Oxygen-Releasing Antioxidant Cryogel Scaffolds with Sustained

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Biological and Medical Applications of Materials and Interfaces

Oxygen releasing antioxidant cryogel scaffolds with sustained oxygen delivery for tissue engineering applications Parvaiz Ahmad Shiekh, Anamika Singh, and Ashok Kumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01736 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Oxygen Releasing Antioxidant Cryogel Scaffolds with Sustained Oxygen Delivery for Tissue Engineering Applications Parvaiz A. Shiekh, Anamika Singh, and Ashok Kumar* Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, UP, India

*Correspondence should be addressed to: Ashok Kumar Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, UP, India E-mail: [email protected] (A. Kumar) Tel: +91 512 3594051

Key words: antioxidant, calcium peroxide, cryogel, oxygen release, controlled release.

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Abstract With the advancement in biomaterial sciences, tissue engineered scaffolds are developing as a promising strategy for the regeneration of damaged tissues. However, only a few of these scaffolds have been translated into clinical applications. One of the primary drawbacks of the existing scaffolds is the lack of adequate oxygen supply within the scaffolds. Oxygen producing biomaterials have been developed as an alternate strategy but are faced with two major concerns. One is controlling the rate of oxygen generation, and the other is the production of reactive oxygen species. To address these concerns, here, we report the development of an oxygen releasing antioxidant polymeric cryogel scaffold (PUAO-CPO) for sustained oxygen delivery. PUAO-CPO was fabricated using the cryogelation technique by incorporation of calcium peroxide in the antioxidant polyurethane scaffolds. PUAO-CPO cryogels attenuated reactive oxygen species and showed a sustained release of oxygen over a period of 10 days. An in vitro analysis of the PUAO-CPO cryogels showed their ability to sustain H9C2 cardiomyoblast cells under hypoxic conditions, with cell viability being significantly better than normal polyurethane scaffolds. Furthermore, in vivo studies using an ischemic flap model showed the ability of the oxygen releasing cryogel scaffolds to prevent tissue necrosis upto 9 days. Histological examination indicated maintenance of tissue architecture and collagen content, whereas immunostaining for proliferating cell nuclear antigen (PCNA) confirmed the viability of the ischemic tissue with oxygen delivery. Our study demonstrated an advanced approach for the development of oxygen releasing biomaterials with sustained oxygen delivery as well as attenuated production of residual reactive oxygen species and free radicals either due to ischemia or oxygen generation. Hence, the oxygen releasing PUAO-CPO cryogel scaffolds may be used with cell based therapeutic approaches for regeneration of damaged tissue, particularly with ischemic conditions such as myocardial infarction and chronic wound healing.


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1. Introduction Despite tremendous advances in tissue engineering, vascularization continues to remain a challenge for the regeneration of clinically relevant tissues. Three dimensional (3D) tissue engineering scaffolds face a major problem in terms of nutrient and oxygen availability to the cells after transplantation.1 Sufficient oxygen availability is essential for cell survival, growth, and differentiation.2,3 Inadequate oxygen diffusion critically restricts the development of a functional tissue post implantation.4 This is especially important for large size defects, which require adequate blood and nutrient supply for cell viability. Sufficient nutrient and oxygen diffusion within the scaffold facilitates cellular migration, neovascularization, and tissue ingrowth.5 Oxygen deprivation, mostly within the scaffold, leads to hypoxia, which causes tissue necrosis and the lack of development of neo-vascularization.6 It has been reported that 3D scaffolds should have large and interconnected pores of around 100 µm for sufficient oxygen and nutrient diffusion.7 If the distance between the blood vessel and the cells seeded within a bioengineered scaffold is more than 100 – 200 µm, the cells will not survive, and this will ultimately lead to cell and tissue necrosis.1 In particular, cells with a high oxygen demand, such as cardiac cells, neurons, hepatocytes, and β cells within the pancreas, are highly sensitive to oxygen deprivation and cease to function under hypoxia conditions.8 Thus, oxygen diffusion is more vital for tissue survival especially under ischemic conditions such as myocardial infarction and chronic wound healing.9,10 Hypoxia also leads to the formation of free radicals, which further aggravate cellular damage.8 These free radicals react with biological entities such as proteins or DNA, making them inactive. To overcome the diffusion limitations of oxygen within the scaffolds under in vivo conditions, an imperative solution would be to facilitate the infiltration of the host vasculature within the scaffold. However, host vasculature takes 1-2 weeks to infiltrate the interior of a 3 ACS Paragon Plus Environment


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scaffold of 3 mm thickness.11 During this period the availability of oxygen inside the scaffold and in surrounding tissue plays a critical role in cellular and tissue viability. Increasing the availability of oxygen inside the scaffolds will help to overcome these effects and is a key issue while designing next generation scaffolds for tissue engineering applications. To augment this, oxygen releasing biomaterials may provide as a potential rescue therapy to attenuate these conditions and maintain cellular viability and signaling during this period. Recent studies have focused upon the development of oxygen releasing biomaterials for providing prolonged and sufficient oxygen both in in vitro and in vivo conditions.12–17 These biomaterials produce oxygen through chemical reactions of the encapsulated oxygen releasing materials. The most commonly used materials for oxygen generation are sodium percarbonate (Na2CO3), calcium peroxide (CaO2), magnesium peroxide (MgO2) and hydrogen peroxide (H2O2).12,15,18 One of the major challenges for the design of oxygen releasing biomaterials is the controlled release of oxygen from the scaffolds-an important parameter for the viable outcome of the tissue engineered scaffold. Most of the oxygen releasing materials produces oxygen by chemical decomposition, when exposed to water. The rate of oxygenation depends on various factors such as pH, temperature, solubility of the oxygen releasing material, diffusion of water inside the scaffold, and exposure to an aqueous environment.19 A burst release of oxygen can result in oxidative damage to the cells. Encapsulation of this oxygen releasing materials in a hydrophobic polymer could slow down the release of oxygen.15 Another major challenge is the production of free radicals. Some oxygen releasing biomaterials such as peroxides, produce free radicals as a reaction intermediate during oxygen generation.13 Accumulation of these free radicals increases oxidative stress, thus reducing cell viability. To counter that, an antioxidant such as catalase-a naturally occurring antioxidant enzyme, can be incorporated inside the scaffold to attenuate


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the production of the free radicals.20 However, incorporation of an enzyme inside the scaffold complicates the design of the scaffold and does not guarantee stability of the enzymes. Development of oxygen releasing scaffolds with antioxidant properties will offer a unique solution to prevent hypoxia induced cell death by providing sufficient oxygen and attenuating the free radicals produced during oxygen generation. Considering these points, in this work, we hypothesized that the incorporation of solid peroxides into an antioxidant hydrophobic polymer will yield an oxygen generating 3D scaffold with the sustained oxygen release as well as attenuating the effect of free radicals. By doing so, this oxygen releasing scaffolds may increase cell survival and minimize tissue necrosis under hypoxic conditions. To achieve this, calcium peroxide (CPO) as an oxygen generating material is incorporated in an antioxidant polyurethane polymeric material (PUAO). Previously, our group has reported the fabrication of

an antioxidant polyurethane polymer PUAO, which has shown adequate

antioxidant properties.21 The polymer could attenuate free radicals and oxidative stress induced cell death. CPO incorporated PUAO-CPO cryogel could provide sustained release of oxygen as well as combat the oxidative stress leading to better survival of critically perfused tissues. To the best of our knowledge, this is the first study in which an oxygen generating antioxidant scaffold has been developed to successfully maintain cell survival under hypoxia conditions and prevent tissue necrosis in an in vivo skin flap model.

2. Materials and Methods 2.1. Polyurethane synthesis All chemicals were of ACS grade and procured from Sigma Aldrich (St. Louis, MO, USA) unless otherwise mentioned. Antioxidant polyurethane (PUAO) was synthesized as reported previously.21 Briefly, Polycaprolactone diol 2000 (PCL diol, 8g) was dissolved in anhydrous dimethyl sulfoxide (DMSO, 60 ml). Hexamethylene diisocyanate (HDI 1.34, g) was charged 5 ACS Paragon Plus Environment


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into a three-necked flask equipped with magnetic stirrer and inlet for nitrogen, at 80 °C with tin(II) 2-ethylhexanoate as a catalyst. After 4 h, ascorbic acid in a molar ratio of 1: 0.8 with PCL diol, dissolved in DMSO was added and the reaction continued for another 2 h for final polyurethane formation. The polymer was precipitated in cold distilled water, dried in a vacuum oven at 40 °C and further lyophilized for 24 h for solvent removal. PUAO was stored at room temperature until further use. Polyurethane without antioxidant properties (PU) was synthesized using the same methodology as described above, but replacing ascorbic acid with butanediol as a chain extender. 2.2. Fabrication of oxygen releasing PUAO-CPO cryogels PUAO solution (5% w/v) was prepared by dissolving PUAO in DMSO at 60 °C. Calcium peroxide (CPO, 75% purity) was ground and passed through a 40-micron sieve to get uniform size particles of less than 40 µm. CPO was added at three different concentrations of 1%, 2% and 3% (w/v) and stirred overnight for homogenous distribution of CPO in PUAO. PUAOCPO solution was cast in glass petri dishes, frozen overnight at -20 °C and thawed in absolute ethanol/cold water for cryogel formation. The formed cryogels were washed extensively with cold distilled water, lyophilized and stored in a vacuum desiccator to avoid CPO degradation until further use. 2.3. CPO incorporation and distribution in PUAO cryogels To confirm incorporation of CPO in PUAO cryogels, FTIR analysis was performed at room temperature within the range of 4000 cm-1 to 400 cm-1 using PerkinElmer spectrum version 10.03.06. Distribution and quantification of CPO inside the PUAO-CPO cryogels was studied by alizarin red staining which specifically stain’s calcium. Briefly, cryogels were incubated with freshly prepared alizarin red solution (2% w/v) in distilled water (pH 4.2) for 20 min, followed by rinsing with running distilled water to remove the unbound dye. The cryogels


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were microphotographed using a bright field microscope (Leica DM2500) fitted with CCD camera. 2.4. Antioxidant assay To study the antioxidant properties of the PUAO and PUAO-CPO cryogels; DPPH assay was carried out. Briefly 50 mg of PUAO and 1% PUAO-CPO cryogel after lyophilization were immersed in 1 ml of 200 µM DPPH solution in ethanol and incubated at 37 °C. Change in absorbance at 517 nm as compared to control DPPH solution was monitored over a period of 180 min, which gave the antioxidant power of PUAO-CPO cryogels. The antioxidant capacity was measured as percentage inhibition and calculated by the following formula, % inhibition = (AB – AS)/AB x 100, where AB is absorbance of control (DPPH only) and AS is absorbance of the sample (DPPH + PUAO-CPO cryogels).21 2.5. Microstructure of PUAO-CPO cryogels The microstructure of PUAO-CPO cryogels was determined using Scanning Electron Microscopy (SEM) analysis. Freeze dried PUAO-CPO cryogels were gold-coated for 90 sec using a sputter gold coater machine (Vacuum Tech, Bangalore, India) and observed using WSEM (FEI Quanta 200) at an accelerating voltage of 12.5 kV. The micrographs obtained were used for microstructure and pore size determination by ImageJ software. (NIH, USA). A total of 10 images were processed for pore size determination, and the average size was calculated. 2.6. Swelling studies and porosity measurement Dried PUAO-CPO cryogels of 1cm x 1cm sizes were immersed in PBS for 12 h at room temperature and the change in weight of swollen cryogel was determined. The percentage swelling was calculated using the formula. % Swelling = (Weight of swollen gel - weight of dry gel)/ weight of dry gel x 100



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Furthermore, porosity was measured using the liquid displacement method with ethanol as a non-solvent.22 The PUAO-CPO cryogel was immersed in a cylinder containing a known volume of ethanol (V1). The sample was then saturated with ethanol for 5 min and the total volume of ethanol along with the ethanol saturated scaffold was measured as V2. Later, the ethanol saturated scaffold was removed from the cylinder, and the residual volume measured as V3. Porosity was calculated using the formula p = (V1-V3)/V2-V3). 2.7. Degradation analysis Degradation analysis of the PUAO-CPO cryogels was carried out in DMEM at 37 °C. Briefly PUAO-CPO cryogels were lyophilized, weighed and incubated in DMEM containing 0.1% antimycotic solution (Himedia, India) after sterilization under UV for 30 min. The samples were removed at different time points, washed with cold PBS, lyophilized for 24 h and weighed again to determine the weight loss due to degradation. The percentage weight loss was calculated as (W1-W2)/W1, where W1 is the initial weight of the PUAO-CPO scaffold, and W2 represented the final weight of the scaffold after degradation. An average value of the triplicates was calculated and reported as mean ± s.d. To determine the morphological change in structure of the scaffold upon degradation, scaffolds were dehydrated in gradient ethanol (70%, 80%, 90%, 95%, 100%) followed by critical point drying in hexamethyldisilazane (HMDS). The dried scaffolds were gold coated for 90 sec and imaged in FE SEM (Carl Zeiss EVO18). 2.8. Oxygen releasing kinetics Oxygen releasing kinetics of PUAO-CPO cryogels was measured using a platinum based oxygen sensor probe (OpTech®- O2 Model P, MOCON, Inc. USA). PUAO-CPO cryogels of 1 cm x 1 cm dimensions were incubated in 3 ml of serum-free DMEM under hypoxia conditions (1% O2, 94% N2, 5% CO2) in a 6 well multiwell plate in a humidified incubator


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(ESCO, CCL-170T-8, Denmark) at 37 °C. Oxygen was measured by epifluorescence noninvasively using an adhesive probe inside the media which was illuminated by visible light to measure oxygen concentration. 2.9. Cell seeding on PUAO-CPO cryogel H9C2 cardiomyoblast cells were used as a model cell line for evaluation of oxygen releasing cryogel scaffolds. Briefly, the cells were grown in T-75 flasks until 70-80% confluency and passaged before seeding. PUAO-CPO cryogels were first washed with chilled PBS-antibiotic (2% penicillin-streptomycin) for 10 min followed by sterilization in absolute ethanol for 1 h under UV light. Again, scaffolds were washed with cold PBS-antibiotic solution for 10 min and repeated 3 times. Excessive washing with PBS was avoided to reduce the premature release of oxygen. After equilibration with DMEM containing 10% FBS for 4 h, the cells were seeded on each cryogel scaffold, and media was added after 4 h of incubation to avoid cell loss. Cells were cultured in DMEM supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (Himedia) and maintained at 37 °C in a humidified CO2 incubator with 1% or 21% O2 as desired. 2.10. Scaffold cytotoxicity assay Scaffold cytotoxicity was analysed by MTT assay. Briefly, extract media was obtained by immersing 1 cm x 1 cm PUAO-CPO cryogels in culture media for 5 days. H9C2 cells were seeded at a density of 5 x 104 cells/ well in a 96 well plate. After 24 h of culture, 200 µl of extract media was added into each well and cell viability was assayed after 24 h and 48 h of culture. Scaffold cytotoxicity was analysed by MTT assay. For this, the cell culture media was removed, and cells were washed with 1X PBS. MTT (0.5 mg/ml) in DMEM was added to the cells and incubated for 4 h at 37 °C in a humidified CO2 incubator. Post incubation, the residual MTT solution was removed and the formazan crystals formed were dissolved in



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DMSO and absorbance measured at 570 nm. Cells cultured in tissue culture plate (TCP) with fresh culture media was used as control. The amount of calcium/calcium hydroxide released into the media was estimated by modified alizarin red assay as reported earlier.23 Briefly, PUAO-CPO scaffolds of dimensions 1 x 1 cm were incubated in cell culture media. To estimated calcium released into media, 100 µl of 40mM Alizarin Red S solution (pH 4.2) was added to 1mL solution of media collected at day 5 and incubated for 30 min to form a red precipitate. The red precipitate was centrifuged at 13000 rpm for15 min, washed repeatedly to remove excess stain and solubilized in 800 µl of 10% acetic acid by heating at 80 °C for 10 min. The solution was neutralized with 200 µl of 10% ammonium hydroxide and the calcium released was quantified by measuring OD at 405 nm. 2.11. Metabolic activity of H9C2 cells on PUAO-CPO cryogels under hypoxia The effect of oxygen released from the PUAO-CPO cryogels on the metabolic activity of H9C2 cells under hypoxia conditions was evaluated by resazurin assay. Briefly H9C2 cells at a density of 2 x 105 were seeded on PU, PUAO and PUAO-CPO cryogels and cultured for 10 days under hypoxia conditions (1% O2, 94% N2, 5% CO2) in a humidified incubator (ESCO, CCL-170T-8, Denmark) at 37 °C. Each cell seeded scaffold of 1 cm x 1 cm size was cultured in 3 ml of DMEM with 10% FBS in a 24 well plate. At each time point, after removing the media, cells were washed with PBS. Scaffolds were removed and placed in a new multiwell plate. To each scaffold, 200 µl of 10 µg/ml resazurin in culture media was added and incubated in a humidified incubator (1% O2, 94% N2, 5% CO2). After 12 h of incubation, the reduced solution was collected, and fluorescence intensities were measured in Thermo Scientific Varioskan™ Flash Multimode Reader at an excitation wavelength of 540 nm and emission of 590 nm. Cells cultured in normoxia conditions were used as a control. All the values were normalized with day 0 and reported as mean ± s.d.


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2.12. Live dead viability assay of H9C2 cells on PUAO-CPO cryogels Cell survival of H9C2 cells under hypoxia was evaluated using the live/dead assay. Briefly, scaffolds at the end of day 3 and day 5 were incubated with 5 µM Calcein AM (Invitrogen) and 3 µM Propidium Iodide (PI) and incubated for 30 min. The scaffolds were then washed with PBS and imaged using Leica DM2500 fluorescent microscope with appropriate filters. The representative images were collected and quantified using the ImageJ software.24 2.13. In vivo studies All animal experiments were approved and performed under the supervision of an animal ethics committee of Indian Institute of Technology Kanpur (IITK/IAEC/2014/1023). Female mice (n = 28; Swiss albino, 25 g-30 g) were randomly selected in four groups. Group I (n = 4) did not receive any scaffold. Group II (n=8) was implanted with normal polyurethane scaffold while Group III (n=8) received antioxidant scaffold PUAO. Finally, Group IV (n=8) was implanted with oxygen releasing antioxidant scaffold PUAO-CPO. We used an established dorsal skin flap model to produce critically ischemic skin flaps.18 Mice were anesthetized using 2% isoflurane. The back of the skin was shaved, wiped with 70% ethanol, and disinfected with povidone iodine solution. A U-shaped skin flap of 30 x 10 mm size was produced on the back of the mice and scaffolds of 25 x 10 mm sizes were implanted. PU, PUAO and PUAO-CPO (1%) cryogels were implanted under the skin flap and sutured using 5-0 degradable sutures. In the sham group, the skin flap was sutured without any scaffold implantation. Animals received 12.5 mg/kg body weight tramadol as an analgesic for first 24 h. All the animals were provided free access to food, water and were housed in a 12 h day/night cycle. A 100% survival rate was observed during the entire experimental period without any complications. 2.14. Quantification of the necrosis 11 ACS Paragon Plus Environment


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Mice were anesthetized at day 3 and day 9 using 2% isoflurane and photographed under standard lighting conditions. Necrosis was observed by the visible darkening of the skin. The observed necrosis was quantified using ImageJ software (NIH, USA) and reported as percentage necrosis of the total flap area.24 2.15. Histology studies Animals were sacrificed at day 3 and day 9 using high doses of isoflurane followed by cervical dislocation. Tissue samples as well as implanted scaffolds were collected, fixed in 10% formalin in DPBS for 48 h and embedded in paraffin wax for sectioning. Cross sections of 7 µm thickness were collected on poly-L-lysine coated slides. The sections were stained with Hematoxylin & Eosin, and Picrosirius red for histological analysis. For scanning electron microscopic analysis, implanted scaffolds were washed with PBS, dehydrated with gradient ethanol treatment followed by critical point drying in hexamethyldisilazane (HMDS). Dried samples were gold coated using a sputter coater and imaged by scanning electron microscopy (Carl Zeiss EVO18) Immunostaining of PCNA was performed on the paraffin embedded samples. Briefly, wax embedded sections were hydrated, after wax removal in xylene, permeabilized with 0.1% triton x-100, blocked with 1% BSA in PBST (PBS+Tween-20). Antigen retrieval before permeabilization was done in citrate buffer and samples were incubated overnight at 4 °C with PCNA antibody (PC10, Santacruz 1:50) in 1% BSA. Post incubation, the samples were treated with secondary antibody (Anti mouse FITC conjugated, Invitrogen; 1:200) for 2 h, counterstained with PI and imaged under fluorescent microscopy (Leica DM 2500) using appropriate filters. 2.16. Statistical analysis


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All the quantitative experiments were performed in triplicates and expressed as mean ± standard deviation. All the analysis was carried out using GraphPad Prism 5 using 2 tailed student’s T test. A one-way ANOVA followed by Bonferroni post-tests was carried to compare control with different groups. A p value of less than 0.05 was considered statistically significant.

3. Results and Discussion 3.1. Fabrication and characterization of PUAO-CPO cryogels PUAO-CPO cryogels were prepared by cryogelation of PUAO in anhydrous DMSO (Figure 1). The frozen PUAO-CPO solution was thawed in cold water or ethanol to remove DMSO with the subsequent cryogel formation. Cold water/ethanol does not readily react with calcium peroxide thus preventing the premature release of oxygen from PUAO-CPO cryogels. Although different peroxides such as magnesium oxide (MgO2), calcium peroxide (CPO) and sodium percarbonate (Na2CO3) have been used for oxygen production, CPO is preferred as an oxygen releasing compound due to its higher purity and optimum reaction rate.25 Nevertheless, CPO has been shown to be most promising oxygen generating compound for tissue engineering applications. Earlier studies reported the incorporation of calcium peroxide into PLGA scaffolds wherein the oxygen release was observed upto 10 days.18 Further, PDMS- a hydrophobic polymer, when incorporated with calcium peroxide also showed the oxygen release

upto 7 weeks, but has certain limitations in tissue

engineering due to its non-degradable nature.15,26 Here, CPO was dispersed in an antioxidant polyurethane polymer PUAO and fabricated into a 3D cryogel scaffold. PUAO has earlier been synthesized by our group, which demonstrated excellent antioxidant properties of the polymer and being biocompatible, moderately hydrophobic and degradable in nature.21 13 ACS Paragon Plus Environment


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PUAO being hydrophobic in nature allows slower diffusion of water inside the scaffold which in turn will lead to sustained release of oxygen from the scaffold. To demonstrate the incorporation and distribution of CPO inside the PUAO scaffold, PUAOCPO cryogels were stained with Alizarin Red S (Figure 2 A-D). Alizarin Red S stain’s inorganic calcium and its intensity increases with growing concentration of calcium. The staining intensity of the PUAO-CPO cryogels increased with the increasing concentration of CPO showing successful incorporation of calcium peroxide inside the cryogels. Moreover, CPO particles were distributed uniformly inside the cryogels, which is important for the uniform release of oxygen during implantation. This was in addition confirmed by energydispersive X-ray spectroscopy (EDS) analysis. PUAO cryogel without CPO does not show any traces of calcium peroxide, whereas, calcium was observed in PUAO-CPO cryogels (Figure 2 E-H). Additionally, an increase in the mass percentage of calcium showed successful incorporation of calcium peroxide inside the PUAO-CPO cryogels in accordance with the percentage of calcium peroxide incorporated. Next, we determined the effect of CPO incorporation on the morphology and porosity of the PUAO-CPO cryogels. Porosity of the scaffold is important for efficient diffusion of nutrients and oxygen as well as for the removal of cellular wastes inside the scaffold.27 It also aids in the migration of cells within the scaffold. SEM analysis of the cryogels indicated that all the scaffolds are highly porous in nature. Also, CPO incorporation does not have any major effect on the porosity of the PUAO-CPO cryogels (Figure 3). This was further corroborated by the quantitative analysis for porosity, which showed a slight decrease in porosity from 85 ± 2.5% in 0% PUAO-CPO cryogels to 77 ± 1.5% in 3% PUAO-CPO cryogels respectively (Figure 4 D). The porosity of 1% PUAO-CPO and 2% PUAO-CPO cryogels was 82.23 ± 1.65 and 78.73 ± 0.86 %, respectively.


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Fourier-transform infrared spectroscopy (FTIR) was used to analyze the effect of CPO incorporation on PUAO polymer structure. A strong absorption band around 3425 cm-1 and 400 cm-1, characteristic for CPO, showed the incorporation of calcium peroxide into the PUAO cryogel scaffold. The FTIR analysis indicated that CPO does not chemically react with PUAO and is present as a blend inside the PUAO-CPO cryogel (Figure 4A). Swelling ratio is an important parameter for tissue engineered scaffolds and in particular, for oxygen releasing scaffolds as it effects the sustained and slow release of oxygen. PUAO cryogels showed a significantly low swelling ratio of 5.3 ± 0.96 (Figure 4B). This was expected due to the low hydrophilicity of PUAO, which has a water contact angle of 65 ± 5.21 This low hydrophilicity decreases the diffusion of water inside the PUAO cryogel, hence, will lead to slow release of oxygen. The swelling ratio further decreased by an increase in concentration of CPO inside the PUAO-CPO cryogels. The swelling ratio for 1%, 2% and 3% PUAO-CPO cryogels was 4.99 ± 0.42, 4.823 ± 0.24 and 3.655 ± 0.45, respectively. A major challenge with the peroxide based oxygen generating materials is that they produce ROS as a reaction intermediate on reacting with water. The reaction of calcium peroxide with water releases H2O2- a type of ROS, which decomposes to release oxygen. This decomposition of H2O2 is known to be accelerated by catalase leading to the release of oxygen at a higher rate. A burst increase in oxygen release may lead to hyperoxia, which may further lead to cellular damage.28,29 Furthermore, burst release of oxygen also means that the oxygen will not be released for longer periods of time. In addition, in the absence of enzymes such as catalase, the presence of H2O2, which acts as a free radical, leads to the generation of oxidative stress and subsequent cellular damage. To compensate for both of these effects, we used a hydrophobic antioxidant polymeric scaffold PUAO, which can slow down the release of oxygen and attenuate any oxidative stress produced due to H2O2. To substantiate the effect of CPO incorporation on antioxidant properties of PUAO, DPPH assay was carried out, 15 ACS Paragon Plus Environment


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which indicated that PUAO-CPO cryogels are antioxidant in nature. DPPH*, a violet colored stable free radical solution in ethanol, is reduced by antioxidant substances into a colorless DPPH solution, that gives the antioxidant potential of the material. The DPPH assay here showed scavenging of free radicals by both PUAO as well as PUAO-CPO cryogels over a period of 180 min. PUAO showed rapid scavenging as compared to PUAO-CPO, which became almost stable after 60 min. However, PUAO-CPO showed gradual scavenging of free radicals but the % inhibition was equal to that of PUAO after 180 min (p≥0.05). (Figure 4C). This is due to a decrease in the integral amount of antioxidant PUAO in PUAO-CPO cryogels. PUAO-CPO showed a total percentage inhibition of 88% ± 1.1 after 180 min in comparision to 92% ± 2.3 in PUAO cryogel and was significant for free radical attenuation. Oxygen releasing scaffolds for tissue engineering applications needs to be degradable in nature. Although PUAO is biodegradable in nature, we studied the degradation kinetics of PUAO-CPO cryogels. To understand this, in vitro degradation of PUAO-CPO cryogels was evaluated in DMEM for 4 weeks. All the scaffolds showed progressive weight loss over a period of 4 weeks. Incorporation of CPO inside the PUAO scaffold increased the degradation rate of PUAO-CPO cryogels. Polyurethanes due to presence of ester linkages show enhanced degradation under hydrolytic and oxidative conditions.30 Hence, the increased degradation of PUAO-CPO cryogels can be attributed to the release of oxygen, which accelerated the degradation of these cryogel scaffolds. PUAO cryogels without CPO showed a percentage degradation of 11.00 ± 1.01 after 4 weeks. Pertinently, as the concentration of CPO in PUAO increased, the degradation rate of the 1%, 2% and 3% PUAO-CPO cryogels also increased to 17.55 ± 3.70, 24.42 ± 2.93 and 32.26 ± 1.20, respectively after 4 weeks (Figure 5). SEM analysis confirmed the increased degradation of PUAO-CPO cryogels. Significant morphological changes were observed in all scaffolds after 28 days in DMEM. The PUAO-


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CPO cryogels have lost their overall morphology after 28 days of degradation as compared to only PUAO cryogels (Figure 5). 3.2. Oxygen releasing behavior of the PUAO-CPO cryogels To determine the oxygen releasing kinetics, PUAO-CPO cryogels were incubated in 3 ml of serum-free media under hypoxia conditions, and the oxygen concentration was measured over a period of 10 days. PUAO-CPO cryogels without CPO incorporation (0% CPO) showed basal levels of oxygen throughout the experimental period of 10 days. This indicated that PUAO, as such, does not produce any oxygen. However, PUAO-CPO cryogels showed a sustained release of oxygen over the period of 10 days in a dose dependent manner (Figure 6A). After day 1, 1%, 2% and 3% CPO cryogels showed increased oxygen levels of up to 24.26 ± 0.30% (7.74 ± 0.09 mg/L), 24.65 ± 0.30% (7.86 ± 0.09 mg/L), and 24.79± 0.46% (7.91 ± 0.14 mg/L), respectively. Later, sustained release of oxygen was observed, with concentrations reaching up to 10.23 ± 0.13% (3.26 ± 0.04 mg/L), 11.52 ± 0.37% (3.76 ± 0.11 mg/L, and 13.00 ± 0.26% (4.14 ± 0.08 mg/L), respectively after 10 days in hypoxia. Oxygen released by 3% PUAO-CPO cryogels was significantly higher as compared to 1% (p ≤ 0.05) and 2%(p ≤ 0.05) PUAO-CPO cryogels over a period of 10 days. This oxygen concentration may be sufficient to sustain cell viability for over 10 days. Earlier CPO incorporated into GelMA hydrogels showed an oxygen release for 5 days.20 CPO, when incorporated in PDMS, a hydrophobic but nondegradable polymer showed a sustained release of oxygen for upto 7 weeks.15 Similarly, oxygen releasing gellan gum hydrogels showed a release upto 2 days using 1% CPO.31 These studies indicate that the hydrophobicity of the polymeric scaffold is one of the important factors for sustained release of oxygen. Similarly, PUAO-CPO cryogels, due to their hydrophobic nature prolonged the release of oxygen for over 10 days, which may be beneficial for maintaining cell viability under hypoxic conditions. This may also provide a time window for vascularization of the implanted scaffold. 17 ACS Paragon Plus Environment


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It is important for oxygen releasing scaffolds not to affect the normal tissue at the site of implantation while providing oxygen to cells at the site of the defect. Therefore, we estimated the cell cytotoxicity of H9C2 cells in contact with PUAO-CPO cryogels under normoxia conditions for 48 h by MTT assay (Figure 6B). The metabolic activity of H9C2 cells with PUAO-CPO cryogels was statistically equivalent to that of the PUAO cryogels after 24 h and 48 h in culture. This indicated that PUAO-CPO cryogels do not show any cellular cytotoxicity under normoxia conditions. However, after 48 h, the viability at higher concentration of CPO (2% and 3%) were relatively less compared to 1% CPO. 3.3. Improved cell survival on the PUAO-CPO cryogel under hypoxia conditions To demonstrate the effect of oxygen release on metabolic activity of cells cultured under hypoxia conditions, H9C2 cells were seeded on PUAO-CPO cryogels and metabolic activity was studied over a period of 10 days (Figure 7). The metabolic activity of the H9C2 cells on PUAO-CPO cryogel, estimated by the resazurin assay, increased with time and was comparable to that of the cells grown under normoxia conditions. PUAO-CPO cryogels with 1% and 2% calcium peroxide showed increased metabolic activity while 3% CPO showed less metabolic activity. At day 3, cells cultured on PU cryogel under hypoxia showed a significant decrease in metabolic activity as compared to cells grown on PU cryogels in normoxia (p = 0.0295). However, cells grown on PUAO as well as 1% and 2% PUAO-CPO cryogels maintained their viability and showed increased metabolic activity than PU cryogels (p ≤ 0.01). There was no significant difference between the metabolic activity of PUAO vs 1% and 2% PUAO-CPO cryogels. The cells grown on 3% PUAO-CPO cryogels showed significant cell toxicity with decreased metabolic activity. This effect was more prominent after 5 days of culture, where, the metabolic activity of the cells cultured on 1% and 2% PUAO-CPO cryogels under hypoxia conditions increased and became indistinguishable from those grown on PUAO cryogels under normoxia conditions (p = 0.186 and 0.5498, 18 ACS Paragon Plus Environment

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respectively). At day 7, the metabolic activity further increased in PUAO-CPO cryogels although 3% PUAO-CPO cryogels showed less metabolic activity. This may be due to an increased release of oxygen as well as free radicals alongside the production of calcium hydroxide generated through the reaction of calcium peroxide with water. This calcium hydroxide along with free radicals has been reported to have detrimental effects on cell growth.32 Infact, we observed an increase concentration of calcium hydroxide at day 5 in 3% PUAO-CPO (316 ±1 µg/mg scaffold) with respect to 2% PUAO-CPO (261 ± 6 µg/mg scaffold) and 1% PUAO-CPO (229 ±7 µg/mg scaffold). Similar effect was observed in earlier studies where an increase in calcium peroxide and sodium percarbonate concentrations in oxygen generating materials were detrimental to cell viability.17 It is important to note here that during initial days, the PUAO cryogel scaffold, due to its antioxidant properties, could show increased metabolic activity as compared to normal polyurethane (PU) cryogels under hypoxia conditions. This effect may be due to the antioxidant properties of PUAO cryogels. Hypoxia besides oxygen deprivation leads to increase in oxidative stress, which also causes cell death. During initial days, PUAO due to its antioxidant potential could attenuate this oxidative stress, however, the effect disappeared at later time points. This was also observed in our previous study where PUAO films attenuated hypoxia induced cell death and showed increased viability of cells during oxidative stress.21 The PUAO-CPO cryogels could support cell growth of H9C2 cardiomyoblast upto 10 days under hypoxia. This time interval may provide a sufficient time frame for cell survival and more importantly neovascularization of the transplanted implant. Next, we determined the effect of the oxygen releasing scaffolds on cell survival under hypoxia conditions. The amount of live and dead cells was estimated by Calcein AM (live) and propidium iodide (PI, dead) staining and quantified through ImageJ software. The images were split into the green (live cells) and red (dead cells) channels and the number of cells in 19 ACS Paragon Plus Environment


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each channel were quantified. The percentage cell viability was obtained as follows; Percentage of live cells = cells in green channel/total number of cells in green and red channel. Cell viability results corroborated with the metabolic studies where cells grown on oxygen releasing scaffolds showed increased cell viability (Figure 8). Cells grown on PU cryogels exhibited increased cell death (up to 28.41 ± 6.91% cell survival) after day 3. Cells grown on 1%, 2%, and 3% PUAO-CPO cryogels showed 91±6 %, 83±8 % and 73±6 % cell survival, respectively after day 3 in culture. Cells under hypoxia besides experiencing low oxygen availability are constantly challenged by free radicals generated due to hypoxia. So, antioxidant polymer itself can rescue cell death to some extent. This was evident in our study where antioxidant polyurethane cryogel (PUAO) showed increased resistance to cell death than normal polyurethane, PU. On day 3, PUAO cryogel showed a viability percentage of 44 ± 9 % compared to 28.41 ± 6.91 % in PU. This effect was more severe on day 5, where only 11.4 ± 3.4 % cell viability was observed in PU. The cell viability does not show any significant change in PUAO-CPO cryogels after 5 days of culture and is expected to be maintained for subsequent days. However, cellular viability decreased at concentration of 3% CPO as observed earlier. All these results indicated an increase in metabolic activity as well as cell viability with oxygen releasing scaffolds under hypoxia conditions. PUAO-CPO cryogels with 1% CPO showed optimum metabolic activity and viability; hence was further used for in vivo studies. 3.4. In vivo studies To study, how the oxygen releasing scaffolds will affect the survival of critically perfused tissues, we used a skin flap model. This model is well established for studying ischemic tissue survival.18 We developed this model in mice and implanted oxygen releasing scaffold on the back of the mice (Figure 9). The vascular pedicles were trimmed to circumvent blood


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supply to the skin flap and the oxygen releasing scaffolds were implanted beneath the skin and monitored over a period of 9 days. Polyurethane cryogel (PU) without any CPO were used as a control. Tissue necrosis was identified as a discoloration of the skin in all the animals after day 3 of implantation (Figure 10). Quantification of the graft necrosis, indicated significantly increased necrosis of up to 41.6±12.4 % in the PU group as compared to only 6.7±0.2% in the PUAO-CPO group (p ≤ 0.5). PUAO group showed significantly better survival with 21.2±8.9% necrosis as compared to 41.6±12.4% in PU group. However, there was no significant difference in tissue survival between PUAO and PUAO-CPO groups at day 3(p ≥ 0.05). PUAO, as discussed earlier, due to its antioxidant behavior, can rescue the cellular damage during the initial stages of hypoxia. At day 9, necrosis in the PU group increased to 68±7% while only 41±7.5% and 12±2% necrosis was observed in the PUAO and PUAO-CPO group, respectively (Figure 10). PUAO showed attenuation of necrosis at day 3; however, by day 9 the effect significantly diminished as the hypoxia conditions further aggravated the condition. By providing oxygen using PUAO-CPO cryogel scaffold, this condition was alleviated as compared to PUAO cryogel scaffold (p = 0.0028). Earlier studies showed that CPO incorporated PLGA gels delayed necrosis for 3 days, but the effect was reversed on day 7.18 However, in the present study, the effect was maintained for 9 days. These results indicate that the oxygen releasing PUAO-CPO cryogels delayed the onset of skin necrosis (p = 0.008) as compared to control groups for up to 9 days. The animals were monitored continuously and were sacrificed on day 3 and day 9 post implantation. Implanted scaffolds and skin samples were harvested and further characterized at the cellular level through histological analysis. Tissue sections were stained with the hematoxylin and eosin after 3 and 9 days of implantation. Histological analysis showed a clear beneficial effect in the case of PUAO-CPO cryogels as compared to the control groups (Figure 11 A-B). At day 3, the PUAO-CPO group showed proper morphology with 21 ACS Paragon Plus Environment


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preservation of the tissue architecture, epidermis and dermis height, and presence of hair follicles. However, in the PU group, necrosis of the tissue with degeneration of epidermis, as well as hair follicles, was observed. Some amount of necrosis and hair follicle degeneration was also observed in the PUAO group at day 3. At day 9, complete degeneration of skin layers was observed in the PU group with no demarcation between different skin layers. Both PUAO and PUAO-CPO groups showed slower progression of necrosis with the preservation of dermal and epidermal tissue architecture with better effects observed in PUAO-CPO group compared to PUAO group. The beneficial effect of oxygen releasing PUAO-CPO scaffolds over the period of 9 days suggests that oxygen is produced from the scaffolds in a controlled manner. Collagen architecture was assessed by picrosirius red staining which specifically stains for collagen (Figure 11 C-D). Collagen architecture and fiber orientation was maintained in both the PUAO and PUAO-CPO groups at day 3 and day 9. In the PU group, collagen fiber architecture was affected with visible fiber disorganization at day 3. By day 9 the collagen matrix was completely lost and replaced with the non-collagenous tissue mass. The retrieved scaffolds stained with hematoxylin and eosin showed the infiltration of cells inside the scaffolds (Figure 12A). All the scaffolds were well integrated with the skin tissue. After 9 days, the implanted scaffolds also showed some visible signs of degradation. This is in agreement with in vitro degradation studies, where, very less degradation was observed after 1 week. However, the oxygen releasing scaffolds exhibited increased degradation as observed in both H & E and SEM images. SEM micrographs also indicated infiltration of cells inside the scaffold. PUAO-CPO cryogels displayed increased cellular infiltration compared to PUAO and PU cryogels (Figure 12B). Further, to demonstrate the viability and proliferation of the cells in the skin tissue in presence of an oxygen releasing scaffold, the skin samples were immunostained at day 9 for PCNA, a marker for cell proliferation. The PUAO-CPO group showed expression of PCNA 22 ACS Paragon Plus Environment

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indicating viable and proliferating cells in the skin flap (Figure 13). In the PU group, however, differential expression of PCNA was observed with few proliferating cells and a prominent necrosis around the hair follicles. This further strengthened the fact that oxygen releasing scaffolds could provide sufficient oxygen to the proliferating cells in the skin flap. At the same time, cell death in the PU group due to the absence of oxygen, led to death and necrosis of the tissue. All these results suggest that the oxygen releasing PUAO-CPO cryogel prevents cellular damage, and necrosis of critically perfused tissue. The antioxidant PUAO cryogel scaffold, on the other hand, could delay necrosis due to its antioxidant properties. Hypoxia leads to the production of free radicals causing cell death, which accelerate the process of necrosis.8 The beneficial effect of the antioxidant scaffold may be due to attenuation of free radicals generated, which delayed the onset of necrosis. This effect was further enhanced in the presence of oxygen delivery, where, the oxygen released from the PUAO-CPO cryogel scaffold increased cell survival, and the effect of any oxidative stress produced was negated by the antioxidant behavior of the PUAO-CPO scaffold. The oxygen releasing antioxidant scaffold shows potential in delaying and preventing the normal tissue degradation process upon hypoxia. Sustained release of oxygen along with antioxidant properties led to the prevention of necrosis and cell death for a longer period, which is of immense importance in tissue engineering applications in general and especially in pathological conditions such as ischemia, wound healing and myocardial infarction. Thus, the oxygen releasing cryogel scaffold is expected to augment tissue engineering strategies by enhancing cell survival and preserving tissue architecture by preventing the cell death and onset of necrosis.

4. Conclusion



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Oxygen releasing antioxidant cryogels were fabricated by incorporation of calcium peroxide in the antioxidant polyurethane cryogel scaffold. These scaffolds showed antioxidant behavior with the sustained release of oxygen over a period of 10 days. PUAO-CPO cryogels could alleviate the effect of hypoxia and increased cell survival under in vitro conditions. In an in vivo skin flap model, PUAO-CPO oxygen releasing scaffold prevented necrosis while PUAO antioxidant scaffold delayed the onset of necrosis. This is an important outcome and will help to develop better strategies for the fabrication of tissue engineering scaffolds for regeneration. In general, these scaffolds will have potential applications to develop strategies for fabricating next generation tissue engineering scaffolds and particularly for regeneration of tissue under ischemic conditions such as myocardial infarction and chronic wound healing.

Acknowledgement The authors would like to acknowledge the Department of Biotechnology, Govt. of India, DBT (BT/IN/SWEDEN/08/AK/2017-18; BT/PR13561/MED/32/392/2015), Department of Science and Technology, Ministry of Science and Technology, Govt. of India, DST (DST/TSG/AMT/2015/329; DST/SSTP/UP/428, DST-VR 2015-06717) AND MHRD/ICMR (IMPRINT-6714) for providing financial assistance for this work. P.A.S. and A.S. would like to acknowledge IIT Kanpur for fellowship. A. K. also acknowledges DBT-TATA Innovation Fellowship from the Department of Biotechnology, Govt. of India.

Conflict of interest The authors declare no conflict of interest.

Disclosure The work reported here has been duly filed for a patent with application number 201711040202.


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Author contribution A.K. and P.A.S. conceived and designed the study. P.A.S. and A.S. carried out the experimental work and analysis. The manuscript was written by P.A.S. and reviewed by A.K.

Abbreviations PUAO: Polyurethane with antioxidant properties PUAO-CPO: Oxygen releasing PUAO CPO: Calcium peroxide PU: Polyurethane without antioxidant properties PCL diol: Polycaprolactone diol 2000 HDI: Hexamethylene diisocyanate EDS: Energy-Dispersive X-ray Spectroscopy SEM: Scanning Electron Microscopy FTIR: Fourier-transform infrared spectroscopy DPPH: 2, 2-Diphenyl-1-picrylhydrazyl DMEM: Dulbecco’s Modified Eagle’s Medium FBS: Fetal Bovine Serum MTT: Thiazolyl Blue Tetrazolium Bromide: TCP: Tissue culture plate PI: Propidium Iodide DPBS: Dulbecco’s Phosphate Buffered Saline BSA: Bovine Serum Albumin 25 ACS Paragon Plus Environment


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HMDS: Hexamethyldisilazane PLGA: Poly (D, L-lactide-co-glycolide) PDMS: Poly (dimethyl siloxane) ROS: Reactive Oxygen Species H2O2: Hydrogen peroxide PCNA: Proliferating Cell Nuclear Antigen GelMA: Gelatin methacryloyl

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Figures

Figure 1: Schematic representation of the fabrication of PUAO-CPO cryogels and its implantation in mice.


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Figure 2: Distribution of calcium peroxide inside PUAO-CPO cryogels. Alizarin red staining of PUAO-CPO cryogel (A) 0% CPO. (B) 1% CPO. (C) 2% CPO. (D) 3% CPO. EDAX analysis of PUAO-CAO (E) 0% CPO. (F) 1% CPO. (G) 2% CPO. (H) 3% CPO showing



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concentration dependent incorporation of calcium peroxide in PUAO-CPO cryogels. Scale bar: 200 µm

Figure 3: SEM micrographs PUAO-CPO cryogels (A) 0% CPO. (B) 1% CPO. (C) 2% CPO. (D) 3% CPO. Scale bar: 500 µm


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Figure 4: (A) FTIR analysis of calcium peroxide incorporated scaffolds. (B) Swelling ratio of CPO incorporated PUAO cryogels. (C) DPPH assay showing antioxidant properties of 1% PUAO-CPO cryogels after incorporation of calcium peroxide. (D) Quantification of porosity of CPO incorporated PUAO cryogels.



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Figure 5: Degradation of oxygen releasing PUAO-CPO cryogels in DMEM. Scanning electron microscopic images showing the bulk degradation of PUAO-CPO cryogels after a period of 28 days. Scale bar: 100 µm 34 ACS Paragon Plus Environment

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Figure 6: (A) Oxygen releasing kinetics of PUAO-CPO cryogel scaffold with 0%, 1%, 2%, 3% calcium peroxide. (B) Scaffold cytotoxicity assay of different PUAO-CPO cryogels on H9C2 cardiomyocytes under normoxia conditions.



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Figure 7: Cell metabolic activity of H9C2 cardiomyocytes under hypoxia conditions (A) Day 3 (B) Day 5 (C) Day 7 (D) Day 10. Cells grown under normoxia were used as control. All the values were normalized with day 0. H: Hypoxia; N: Normoxia; ns: non-significant; *p

Oxygen-Releasing Antioxidant Cryogel Scaffolds with Sustained

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