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Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects Hyun-Ji Park, Yoonhee Jin, Jisoo Shin, Kisuk Yang, Changhyun Lee, Hee Seok Yang, and Seung-Woo Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01670 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects Hyun-Ji Park,1‡ Yoonhee Jin,1‡ Jisoo Shin,1‡ Kisuk Yang,1 Changhyun Lee,1 Hee Seok Yang,2 Seung-Woo Cho1,3*

1

Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea

2

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for

Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea 3

Department of Neurosurgery, Yonsei University College of Medicine, Seoul 120-752,

Republic of Korea

*Corresponding author: Prof. Seung-Woo Cho, Ph.D. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea Tel: +82-2-2123-5662; Fax: +82-2-362-7265; E-mail: [email protected]

KEYWORDS: hyaluronic acid hydrogel, catechol, stem cell transplantation, angiogenesis, bone reconstruction 1 ACS Paragon Plus Environment

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ABSTRACT

Over the last few decades, stem cell therapies have been highlighted for their potential to heal damaged tissue and aid in tissue reconstruction. However, materials used to deliver and support implanted cells often display limited efficacy, which has resulted in delaying translation of stem cell therapies into the clinic. In our previous work, we developed a musselinspired, catechol-functionalized hyaluronic acid (HA-CA) hydrogel that enabled effective cell transplantation due to its improved biocompatibility and strong tissue adhesiveness. The present study was performed to further expand the utility of HA-CA hydrogels for use in stem cell therapies to treat more clinically relevant tissue defect models. Specifically, we utilized HA-CA hydrogels to potentiate stem cell-mediated angiogenesis and osteogenesis in two tissue defect models: critical limb ischemia and critical-sized calvarial bone defect. HA-CA hydrogels were found to be less cytotoxic to human adipose-derived stem cells (hADSCs) in vitro compared to conventional photopolymerized HA hydrogels. HA-CA hydrogels also retained the angiogenic functionality of hADSCs and supported osteogenic differentiation of hADSCs. Due to their superior tissue adhesiveness, HA-CA hydrogels were able to mediate efficient engraftment of hADSCs into the defect regions. When compared to photopolymerized HA hydrogels, HA-CA hydrogels significantly enhanced hADSC-mediated therapeutic angiogenesis (promoted capillary/arteriole formation, improved vascular perfusion, attenuated ischemic muscle degeneration/fibrosis, and reduced limb amputation) and bone reconstruction (mineralized bone formation, enhanced osteogenic marker expression, and collagen deposition). This study proves the feasibility of using bio-inspired HA-CA hydrogels as functional biomaterials for improved tissue regeneration in critical tissue defects.

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INTRODUCTION

Hydrogels show great promise as biomaterial platforms for tissue engineering, cell therapy, and drug delivery. Hydrogels generally display high permeability to oxygen, nutrients, and water-soluble biofactors, and they induce beneficial tissue responses dependent upon their physical and chemical properties.1 Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan present in vertebrate tissue and body fluid that is known to interact with cell surface receptors including CD44, ICAM-1, and RHAMM.2-4 HA-receptor binding triggers intracellular signals that influence cellular proliferation, differentiation, migration, and survival.5-7 Due to these advantages, HA hydrogels are commonly utilized as regenerative and drug delivery scaffolds.5, 8 HA hydrogels have also shown applicability as culture substrates and tissue-engineered scaffolds for various types of stem cells including embryonic stem cells, mesenchymal stem cells, and neural stem cells.9-13 Currently available HA hydrogel systems often cause significant biocompatibility issues, limiting their therapeutic potential.14 Specifically, the chemical and physical crosslinking methods used to create HA hydrogels with HA conjugates containing functional groups such as methacrylate, aldehyde, and thiols damage cells during encapsulation and cause cytotoxicity.14-16 For example, HA-methacrylate (HA-ME) hydrogels are prepared by photopolymerization in the presence of a photoinitiator using ultraviolet (UV) irradiation. Although photopolymerization is a widely used technique for gelation, free radicals generated during the process can cause unwanted cellular damage directly, by reacting with cellular components, or indirectly, via formation of reactive oxygen species.17-19 An alternative crosslinking method is vital for production of highly biocompatible HA hydrogels. Functionalization of biopolymers with a catechol moiety, as observed in mussel adhesion, has been used to improve biocompatibility and enhance adhesive properties of 3 ACS Paragon Plus Environment

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three-dimensional (3D) hydrogels. Adhesive proteins in marine mussels, Mytilus edulis, contain repeated catecholic amino acids, 3,4-dihydroxy-L-phenylalanine (L-DOPA) and lysine residues.20, 21 L-DOPA possesses redox activity that allows oxidative crosslinking of 3D hydrogels.22-24 The pH-induced oxidation of the catechol group of dopamine, a catecholamine containing the catechol group of L-DOPA and the amine group of lysine, results in transition of catechol into o-quinone, which subsequently forms catechol–catechol adducts.25 We previously demonstrated that HA-catechol (HA-CA) hydrogels form via oxidative crosslinking of dopamine-conjugated HA and show high biocompatibility both in vitro and in vivo.26, 27 In our previous studies, HA-CA hydrogels did not induce cytotoxicity in a range of cell types and we successfully utilized them for minimally invasive cell transplantation studies.26, 27 HA-CA conjugate was also applied for polymer surface coating to enhance cell adhesion and proliferation.27, 28 In the present study, we further expand upon the utility of HA-CA hydrogels to test their efficacy in critical tissue defect models which have high clinical relevance. To this end, HA-CA hydrogels containing human adipose-derived stem cells (hADSCs) were transplanted into mouse hindlimb ischemia and critical-sized bone defect models. The improved biocompatibility and strong tissue adhesiveness of HA-CA hydrogels allowed them to promote engraftment of stem cells into the defect regions and significantly enhance angiogenesis and osteogenesis of the transplanted stem cells at the site of the defect.

EXPERIMENTAL SECTION

Synthesis and Gelation of the HA-CA Conjugate. HA-CA was synthesized chemically by conjugating dopamine hydrochloride (Sigma, St. Louis, MO, USA) to HA (MW = 200 kDa, Lifecore Biomedical, Chaska, MN, USA) using 1-ethyl-3-(3-dimethylaminopropyl)


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carbodiimide (EDC) (Thermo Scientific, Rockford, IL, USA) and N-hydroxysulfosuccinimide (NHS) (Sigma) as previously described.26, 27 Briefly, HA was dissolved in distilled water at a concentration of 1% (w/v). EDC and NHS were then added at an equal molar ratio to HA and stirred for 30 minutes at pH 5.0. Dopamine hydrochloride was added to the solution at a 1:1 molar ratio to HA and stirred overnight at room temperature while maintaining a pH of 5.0 using 1 M hydrochloride. Unreacted dopamine hydrochloride was removed by four six-hour cycles of dialysis (Cellu Sep T2, MW cut-off 6–8 kDa, Membrane Filtration Products Inc., Seguin, TX, USA) against 1× phosphate buffered saline (PBS, Sigma) at pH 5.0 followed by dialysis against distilled water for 4 hours at pH 5.0. The final product was lyophilized and stored at 4°C until use. The percentage of catechol conjugated to the HA backbone was calculated by measuring the absorbance of the conjugate sample at 280 nm using a UV-visible spectrophotometer (GloMax-Multi Microplate, Promega, Madison, WI, USA). The HA-CA conjugate product was confirmed using proton nuclear magnetic resonance (1H NMR) (Bruker 400 MHz, Bruker, Billerica, MA, USA). To form the HA-CA hydrogel, the conjugate was dissolved in 1× PBS and then mixed with a solution containing sodium periodate (NaIO4, Sigma) at an equal molar ratio to catechol and 0.4 M sodium hydroxide (NaOH, Sigma). The HA-CA hydrogel was formed at a final concentration of 2% (w/v).

HA-ME Synthesis and Gelation. The HA-ME conjugate for creation of a photopolymerized hydrogel was synthesized as previously described.26, 27 Briefly, HA (MW = 200 kDa) was dissolved in distilled water at a concentration of 1% (w/v). Methacrylate anhydride (Sigma) was added to the HA solution at a molar ratio equal to HA and incubated overnight in the dark. Unreacted methacrylate was removed by ethanol precipitation. The reaction mixture was then dialyzed (MW cut-off 6–8 kDa) for four days against distilled water at neutral pH. It was subsequently freeze-dried. The HA-ME hydrogel was formed by dissolving HA-ME


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conjugate at 2% (w/v) in 1× PBS with 0.3% of the photoinitiator Irgacure 2959 (I2959 Ciba Specialty Chemicals, Basel, Switzerland). The solution was subsequently exposed to UV light (10 mWcm-2, Blak-Ray B-100AP high-intensity UV Lamp, UVP, Upland, CA, USA) for 2-5 minutes to induce radical polymerization.

Characterization of HA Hydrogels. The physical and mechanical properties of the HA hydrogels were characterized as previously reported.26 Briefly, to determine the swelling properties of the hydrogels, they were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) at 37°C. The wet weight of the hydrogel constructs was measured at several time points (days 0, 1, 3, 5, 10, and 14) during incubation. The swelling ratio of the hydrogel was calculated using the following equation, (Ws - Wi)/Wi × 100, where Ws indicates the weight of the swollen hydrogel at each time point and Wi indicates the weight of the dried hydrogel on day 0.29 The elastic modulus of the hydrogel was measured using a rheometer (Bohlin Instruments, Worcestershire, UK) at a frequency sweep mode in the range of 0.1 Hz to 10 Hz. To determine the degradation rate of the hydrogels, the constructs were pre-incubated in PBS (pH = 7.2) for 3 days and then treated with hyaluronidase (100 unit/ml, Sigma). The remaining weight was measured up to 18 hours after enzymatic treatment. The microporous structure of the hydrogels was observed in fluorescently labeled HA hydrogels using confocal microscopy. Fluorescently labeled HA-CA and HA-ME conjugates were synthesized by conjugating 5-Aminofluorescein [fluorescein isothiocyanate (FITC), Sigma] to HA backbone of HA-CA and HA-ME. In brief, HA-CA or HA-ME was dissolved in distilled water at a concentration of 1% (w/v). EDC, NHS, and FITC were added to the HA solutions at an equal molar ratio to HA-CA or HA-ME and stirred overnight in the dark at 4°C. Unreacted FITC was removed through dialysis against PBS for 48 hours and distilled 6 ACS Paragon Plus Environment

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water sequentially. The final products were lyophilized. After inducing oxidative crosslinking of FITC-labeled HA-CA hydrogel and photopolymerization of FITC-labeled HA-ME hydrogel, the hydrogels were mounted in optimal cutting temperature (OCT; CellPath Ltd., Wales, UK) compound and cryosectioned in 10 µm-thickness. To observe the microporous structures of HA hydrogels, the sections were examined using a confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany).

Cell Culture and Encapsulation. Cell culture and encapsulation were conducted following previously described procedures.26 hADSCs were purchased from Invitrogen and expanded up to ~5–6 passages in growth medium comprised of low glucose DMEM supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen). For cell encapsulation and culture within the HA hydrogels, cells were suspended at a density of 1.0 × 106 cells per 100 µl (or 50 µl for Live/Dead viability test) of pre-gelation solution. After gelation, the hydrogel constructs were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. The viability of cells encapsulated in the HA hydrogels was evaluated using a Live/Dead viability/cytotoxicity kit (Invitrogen) following the manufacturer’s protocol. The stained cells were observed using a fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan) and the percentage of viable cells (green) was calculated by manual counting from the acquired images (n = ~4–8). To evaluate paracrine secretion of hADSCs in HA hydrogels, conditioned medium was collected from the culture medium of hADSCs encapsulated in HA hydrogels (1.0 × 106 cells in 100 µl of hydrogel) in non-adherent 24-well plates. At each time point in culture (days 3, 7, and 14), 1 ml of medium was collected from the well plates and stored at -80°C. Fresh medium was then added to the wells and the cells were cultured until the next medium collection time point. The level of vascular endothelial


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growth factor (VEGF) in the supernatant was determined using a human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA).

hADSC Differentiation in HA-CA Hydrogels. To test the osteogenic differentiation capacity of encapsulated hADSCs in HA-CA hydrogels, bone morphogenetic protein-2 (BMP-2) peptide (sequence: KIPKASSVPTELSAISTLYLGGK,30, 31 100 µg in 100 µl of hydrogel) was added to the pre-gelation solution. After gelation, the hydrogel constructs containing hADSCs with or without BMP-2 peptides were maintained either in growth medium or osteogenic induction medium composed of low glucose DMEM supplemented with 10% FBS, 0.1 µM dexamethasone (Sigma), 50 µM ascorbic acid (Sigma), and 10 mM βglycerol phosphate (Sigma) for 21 days. The medium was changed every 2 or 3 days. Gene expression of osteogenic markers in differentiated hADSCs cultured in the HA-CA hydrogels was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell-hydrogel constructs were washed with PBS and incubated in DMEM containing 5000 U hyaluronidase (Sigma) per 100 µl of gel construct for 1 hour. Total RNA was extracted from the cells in the degraded hydrogel solution (n = 3) using an RNeasy mini prep kit (Qiagen, Valencia, CA, USA). The RNA samples were reverse-transcribed into cDNA using a PrimeScriptTM 1st strand cDNA synthesis kit (TaKaRa, Shiga, Japan). Subsequently, qRT-PCR was carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays (human osteocalcin (OCN): Hs01587814_g1 and human runt-related transcription factor 2 (RUNX2): Hs00231692_m1). The level of target gene expression was calculated using the comparative Ct method and normalized to an endogenous reference, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

BMP-2 Release Profile of HA-ME Hydrogel. To check the release profile of BMP-2 peptide 8 ACS Paragon Plus Environment

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from HA-ME hydrogel, BMP-2 peptides were encapsulated within 2% (w/v) HA-ME hydrogel at a final concentration of 200 µg/ml. The HA-ME hydrogel loaded with BMP-2 peptides was incubated in PBS for 48 hours at 37°C. The buffer solution was retrieved at each time point and used as a sample solution for quantifying the amount of released BMP-2 peptides using fluorescamine assay following the manufacture’s protocol. In brief, the sample solution was mixed with the fluorescamine solution (Sigma) (3 mg/ml in dimethyl sulfoxide) at 1:3 (v/v) ratio in a 96-black-well plate. After incubation for 15 minutes at room temperature, the fluorescence intensity was measured by using a microplate reader (Infinite® 200 PRO, Tecan Trading AG, Männedorf, Switzerland) and the released amount of BMP-2 was calculated from the standard curve of BMP-2 peptides.

Transplantation of hADSCs within the HA Hydrogel in a Hindlimb Ischemia Mouse Model. All animal experiments were performed in accordance with Korean Food and Drug Administration (KFDA) guidelines. All surgical procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Yonsei Laboratory Animal Research Center (YLARC; permit number: 2010-0049). Hindlimb ischemia was induced in a mouse model as previously described.32 Female athymic mice at 6 weeks of age (Balb/c-nu, Nara Biotech) were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and the operating area was pre-sterilized. After skin incision, the left iliac and femoral arteries were ligated with a 6-0 prolene suture (Ethicon, Somerville, NJ, USA) and excised from beneath the external iliac artery to the distal point of bifurcation of the saphenous and popliteal arteries. Immediately after arterial dissection, hADSC-encapsulated hydrogels (1.0 × 106 hADSCs per 100 µl hydrogel) were intramuscularly administered at two sites of the gracilis muscle in the medial: PBS, HA-ME, HA-CA, HA-ME-hADSC, and HA-CA-hADSC.


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Mice were monitored by serial scanning of hindlimb surface blood flow using a laser Doppler perfusion imager (Moor Instruments, Devon, UK) on days 0, 7, 14, and 21 after treatments.32 Digital color-coded images were analyzed to quantify blood flow from the knee joint to the toe region, and perfusion rate was calculated using the ratio of the ischemic limb to the normal limb. The ischemic muscles were harvested 21 days after hADSC transplantation, fixed with 4% paraformaldehyde (Sigma) overnight, and embedded in paraffin and sectioned (4 µm). The sectioned specimens were stained using hematoxylin & eosin (H&E) and Masson’s trichrome. Immunohistochemistry was performed on the capillaries and arterioles using anti-von Willebrand factor (vWF) (1:200 dilution; Abcam, Cambridge, UK) and anti-smooth muscle α-actin (SMA) (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) following standard histological procedures. The density of the capillaries and arterioles was quantified by counting vWF-positive microvessels and SMA-positive microvessels, respectively. To assess cell survival and migration in ischemic muscle, hADSCs were tracked with Vybrant DiI Cell-Labeling solution (Invitrogen) prior to encapsulation following the manufacturer’s protocol. Labeled cells were transplanted within the hydrogels into ischemic regions. The ischemic muscles were then harvested, embedded in OCT compound (Sakura Finetek, Torrance, CA, USA), and cryosectioned. The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) and examined using a fluorescence microscope (Olympus).

Transplantation of hADSC-encapsulated HA Hydrogels into a Calvarial Bone Defect. Protocols for the calvarial bone defect experiments were reviewed and approved by the IACUC of the YLARC (permit number: 2011-0059). Female athymic mice (Balb/c-nu, 6 weeks old, Nara Biotech) were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg). After skin incisions were made, two defect sites (4-mm diameter defect) were created in the skull using a micro bone drill (Strong 102L, Saeshin, Daegu, Korea) as previously 10 ACS Paragon Plus Environment

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described 30. HA-CA or HA-ME hydrogels containing 10% hydroxyapatite and undifferentiated hADSCs (6.0 × 105 cells per 30 µl hydrogel) were placed into the defect regions and the skin was sutured with 6-0 sutures (Ethicon). The mice were divided into seven groups: (i) no treatment, (ii) HA-ME, (iii) HA-CA, (iv) HA-ME-hADSC, (v) HA-CA-hADSC, (vi) HA-ME-hADSC-BMP-2, and (vii) HA-CA-hADSC-BMP-2. The BMP-2 peptide concentration in hydrogel constructs was 1 mg/ml (30 µg BMP-2 peptides per 30 µl hydrogel). After 8 weeks, mice were sacrificed for gross and histological analysis. Micro-computed tomography (micro-CT) analysis was conducted using a micro-CT system (SkyScan-1172, SkyScan, Kontich, Belgium) and the SkyScan CT analyzer program (SkyScan). The area of regenerated bone within the defects was quantified using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA). Calvarial bone samples were treated with Decalcifying Solution (Sigma, 20:1 ratio (v/v) of Decalcifying Solution to tissue sample) at room temperature for 6 hours and then embedded in paraffin. The embedded tissue specimens were sliced into sections in 5-µm thickness and stained with Goldner’s trichrome method. Hematoxylin was used to stain nuclei. Collagen regeneration in the defect sites was determined as a percentage ratio of the collagen-stained area to the total defect area using ImageJ software (NIH). Tissue sections were immunofluorescently stained for bone markers, osteopontin (OPN) (1:100 dilution; Santa Cruz Biotechnology) and collagen type I (COL I) (1:50 dilution, Calbiochem, San Diego, CA, USA). The stained sections were observed using a confocal microscope (LSM 700, Carl Zeiss).

Statistical Analyses. GraphPad Prism Software (GraphPad Software, San Diego, CA, USA) was used to perform statistical analyses. Data are shown as means ± standard deviations. Unpaired Student’s t-tests were used to determine statistical significance. A p-value < 0.01 and 0.05 was considered statistically significant.


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RESULTS AND DISCUSSION

Characterization of HA-CA Hydrogels. HA-CA conjugate was synthesized by modifying the HA backbone with dopamine (Figure 1A) via a carbodiimide coupling reaction using EDC and NHS. Through the reaction with an equal molar ratio of dopamine to HA backbone, ~7– 11% of the HA backbone was functionalized with the catechol group of dopamine. NMR analysis also confirmed dopamine conjugation to HA backbone by showing the appearance of catechol proton peaks at around 7 ppm (Supplementary Figure S1). The gelation of HA-CA conjugate was induced by crosslinking the conjugate with an oxidizing agent, sodium periodate (NaIO4), at an equal molar ratio of NaIO4 to the catechol group of HA-CA conjugate under alkaline condition (pH = 8) as previously described.26, 27 It is thought that HA-CA conjugates form a hydrogel via generation of catechol-catechol adducts, which is reminiscent of monomer polymerization observed in melanin formation.33 The color of pregel solution turned brown immediately upon gelation (Figure 1B) due to generation of oquinone by oxidative conversion of the catechol moiety. HA-ME conjugate was synthesized by conjugating methacrylate anhydride to the hydroxyl group of HA. The HA-ME hydrogel was formed via UV light-mediated photopolymerization in the present of a photoinitiator, Irgacure 2959. The swelling property of the 2% HA-CA hydrogel was examined by measuring wet weight changes of the hydrogel constructs during 2-week incubation under physiological conditions (i.e., in culture medium at 37°C) (Figure 1C). Photopolymerized HA-ME hydrogel, a widely used hydrogel in biomedical applications, served as a control group. Both HA hydrogels (2%) did not exhibit significant weight loss or structural deformation (Figure 1C). The swelling reached an equilibrium state in both HA hydrogels after 3 days of incubation and persisted throughout the incubation period (Figure 1C). When compared with the HA-ME 12 ACS Paragon Plus Environment

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hydrogel, the HA-CA hydrogel showed more rapid increase in wet weight due to greater swelling capacity (163.4 ± 8.7% for HA-CA versus 102.3 ± 21.9% for HA-ME on day 14). The rheological property of the 2% HA-CA hydrogel was examined by dynamic mechanical analysis (Figure 1D and E). Storage moduli (G′) and loss moduli (G″) of the HA hydrogels were measured in a frequency sweep mode 10 minutes after gelation induction. In frequency ranges tested (from 0.1 to 10 Hz), G′ (~1.5 kPa) was constantly higher than G″, indicating that the formed HA-CA hydrogel was quite stable as a viscoelastic solid (Figure 1D). The average storage moduli of the HA-CA and HA-ME hydrogels (2%) measured at 1 Hz frequency were 1.7 ± 0.2 kPa and 4.3 ± 0.6 kPa, respectively (Figure 1E), suggesting that the mechanical property of the HA-CA hydrogel was softer than that of the HA-ME hydrogel. Rheological analysis in a time sweep mode performed in our previous study confirmed that the HA-CA conjugate formed a hydrogel around 6−7 minutes after inducing gelation.26 The assay of HA hydrogel degradation by the treatment of hyaluronidase revealed that the weight decrease of the HA-CA hydrogel was much faster than that of the HA-ME hydrogel (Figure 1F). After 3 hours of enzymatic treatment, the HA-CA hydrogel underwent a 50% loss of its initial weight. In contrast, the weight of HA-ME hydrogel decreased to 50% of its initial level 8 hours after treatment (Figure 1F). HA-CA and HA-ME hydrogels underwent complete degradation approximately 10 and 18 hours after hyaluronidase treatment, respectively (Figure 1F). More rapid degradation of the HA-CA hydrogel compared to the HA-ME hydrogel after hyaluronidase treatment may be due to the difference in the degree of chemical modification on the HA hydrogel backbone. The conjugation efficiency of catechol group to HA backbone was ~7–11% while the conjugation efficiency of methacrylate group to HA backbone was ~20%, and therefore it is assumed that crosslinking density of HA-CA hydrogel was lower than that of HA-ME hydrogel, which leads to a faster biodegradation of HA-CA hydrogel compared to HA-ME hydrogel. The increased degradation susceptibility of HA-CA hydrogel to hyaluronidase might also be due to the reactivity of catechol group 13 ACS Paragon Plus Environment

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towards hyaluronidase, as catechol group is well known to have high affinity to various nucleophiles present in proteins.26, 27 Thus, it is highly possible that the catechol group in HACA hydrogel could covalently tether hyaluronidase, which consequently results in faster degradation of the HA chains than HA-ME hydrogel. The porous and interconnected microstructures within the HA-CA hydrogel were confirmed by confocal microscopy observation of fluorescently labeled HA hydrogels (FITC-HA-CA and FITC-HA-ME hydrogels) (Figure 1G). The average pore sizes in the HA-CA and HA-ME hydrogels were 24 ± 10 µm and 16 ± 5 µm, respectively (Figure 1H).

Improved Biocompatibility of HA-CA Hydrogels. To confirm improved biocompatibility of HA-CA hydrogels, the viability of hADSCs cultured in HA-CA hydrogels was compared with that of hADSCs in photopolymerized HA-ME hydrogels. hADSCs in HA-CA hydrogels were highly viable (~93%) up to 7 days in culture, but the viability of cells in HA-ME hydrogels decreased over the 7 days in culture (Figure 2A and B). Interestingly, in only the HA-CA hydrogels, hADSCs spread out and exhibited highly extended cellular morphology (Figure 2A). Our previous studies confirmed marginal cytotoxicity of HA-CA hydrogels during in vitro 3D culture of diverse cell types such as neural stem cells and hepatocytes.26, 27 These results suggest that HA-CA hydrogels are less cytotoxic and can provide more favorable microenvironments for stem cells compared with conventional HA hydrogel systems. An oxidizing agent used for oxidative crosslinking of HA-CA conjugate, sodium periodate (NaIO4), did not affect human stem cell behaviors at the crosslinking concentration. In our current study, crosslinking of HA-CA hydrogel was induced at an equal molar ratio of NaIO4 to catechol and it seems that NaIO4 at this concentration (4.5 mg/ml) has an insignificant effect on viability and proliferation of hADSCs (Figure 2A and B). Our previous study also demonstrated that NaIO4 at the same HA-CA crosslinking concentration did not 14 ACS Paragon Plus Environment

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affect the viability and proliferation of human neural stem cells.27 Actually, Burke et al. previously reported that periodate ion is reduced to iodate ion, a less toxic ion after gaining electrons by inducing oxidative conversion of catechol group in L-DOPA-functionalized poly(ethylene glycol) (PEG) into o-quinone.34 Several other studies have also revealed that in general NaIO4 in the ranges of the concentrations to induce oxidative crosslinking for hydrogel formation did not affect cell behaviors during the culture.35-37 The equal molar ratio of NaIO4 to aldehyde-modified alginate or 1~3:5 ratios of NaIO4 to aldehyde-modified HA for hydrogel formation did not show cytotoxicity to varied cell types including chondrocytes, endothelial cells, and fibroblasts.35, 36 Oxidative crosslinking of modified alginate by NaIO4 did not alter adhesion, proliferation, and differentiation of progenitor cells (e.g., myoblasts).37 These previous literatures may further explain negligible effect of NaIO4 on cell viability, proliferation, and differentiation. In addition to viability, the function of stem cells can be retained in 3D culture using HA-CA hydrogels. hADSCs are regarded as effective therapeutics for the treatment of ischemic diseases because of their ability to secret various angiogenic factors including VEGF.38, 39 Therefore, we compared the level of VEGF secretion from hADSCs cultured in both HA-CA and HA-ME hydrogels at days 3, 7, and 14 after encapsulation (Figure 2C). Although VEGF secretion decreased in hADSCs exposed to either hydrogel, the level of VEGF secretion was retained to some extent in the HA-CA group (1721.67 ± 18.79 pg/ml at day 3, 1161.06 ± 36.36 pg/ml at day 7, and 855.70 ± 71.86 pg/ml at day 14), whereas VEGF levels in the HA-ME group rapidly decreased from days 3 to 7 (1390.81 ± 98.49 pg/ml at day 3 and 166.83 ± 27.11 pg/ml at day 7) and was barely detected at day 14 (3.66 ± 4.90 pg/ml) (Figure 2C). Together, these results suggest that HA-CA hydrogels are more suitable scaffolding biomaterials than photopolymerized HA hydrogels because they retain hADSC angiogenic function and therapeutic potential.


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In this study, it was observed that the levels of VEGF secretion from hADSCs were gradually decreased during in vitro 3D culture in HA hydrogels (Figure 2C). There are several previous studies reporting such similar trend of decreased VEGF secretion and expression during the culture in tissue culture plate. For example, Kim et al., showed that VEGF secretion from genetically engineered mesenchymal stem cells (MSCs) was increased for 8 days but the release profile was gradually decreased for the next 7 days at both hypoxic and normoxic conditions.40 Ding et al., also observed that the mRNA expression levels of VEGF in bone marrow-derived MSCs were gradually decreased during 14 days of culture in vitro.41 The decrease in growth factor secretion from MSCs was also observed during 3D culture in hydrogel scaffolds. As one of those examples, Marklein et al., reported gradual decrease in secretion of several growth factors including VEGF from human MSCs encapsulated in hydrogel constructs during the culture.42 Although functional scaffolds could help retain the viability of stem cells during the culture, the cells cultured in vitro are prone to somewhat lose the activity of paracrine secretion of growth factors.

hADSC Therapy using HA-CA Hydrogels Enhanced Angiogenesis in Critical Limb Ischemia. To confirm the feasibility of using HA-CA hydrogels for scaffolding to enhance the therapeutic efficacy of stem cells, hADSCs were intramuscularly transplanted within the HA-CA hydrogel into a mouse model of hindlimb ischemia.32 After 3 weeks, the HA-CAhADSC group showed significantly reduced foot/limb loss or necrosis and greater salvage of the ischemic limb (64%) compared to the PBS group (23%), hydrogel alone groups (29% for HA-ME and 40% for HA-CA), and the HA-ME-hADSC group (36%) (Figure 3A and B). Although HA-ME hydrogel could mediate hADSC transplantation exhibiting therapeutic effects in hindlimb ischemia models as confirmed in several previous studies reporting the utility of HA-ME hydrogel for tissue regeneration,11, 43-45 hADSC therapy using HA-CA hydrogel further improved ischemic limb salvage (Figure 3A and B). Blood perfusion 16 ACS Paragon Plus Environment

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assessment of the ischemic limbs by Doppler imaging analysis revealed the highest blood flow recovery in the HA-CA-hADSC group (Figure 3C and D). This improvement may be attributed to an increase in survival and localization of transplanted hADSCs by the HA-CA hydrogel in ischemic tissue. Fluorescent DiI-labeled cell tracking indicated that a significantly larger number of hADSCs survived in the HA-CA-hADSC group and migrated into the ischemic region (Figure 4A and B). Accordingly, the densities of capillaries and arterioles were highest in the HA-CA-hADSC group, as indicated by double immunofluorescent staining for endothelial (vWF) and smooth muscle (SMA) markers (Figure 4C-E). Histological analysis of H&Estained ischemic muscles 3 weeks after treatments revealed significantly attenuated muscle degeneration in the HA-CA-hADSC group (Figure 4F, first row). Masson’s trichrome staining showed reduced fibrosis in ischemic muscle of the HA-CA-hADSC group compared with that of the control groups (PBS, hydrogel alone, and HA-ME-hADSC) (Figure 4F, second row and Figure 4G). These results suggest that the therapeutic effects of hADSCs are more dramatic when transplanted within HA-CA hydrogels compared with HA-ME hydrogels.

hADSC Transplantation using HA-CA Hydrogels Promoted Bone Regeneration in a Critical-sized Bone Defect. The regenerative potential of transplanted hADSCs within the HA-CA hydrogel was evaluated in a critical-sized calvarial bone defect model. We first examined the viability of hADSCs encapsulated in HA-CA hydrogels under osteogenic and non-osteogenic conditions for up to 2 weeks. The cells remained highly viable under both culture conditions (Figure 5A). The peptides or proteins with functional groups including amines, thiols, and imidazoles could be efficiently incorporated into the 3D HA-CA hydrogel constructs due to high affinity of the oxidized catechol group to these nucleophiles.26, 30, 46-48 Thus, incorporation of BMP-2 peptides (sequence: KIPKASSVPTELSAISTLYLGGK)30, 31 into the HA-CA hydrogel further increased gene expression of osteogenic markers (RUNX2 17 ACS Paragon Plus Environment

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and OCN) in hADSCs after 3 weeks of culture under both osteogenic and non-osteogenic conditions (Figure 5B), indicating enhanced osteogenic differentiation of hADSCs by osteoinductive signals from incorporated BMP-2 peptides in the HA-CA hydrogel. Undifferentiated hADSCs were transplanted within HA hydrogels into critical-sized calvarial defects. Bone tissue is largely composed of organic components and inorganic components mainly mineral hydroxyapatite.49 HA hydrogels could provide organic microenvironments for bone formation, but it is still difficult to efficiently induce in vivo bone regeneration with HA hydrogel alone due to the lack of inorganic components required to improve the mechanical properties and mineralization of the implants. Therefore, in this study HA-CA hydrogel system was supplemented with hydroxyapatite, mimicking native bone tissue by providing both organic and inorganic components. Incorporation of hydroxyapatite into HA-CA hydrogel was expected to improve the mechanical properties and mineralization of the formed tissues, consequently enhancing bone regeneration in a critical-sized calvarial bone defect model. The HA-CA hydrogel construct was still in place eight weeks after surgery and transplantation due to its adhesive properties, whereas the HA-ME hydrogel was often displaced from the transplanted site (Figure 5C). Micro-CT analysis indicated that more extensive osteogenesis occurred in hADSCs transplanted within the HA-CA hydrogels compared with the HA-ME hydrogels (Figure 5D). The greatest bone regeneration at the site of the defect was observed for the HA-CA-hADSC-BMP-2 group (Figure 5D and E). Furthermore, Goldner’s trichrome staining revealed that the highest regeneration of collagen tissue in the defect regions was achieved in the HA-CA-hADSC-BMP-2 group (Figure 6A and B). These results were further supported by double immunofluorescent staining of bone markers (OPN and COL I), which showed that the greatest enhancement of bone tissue in the defect region occurred for the HA-CA-hADSC-BMP-2 group (Figure 6C). The osteogenic effect of BMP-2 peptides incorporated into HA hydrogel on bone formation was more dramatic in HA-CA hydrogel than in HA-ME hydrogel. The addition of 18 ACS Paragon Plus Environment

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BMP-2 peptides in HA-CA hydrogel substantially promoted hADSC-mediated bone regeneration (HA-CA-hADSC group versus HA-CA-hADSC-BMP-2 group), but HA-MEhADSC group with BMP-2 peptides did not show the enhanced bone regeneration as compared to HA-ME-hADSC group without BMP-2 peptides (Figure 5E). In contrast to HACA hydrogel with catechol group exhibiting a high binding affinity to various nucleophiles in peptides and proteins, HA-ME hydrogel does not have any functional moieties to tether the peptides and proteins. The release assay of BMP-2 peptides incorporated into HA-ME hydrogel revealed that BMP-2 was released out completely within 12 hours under in vitro incubation in a physiological condition (in PBS solution at 37°C) (Supplementary Figure S2). Thus, BMP-2 incorporated into HA-ME hydrogel might had released out within several hours in vivo that was not retained long enough to enhance osteogenesis of hADSCs encapsulated within the hydrogel. This may explain why there was no significant difference in bone regeneration between HA-ME-hADSC-BMP-2 group and HA-ME-hADSC group without BMP-2.

CONCLUSIONS

In summary, we demonstrate the feasibility of using bio-inspired HA-CA hydrogels for human stem cell therapies to treat critical tissue defects. HA-CA hydrogels were shown to be less cytotoxic and more effective at retaining cellular function compared with conventional photopolymerized HA hydrogels. Ultimately, these highly biocompatible HA-CA hydrogels could enhance the therapeutic and regenerative capacity of transplanted stem cell for angiogenesis and osteogenesis by supporting the viability, functionality, differentiation, and in vivo engraftment of stem cells. Although we have previously reported the synthesis and application of mussel-inspired HA-CA hydrogel,26, 27 our previous studies did not prove fully 19 ACS Paragon Plus Environment

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the clinical feasibility of HA-CA hydrogel in diseased and defected models. Based on our previous studies, we further extend our bio-inspired approach to clinically relevant animal models with critical tissue defects and our current study suggests the clinical feasibility of HA-HA hydrogel as a real cell carrier by providing the evidences of enhanced angiogenesis and osteogenesis by HA-CA hydrogel-based stem cell therapy in animal models of critical tissue defects.

SUPPORTING INFORMATION Supporting Information; NMR data of the synthesized HA conjugate, release profile of BMP2 peptides from the hydrogel.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (S.-W.C.)

Author Contributions ‡These authors contributed equally to this work. (H.-J.P., Y.J., and J.S.)

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by a grant (2009-0083522) from the Translational Research Center for Protein Function Control (TRCP) funded by the Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea. This work was also supported by a grant (NRF-


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2013R1A1A2A10061422) from the National Research Foundation of Korea (NRF) and a grant (HI13C1479) from the Korea Health Technology R&D Project funded by the Ministry of Health and Welfare, Republic of Korea.

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Figure 1. Physical and mechanical characterization of HA-CA hydrogels. (A) Schematic representation of an engineered mussel-inspired HA hydrogel. The major component of the mussel adhesive protein, Mytilus edulis foot protein-5 (Mefp-5), is located in the adhesive pads. L-Dopa and lysine are the primary amino acids that give the protein adhesive properties. Dopamine, a catecholamine containing a catechol group of L-Dopa and an amine group from lysine, was conjugated with HA for hydrogel formation. (B) The gross view shows the HACA hydrogel before (top) and after (bottom) gelation. (C) Swelling properties of the HA-CA and HA-ME hydrogels were determined upon incubation at 37°C (n = 3). (D) Rheometric analysis of the HA-CA hydrogel was performed by a frequency sweep mode. The black symbols represent storage moduli G′ and the white symbols represent loss moduli G″. (E) The average elastic moduli of HA-CA and HA-ME hydrogels at 1 Hz are shown (n = 3, **p < 0.01). (F) Enzymatic degradation of the HA hydrogels was performed by hyaluronidase (100 24 ACS Paragon Plus Environment

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U/ml). The remaining wet weights were measured at each time point (n = 3). (G) Confocal microscopic observation of the FITC-HA-ME and FITC-HA-CA hydrogels indicated highly porous structures within the hydrogels (scale bar = 50 µm). (H) The internal pore size of the HA hydrogels were calculated from confocal microscopy images (n = 32, **p < 0.01).


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Figure 2. In vitro biocompatibility of the HA-CA hydrogels. (A) Representative Live/Dead fluorescence images of hADSCs cultured within the 3D constructs, HA-CA or HA-ME hydrogel, are shown (scale bars = 200 µm). The green indicates live cells and the red indicates dead cells. (B) Quantification of cell viability was performed by manual counting of live and dead cells (n = ~4–8, **p < 0.01 versus HA-ME group). (C) The level of VEGF secreted from hADSCs encapsulated in HA hydrogels was determined by ELISA quantification (n = 3-4, **p < 0.01 versus HA-ME group).


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Figure 3. Improvement of ischemic limb salvage by transplantation of hADSCs within HACA hydrogels in a critical hindlimb ischemia mouse model. (A) Representative photographs are shown for ischemic hindlimbs on days 0 and 21 after hADSC transplantation. A hindlimb ischemia model was prepared by ligating and removing the femoral artery located nearer to the ventral skin in the left limb. The photographs of the animals were taken dorsally. (B) Physiological status scores for ischemic limbs are shown for day 21 (n = ~7–14). (C, D) Serial analysis was performed on laser Doppler perfusion imaging after hADSC transplantation within the HA-CA and HA-ME hydrogels or PBS injection. The pictures for Doppler imaging were taken ventrally. The ratio of ischemic to normal limb blood perfusion was improved in mice that received HA-CA-hADSC transplantation compared with controls on day 14 and 21 after treatment (n = 6, *p < 0.05 versus PBS group).


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Figure 4. Enhancement of angiogenesis by transplantation of hADSCs within HA-CA hydrogels in a critical hindlimb ischemia mouse model. (A) DiI-fluorescently labeled hADSCs transplanted within HA-CA hydrogels were tracked in the ischemic limb tissue 2 days after transplantation, scale bar = 200 µm. (B) Quantification of hADSCs surviving within ischemic limb tissue was performed 2 days after transplantation (n = 10, **p < 0.01 versus HA-ME-hADSC group). (C-E) Enhanced angiogenesis was observed within the ischemic limb muscle after transplantation of hADSCs within HA-CA hydrogels. (C) Arterioles (SMA; red) and capillaries (vWF; green) in ischemic muscle tissue were double immunofluorescently stained 21 days after hADSC transplantation, scale bar = 100 µm. Quantification of (D) vWF-positive microvessel density and (E) SMA-positive arteriole density was performed in the ischemic regions (n = 10, *p < 0.05 and **p < 0.01 versus the HA-CA-hADSC group). (F) H&E and Masson’s trichrome staining was performed 21 days after cell transplantation (scale bars = 100 µm) and (G) the fibrotic area in ischemic regions was quantified (n = 5, **p < 0.01 versus the HA-CA-hADSC group).


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Biomacromolecules

Figure 5. Enhanced bone formation by transplantation of hADSCs within HA-CA hydrogels in a critical-sized calvarial bone defect mouse model. (A) Live/Dead staining was performed in hADSCs encapsulated in HA-CA hydrogels under non-osteogenic or osteogenic conditions on day 14, scale bar = 200 µm. (B) qRT-PCR analysis was performed to examine the expression of osteogenic markers (OCN and RUNX2) in hADSCs cultured in the HA-CA hydrogel under non-osteogenic and osteogenic conditions on day 21 (n = 3, *p < 0.05 and **p < 0.01 versus the hADSC group under non-osteogenic conditions, ++p < 0.01 versus the hADSC-BMP-2 group under non-osteogenic condition). (C) The gross view shows calvarial bones retrieved 8 weeks after hADSC transplantation within HA-CA and HA-ME hydrogels. (D) Micro-CT images were acquired for the calvarial bones from the seven groups (no treatment, HA-ME, HA-CA, HA-ME-hADSC, HA-CA-hADSC, HA-ME-hADSC-BMP-2, and HA-CA-hADSC-BMP-2). (E) The regenerated bone tissue in the defect area after hADSC transplantation was quantified (n = ~4–5, *p < 0.05 and **p < 0.01 versus the HA-CAhADSC-BMP-2 group).


Biomacromolecules

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Figure 6. Enhanced osteogenesis by hADSCs transplanted within HA-CA hydrogels into critical-sized calvarial bone defects in a mouse model. (A) Goldner’s trichrome staining was performed on the calvarial bones retrieved 8 weeks after hADSC transplantation within HACA and HA-ME hydrogels in the absence or presence of BMP-2 peptides. Collagen was stained green, and purple arrowheads in the left columns indicate the defect regions, scale bars = 1 mm (left column) and 50 µm (right column). (B) The regenerated collagen within the defect area was quantified by calculating the percentage of tissue area with regenerated collagen relative to the total defect area (n = ~8–11, **p < 0.01 versus the HA-CA-hADSCBMP-2 group). (C) Double immunofluorescence staining was performed for the bone markers osteopontin (OPN; green) and collagen I (COL I; red) in calvarial bone defects 8 weeks after hADSC transplantation. The yellow arrowheads indicate the defect regions, scale bar = 1 mm.


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Biomacromolecules

For Table of Contents (ToC) Use Only

Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects Hyun-Ji Park,1‡ Yoonhee Jin,1‡ Jisoo Shin,1‡ Kisuk Yang,1 Changhyun Lee,1 Hee Seok Yang,2 Seung-Woo Cho1,3*


Catechol-Functionalized Hyaluronic Acid ... - ACS Publications

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