 weeks, and less than 10% recovery in blood flow was observed after 4 weeks. In the bead group, the blood flow was higher than that reported in the control and cell-treated groups, but only 20% recovery was observed after 4 weeks (Fig. 5B). On the other hand, the bead + cell group showed a significant increase in blood flow in the ischemic hindlimb as compared to other groups throughout a period of time. In addition, the bead only group showed a significant increase compared with PBS or Cell group (Fig. 5B). Especially, in the bead + cell group, after 1 week, the blood flow increased from 20% to over 40% and up to 60% recovery was reported after 4 weeks. We scored the degree of necrosis after 4 weeks. The bead + cell group showed reductions in tissue necrosis and limb loss, while the PBS-injected control group and cell group showed severe necrosis and loss of the hindlimb after 4 weeks (Fig. 5C). In the bead + cell group, only 10% of mice lost hindlimb, while 30% of mice maintained healthy limb. In the control and cell groups, hindlimb was rotten and amputated over 40% and 50% animals, respectively. In the bead group, 16.6% of animals retained the healthy limb but 25% lost hindlimbs. To assess the therapeutic effect in the bead + cell group, H&Estained muscle tissue and CD-31-positive capillaries and a-SMApositive arteries/arterioles were evaluated on day 28 after the induction of ischemia (Fig. 6A). Histological analysis revealed the presence of red blood cells in the muscle of these mice. The muscle

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Fig. 3. Physicochemical characterization of hydrogels at different concentrations of GelMA. (A) Scanning electron microscopy (SEM) images of hydrogels (scale bar = 100 lm). (B) The Young’s modulus of hydrogels derived from the stress-strain curve. (C) The swelling ratio of hydrogels. (D) To verify the success of the synthesis of the microhydrogels (GelMA), X-ray diffraction (XRD) patterns of micro-hydrogels were recorded using Cu Ka radiation source (D8 Advance, Bruker, Germany). Alginate composition was clearly removed by chelation process and the crystal structure of GelMA was retained. (E) Evaluation of degradation profiles of GelMA micro-hydrogels in varying conditions. The degradation forces of GelMA microhydrogels dominantly depended on external enzymatic activity, not the endogenic MMP from HUVECs.

tissues from the other groups showed necrotic morphology. Intramuscular injection of bead + cell significantly increased the number of CD-31-positive and a-SMA-positive cells, indicative of increased vascular densities (Fig. 6B, C). 4. Discussion Micro-hydrogel-based cell therapy aims to deliver cells locally to the trauma site, and keep therapeutic cells in that site without clearance facilitating the regeneration of impaired native tissues. This strategy offers several advantages than the other cell delivery methodologies (e.g., bolus injection) by reducing the clearance and entrapment rate of the transplanted cells in the lungs and the liver and enhancing the survival of cells at the injected site. Therefore, in this study, we developed a high-throughput system that fabricated GelMA-based micro-hydrogels to deliver genetically engineered cells for therapeutic applications. There are a few approaches for the production of micro-hydrogels in a high-throughput manner.

For instance, in microfluidic systems, the microfluidic chip was designed by 3D modeling, and the silicon-based chip was fabricated through soft lithography [12]. The microfluidic chip was precise and size controllable. However, the process of chip design and fabrication is complex and needs expensive equipment [12]. In addition, the process demands a lead-time of a few days. The emulsion approach employs agents that remove hydrophobic oils; such agents are cytotoxic and affect the stability of cell membrane [14]. To overcome these limitations, we used the electrospraying method that was modified with electrospinning. Electrospinning has been recognized as a novel method for the fabrication of homogenous micro- to nano-sized fibers with controlled morphology [35]. This method has been widely used in tissue engineering, owing to its simplicity and versatility. In the present study, we slightly modified the collector and spinning directions of electrospinning to produce micro-sized hydrogels following previous studies [13]. With simple equipment and a single spraying step, GelMA-based micro-hydrogels could be fabricated in a few minutes

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Fig. 4. Characteristics of HUVECs coated on the GelMA micro-hydrogels. (A-C) To determine the angiogenic response of HUVECs, the expressions of angiogenesis-specific genes (angiogenin: ANG, angiopoietin-1: ANGPT-1, vascular cell adhesion molecule 1: VCAM-1) were investigated at different GelMA concentrations of micro-hydrogels. The expression level of all genes was maximum in 3.5% group (w/v % of GelMA). (D) Cumulative amount of vascular endothelial growth factor (VEGF) secreted from the genetically engineered HUVECs. The amount of VEGF was quantified using an ELISA kit during a period of 7 days. (E) Release amount of VEGF at each day. The amount of VEGF secreted from engineered HUVECs was similar to unmodified HUVECs after 4 days. (F) The genetically engineered HUVECs coated on the GelMA micro-hydrogels was observed with the optical microscope (scale bar = 1000 lm [left] and 200 lm [right]). Genetically engineered HUVECs were coated on GelMA micro-hydrogels by incubating on a rotating shaker. (G) To evaluate whether the GelMA micro-hydrogels coated with genetically engineered HUVECs can activate the endothelial cells, tube formation assay was performed (scale bar = 1000 lm). Control bead group (GelMA micro-hydrogels), Bead + cell group (GelMA micro-hydrogels coated with unmodified HUVECs), Bead + modified cell group (GelMA micro-hydrogels coated with genetically engineered HUVECs). Cells were stained by Calcein-AM. (H-I) Bead + modified cell group induced formation of the largest tubule area and the highest number of tube loops. The Bead + cell group significantly induced tube formation of endothelial cells compared with Control bead group.

(Figs. 1 and 2). Alginate has easy and rapid gelation properties when mixed with divalent cationic cross-linkers such as Ca2+ [36]. The structural stability of alginate hydrogel depends on the stable existence of Ca2+ between the alginate polymer chains. Since the chelating agents such as disodium ethylenediaminetetraacetate (EDTA) or sodium citrate could spontaneously and strongly bind calcium ions, alginate hydrogel is rapidly solubilized when mixed with sodium citrate [37]. In this way, GelMA/ALG micro-hydrogels were immersed in the sodium citrate solution. As a result, solely GelMA micro-hydrogel remained and this facilitated adhesion, spreading, and proliferation of cells on the surface of micro-hydrogel. We have monitored the removal of fluorescent labeled ALG from GelMA/ALG micro-hydrogel as result of sodium citrate treatment (Fig. 2C, D). Similarly, several recent studies have utilized GelMA along with alginate as bioinks for bioprinting [38,39]. In these studies, alginate has been used to maintain of the initial bioprinted constructs via its fast ionic crosslinking property and used as a sacrificial material after EDTA treatment. Chelation of ALG with sodium citrate resulted in the formation of a homogenous micro-hydrogel with a uniform size of 500 mm. As a high-throughput system, electrospraying is suitable for the fabrication of mono-dispersed

micro-hydrogel, as it is simple, quick, cost-effective, and free of organic solvents. Even though there are numerous articles on gelatin-based microparticles as carriers for biological compounds, we believe that we present a novel biomanufacturing process to mass produce micro-sized hydrogels in uniform patterns on a high-through put manner using electro-spraying method within several minutes. In addition, we initially applied alginate to fabricate uniform micro-sized hydrogels and then we further utilized as a sacrificial component where we were able to obtain 100% gelatin-based hydrogel in the end (alginate was used to form spherical and micro-sized hydrogel). Previously, gelatin micro-particles were typically formed via water-in-oil emulsion technique. However, this method requires removal of oil, as such it could denature the growth factors and cause cell death. Our strategy to biomanufacture gelatin-based microgel is devoid use of oil, and our strategy to utilized alginate in conjunction with GelMA allowed us to manufacture GelMA micro-hydrogels with different crosslinking density (i.e., varying Alginate/GelMA ratio) to produce microhydrogels with varying properties in facile and rapid manner. The cell metabolism and fate of the transplanted cells depend on various properties such as the composition and mechanical

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Fig. 5. Effects of the genetically engineered HUVEC-coated GelMA micro-hydrogels on blood perfusion and necrosis in the ischemic hindlimb. (A) Representative blood perfusion images of mouse hindlimb injected with PBS (control), GelMA micro-hydrogel only (bead), genetically engineered HUVECs (cell), or genetically engineered HUVECcoated GelMA micro-hydrogels (bead + cell) on day 0 and 28. (B) Quantitative measurement of blood perfusion. Blood perfusion was evaluated on day 0, 7, 14, 21, and 28. LDPI ratio was measured by the ratio of ischemic to contralateral (normal) hindlimb blood perfusion during the observation period. Significant differences between groups were investigated by 2-way/ANOVA analysis (C) Quantitative analysis of the degree of necrosis on day 28.

properties of micro-hydrogels and additive stimulatory factors such as growth factors. GelMA, the key component of our system, has been demonstrated as a cell-responsive hydrogel [40]. It has been widely used for the stimulation and support of endothelial cells, as gelatin is a mixture of proteins and peptides that may induce cell adhesion and proliferation [40,41]. GelMA also may substitute native extracellular matrix (ECM), owing to the presence of matrix metalloproteinase (MMP) moieties that allow cell spreading and degradation [40]. Altogether, our fabrication system may enhance the therapeutic effects on the regeneration of vascular structures. Micro-hydrogels for cell delivery should retain their own structures and resist external stimuli from in vivo dynamic environment to maintain the biological functions of cells. In this regard, GelMA is suitable owing to its easily tunable physical characteristics. Regarding the mechanical property of hydrogels, several studies have identified its influence on activation and differentiation of cells [42,43]. Specifically, several studies have reported the importance of mechanical properties of hydrogels on activation of endothelial cells [44,45]. Furthermore, the swelling characteristics of hydrogel is related with porosities, diffusion rate of drugs and mechanical properties [46–48]. In this regard, we have analyzed the several properties such as surface morphology, mechanical property and swelling ratio of hydrogels as a result of chelation (Fig. 3). According to our analysis, the chelated samples showed more uneven surface morphology than the un-chelated samples (Fig. 3A). Young’s Modulus decreased as the result of chelation process (i.e., as a result of ALG removal). In addition, Young’s Modulus of GelMA micro-hydrogel increased in GelMA concentration dependent manner while the swelling ratio of GelMA microhydrogel decreased (Fig. 3B, C). Our in vitro cell analysis indicate that 3.5 w/v % GelMA hydrogel resulted in enhanced angiogenic response of endothelial cells (Supplementary Figs. 3 and 4A-C).

Multi-factors such as surface morphology, mechanical property and swelling characteristics may have created the optimized cellular microenvironment for angiogenic response. Then, we utilized chelated 3.5 w/v % GelMA hydrogels as cell carrier for further study. One strategy for improving the survival rate and metabolism of transplanted cells includes the incorporation of the growth factors that promote auto and paracrine signal pathways [15–18]. Among the various methodologies such as encapsulation and incorporation of protein-binding moieties to deliver growth factors, we chose genetically engineered cells that may produce the encoded growth factors. Cell-based therapies that are geared to genetically engineer the cells to secrete therapeutic proteins or soluble factors have shown improved tissue regeneration outcomes [21,22,49,50]. For effective neovascularization and tissue regeneration in the hindlimb ischemia model, we transfected HUVECs with GFP-tagged VEGF plasmid through electroporation. VEGF has been demonstrated to induce proliferation, migration, and tube formation in endothelial cells [51] and serves as a survival factor for endothelial cells both in vitro and in vivo [52,53]. The ELISA results showed that the genetically engineered HUVECs (5  104 cells/mL) secreted total of 1,416 pg of VEGF, while the unmodified HUVECs secreted 107.0 pg of VEGF until day 7 (mean of cumulative VEGF release) in vitro (Fig. 4D). Our results indicate that the genetically engineered HUVEC maintained high VEGF production rate until 4 days, and this rate of VEGF production was reduced to the normal amount that is comparable to the unmodified HUVECs (Fig. 4E). The results of flow cytometry analysis showed that 1.42  105 of 1  106 HUVECs were genetically modified (Supplementary Fig. 4). For the in vivo study, genetically engineered HUVECs were coated on GelMA micro-hydrogels for a day. These hydrogels were injected at the ischemic site. Intramuscular injection of GelMA micro-hydrogels coated with genetically engineered HUVECs

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Fig. 6. Effects of genetically engineered HUVEC-coated GelMA micro-hydrogels on neovascularization in the ischemic hindlimb. (A) Hematoxylin and eosin staining of ischemic muscle tissue and immunostaining of CD-31-positive microvessels (red) or a-SMA-positive blood vessels (green) in ischemic muscle tissue. Nuclei (blue) were stained with DAPI (scale bar: 200 mm). (B) Quantitative analysis of microvessels, expressed as the number of CD-31-positive cells. (C) Quantitative analysis of functional blood vessels, expressed as the number of a-SMA-positive vessels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

resulted in the recovery of blood perfusion, reduction in tissue necrosis, and neovascularization at the treatment site (ischemic hindlimb) (Figs. 5 and 6). In previous studies, gelatin was shown to provide cell-binding moieties to support efficient cell adhesion. In addition, gelatin-based scaffolds respond to cell-secreted proteolytic enzymes such as collagenase [54–57]. In our study, we initially demonstrated that HUVECs on GelMA micro-hydrogels can exhibit angiogenic activities in vitro, consistent with the results of the previous studies [24,32,58]. Similarly, HUVECs encoding VEGF gene, carried on the micro-hydrogels, stably survived and facilitated the release of VEGF in vivo. After 1 week of intramuscular injection, the bead + cell group showed recovery in blood perfu-

sion with the assistance of the released VEGF. It was suggested that the transplanted HUVECs stably released VEGF, which stimulated the innate endothelial cells to allow the recovery of the ischemic tissue. These therapeutic effects prolonged until 4 weeks and prevented hindlimb necrosis. In comparison with the other groups, the bead + cell group showed mature vessels and minor necrosis in the muscle tissues. Furthermore, this group recruited native smooth muscle cells to stabilize the newly created vessels and showed recovery of ischemic tissue in a shorter time as compared to other groups. In previous studies, intramuscular injection of only cells resulted in poor cell viability, as these cells were not protected from the in vivo environment [59,60]. In the group treated with

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cells, blood perfusion decreased until 4 weeks of intramuscular injection, consistent with the observation in the PBS-treated group; approximately 50% mice showed limb necrosis. The treatment with GelMA bead, on the other hand, showed therapeutic effects. GelMA has the innate potential to enhance cell metabolic activity, owing to the presence of the cell-responsive moieties [55,61]. It may be suggested that in the presence of only GelMA micro-hydrogels, the native cells in the ischemic environment would show enhanced migration and proliferation abilities through gelatin and increase the paracrine effects on the neighboring cells. Moreover, gelatin-based hydrogels are used in a clinical trial for peripheral ischemia disease, as these gels undergo degradation in vivo and show cell-responsive moieties [54,62]. Furthermore, recently, Griffin et al, have devised microporous gel scaffold composed of microgels for acceleration of wound healing [63]. They have demonstrated that the scaffolds composed of microgels induced low level of immune response, and facilitated endogenous cell migration and tissue regeneration. We hypothesize that injected micro-hydrogel may have provided template where surrounding cells could migrate and engage in tissue remodeling process. These results indicate that HUVECs with GelMA micro-hydrogels showed a synergic therapeutic effect in ischemic tissues to support HUVEC metabolic activity and stimulate and recruit the innate smooth muscle cells to stabilize angiogenic responses. 5. Conclusion We fabricated a GelMA-based micro-hydrogel using an electrospraying method and confirmed its therapeutic effect as an endothelial cell carrier for neovascularization in a mouse hindlimb ischemia model. GelMA micro-hydrogels were easily produced with simple chelating of ALG. GelMA micro-hydrogels had stable mechanical properties and were resistant to external stimuli in vivo. These hydrogels supported the angiogenic activity of endothelial cells. To maximize the angiogenic effect, we encoded the VEGF gene in endothelial cells to facilitate VEGF protein overexpression. The optimal ratio of GelMA to ALG was determined to maximize neovascularization. This combination effectively recovered the blood flow and reduced tissue necrosis and limb loss until 4 weeks in the mouse hindlimb ischemia model. It was demonstrated that GelMA-based cell carriers showed therapeutic effects in the diseased model and exhibited robust potential in translational medicine for the treatment of ischemia. Acknowledgements This work was supported by the Ministry of Science, ICT and Future Planning, Republic of Korea (NRF-2016R1E1A1A01943393 and NRF-2017M3A9C6031786). The Institute of Engineering Research at Seoul National University provided research facilities for this work. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1277). Disclosure The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.actbio.2019.01.057.

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