Heparin Functionalized Injectable Cryogel with Rapid Shape

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Heparin Functionalized Injectable Cryogel with Rapid Shape-Recovery Property for Neovascularization Inseon Kim, Seunghun S. Lee, Sunghoon Bae, Hoyon Lee, and Nathaniel S. Hwang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00331 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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Biomacromolecules

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Heparin Functionalized Injectable Cryogel with

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Rapid Shape-Recovery Property for

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Neovascularization

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Inseon Kim1, ‡, Seunghun S. Lee2, ‡, Sunghoon Bae1, Hoyon Lee1 and Nathaniel S. Hwang1, 2, 3*

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National University, Seoul, 08826, Republic of Korea

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Republic of Korea.

School of Chemical and Biological Engineering, the Institute of Chemical Processes, Seoul

Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826,

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‡These authors contributed equally.

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*Corresponding author

BioMAX/N-Bio Institute, Seoul National University, Seoul, 08826, Republic of Korea

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ACS Paragon Plus Environment

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Cryogel based scaffolds have high porosity with interconnected macropores that may provide

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cell compatible microenvironment.

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minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and

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injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a

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carrier for vascular endothelial growth factor (VEGF) and fibroblasts in hindlimb ischemic

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disease. Our gelatin/heparin cryogel showed gelatin concentration-dependent mechanical

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properties, swelling ratios, interconnected porosities, and elasticities. In addition, controlled

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release of VEGF led to an effective angiogenic responses both in vitro and in vivo. Furthermore,

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its sponge-like properties enabled cryogels to be applied as an injectable carrier system for in

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vivo cells and growth factor delivery. Our heparin functionalized injectable cryogel facilitated

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the angiogenic potential by facilitating neovascularization in hindlimb ischemia model.

In addition, cryogel based scaffolds can be utilized in

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Keywords: heparin, cryogel, injectable, VEGF, neovascularization

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1. Introduction

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Cryogel based scaffolds are characterized as hydrogels with high porosity and interconnected

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macropores.1, 2 Due to its highly interconnected and macroporous structure, cryogels provide

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cell compatible microenvironment that promotes cell proliferation and host cell infiltrating into

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the cryogel upon implantation.3-8 In addition, cryogels have been investigated for drug or

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therapeutic protein carriers for a site-specific release.9 However, unintended and uncontrolled

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burst release of the therapeutic agents from cryogels have been considered as a major challenge

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due to their high porosity.9, 10

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For controlled release of growth factors from cryogels, several biomaterials moieties can

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be incorporated in cryogels for sustained release of growth factors 11. For example, heparin is a

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sulfated glycosaminoglycan of the extracellular matrix (ECM), and it has been widely used in

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protein delivery due to its affinity to several growth factors. Vascular endothelial growth factor

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(VEGF),12-14 bone morphogenic protein-2 (BMP-2),15-17 and basic fibroblast growth factor

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(bFGF) have heparin binding domain that allow specific affinity with heparin functionalized

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biomaterials.18-20 This characteristic capacity of heparin helps to minimize the undesirable burst

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release of proteins from heparin functionalized cryogels, which improve therapeutic effect by

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controlled release of the therapeutic proteins for a long period. In particular, heparin induced

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controlled release of VEGF has been reported as a promising method to promote vascularization

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in ischemic hind limb model.21-23

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In recent years, injectable biomaterials are in high demand in regenerative medicine

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owing to the need for minimally invasive surgery.24 However, injectable biomaterials based on

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hydrogel with small pores inhibit cell infiltration into the hydrogel, leading to an ineffective

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tissue regeneration.25 In contrast, spongy-like injectable cryogel with uniformly formed


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macropores may promote effective cellular proliferation and host cell infiltration. In addition, the

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use of injectable cryogel prevents tissue injury from remained crosslinking reagents in the

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hydrogel because remained crosslinking reagents will be released from swelling and drying

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process prior actual usage

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injectable cryogel system, including dibenzaldehyde-terminated poly (ethylene glycol) (PEG-

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DA) 27, methacrylated gelatin (Gel-MA) 28 and hyaluronic acid (HA).29

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. Following this trend, numerous macromolecules were utilized in

In this study, we have developed an injectable heparin/gelatin based cryogel for the

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treatment of in vivo hind-limb ischemia model.

Furthermore, cryogels were designed and

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fabricated to have (i) optimal VEGF release profile for functional angiogenesis and (ⅱ) optimal

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injectability with viable cells.

Finally, we applied this cryogel to in vivo hind-limb ischemic

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mouse model and demonstrate the recovery of blood flow and vascularization through the

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necrotic muscle tissue.

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2. Experimental Section

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2.1 Preparation of gelatin-heparin cryogels

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Type A gelatin (from porcine skin, Sigma-Aldrich, G2500) and heparin (Merck Millipore,

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375095) were respectively dissolved in deionized water and mixed together at each final

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concentration of 1, 1.5 % (w/v) gelatin and 0.1, 0.3, 0.5 % (w/v) heparin. 50mM of 1-Ethyl-3-(3-

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dimethylaminopropyl)carbodiimide (EDC) (Thermo Scientific, PG82073) and 25mM of sulfo-

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Hydroxysuccinimide (NHS) (Thermo Scientific, PG82071) dissolved in deionized water were

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respectively added into the gelatin/heparin solution as crosslinking reagents. After mixing all the

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compounds, 85µL of the mixed solution was immediately pulled on pre-cooled cryogel mold and

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placed on -20℃ overnight for cryogelation. During cryogelation, ice crystals were formed and

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crosslinking reaction between gelatin and heparin was processed in the entire regions except for

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ice crystals. When the cryogelation finished, scaffolds were lyophilized overnight and stored at -

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20℃. All the followed experiments were processed with 5mm diameter, 3mm height cylindrical

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cryogels.

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2.2 Characterization of gelatin-heparin cryogels

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For scanning electron microscopic (SEM) images, each cryogels dehydrated at least for 24hrs

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fixed onto the stubs using carbon tape and coated with platinum scatter. Microstructure and pores

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of the cryogels were visualized at 10kV with a microscope (Hitachi S3500-N, Hitachi Science

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Systems Ltd., Tokyo, Japan). The area of pores was quantified by ImageJ. For swelling property,

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lyophilized cryogels were weighed on a scale and incubated in PBS at RT for 24hrs. When

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cryogels were fully swelled, the weights were measured and the swelling ratio of each cryogels

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was calculated by the followed equation: Swelling ratio = (Ws-Wi)/Wi , where Wi means the


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initial weight of lyophilized cryogel and Ws means the weight of fully swelled cryogel. The pore

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connectivity analysis was examined as previously described by Bencherif et al.

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cryogels were dehydrated on kimwipe until the water was completely removed away. The

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percentage of interconnected porosity was calculated by the following equation: %

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interconnected porosity = (Ws-Wde)/Ws, where Wde means the weight of dehydrated cryogels on

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kimwipe. For the mechanical property, compression test was carried out using universal-testing-

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machine (Shimadzu EZ-SX, Japan) with fully swelled cryogels. Gels were compressed with the

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loading rate of 1mm/min. The result was obtained by the stress-strain curve and Young's

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modulus was calculated by the measurement of the slope linearly increased in the region of the

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Swelled

stress-strain curve.

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For enzymatic degradation test, each scaffold was incubated in 1 unit/mL or 20 unit/mL

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of Type ℃ collagenase (Worthington, LS004176) solution at 37℃. Collagenase solution was

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replaced with fresh solution every 2-3 days. The degree of gel maintenance was calculated by the

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percentage of the weight of remained cryogel to initial weight of the fully swelled cryogel.

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2.3 Rheology test

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Rheological behavior test was carried out in Demo lab (Anton Paar Korea) using rheometer

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(MCR 302, Measuring cell: P-PTD & H-PTD 200, Measuring System: PP 25, Anton-Paar,

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Austria). All the cryogels used in experiments had 8mm diameter and 2mm thickness cylindrical

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shape. Firstly, amplitude sweep (strain sweep) test was applied from 0.01 to 100% at the

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controlled frequency of 1Hz to determine the rheological change related to the shear stain.

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Secondly, frequency sweep measurement was performed from 0.01 to 100Hz at a constant strain

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of 0.2% to investigate the modulus change related to the oscillatory frequency. Finally,


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continuous step strain was measured by alternating 0.2% and 25% for 100s at a constant

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frequency of 1Hz to confirm the shape recovery behavior.

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2.4 Heparin detection via alcian blue assay

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Unreacted heparin during cryogelation was detected by alcian blue assay as followed under the

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modified standard protocol.31 Firstly, after cryogels swelled in PBS for 24hrs, the PBS was

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transferred to 1.5mL tube. 20µL of collected PBS was mixed with 0.05% (w/v) alcian blue 8GX

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(Sigma-Aldrich, A5268) in DW. After 3hrs incubation at RT, the precipitate of heparin was

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formed and collected by centrifuging at 10,000rpm for 10min as a pellet. The supernatant was

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removed and the pellet was washed with 500µL of 99.9% ethanol under centrifugation. After

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that, ethanol was completely aspirated and the pellet was dissolved in 500µL of 7.5% (w/v)

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sodium dodecyl sulfate (SDS) (Bio-Rad, 1610302) solution for an hour at 60℃. The absorbance

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of the dissolved solution was detected at 678nm and then, analyzed by the standard curve with 0-

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0.25mg/µL of heparin solution.

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2.5 Synthesis of RITC-heparin-conjugated cryogel

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Rhodamine B isothiocyanate (RITC) conjugated heparin was synthesized by adding 1mg of

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RITC (Sigma-Aldrich, R1755) dissolved in 0.1mL of Dimethyl sulfoxide (DMSO) (Sigma-

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Aldrich, M81802) to 2% (w/v) heparin solution for 1hr at 60℃. After the reaction, RITC-heparin

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was placed in 3.5K MW dialysis tube and dialyzed in distilled water for 3days to remove

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unbound RITC. Dialysis water was freshly replaced every day. In the last day, RITC-heparin was

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freeze-dried and used for the fabrication of cryogels as described in 2.1. The microstructure of


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RITC-conjugated cryogel was visualized with a confocal microscope (Olympus FluoView

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FV100).

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2.6 Heparin mediated protein release kinetics

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For the observation of heparin-mediated sustained release of VEGF, 30µL of 0.01% (w/v) FITC-

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BSA (Sigma-Aldrich, A9771) or 100ng/mL human VEGF (Pepro tech, 100-20) was soaked to

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each dehydrated cryogels. After 5min to bind proteins to heparin, 500µL of PBS was added to

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the gels and the PBS was collected at each time point. The amount of released FITC-BSA was

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measured by fluorescence spectroscopy at excitation wavelength 490nm, emission wavelength

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525nm. The amount of released VEGF was measured by Human VEGF ELISA Kit

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(KOMABIOTECH, K0331132).

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2.7 HUVEC migration and tube formation assay

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Firstly, 1x104 Human Umbilical Vein Endothelial Cells (HUVEC) were seeded in gelatin coated

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24-well plate. Then, HUVECs were primed in daily collected assay medium (EBM-2 with 10%

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FBS containing VEGF released from cryogels) for a week. For wound migration assay, 100%

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confluent HUVECs were scratched by 1000µL pipet tip, which made vertical empty space. Cells

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were washed with PBS and incubated in the assay medium. After 2days, the cells were fixed in

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4% PFA for 10min and stained with DAPI. The rate of migration healing was quantified by

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measuring the gap distance compared to the beginning interval. For tube formation assay, 150µL

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of BD matrigelTM matrix (BD Biosciences, 354234) in ice was transferred to a pre-cooled 48-

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well plate and incubated for 30min at 37℃. 2.5x105 VEGF primed HUVECs were trypsinized,


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Biomacromolecules

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suspended in 125µL of assay medium and seeded onto the matrigel. After 8hrs incubation, cells

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were visualized using a light microscope.

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2.8 Injectability test

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To confirm the injectability of the scaffolds, cryogels were loaded in 5ml syringe with 1mL of

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PBS and injected through the 17G needle to 50mL tubes. The remained cryogels after injection

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were weighed and the degree of weight loss was calculated by the ratio of lost weight to initial

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weight of cryogels. For the degree of FITC-BSA retention, 0.01 % (w/v) FITC-BSA loaded

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cryogels were utilized. Original cryogels and remained gels after injection was completely

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digested in Papain (Worthington, LS003127) for 16hrs at 60℃. The digested solution was

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analyzed using fluorescence spectroscopy at excitation wavelength 490nm, emission wavelength

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525nm. The ratio was calculated by the same method of weight retention.

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2.9 Preparation of cell-seeded scaffolds

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Freeze-dried cryogels were swelled in PBS for 24hrs to remove excess EDC. Swelled cryogels

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were washed 2 more times in PBS and sterilized by UV irradiation for 3hrs. Then 5x104 NIH-

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3T3 fibroblast cells, mouse embryo fibroblasts, were suspended in 30µL of growth medium

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(DMEM, 10% FBS, 1% L-glutamate, 1% Pen-strep). Cell solution was soaked to the sterile

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dehydrated cryogel and incubated at 37℃ for 2-3hrs for cell attachment. After that, 300µL of

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fresh medium was added to the cryogels to provide sufficient nutrients for cell growth.

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2.10 PicoGreen and live/dead assay


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For the confirmation of the cell number preserved in the scaffold after injection, injected cell-

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seeded scaffolds were degraded by papain digestion as described in 2.8. Remained cells were

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quantified with PicoGreen™ dsDNA Assay Kit (Invitrogen, P11496). This method was also

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applied to original cell-seeded scaffolds as a control. For the viability test, cell-seeded scaffolds

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before, after injection were stained for 30min in 0.5µL/mL of calcein-AM and 2µL/mL of

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ethidium homodimer-1 (Eth-1) from LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian

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cells (Invitrogen, L3224). GFP positive live cells and RFP positive dead cells were visualized

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with a confocal microscope and fluorescent cells were counted for the quantification of the cell

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viability.

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2.11 In vivo ischemic hindlimb mouse model

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Balb/c nude mice (female, 6-weeks) was used for the animal experiments with the protocols

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approved by the Seoul National University. All the cryogels utilized for in vivo experiments was

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G1H0.3. In this study, 5 types of experimental groups were developed as follows: sham cryogels

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(Control), an acellular cryogel (Gel), cryogel with 100ng/mL VEGF (VEGF), cryogel with 1x106

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NIH-3T3 cells (NIH) and cryogels with 1x106 NIH-3T3 cells and 100ng/mL VEGF

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(NIH_VEGF). The ischemic hind limb was induced on the left leg excising three vessels

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followed by the reference.32 After the development of the animal model, cryogels were injected

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into the surgical region through 17G syringe needle with sterile PBS. Blood perfusion was

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evaluated using a laser Doppler perfusion imaging (LDPI) system (Moor instrument, Devon,

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UK) right after surgery and every week for 28days to observe the recovery rate of the blood. The

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rate of blood flow recovery was expressed as the LDPI ratio of ischemic to non-ischemic hind

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limb blood perfusion. And a limb necrosis was scored as the following scale: entire recovery


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(score 6), minor necrosis or nail loss (score 5), partial toe amputation (score 4), total toe

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amputation (score 3), partial/total foot amputation (score 2), or partial/total limb amputation

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(score 1).33

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2.12 OCT embedding

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Muscle tissues or ex vivo cryogels collected after in vivo study were embedded in OCT (Sakura

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Fineteck) to determine the degree of angiogenesis via histology and immunostaining. Firstly,

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tissues or gels were fixed in 4% PFA at 4℃ overnight. Then, samples washed twice with PBS

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and stored in 20 % (w/v) sucrose solution overnight to maintain the structures. In addition,

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sucrose solution was replaced to OCT/ sucrose=1:1 solution and placed at 4℃ overnight. Finally,

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the cross-sectioned area of the samples was located in the bottom of a cryomold and filled with

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OCT. After removing bubbles, the cryomold was frozen in the liquid nitrogen. Constructed

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cryoblocks were stored at -80℃. For the histology or immunostaining, samples were

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cryosectioned at 7µm thickness.

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2.13 Histology & immunostaining

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Cryosectioned samples were washed 3 times with PBS containing 1% (w/v) BSA (BSA/PBS) to

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remove OCT. For H&E staining, samples were stained with hematoxylin (Sigma-Aldrich,

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HHS16) for 2min and then, washed in the running water for 5 min. If hematoxylin was

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overstained, washed with 1% HCl in 70% EtOH for the 30s. Finally, samples were stained with

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eosin (Sigma-Aldrich, HT110332) for 1min and washed with 95% EtOH 4 times for 1min in

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series. Stained samples were mounted with Canada balsam (Sigma-Aldrich, C1795) in xylene.

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For immunofluorescence staining, samples were permeabilized in the blocking solution based on


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PBS containing 1% (w/v) BSA, 10% normal goat serum and 0.1% Triton X-100 (Sigma-Aldrich,

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X100) for 45min at RT and then, incubated in primary Rat Anti-Mouse CD31 (BD Biosciences,

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550274) diluted to 1:40 in the blocking solution overnight at 4℃. The samples were washed 3

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times with BSA/PBS. 1:500 diluted 594-conjugated secondary antibody in BSA/PBS was

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applied to the samples for 2hrs at RT in the dark. The samples were washed 3 times and stained

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with 1:200 diluted DAPI for 10min. Fluorescence was visualized by EVOS FL Imaging System

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AMF4300 (Life Technologies).

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Biomacromolecules

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3. Results and discussion

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3.1

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Gelatin has sufficient primary amine residues (e.g. arginine, histidine, and lysine) which can be

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conjugated with carboxyl groups on heparin polysaccharide chain via EDC/NHS reaction,

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resulting in the formation of amide cryogels by a covalent conjugation.34 We performed the

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gelatin/heparin cyrogel via mixing the solutions and EDC/NHS crosslinking reactions at -20 ℃.

Fabrication of gelatin/heparin cryogel

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In the process of cryogelation, macroporous structures within cryogels were formed by

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ice crystals that were formed. Furthermore, completely frozen cryogels were then lyophilized

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prior to the cell seeding and cryogel characterizations (Figure. 1A). We confirmed the EDC/NHS

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reaction during sub-zero degree condition (i.e., formation of amide groups between gelatin and

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heparin), by FT-IR analysis (Figure. S1). In FT-IR analysis, a single peak of amide A at 3300cm-

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1

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1550cm-1 attributed to the bending frequency of N-H conjugated with C-O was observed with

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amide I peak around 1630cm-1 which also indicated stretching vibrations of the C-O in amide

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groups. All these peaks indicated successful conjugation of gelatin and heparin forming amide

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groups via EDC/NHS crosslinking reaction during cryogelation process.

presented the stretching frequency of N-H in amide groups. A strong amide II peak around

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Figure 1. Fabrication and experimental strategy of gelatin/heparin cryogel. (A) Schematic illustration of gelatin/heparin cryogel developing procedure. Gelatin was crosslinked with heparin under EDC/NHS mediated cryogelation process. (B) Photographs of gelatin/heparin cryogels fabricated with the combination of 1, 1.5% (w/v) gelatin (G1, G1.5) and 0.1, 0.3, 0.5% (w/v) heparin (H0.1, H0.3, H0.5). Lyophilized gelatin/heparin cryogels were fully swelled in PBS for 24hrs (right) and dehydrated on the kimwipes (left).

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In this study, we fabricated 6 different types of cryogels with varying amount of gelatin

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and heparin concentration as depicted Table 1. As shown in Figure. 1B, cryogels based on 1%

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gelatin (G1 cryogels) were more transparent than cryogels based on 1.5% gelatin (G1.5 cryogels)

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when completely swollen in PBS for 24hrs (right). In addition, G1 cryogels had a skinnier shape

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when mechanically dehydrated on kimwipe (left) showing a more sponge-like property.

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Interestingly, cryogels synthesized without heparin displayed a crumbled shape and different

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property compared to the heparin incorporated cryogels (data was not shown). Although Type A

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gelatin can be polymerized without heparin, because gelatin has 0.077 ratio of carboxyl groups to

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amine groups, the crosslinking efficiency is less than 26% in lower EDC, NHS concentration

35,


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36

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reaction for gelation of our injectable cryogel system.

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. As a result, heparin is a key material as it enables to build up carboxyl acids for crosslinking

Table 1. Weight/volume (w/v) percent composition of each gelatin/heparin cryogel.

Sample name Gelatin % (w/v) Heparin % (w/v)

G1 cryogels G1H0.1 G1H0.3 1 1 0.1

0.3

G1H0.5 1

G1.5 cryogels G1.5H0.1 G1.5H0.3 1.5 1.5

G1.5H0.5 1.5

0.5

0.1

0.5

0.3

4 5

3.2

Characterization of gelatin/heparin cryogels

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To analyze the microarchitecture of gelatin/heparin cryogels, cross-sectional areas of

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lyophilized cryogels were visualized by scanning electron microscopy (SEM). All cryogels

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showed monodisperse structures with interconnected pores in a whole area, indicating uniformly

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constructed network by conjugating heparin to gelatin in the slow crosslinking reaction at a low

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temperature (Figure. 2A). In addition, the pore area was calculated from SEM images of cross

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sections of each crygels via ImageJ program following the method from previous paper. 7 There

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was a trend of smaller average pore area as the centration of gelatin and heparin increased due to

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the higher degree of crosslinking between gelatin and heparin; however, the difference between

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each samples were not significant due to the limited varying amount of gelatin and heparin

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utilized in our study (Figure. S2). As shown in the figure S2, all cryogels had over 2000 um2 of

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average pores. This is probably due to the similar crygelation conditioned applied for all the

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samples. For example, even though each cryogel sample had varying amount of gelatin and

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heparin, they were all crosslinked under the same freezing condition. This condition may have

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contributed to the similar degree of ice crystal and macropore structure formation.


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Cryogels are interconnected supermacroporous gels prepared at sub-zero temperatures

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having applications in various research fields. The process of cryogelation is ideally thought to

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take place via following steps: phase separation with ice-crystal formation, cross-linking and

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polymerization followed by thawing of ice-crystals to form an interconnected porous cryogel

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network. Thus, factors such as the concentration of reactants and nonfrozen liquid microphase

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are considered to be important for cryogelation.37, 38 Among them, the condition of sub-zero

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temperature is crucial for cryogels since the reaction rate affects the porosity. Lozinsky et al.

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reported that decrease in temperature lowers the mobility of the solutes which decelerates the

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reaction by Arrhenius principle.37 Therefore, since the cryogelation proceeds much faster than

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the usual reaction at elevated temperature, the EDC/NHS chemistry mediated crosslinking of

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heparin to gelatin was conducted in sub-zero temperature in order to synthesize the injectable

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cryogel with optimized porosity.


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Biomacromolecules

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Figure 2. Characterization of gelatin-heparin cryogels with various combination of gelatin and heparin concentration. (A) Representative SEM images of microstructure of macroporous cryogels (n=3). (B) Swelling ratio (n=3), (C) Interconnected porosity (n=3) and (D) Young’s modulus (n=3) of cryogels. (E) An enzymatic degradation test in 1 unit/ml or 20 unit/ml collagenase solution (n=3). All groups were applied to 500µL of collagenase freshly replaced every 2-3 days. Error bars indicate SD. Next, we monitored swelling ratio and interconnected porosity of swollen gelatin/heparin

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cryogels. First, for the swelling ratio, G1 cryogels showed higher swelling ratio than G1.5

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cryogels except the H0.1 group (Figure. 2B). In addition, G1 cryogels showed higher


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interconnected porosity than G1.5 cryogels (Figure. 2C). Interestingly, as heparin concentration

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increased, swelling ratio and interconnected porosity increased in G1 cryogels while both

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properties decreased in G1.5 cryogels. However, there was no significant difference among the

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cryogels in G1 cryogels. Besides, in the mechanical compression test, G1 cryogels exhibited a

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lower Young's modulus than G1.5 cryogels did (Figure. 2D). This indicates that G1.5 cryogels

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are mechanically stronger and able to endure higher strain against the structure deformation than

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G1 cryogels are.

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From these investigations, we found that various properties including swelling property,

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porosity and mechanical property were determined by gelatin concentration, which means the

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extent of crosslinking was determined by gelatin concentration.39 Due to the lower crosslinking

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extent, G1 cryogels had a mesh structure with larger pores than G1.5 cryogels, resulting in higher

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swelling ratio, interconnectivity, and lower modulus. In addition, an increase of the heparin

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concentration in G1.5 cryogels causes much chemical conjugation of heparin to gelatin, leading

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to lower swelling ratio, interconnected porosity, and higher Young's modulus. This phenomenon

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can be explained from the higher extent of crosslinking in H0.5 cryogels which lead to the

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thicker rigid polymer network that shows lower water uptake than empty pore space.40

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Additionally, other several factors have contributed to the overall crosslinking density of the

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cryogel. This chemistry not only contributed to the conjugation of heparin to gelatin, it also has

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contributed to the intermolecular crosslinking of gelatin which would increase the Young’s

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modulus. Therefore, we measured not only the physical crosslinking formed by gelatin, but also

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covalent intermolecular crosslinking formed due to the EDC/NHS mediated intermolecular

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crosslinking.


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Biomacromolecules

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To observe scaffold stability in enzyme solution, in vitro enzymatic degradation test was

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performed by incubating cryogels in two different concentrations of collagenase solution. When

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1 unit/mL collagenase was applied, the weight of G1 cryogels remained over 80% until 36 days

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and in G1.5 cryogels, over 90% left due to a higher degree of crosslinking of G1.5 cryogels

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(Figure. 2E). Similarly, in 20 unit/mL collagenase solution, G1 cryogels was slightly more

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degraded than in 1unit/mL, but still, over 70% remained. This indicates that our gelatin/heparin

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cryogels are able to maintain their structure with high stability in enzyme solution for a long

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period against the enzymatic hydrolysis even in high enzyme concentration.

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3.3

Rheological properties of gelatin/heparin cryogels

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We also characterized our GHs by investigating the rheological properties to confirm the

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dimensional stability of the inner network. In the rheological study, viscoelastic solids display

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higher storage modulus (G’) than loss modulus (G’’), while viscoelastic liquids have higher G’’

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than G’. Therefore, chemically cross-linked cryogels exhibited G’>G’’. In the frequency sweep,

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G1.5 cryogels showed higher G’ than G1 cryogels, indicating that G1.5 cryogels had higher

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degree of crosslinking with a stiffer property (Figure. 3A). In addition, as the frequency

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increases, G’ and G’’ of both G1 and G1.5 cryogels increased as well which indicate the

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relaxation process of cryogel. In the Figure. 3B, the strain sweep test shows that G1.5 cryogels

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maintained the stable network against a deformation showing stable G’ and G’’ below 2% of

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strain, while structural deformation was detected above 30% of strain which is resulted from the

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crossover of G’ and G’’. In contrast, G1 cryogels preserved the balanced network in the wider

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range of shear strain (

Heparin Functionalized Injectable Cryogel with Rapid Shape

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