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Engineering Bioinspired Antioxidant Materials Promoting Cardiomyocyte Functionality and Maturation for Tissue Engineering Application Article in ACS Applied Materials & Interfaces · January 2018 DOI: 10.1021/acsami.7b14777
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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Engineering Bioinspired Antioxidant Materials Promoting Cardiomyocyte Functionality and Maturation for Tissue Engineering Application Parvaiz A. Shiekh, Anamika Singh, and Ashok Kumar* Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, Uttar Pradesh, India S Supporting Information *
ABSTRACT: Oxidative stress plays an important role in various pathological conditions, such as wound healing, inflammation, myocardial infarction, and biocompatibility of the materials. Antioxidant polymers to attenuate oxidative stress is an emerging field of biomaterial research with a huge impact in the field of tissue engineering and regenerative medicine. We describe here the fabrication and evaluation of an elastomeric antioxidant polyurethane (PUAO) for tissue engineering applications. Uniaxial and cyclic tensile testing, thermal analysis, degradation, cytotoxicity and antioxidant analysis was carried out. An in vitro oxidative stress model demonstrated that PUAO reduced intracellular oxidative stress in H9C2 cardiomyocytes (p < 0.05) and attenuated reactive oxygen species (ROS) induced cell death (p < 0.001). Under simulated ischemic reperfusion, PUAO could rescue hypoxia induced cell death. Further as a proof of concept, neonatal rat cardiomyocytes cultured on PUAO film displayed synchronous beating with mature phenotype showing expression of cardiac specific α-actinin, troponin-T, and connexin-43 proteins. Intracellular calcium transients established the functionality of cultured cardiomyocytes on PUAO film. Our study demonstrated the potential of this biomaterial to be developed into tissue engineered scaffold to attenuate oxidative stress for treatment of diseased conditions with increased oxidative stress, such as cardiovascular diseases, chronic wound healing, and myocardial infarction. KEYWORDS: free radicals, ischemia, bioinspired, cardiac, biomaterials, regeneration
1. INTRODUCTION Tissue engineering strategies aim at the development of biocompatible materials to repair and restore the damaged tissue. From the classical definition of biocompatibility being an inert material that does not produce any deleterious effect on the ability of a biomaterial to perform its desired function without eliciting any undesired effect while generating a beneficial cellular or tissue response, the understanding of the biological response of the materials in terms of their chemistry, morphology, and their degradation products has also been reformed.1 Even materials that are supposed to be biocompatible and FDA approved have been seen to induce a pathological response.2 Oxidative stress generated at the site of implantation is often overlooked when developing materials for tissue engineering applications. Oxidative stress is a pathophysiological condition because of the imbalance between the generation of reactive oxygen species (ROS) and the body’s © XXXX American Chemical Society
ability to scavenge them. ROS react with cell membrane proteins and DNA causing cellular death.3 Oxidative stress has been also implicated in progression of various diseases, such as chronic wound inflammation, cardiovascular diseases, such as atherosclerosis and myocardial infarction, and neurodegenerative diseases, as well as host tissue inflammatory response to biomaterial implant or scaffold.4−7 Myocardial infarction (MI), which is the result of occlusion of the coronary artery in cardiac tissue and subsequent ischemia is one of the leading causes of death worldwide.8 MI generates a very hostile environment, resulting in the search for strategies to combat this hostile environment. Oxidative stress is often overlooked when biomaterial strategies are developed to treat Received: September 28, 2017 Accepted: January 5, 2018 Published: January 5, 2018 A
DOI: 10.1021/acsami.7b14777 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
2. EXPERIMENTAL SECTION
myocardial infarction. In fact, reactive oxygen species (ROS) have been seen to modulate the cardiac remodelling process after myocardial infarction.9,10 It is well established that there is an increase in oxidative stress because of ROS production in both surgically induced myocardial infarction models and animal models for reperfusion.9,11 ROS modulate membrane lipids, proteins and DNA of the transplanted cells causing massive cell death and is one the primary obstacles in treating MI.6,12 Recent attempts to attenuate oxidative stress were accomplished by conjugating small antioxidant molecules, such as ascorbic acid, Vitamin E, GSH, etc., to polymers. These molecules attenuated oxidative stress but have a limited percentage of antioxidant content compared to bulk material.13−15 Thus, development of biomaterials that have intrinsic antioxidant properties in their backbone structure may be more beneficial to attenuate oxidative stress because of relatively high antioxidant content and sustained attenuation when the polymer is present at the site of implantation. A biomaterial to be used as a scaffold to attenuate oxidative stress should have intrinsic antioxidant properties, have simple processability, and be biodegradable in nature with robust and tunable mechanical properties. Previous studies have shown that such polymers can be synthesized by incorporating the small antioxidant molecules, such as vitamin C, Trolox (a synthetic and water-soluble analogue of vitamin E), etc., into polymeric chains.2 Poly(1,8octanediol-co-citrate-co-ascorbate) (POCA) was synthesized and showed suitable antioxidant properties; however, these polymers need to be pretreated for acid leaching and cannot be further processed after polymerization.16 In another study, antioxidant multi-acrylates were synthesized from phenolic antioxidants, such as curcumin and quercetin and developed into hydrogels.17 But, their poor mechanical properties and faster degradation limits their application as a cardiac tissue engineering scaffold. Elastomeric materials with robust, tunable mechanical properties that have antioxidant properties in their backbone structure will be suitable for development of tissue engineering regenerative scaffold to attenuate oxidative stress. In this study, a biodegradable elastomeric polyurethane material with intrinsic antioxidant properties (PUAO) was synthesized and evaluated for tissue engineering applications. Our study proposed that these polymers can be easily synthesized with robust mechanical and antioxidant properties by incorporating ascorbic acid into polymeric backbone. Ascorbic acid is a natural antioxidant and has cell proliferation and differentiation properties.18 We hypothesized that incorporation of ascorbic acid into polyurethane backbone will provide sustainable antioxidant properties, in addition to the polymer being degradable and mechanically strong in nature. Oxidative stress due to generation of ROS is one of the primary causes of cardiomyocyte death during myocardial infarction. We speculated that attenuation of oxidative stress by PUAO will rescue cardiomyocyte death in an in vitro oxidative stress model, as well as in an in vitro simulated ischemic reperfusion model, and will enhance their proliferation and viability. As a proof of concept for the feasibility of PUAO scaffold for cardiac regeneration, we expected that PUAO films should support growth, differentiation, and maturation of neonatal primary cardiomyocytes. To comprehend this, growth, differentiation, and calcium cycling of the neonatal rat cardiomyocytes on PUAO films was studied.
2.1. Polyurethane Synthesis. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Polyurethane was synthesized by a two-step polymerization reaction using poly(ε-caprolactone) diol (Mn = 2000 Da) as soft segment and hexamethylene diisocyanate and ascorbic acid as a chain extender. Briefly, 8 g of PCL-diol was dissolved in DMSO and refluxed over molecular sieves under a nitrogen atmosphere for 3 h. Hexamethylene diisocyanate (1.344 g) was added to this solution and reacted with PCL-diol in the presence of catalyst tin(II) 2-ethylhexanoate to form the prepolymer (4 h, 80 °C). At the end of the first step, the prepolymer solution was cooled to room temperature and reacted with ascorbic acid in a molar ratio of 1:0.8 with macrodiol (2 h, 70 °C). The reaction was stopped after 2 h by the addition of methanol. The polymer was collected by precipitation in distilled water and purified in excess of distilled water, followed by washing in 70% ethanol. The obtained powder was dried under vacuum at 40 °C for 48 h and further lyophilized for 24 h to remove any residual solvent. The synthesized polyurethane was designated as PUAO. 2.2. Characterization of the Synthesized Polyurethane. The synthesized polyurethane was characterized by FTIR and NMR analysis, which showed characteristic peaks for antioxidant polyurethane. FTIR was obtained at room temperature in the range of 4000− 400 cm−1 using PerkinElmer spectrum version 10.03.06. Proton NMR spectra was obtained with a 400 MHz JEOL spectrometer (JEOL JNM-ECS400 USA, Inc.) using DMSO-d6 as solvent and trimethylsilane as an internal standard. A total of 16 scans were obtained at a resolution of 0.45 Hz and acquisition time was 2.18 s. To estimate the presence of free ascorbic acid groups, a modified version of toluidine blue dye assay was done.19 Polymer films of 1 cm2 were immersed in 0.5 mM toluidine blue, pH 10.0 for 10 min. Polymeric films were rinsed in sodium hydroxide pH 9.0 to remove unbound dye. The dye was extracted in 50% acetic acid and quantified at 633 nm against the different dye concentrations. Molecular weight was determined by gel permeation chromatography (GPC) using Viscotek GPC-SEC system (Malvern Instruments Ltd. UK) against polystyrene standard molecular weights in tetrahydrofuran (THF). Thermogravimetric analysis was done using STA 8000 PerkinElmer, USA. Approximately 15 mg of the sample was weighed and heated in an alumina crucible from 50 to 600 °C under ambient atmosphere and change in weight with respect to temperature was recorded. Differential scanning calorimetry (DSC) of the polyurethane samples was performed using DSC 8500 PerkinElmer (Waltham USA). Each sample weighing approximately 10 mg, encapsulated in a hermetic aluminum pan was heated from −60 to +200 °C at 10 °C/ min, isothermally maintained at 200 °C for 3 min, and then cooled from 200 to −60 °C at 10 °C/min under nitrogen atmosphere. 2.3. Mechanical Testing of Polyurethane Films. Mechanical properties of the thin polyurethane films were evaluated using tensile tester Instron 1195 (see Supporting Information). 2.4. Contact Angle Measurements and Swelling Kinetics. For contact angle measurement, the polymer solution was cast on the rectangular coverslip. The water contact angle was measured by contact angle goniometer (Dataphysics OCA 35, Germany) using sessile drop method. Swelling kinetics of the polyurethane films was studied to know the bulk hydrophilicity as described in Supporting Information. 2.5. Morphological Analysis, in Vitro Degradation, and Cytotoxicity Assay of Degradation Products. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to determine the surface morphology of the thin polyurethane films. In vitro degradation of polyurethane films was studied in simulated body fluid (SBF, pH 7.4) over a period of 8 weeks The toxicity of the degradation products was measured as described earlier (see Supporting Information).20 2.6. Cell Culture. C2C12 myoblasts and H9C2 cardiomyoblasts were obtained from National Centre for Cell Science (Pune, India) and were cultured in high-glucose DMEM supplemented with 10% (v/ v) fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 U/ B
DOI: 10.1021/acsami.7b14777 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces mL streptomycin (Himedia) at 37 °C in a 5% CO2 atmosphere. Before seeding, each PUAO film was cut into dimensions of 0.5 × 0.5 mm and sterilized by ethanol serial dilution and subsequent UV exposure. The films were treated with DMEM + 10% FBS and incubated overnight in a CO2 incubator at 37 °C. Next day, media was aspirated, and each film was seeded with 3 × 105 C2C12 cells and 5 × 104 H9C2 cells. Media was changed every third day. 2.7. In Vitro Cell Viability and Morphological Studies. To assess cell viability, cells were cultured on PUAO films for different intervals of time. At each predefined time interval, the cell culture media was aspirated from each well, followed by washing with PBS. MTT solution (0.5 mg/mL) was added to each well and incubated for 4 h in a CO2 incubator at 37 °C. After 4 h, the media was removed, and the formazan crystals formed were dissolved in DMSO and quantified by measurement of the absorbance at 570 nm. Experiments were conducted in triplicates. Cells cultured on tissue culture plates (TCP) were used as a control. For cell morphology and adherence analysis, PUAO films seeded with cells were evaluated by scanning electron microscopy (SEM) and fluorescent microscopy (see Supporting Information). 2.8. Intrinsic Antioxidant Activity. The antioxidant activity of the synthesized polyurethane was evaluated through DPPH assay. Briefly, after lyophilization, PUAO films were treated with a 400 μM solution of DPPH in ethanol and incubated at 37 °C. Antioxidant capacity was measured by change in absorbance at 517 nm after different intervals of time compared to control DPPH solution. All the measurements were taken in triplicates and results expressed as mean ± s.d. The antioxidant capacity was expressed as percentage inhibition and assessed by the following formula, % inhibition = (AB − AS)/AB × 100, where AB is absorbance of control (DPPH only) and AS is absorbance of the sample (DPPH + polymer). 2.9. Oxidative Stress in Cells and Cellular Viability. To understand the effect of polymer on internal oxidative stress upon an oxidative challenge, H9C2 cardiomyocytes were treated with H2O2 in the presence of PUAO extract as described earlier with slight modifications. PUAO (50 mg) was incubated in 3 mL of DMEM without FBS for 7 days at 37 °C to obtain PUAO extract. H9C2 cells were seeded at a density of 5 × 104 cells/well in a 48-well plate and cultured for 24 h. Cells were treated with PUAO extract in DMEM for 2 h. Media was removed and cells were treated with 10 μM DCF-DA for 30 min. After the cells were washed with HBSS (3×), they were treated with 200 μM H2O2 to generate oxidative stress. Fluorescence developed due to oxidative stress was measured after 10, 20, and 30 min at ex/em of 485/535 nm. To validate the effect of antioxidant properties of PUAO on cell viability upon ROS challenge, an in vitro system was developed. Briefly, PUAO films after overnight sterilization in 70% ethanol under UV light were incubated with DMEM + 10% FBS for 24 h. On next day, H9C2 cardiomyocyte cells at a density of 5 × 104 cells were seeded onto each film and cultured for 24 h. After 24 h of culture, cells were treated with 20 μM menadione (TCI Chennai, India)) to induce oxidative stress. Cells were monitored over a time period under light microscopy for characteristic morphological changes and imaged using phase contrast microscopy. MTT assay was done after 12 h of treatment to check the cell viability. Lactate dehydrogenase (LDH) assay was done to further confirm the attenuation of cellular death as reported earlier.21 2.10. Simulated Ischemic Reperfusion. To simulate ischemic reperfusion, H9C2 cardiomyocytes at a density of 5 × 104 were grown in hypoxia in serum-deprived conditions for 12 h, followed by reoxygenation for 12 h. To evaluate the effect of PUAO in an ischemic reperfusion injury, H9C2 cells seeded on PUAO and TCP were grown in hypoxia (
