Hydrogen peroxide-releasing hydrogels for enhanced endothelial cell

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Applications of Polymer, Composite, and Coating Materials

Hydrogen peroxide-releasing hydrogels for enhanced endothelial cell activities and neovascularization Yunki Lee, Joo Young Son, Jeon Il Kang, Kyung Min Park, and Ki Dong Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04522 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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ACS Applied Materials & Interfaces

Hydrogen peroxide-releasing hydrogels for enhanced endothelial cell activities and neovascularization

Yunki Lee1, *, Joo Young Son1, *, Jeon Il Kang2, Kyung Min Park2, #, Ki Dong Park1, #

1

Department of Molecular Science and Technology, Ajou University, Suwon 16499,

Republic of Korea 2

Department of Bioengineering and Nano-bioengineering, College of Life Sciences and

Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

* These authors contributed equally to this work. #

Co-correspondence to:

K.M. Park, Department of Bioengineering and Nano-bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea. Tel.: +82-32835-8835; E-mail: [email protected]; Fax: +82-32-835-0804 K.D. Park, Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong, Suwon 16499, Republic of Korea. Tel.: +82-31-219-1846; E-mail address: [email protected]

KEYWORDS: Polymeric hydrogels, reactive oxygen species, hydrogen peroxide, angiogenesis, tissue regeneration. ACS Paragon Plus Environment

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ABSTRACT Reactive oxygen species (ROS) have been implicated as a critical modulator for various therapeutic applications such as treatment of vascular disorders, wound healing, and cancer treatment. Specifically, growing evidence has recently demonstrated that transient or low levels of hydrogen peroxide (H2O2) facilitates tissue regeneration and wound repair through acute oxidative stress that can evaluate intracellular ROS levels in cells or tissues. Herein, we report a gelatin-based H2O2-releasing hydrogel formed by dual enzyme-mediated reaction using horseradish peroxidase (HRP) and glucose oxidase (GOx). The release behavior of H2O2 from the hydrogel matrices can be precisely controlled by varying GOx concentrations. We demonstrate that H2O2-releasing hydrogels with the optimal condition increase transient upregulation of intracellular ROS levels in the endothelial cells (ECs) and enhance proliferative activities of ECs in vitro, and facilitate neovascularization in ovo. We suggest that our H2O2-releasing hydrogels hold a great potential as an injectable and dynamic matrix for the treatment of vascular disorders as well as tissue regenerative medicine.

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1. INTRODUCTION Polymeric hydrogels have attracted substantial attention in a wide range of biomedical applications such as tissue engineering and regenerative medicine as well as drug delivery because of their biocompatibility and multi-tunable properties.1-3 In particular, hydrogels formed in situ have been widely utilized as injectable delivery carriers of therapeutic agents (e.g., cells or growth factors) in minimally invasive manners or as artificial extracellular microenvironments to create engineered tissues for tissue regeneration and for the study of basic cell biology.4-9 In addition to these therapeutic approaches, many researchers have recently endeavored to develop dynamic hydrogel matrices that can induce in situ physicalchemical stimuli (e.g., pH, chemical composition, oxygen tension) to surrounding cells or tissues when implanted in vivo for enhancing cellular activities or facilitating tissue regeneration.10-11 Reactive oxygen species (ROS) play pivotal roles in cell signaling and homeostasis.12 ROS include hydrogen peroxides (H2O2), superoxide, hydroxyl radicals, and singlet oxygen, which have been implicated as critical signaling molecules for versatile therapeutic applications, such as vascular disorders, wound healing, anti-cancer therapies, and anti-bacterial treatment.13-17 These bioactive molecules are well known as a double-edged sword. While an appropriate concentration of ROS is essential to maintain homeostasis and cellular activity, it has been demonstrated that prolonged exposure to high levels of ROS can cause significant damage to cells.18-19 In particular, H2O2, among these active molecules, is well known as a signaling molecule that regulates the activities of vascular cells.13,

18

Growing evidence has recently demonstrated that transient or low levels of H2O2 (ranging from 0.1 to 10 µM) facilitate angiogenic activities of endothelial cells (ECs) and regulate their function through acute oxidative stress via evaluated intracellular ROS levels in the cells.20-22 3 ACS Paragon Plus Environment


In this study, we developed H2O2-releasing hydrogels as a dynamic matrix via dual enzymatic crosslinking reaction using horseradish peroxidase (HRP) and glucose oxidase (GOx). We demonstrate that H2O2 release behaviors were accurately controlled by varying GOx concentrations. The H2O2-controllable hydrogels with the optimal condition evaluate transient intracellular ROS levels in ECs and enhance proliferative activities of ECs in vitro. In addition, the H2O2-releasing dynamic matrices facilitate host tissue infiltration and neovascularization in ovo. Although H2O2 plays a pivotal role in vascular development and angiogenesis, as far as we know, it still remains a challenge to develop the H2O2-releasing hydrogels with precisely controlled H2O2 levels, which could stimulate endothelial cell activities and neovascularization without compromising cytocompatibility. We suggest that our H2O2-releasing hydrogels have great potential as injectable and dynamic matrices for the treatment of vascular disorders as well as in tissue regenerative medicine.

2. MATERIALS AND METHODS 2.1. Materials For polymer synthesis, gelatin (Gtn, type A from porcine skin, less than 300 bloom), peroxidase from horseradish (HRP, type VI, salt-free, 250–330 units per milligram solid), hydrogen peroxide (H2O2, 30% w/v in H2O), 3-(4-hydroxyphenyl) propionic acid (HPA), 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), and deuterium oxide (D2O) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dimethylformamide (DMF) was obtained from Junsei (Tokyo, Japan). The chemical reagents were used as obtained without purification. Dialysis membrane (molecular cutoff = 3,500 Da) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA)


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For in vitro cell culture, human umbilical vein endothelial cells (HUVECs) and endothelial cell growth medium-2 (EGM-2 Bullet Kit, cat. no. CC-3162) were purchased from Lonza (Walkersville, MD, USA). Dulbecco’s phosphate-buffered saline (DPBS), penicillin-streptomycin (P/S), and 0.05% trypsin-0.1% ethylene-diamine tetraacetic acid (trypsin-EDTA) was purchased from Gibco (CA, USA). Live/dead kit was purchased from Molecular Probes (Eugene, OR, USA). WST-1 Cell Proliferation kit was supplied by SigmaAldrich (Saint Louis, MO, USA). For in ovo angiogenesis models, fertilized eggs of the domestic fowl (Gallus domestics, strain high line brown) were purchased from saenal egg farm (Na-Ju, South Korea). Anti-alpha smooth muscle actin (α-SMA) antibody was purchased from Abcam (cat. no. ab64261, Cambridge, UK). Formaldehyde (10%), paraformaldehyde, Triton X-100, bovine serum albumin, Harris hematoxylin solution, and Eosin Y solution were all purchased from Sigma-Aldrich (Saint Louis, MO, USA) 2.2. Synthesis of gelatin-g-hydroxyphenyl propionic acid (GH) Gelatin-g-hydroxyphenyl propionic acid (GH) polymer was synthesized using EDC/NHS as coupling reagents, as previously described.23 For synthesis, a mixture of water and DMF at a volume ratio of 3:2 was prepared as a solvent. Gtn (5 g) was dissolved in 150 mL of the solvent at 40 °C. HPA (0.913 g, 6 mmol) was dissolved in 30 mL of the solvent and reacted with EDC (0.932 g, 6 mmol) and NHS (1.036 g, 9 mmol) at 25 °C for 30 min to activate the terminal carboxyl groups of HPA. The activated HPA solution was then applied to the Gtn solution, and a conjugative reaction was conducted at 40 °C for 24 h. After the reaction, the solution was dialyzed against distilled water for three days (molecular cutoff = 3,500 Da). After dialysis, the GH polymer was obtained by freeze-drying and the product was kept in a refrigerator before use. The degree of substitution (DS) of HPA was measured using a UV/Vis spectrometer (V-750, Jasco, Japan). For the UV measurement, GH polymer (1 mg) 5 ACS Paragon Plus Environment


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was dissolved in 1 mL of a mixture of the solvent, and the absorbance was measured at a wavelength of 275 nm. The concentration of the conjugated HPA molecules was calculated from a calibration curve given by monitoring the absorbance of a known concentration of HPA, standardized with the baseline measured using Gtn solution (1 mg/mL). The chemical structure of GH was characterized using a 1H NMR spectrometer (AS400, OXFORD Instruments, UK). The GH polymer solution (25 mg/mL in D2O) was prepared for the measurement. 2.3. Preparation of H2O2-releasing hydrogels H2O2-releasing hydrogels were prepared by simply mixing two types of solutions (sol A and sol B), as previously reported.24 Briefly, to fabricate solution A, HRP (0.05 mg/mL) and GOx (0–500 µU/mL) were dissolved in DPBS, followed by mixing with 2.5 wt% GH solution (volume ratio of GH:HRP: GOx = 8:1:1). To prepare solution B, 2.5 wt% GH was dissolved with H2O2 (9.81 mM) and glucose (500 mM) (volume ratio GH:H2O2: glucose = 8:1:1). In order to fabricate the H2O2-releasing hydrogels, the solutions (A and B) were mixed in a volume ratio of A: B = 1:1 and gently shaken at room temperature. The detailed final concentrations of each component are given in Table 1.

Table 1. Experimental conditions for the characterization of H2O2-controllable hydrogels Polymer

HRP

H2O2

GOx

Glucose

Elastic modulus

(wt%)

(µg/mL)

(mM)

(µU/mL)

(mM)

(G’, Pa)

GOx 0

2

2.5

0.49

0.00

25

250

GOx 0.31

2

2.5

0.49

0.31

25

248

GOx 0.63

2

2.5

0.49

0.63

25

254

GOx 1.25

2

2.5

0.49

1.25

25

251

GOx 2.5

2

2.5

0.49

2.50

25

248

GOx 5

2

2.5

0.49

5.00

25

249

Sample code


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2.4. Quantification of H2O2 released form hydrogels To analyze the release behavior of H2O2 from the GH hydrogel matrices, the amounts of H2O2 released from the hydrogels were quantified by Quantitative Peroxide Assay Kit (Pierce, Rockford, IL, USA) using the ferrous ion (Fe2+) oxidation xylenol orange (XO) assay, as previously described.16, 24 Briefly, 100 µL of a GH hydrogel was prepared in a 48-well plate and incubated with 900 µL of EGM-2. At predetermined time points, 20 µL of each medium (including H2O2 released from the hydrogel matrices) was collected and added to a 96-well plate. A 200-µL aliquot of each assay kit was added to the collected sample and incubated at room temperature for 15 min. The absorbance was measured at 595 nm using a multimode microplate reader (BioTek Instruments, Inc., VT, USA). Using the difference in absorbance between the samples and blank solutions, a calibration curve was generated. 2.5. Rheological analysis We performed rheological analysis of the H2O2-releasing hydrogels using an Advanced Rheometer (GEM-150-050, Bohlin Instruments, USA) in oscillatory mode. The hydrogel samples were prepared on the plate in the instrument and we performed dynamic time sweep on the samples in various conditions (Table 1). We monitored the changes in the elastic modulus (G’) at a frequency of 0.1 Hz using parallel plate geometry (diameter = 20 mm, gap = 0.5 mm, stress = 10 Pa). A solvent trap wetted with deionized water was used to prevent sample evaporation. 2.6. In vitro HUVEC culture For in vitro HUVEC culture, all solutions were prepared using DPBS and filtered for sterilization using a syringe filter with a pore size of 200 nm. HUVECs (Lonza, Walkersville, MD, USA) were cultured in endothelial growth medium supplemented with 2% fetal bovine 7 ACS Paragon Plus Environment


serum, endothelial growth supplemental mix (EGM-2 SingleQuot Kit), and 1% P/S (Gibco) under standard culture conditions (37 °C and 5% CO2). For 2D HUVEC proliferation study, we performed WST-1 assays (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, HUVECs (1.5 × 104 cells/well in a 48well plate, SPL Life Sciences (SPL), cat. no. 30096) were cultured for 20 h, following by incubation in EBM-2 with 60 µL of hydrogel disc. After pre-incubation, the hydrogels and culture medium were removed, and the cells were then cultured in 300 µL of medium containing 10% WST-1 solution for 30 min. For the quantitative analysis, the medium (100 µL) was removed, placed in a 96-well plate, and measured using a microplate reader at a wavelength of 450 nm. Cell viability and proliferation were determined as a percentage of those of control cells (HUVECs cultured on tissue culture plate with GHPA hydrogel disc without GOx were defined as 100%). For 3D HUVEC culture, we prepared two types of polymer solutions (solution A and B) as described above. Solution A was mixed with HUVEC pellets to create a cell suspension (3.5 × 106 cells/mL), and then solution B was added at a volume ratio of 1:1 (A: B) and gently mixed. The mixture (60 µL) was placed in a cylindrical mold (diameter = 5 mm; thickness = 3 mm) and allowed to form hydrogels at room temperature for 10 min. Cells encapsulated within the hydrogel disc were placed in a 48-well plate and cultured with 500 µL of EGM-2 (Lonza) under standard culture conditions (37 °C and 5% CO2) for up to 24 h. The cell morphology was observed using optical microscopy in phase-contrast mode using an inverted phase contrast microscope (Eclipse TS100, Nikon). For cell viability test, after 24 h of incubation, we treated cells with a mixture of 200 µL of 2 µM acetomethoxy derivate of calcein and 4 µM ethidium homodimer-1 at 37 °C for 30 min. The stained samples were washed three times using DPBS and then observed using fluorescence microscopy (inverted microscope, Eclipse Ti-E system, Nikon). For quantitative analysis, WST-1 assay was 8 ACS Paragon Plus Environment

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performed according to the manufacturer’s instruction. In brief, the 3D hydrogel matrices encapsulating the cells were incubated in 300 µL of culture medium containing 10% WST-1 solution for 30 min. For the quantitative analysis of cell viability, the medium (100 µL) was placed in a 96-well plate and measured using a microplate reader at a wavelength of 450 nm. Cell viability and proliferation were determined as a percentage of those of control cells (HUVECs cultured within the hydrogels without GOx were defined as 100%). 2.7. Intracellular ROS assay We performed intracellular ROS assay using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as an oxidant-sensing probe, according to the manufacturer’s instruction. Briefly, HUVECs (8.0 × 103 cells/well in 96-well plate, SPL cat. no. 30096) were cultured for 20 h, following by incubation in culture medium with 30 µL of hydrogel disc. After 24 h of incubation, the hydrogel samples and culture medium were removed, and the cells were then cultured in 100 µL of medium containing 10 µM DCFH-DA solution for 30 min. The cultured cells were observed using optical microscopy in phase-contrast mode using an inverted microscope (Eclipse Ti-E system, Nikon) and fluorescence microscopy (inverted microscope, Eclipse Ti-E system, Nikon) after washing with 100 µL of DPBS and cultured using 200 µL of EBM-2. For quantitative analysis, we calculated the ratio of fluorescencepositive cells to all cells on the plates. 2.8. In ovo chicken chorioallantoic membrane (CAM) models The in vivo angiogenic activity of the H2O2-controllable hydrogels was assessed by an openshell chicken CAM assay, as previously described.25 Fertilized eggs (Gallus domestic, strain high line brown) were incubated at 37 °C for six days at 40–60% humidity, and the eggs were turned every minute. On day six of incubation, the eggs were moved out from the incubator to completely sterilized conditions in a laminar hood after sterilization with a 70% ethanol



pad. We created windows on the eggs (diameter = approximately 20 mm) and the polymer solutions (100 µL) were injected under the CAM. The windows were covered with sterilized parafilms and aluminum foil and incubated at 37 °C at 40–60% humidity in aseptic conditions. At the predetermined time point, we sacrificed the eggs and the hydrogels were removed for histological analysis. The explants were fixed using 10% formalin (SigmaAldrich). The animal study was performed using a protocol approve by Incheon National University Institutional Animal Care and Use Committee. 2.9 Histological analysis The fixed samples were dehydrated in graded ethanol (80–100%), embedded in paraffin. They were serially sectioned (4 µm) using a microtome and stained with either hematoxylin and eosin (H&E) or subjected to immunohistochemistry for α-SMA. 2.10. Statistical analysis All measurements of hydrogel characterization, including H2O2 release kinetics and mechanical strength, were carried out using triplicate samples for each data point. The statistical analyses of in vivo and in vitro studies were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). We also used the software to perform t-test to determine significance. Significant levels and determined post-tests were set at: *P < 0,05, **

P < 0.01, and ***P < 0.001.

3. RESULTS AND DISCUSSION 3.1. H2O2-releasing hydrogel synthesis and controlled H2O2 release In our previous study, we developed dual enzyme-triggered in situ crosslinkable gelatin hydrogels as artificial cellular microenvironments using HRP and GOx.24 We demonstrated that the hydrogels provided engineered cellular microenvironments without compromising 10 ACS Paragon Plus Environment

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cytotoxicity issues. In addition, we noticed that the amount of released H2O2 could be controlled by varying GOx concentrations in the range of 0 to 1000 µM. In the present study, we hypothesized that the down-regulation of H2O2 release amount (0.1–10 µM) from the hydrogels can provide dynamic hydrogel matrices that may stimulate vascular cell activities and neovascularization. We fabricated GH hydrogels through dual enzyme-mediated crosslinking reaction using HRP and GOx as an H2O2-generating enzyme to gradually supply a radical source in HRP-mediated crosslinking reactions (Fig. 1a). Figure 1b shows the digital images of hydrogel formation by simply mixing the polymer solutions. To determine whether the elastic moduli (G’) of the hydrogels was constantly adjusted regardless of the GOx concentrations, we first measured the viscoelastic modulus of the hydrogels using a rheometer. It should be noted that the hydrogels were fabricated with the same concentration of HPA molecules, maintaining the same crosslinking density which affected mechanical properties. We monitored the time-course elastic modulus of the GH hydrogels depending on GOx concentrations (0–5 µU/mL). Interestingly, we observed that the elastic modulus was similar (210–240 Pa) in all groups (Fig. 1b), even with different GOx concentrations, suggesting that hydrogels with constant mechanical strength could be fabricated regardless of the GOx concentrations. We next investigated the effect of GOx concentrations (0–5 µU/mL) on H2O2 release behavior. To examine H2O2 release behavior, we analyzed the amount of H2O2 released from the hydrogel matrices using a ferrous (Fe) ion oxidation xylene orange assay by quantitative peroxide assay kit, as previously reported.16, 24 We found that increasing GOx concentrations (0–5 µU/mL) released higher concentrations of H2O2 from the hydrogel matrices ranged from 0 to 9.62 µM for up to 48 h (Fig. 1c), demonstrating that our hydrogels provide a dynamic hydrogel matrix with precisely controllable properties of H2O2 release amount.



In sum, we fabricated in situ crosslinkable hydrogels through dual enzyme-mediated crosslinking reaction without compromising hydrogel stiffness, which can precisely control H2O2 release behaviors.

Figure 1. H2O2-releasing hydrogel synthesis and characterization. (a) Schematic representation of in situ formation of H2O2-releasing hydrogel through dual enzyme-mediated crosslinking reaction. Newly formed chemical bonds are indicated in red. (b) Elastic modulus (G’) of the hydrogels with varying GOx concentrations. (c) In vitro H2O2 release behavior from the hydrogel matrices with varying GOx concentrations. Results in b, c are shown as average values ± s.d. (n = 4).

3.2. H2O2-releasing hydrogels as dynamic microenvironments for enhanced endothelial cell activities Growing evidence has demonstrated that adequate concentrations of H2O2 play a pivotal role in regulating vascular cell activities, including proliferation, migration, and vascular morphogenesis for vascular development and angiogenesis.13, 18, 20 While many studies have 12 ACS Paragon Plus Environment

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demonstrated that exogenous H2O2, which might elevate intracellular ROS levels, stimulated vascular cell activities, it is not yet clear to accurately determine H2O2 concentrations affecting on the biological activities. Generally, H2O2 is widely used at 10 µM or lower to improve vascular cell activity, and its exact concentration has been reported to vary slightly depending on culture conditions and cell type.20-22 We speculated that the H2O2-releasing hydrogels could provide either dynamic acellular matrices or cellular microenvironments, which would enhance proliferative activities of vascular cells depending on the concentration of the released H2O2. In using H2O2-controllable hydrogels to enhance vascular cell activities, we selected HUVECs, which are widely used as a laboratory model system to study the function and pathology of endothelial cells (e.g., angiogenesis). We first investigated the effect of released H2O2 concentrations on HUVEC viability using WST-1 cell proliferation kit. Toward this, we cultured HUVECs on plates with hydrogel samples at different GOx concentrations (0–2.5 µU/mL) for 24 h. Although we observed elongated structures of HUVECs cultured in all groups, there were no significant differences in vascular morphogenesis between the samples (Fig. 2a). Interestingly, we noticed that the optimal hydrogel condition (GOx 2.5 hydrogels) releasing 7.62 µM H2O2 for 24 h enhanced HUVEC viability (115.58 ± 6.01%) compared to other groups (GOx 0, 100 ± 00%; GOx 1.25, 105.35 ± 14.64%) (Fig. 2b), demonstrating that our H2O2-releasing hydrogels with the optimal condition could stimulate proliferative activities of HUVECs. We also investigated the effect of higher GOx concentration (GOx 5.0), showing the cytotoxicity due to the high concentration of released H2O2 amount. Thus, we used the optimal hydrogels (GOx 2.5) showing higher activities for further studies.



Figure 2. H2O2-releasing hydrogels as a dynamic acellular matrix for enhanced endothelial cell viability. (a) Light microscopic images and (b) quantification of cell proliferative activities of HUVECs cultured on plates with hydrogels (as an acellular matrix) with varying GOx concentrations for 24 h. Scale bars, 100 µm. Results in b are shown as average value ± s.d. (n = 6). Significant levels were set at: *P < 0.05, **P < 0.01, and ***P < 0.001.

In studying the importance of the 3D H2O2-controllable matrices in regulating vascular cell activity, we hypothesized that the hydrogels would enhance proliferative activities of ECs within hydrogels. To test this, we encapsulated HUVECs within hydrogels with different GOx concentrations (0–2.5 µU/mL) and cultured them for up to 24 h. We observed predominately viable cell populations (>80%) with elongated structures of HUVECs cultured in all groups, but there was no significant difference in cell viability between the samples (Fig. 3a). These results demonstrated that our hydrogel systems are cytocompatible and could support vascular cell spreading and elongation regardless of the concentration of released H2O2. It is demonstrated that vascular development (e.g., vasculogenesis and angiogenesis) are very complex and multi-step process that involves matrix remodeling, vascular cell proliferation and migration, lumen formation, cell to cell junctions, and micro-capillary formation.26-27 While we observed cell sprouting and cell-cell junction that is one of the important processes in vascular developments, microvasculature or lumen structures were not identified within the hydrogels. This result may be explained by the fact that the cell culture periods is not enough to form capillary-like tube structure.28-30 Notably, we found that the optimal condition (GOx 0.3 hydrogels) releasing 2.95 µM H2O2 for 24 h enhanced HUVEC viability within the microenvironments (Fig. 3b and c), 14 ACS Paragon Plus Environment

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suggesting that our H2O2-releasing hydrogels could provide engineered cellular microenvironments that stimulate proliferative activities of HUVEC. We noticed that optimal hydrogel conditions were different between 2D and 3D culture systems (in 2D culture, GOx 2.5; in 3D culture, GOx 0.3). The cells encapsulated within the hydrogels (GOx 0.3) shows highest cell proliferative activities in 3D culture systems, while the cells cultured with GOx 2.5 hydrogels in 2D system exhibited higher proliferative activities compared to the others. In fact, it is difficult to directly correlate the results between 2D and 3D culture models as there are more parameters should be considered in the 3D culture systems compared to the 2D systems, including cell density, cell-tomaterials interactions. This results may be explained by the facts that 1) the cells encapsulated within hydrogel matrices are exposed to a relatively high initial amount of H2O2 compared to 2D models, and 2) it is caused by the different cell density in the both in vitro culture systems (2D, 1.5 × 104 cells/well; 3D, 3.5 × 106 cells/mL). Taken together, we demonstrated that the H2O2-releasing hydrogels could provide either bioactive acellular or cellular microenvironments that stimulate HUVEC viability at the optimal H2O2 concentrations.



Figure 3. H2O2-releasing hydrogels as a dynamic cellular microenvironment for enhanced endothelial cell viability. (a) Optical microscopic images and (b) fluorescence microscopic images of HUVECs within the H2O2-releasing hydrogels with varying GOx concentrations (as a cellular matrix). Scale bars, 100 µm. (c) Quantitative analysis of HUVEC proliferation within the hydrogel matrices with different concentrations of GOx. Results in c are shown as average value ± s.d. (n = 3–9) Significant levels were set at: *P < 0.05, **P < 0.01, and ***P < 0.001.


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3.2. Upregulation of intracellular ROS levels in HUVECs by exogenous H2O2 from hydrogel matrices Intracellular ROS levels have been implicated as a critical factor in regulating vascular cell activities, including proliferation, migration, and vascular morphogenesis as well as cell death through apoptosis when proper ROS levels are maintained. To better understand of the effect of exogenous H2O2 released from the hydrogels on intracellular ROS levels, we analyzed intracellular ROS levels in HUVECs cultured with hydrogels with the different condition of hydrogels (GOx 0 and GOx2.5) for 3 and 24 h. We selected tissue culture plate (TCPS) and GOx 0 hydrogels as controls. Interestingly, we found that GOx 2.5 hydrogels exhibited a higher percentage of ROS-positive cells (32.0%) compared with those on TCPS (15.5%) and GOx 0 (16.5%) as a control after 3 h of culture, demonstrating that exogenous H2O2 released from the hydrogels elevated transient ROS levels in HUVECs (Fig. 4) that may stimulate HUVEC activities13,

21

. In order to confirm the time-dependent effect,

intracellular ROS levels were monitored after 24 h of incubation when all of the H2O2 was released from the hydrogel matrices, resulting in no significant different ROS levels in cells cultured with GOx 2.5 hydrogels and those of control groups (TCPS, 7.0 ± 3.5 %; GOx 0, 11.7 ± 0.6 %; GOx 2.5, 6.7 ± 3.2%). This result demonstrates that our H2O2-releasing hydrogels (GOx 2.5) temporally increase the initial intracellular ROS cells in HUVECs, which can stimulate angiogenic activities of ECs. The result is considered as a critical advantage of our hydrogel system because long-term over-expression of intracellular ROS may cause cell death through apoptosis and may exert negative effects on cellular activity.12, 18



Figure 4. Upregulation of intracellular ROS levels by sustained release of H2O2 from the hydrogel matrices. (a) Representative fluorescence microscopy images. Scale bars, 100 µm. (b) quantitative analysis of fluorescence-positive cells. Result in b is shown as average value ± s.d. (n = 3) Significant levels were set at: *P < 0,05, **P < 0.01, and ***P < 0.001; ## indicates not significant.

3.4. Enhanced neovascularization of H2O2-releasinf hydrogels in ovo Although numerous studies have demonstrated that H2O2 stimulates vascular cell activity and angiogenesis in vitro and in vivo13, 19-20, it is still not fully understood how acute oxidative stress induced by exogenous H2O2 stimulates angiogenesis and neovascularization in vivo. In investigating the potential application of H2O2-releasing hydrogels for the treatment of vascular disorders, we hypothesized that our hydrogels could induce acute oxidative stress in surrounding tissues through in situ hydrogel formation with H2O2 release from the hydrogels, which in turn would facilitate neovascularization as well as host tissue invasion. To confirm the angiogenic effect and tissue infiltration of our hydrogels, we injected the hydrogels (100 µL of GOx 2.5) under CAM in ovo and incubated them for up to seven days (Fig. 5a). To investigate the effect of H2O2 released from hydrogel matrices on 18 ACS Paragon Plus Environment

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tissue infiltration and neovascularization, we selected the GOx 0 hydrogels as a control group. Interestingly, we observed that GOx 2.5 hydrogels facilitated tissue infiltration into the hydrogel matrices compared to the control group (Fig. 5b). This result may be caused by the activation of tissue-materials interfaces through temporal oxidative stress.31-32 In fact, many studies have demonstrated that the induction of acute oxidative stress stimulates the surrounding tissues and facilitates blood vessel recruitment as well as neovascularization.10, 15, 18

To

evaluate

neovascularization

and

tissue

infiltration,

immunohistochemistry with α-SMA, as previously reported.29,

33-34

we

performed

Figure 5c shows

histological images of sections stained with α-SMA, indicating mature blood vessels. While α-SMA-positive cells were observed within both hydrogels, higher density of α-SMApositive cells was observed within GOx 2.5 hydrogels (82.0 α-SMA-positive cells in histological field) compared to GOx 0 (34.7 α-SMA-positive cells in histological field) as a control (Fig. 5d), demonstrating that GOx 2.5 hydrogels enhanced vascular cell infiltration from the surrounding tissues. Collectively, our results demonstrated that acute oxidative stress induced by H2O2-releasing hydrogels facilitated angiogenesis and host tissue infiltration in the in ovo models. Therefore, our H2O2-controllable hydrogels can be further utilized as a dynamic acellular matrix for the treatment of vascular disorders and in tissue generative medicine. Growing evidence has demonstrated that controlling ROS levels plays a critical role in facilitating wound healing process and vascular developments, while the essential concentration of ROS has not been reported yet.35-37 It is well-known that the ROS is a double-edged sword. In some previous studies, it has been reported that antioxidants are helpful in regulating wound healing processes and angiogenesis through suppression of the ROS surrounding wound or defect area.35-36 Many others have reported that transients and acute oxidative stress promoted wound healing and repair, as well as vascular recruitments.37 Our hydrogel systems have an advantage, such as precisely controllable H2O2-releasing 19 ACS Paragon Plus Environment


behavior, for the treatment of vascular disorders and wound management. Although the H2O2-controllable hydrogels can induce acute oxidative stress by regulating the ROS levels for enhanced wound healing processes as well as angiogenesis, it should be carefully considered to use the hydrogels in a chronic wound (e.g., diabetic wound) with hypoxic or infected environments that induce higher ROS levels compared to the healthy or normal tissues.38

Figure 5. In ovo angiogenic effect of the H2O2-releasing hydrogels. (a) Schematic representation of CAM assay protocols. Histological sections of the hydrogels seven days after injection, stained with (b) H&E and (c) α-SMA. (d) Quantification of number of blood vessels surrounding and penetrating the hydrogel matrices. H, hydrogels; C, CAM. Scale bars, 100 µm. Result in d is shown as average ± s.d. (n = 3) Significant levels were set at: *P < 0,05, **P < 0.01, and ***P < 0.001.


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4. Conclusions In the present study, we developed gelatin-based H2O2-releasing hydrogels as injectable and dynamic matrices. We successfully fabricated the hydrogels through dual enzymatic crosslinking reaction using HRP and GOx. We could accurately control H2O2 release amount (0–9.62 µM) from the hydrogels by varying the GOx concentrations. The optimal hydrogels (GOx 2.5) enhanced EC viability with transient upregulation of intracellular ROS levels in vitro. In addition, our hydrogels promoted neovascularization and host tissue infiltration via material-tissue interactions in ovo. In conclusion, our H2O2releasing hydrogels have great potential as injectable and dynamic matrices for the treatment of vascular disorders, including diabetic wounds, ischemia, peripheral vascular disease, and myocardial infarction, as well as in tissue regenerative medicine.

5. Acknowledgements This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (2015R1A2A1A14027221) and by the Incheon National University International Cooperative Research Grants in 2015.

6. Conflict of interest The authors declare no conflict of interest.



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Hydrogen peroxide-releasing hydrogels for enhanced endothelial cell

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