Polyurethane-Polycaprolactone Blend Patches - ACS Publications

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Polyurethane-Polycaprolactone Blend Patches: Scaffold Characterization and Cardiomyoblast Adhesion, Proliferation, and Function Shiva Asadpour,†,‡ Hamid Yeganeh,§ Jafar Ai,‡ Saeid Kargozar,† Morteza Rashtbar,‡ Alexander Seifalian,∥ and Hossein Ghanbari*,⊥,#

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 10/16/18. For personal use only.

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Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Azadi Square P.O. Box 917794-8564 Mashhad, Iran ‡ Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine (SATiM), Tehran University of Medical Sciences (TUMS), Italia Street, 14177-55469 Tehran, Iran § Iran Polymer and Petrochemical Institute, Pajuhesh Boulevard, P.O. Box 112/14975, 14977-13115 Tehran, Iran ∥ Nanotechnology and Regenerative Medicine Commercialization Centre (Ltd), The London BioScience Innovation Centre, 2 Royal College Street, London, NW1 0NH, United Kingdom ⊥ Department of Medical Nanotechnology, Regenerative Nanomedicine Research Group, SATiM, TUMS, Italia Street, 14177-55469 Tehran, Iran # Research Center for Advanced Technologies in Cardiovascular Medicine, Tehran Heart Center, North Kargar Ave, Tehran University of Medical Sciences, 14177-55469 Tehran, Iran S Supporting Information *

ABSTRACT: A remarkable challenge in myocardial tissue engineering is the development of biomimetic constructs that can potentially improve myocardial repair and regeneration. Polyurethane (PU) scaffolds are extensively utilized in the cardiovascular system. We have synthesized a new biodegradable poly(ester-ether urethane urea) (PEEUU) using a new and simple method. To enhance mechanical and physicochemical properties, the PEEUU was blended with polycaprolactone (PCL). We then fabricated a series of new PU− PCL scaffolds. The scaffolds were then characterized using SEM, porosity measurement, attenuated total reflectanceFourier transform infrared spectroscopy (ATR-FTIR), DSC, water contact angle measurement, swelling measurement, in vitro degradation rate, and mechanical tests. Expression of the cardiac-specific proteins on the scaffolds was investigated using immunofluorescence staining and quantitative real-time PCR. The elasticity of blends increased with an increase of PEEUU. In the blend scaffolds, the size and interconnectivity of pores were in an appropriate range (142−170 μm) as reported in the literature. These blend scaffolds revealed high cell metabolic activity for cardiomyoblasts and also enabled cells to proliferate and express cardiac marker proteins at higher rates. Histological examination of subcutaneously transplanted scaffolds after two months revealed degradation in the blend scaffolds. It is demonstrated that functionality of cells is sensitive to the composition of biomaterials used, and the effective cell−biomaterial interactions are critical in order to create a functional tissue engineered product that allows seeded cells to develop their normal activity. The PEEUU−PCL blends could potentially provide a versatile platform to fabricate functional scaffolds with an effective cell−biomaterial interaction for cardiac tissue regeneration. KEYWORDS: cardiac tissue engineering, polyurethane, polycaprolactone, polymeric scaffolds

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INTRODUCTION Cardiovascular diseases are the leading cause of mortality worldwide, including approximately 40% of all human mortality despite the progress and developments in the therapeutic methods.1 Myocardial infarction (MI) occurs following the blockage of a coronary artery which leads to a massive cardiomyocyte loss. The heart possesses limited regeneration potential, and the lost cardiomyocytes are © XXXX American Chemical Society

normally replaced with noncontractible scar tissue that reduces cardiac output.2 To overcome the limitations such as shortage of organ donors, complications of immune rejection, and low efficiency of cell delivery systems for optimal clinical Received: July 25, 2018 Accepted: September 24, 2018

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DOI: 10.1021/acsbiomaterials.8b00848 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

engineering.17,28 PCL has been blended with various amorphous and crystalline polymers. In this research, the hypothesis was that blending a biodegradable PEEUU and PCL will give a superior biomaterial with suitable physicochemical properties, biocompatibility, and biological interactions. The various techniques such as solvent casting/salt leaching, electrospinning, decellularization, and microfluidic systems have been developed to fabricate scaffolds.29−31 We used the solution casting/solvent evaporation and solvent casting/salt leaching method to fabricate microporous and macroporous scaffolds, respectively. The method of synthesis, the resultant polyurethane, and new PU−PCL scaffolds are the main novelties of this work. This study was conducted to evaluate the potential of new PU−PCL blends for cardiac tissue engineering application.

application, scientists seek new approaches to induce adequate regeneration of the damaged myocardium. Cardiovascular tissue engineering that includes the vascular network, myocardium, and heart valves is a promising approach.3−5 Critical properties for cardiac tissue constructs include biocompatibility, ability to foster cells, tailored degradation rate, and appropriate mechanical properties to improve cell attachment and growth, allowing cardiomyocytes to accomplish their function.6 Scaffolds should mimic the natural extracellular matrix to endorse cell−biomaterial interactions.7 Here, the cell−biomaterial interactions are referred to as attachment of cardiomyoblasts to the biomaterial surface and their functions such as adhesion, survival, growth, proliferation, and expression of cardiac-specific markers. The regeneration process of heart post-MI usually takes 6−8 weeks. The cardiac patch should degrade over the regenerative period to prevent fibrous capsule formation and chronic inflammatory response in the cardiac tissue.8 Furthermore, matching the mechanical properties of the tissue engineering construct with the heart is an important feature to promote the contraction of the growing cells. The stiffness of the adult human myocardium ranges nonlinearly from 10−20 kPa at early diastole to 200−500 kPa at the end of diastole.9 Choice of material, scaffold fabrication techniques, and design of scaffold microstructure could influence the cell− biomaterial interactions.10 Several synthetic and natural biomaterials have been studied as a possible scaffold for the cardiac tissue. Among the synthetic polymers, the elastomeric polymers better mimic mechanical properties of the dynamic heart, increasing beating rate and enhancing the growth of cardiomyocytes compared with stiffer biomaterials.11 Biodegradable polyurethane elastomers have been used for cardiac tissue repair and regeneration after myocardial infarction.12 Polyurethanes tend to adjust their behavior using the ratio of components and combination of the soft segment and hard segment.13 We have previously developed a family of PU-based nanocomposites for cardiovascular application.14,15 In our previous studies, we were also synthesized various polyurethane elastomers for cardiac tissue engineering application.16−18 Poor mechanical and physicochemical properties and also low electrical and vascular integration of constructs are the major limitations of currently available cardiac scaffolds. In previous work by our group, a new biodegradable poly(ester-ether urethane) urea (PEEUU) with monodisperse hard segment has been developed through in situ generation of AB-type macromonomer using poly(diethylene glycol adipate) (PDEGA), poly(ethylene glycol) (PEG), 1,6-diisocyanatohexane (HDI), benzoic acid, and dimethyl sulfoxide as main constituents according to a one-pot method.19,20 This method creates monodisperse hard segments, eliminates monotonous approaches to adjust the exothermic nature of isocyanate− amine reaction, has less sensitivity to impurities, and implicates no isolation of intermediates. Polymer blending is a very attractive way to obtain new biomaterials exhibiting combinations of properties that could not be obtained by individual polymers.21−24 It has been demonstrated that blending polyurethane with natural or synthetic polymers leads to enhancement of mechanical properties.25−27 Polycaprolactone (PCL) is a biodegradable and biocompatible polymer with good mechanical properties which has been utilized for various tissue engineering applications such as myocardium, vascular, musculoskeletal, skin, and nerve tissue

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EXPERIMENTAL SECTION

Synthesis of PEEUU. The PEEUU was synthesized according to the procedure described in our previous study (Figure 1).32 Briefly,

Figure 1. Synthesis of poly(ester-ether urethane urea) elastomer. dried PDEGA (Mn 2500, Sigma-Aldrich, U.S.A.) and PEG (Mn 2000, Sigma-Aldrich, U.S.A.) at equal molar ratio were placed into a fournecked polymerization reactor equipped with a mechanical stirrer, condenser, dropping funnel, and a nitrogen inlet. The temperature was adjusted at 60 °C. 1,6-diisocyanatohexane (HDI, Merck, Germany) was then poured dropwise into the reactor at a rate such that the maximum reaction temperature reached up to 70 °C (about 1 h). The temperature was then increased to 85 °C, and the NCO content was allowed to reach the theoretical value that was determined according to dibutylamine titration (ASTMD-2572). This isocyanate-terminated polyurethane prepolymer (ITPP) was dissolved in DMSO (Sigma-Aldrich, U.S.A.) and cooled to room temperature. The benzoic acid/DMSO solution was then added dropwise into the polymerization kettle. The polymerization was continued at room temperature for 10 to 15 h to obtain a lucid viscous solution that was then precipitated into an excess volume of cool deionized (DI) water, consecutively washed with warm water and methanol, and dried in a vacuum for 24 h. Preparation of PEEUU−PCL Blends. PCL (Mn 80 000, SigmaAldrich, U.S.A.) and the synthesized PEEUU were dried in a vacuum oven at 40 °C for 5 h. The PEEUU−PCL blends were obtained by solution mixing in a beaker. In brief, they were distinctly dissolved in B


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Water Contact Angle and Swelling Measurements. The water contact angle of the films (n = 3 per sample) was assessed at room temperature on an optical video contact angle system (OCA-15plus, Dataphysics) using the sessile drop method. Four microliters of deionized water were trickled on the surface of films and then measured. The swelling behavior of polymer films was evaluated according to ASTM D570-98. Dried scaffolds were soaked in Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) at 37 °C. After the time intervals were determined, samples were removed and wiped with a filter paper to eliminate excess water. They were then weighed on a digital scale, and the content of DPBS in the swollen specimens was calculated as eq 3:

N,N,-dimethylformamide (DMF, Merck, Germany) at different weight ratios (PEEUU−PCL ratios of 100/0, 25/75, 50/50, 75/25, and 0/100) at 70 °C to obtain ∼6 and 10 wt % solutions. The polymer solutions were put into a vacuum desiccator to remove any air bubble. The composed scaffolds from determined PEEUU−PCL weight ratios are referred to as PEEUU, PEEUU−PCL 1/3, PEEUU− PCL 1/1, PEEUU−PCL 3/1, and PCL, respectively. Fabrication of Films and Macroporous Scaffolds. Films were prepared by solution casting/solvent evaporation method. The ∼6 wt % polymer solutions were cast into glass Petri dishes, and the films were dried under vacuum for 4 days to eliminate solvent. Porous scaffolds were fabricated by a solvent casting/salt-leaching process. The polymers in various weight ratios (PEEUU−PCL ratios of 100/0, 25/75, 50/50, 75/25, and 0/100) were dissolved in DMF to acquire 10% (w/v) solutions. Then 1.5 g salt particles (NaCl, Merck, Germany) were subjoined into each polymer solution (1 mL) and stirred about 15 min at 50 °C to acquire homogeneous mixtures. The sodium chloride salt crystal sizes ranging from 100 to 150 μm were achieved by American Standard Test Sieve Series (ASTM). The salt particles were made dry 24 h in an oven at 115 °C and a 100-mesh screen was initially utilized, NaCl crystals were then poured onto the 140-mesh screen. The PEEUU/NaCl/solvent mixture was cast into a cylindrical glass mold and then dried. The scaffolds were immersed in a 20% ethanol solution for 5 h and then placed in ultrapure deionized water for 3 days to leach the salt particles from the scaffold. Scanning Electron Microscope (SEM) Analysis. To evaluate cell-scaffold morphology, samples were rinsed with 0.1 M phosphate buffered saline solution (PBS, Gibco, U.S.A.) and fixed in 4% PBS/ paraformaldehyde (PF, PH 7.4) solution for 30 min. Afterward, the specimens were washed with PBS and dried in a vacuum oven. The macroporous scaffolds were cut to obtain the cross-section of samples. The microstructure of scaffolds and cell-scaffolds in these cross sections were analyzed by SEM instrument (AIS2100, Seron Technology) at an accelerating voltage of 30 kV. The average pore size was calculated by measuring the dimensions of 30 pores using SEM images. The pores were randomly selected from the samples and analyzed by ImageJ software. To calculate the pore density of the scaffolds, the SEM images (3 different cross sections) were used by the ImageJ software. The pore density was defined according to the eq 1:33

pore density = (N /A)3/2

swelling ratio (%) = [(Ww − Wd)/Ww ] × 100

where Ww and Wd represent the weights of the wet and dried specimens, respectively. In Vitro Polymer Degradation. Rectangular samples from films were weighed (w1 ∼ 40 mg) and then dried in a vacuum oven and sterilized by UV irradiation for 15 min. The samples were immersed into tubes containing DPBS with pH 7.4 at 37 °C. The specimens were removed at determined time intervals, washed with DI water, and dried in a vacuum oven at 40 °C. They were then weighed (W2), and the weight remaining was determined by the eq 4; (W2/W1) × 100%

(4)

Mechanical Properties of Films. Uniaxial tensile testing of the polyurethane films was tested using a mechanical tester (Model 5566; Instron Company, U.S.A.) with a 50 lb load cell at room temperature. According to ASTM D638-98, dumbbell shape strips with dimensions 2 mm × 20 mm were cut from films. The cross head speed was set at 10 mm/min. Maximum stress was defined as the tensile stress and the strain at the maximum stress was considered as a breaking strain in the specimens. The Young’s modulus was obtained as the slope of the linear region of the stress−strain curve corresponding with 0%−13% strain and the crossway point of two regression lines drawn from the collapsed structure and linear elastic tendency, respectively. The assessments were carried out in three runs. Culture of Cardiomyoblasts. Rat embryonic cardiomyoblast cells (H9c2, CRL-1446) were obtained from Cell Bank of Iran. The cells were maintained in Dulbeccos’s Modified Eagle’s Medium-high glucose (DMEM-HG, Gibco, 12800-116) supplemented with 4.5 g/L glucose, 20 μM L-glutamine, 3.7 g/L sodium bicarbonate (NaHCO3), 10% fetal bovine serum (FBS, Gibco, 10270−106), 1 mM pyruvate, and 1% penicillin/streptomycin (Pen-Strep, Gibco, 15140-122). Cells were cultured in 75 cm2 cell culture flasks at 37 °C and 5% CO2 in 95% humidity. The medium was changed once every 2 days, and the cells were subcultured with 0.25% trypsin-EDTA (1×) (Gibco, 25300-054) in 70−80% confluence in order to avoid differentiation into cardiomyocyte by overconfluence. Evaluation of Cell Metabolic Activity. The viability of cardiomyoblasts on the surface of films was determined by direct MTT assay (Sigma-Aldrich, U.S.A.). Strips (6 × 6 mm) from films were punched, rinsed with 1% PBS/streptomycin/penicillin solution (Gibco, U.S.A.) and sterilized using 70% ethanol solution and UV irradiation. The cells were seeded on the film surfaces in 96-well polystyrene tissue culture plates (SPL lifesciences, Korea) at a density of 3 × 103 cells into each well. The culture medium (high-glucose DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.) and 1% penicillin/streptomycin was utilized and replaced every 2 days. At days 2, 3 and 4, the MTT solution was added to each well and incubated in the dark at 37 °C for 4 h. The optical density of the samples was detected at 490 nm using a microplate reader (Epoch, U.S.A.). For quantitative analysis, the cells were observed on the film surfaces after a fluorescent staining. In brief, the cells were fixed using 4% PF solution, rinsed with PBS, and immersed in 0.2% Triton X-100 solution for 20 min at room temperature. Thereafter, the cells were rinsed with PBS, and 4,6-diamidino-2-phenylindole (DAPI, Sigma-

(1)

where N and A are the numbers of the pore and the area in the SEM images, respectively. Porosity Measurements. The porosity of scaffolds (n = 3 per sample) was evaluated using the liquid displacement method.13 Ethanol was utilized as the replacement liquid to permeate easily into scaffold pores. The samples were placed in a vacuum to remove air from the scaffolds. A dried scaffold was then submerged in a measuring cylinder containing a determined volume (V1) of ethanol and incubated at 37 °C. After 20 min, the total volume of ethanol and the ethanol-saturated sample was considered as V2. The scaffold was raised from the measuring cylinder and the residual ethanol volume was considered as V3. The porosity ratio was obtained according to the eq 2: porosity ratio (%) = [(V1 − V3)/(V2 − V3)] × 100

(3)

(2)

Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectra of the polymer films were recorded with a FTIR spectrometer (Bruker IFS 48 instrument). All transmission spectra were collected at a resolution of 4 cm−1 in the wavenumber range of 4000−500 cm−1 in the KBr-diluted medium. ATR-FTIR curves are shown in Figure S1. Thermal Characterization. The glass transition temperature (Tg) and melting temperature (Tm) of PEEUU film were determined by differential scanning calorimetry (DSC3+, Mettlertoledo) according to ASTM D3418-12. The DSC was performed with a temperature range from −80 to 250 °C under a nitrogen atmosphere at the cold source and scanning rate of 10 °C/min. The sample was heated to 250 °C, then cooled to −80 °C, and heated again to 250 °C. C


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ACS Biomaterials Science & Engineering Aldrich, 1:1000) was added to the cells for 5 min to stain cell nuclei. The samples were then washed with PBS. Immunofluorescent Staining of H9c2 Cells. To evaluate the proliferation of H9c2, the cells were seeded at a density of 1 × 104 cells/cm2 onto the films. At day 4 after the cell seeding, the cells were fixed using 4% PF solution, permeabilized (by 0.2% Triton-X), blocked (by 5% BSA/PBS), and finally labeled with the rabbit polyclonal primary antibody Ki67 (Abcam, ab15580) for 24 h at 4 °C. After adding a secondary antibody (Alexa Fluor 488 goat antirabbit, Abcam, ab150077), the cell nucleus was stained with DAPI. The cytoskeletal phenotype of H9c2 cells was identified using immunofluorescent after 4 days. To evaluate cardiac α-actin and GATA4, primary antibodies including mouse monoclonal antisarcomeric α-actin (Sigma, A2172) and rabbit polyclonal anti-GATA4 (Abcam, ab84593) were used, respectively. The secondary antibody was Alexa Fluor 488 goat antirabbit (Abcam, ab150077). Antigen retrieval was performed using boiling samples in 1 mM EDTA (Sigma, ED4SS). Antibodies were diluted in blocking buffer (5% PBS/BSA). RNA Isolation and Quantitative Reverse Transcriptase PCR (qRT-PCR). The gene expression profile of cardiomyogenic markers in H9c2 cells was determined using a qRT-PCR. For this aim, the cells were first cultured onto films for 7 days. The total RNA was extracted from the cultured cells using TRIzol reagent (Ambion, Thermo Fisher Scientific, U.S.A.) and chloroform following the manufacturer’s instructions. In brief, Trizol reagent (1 mL) was added to the cells at room temperature. After 30 min, RNA was isolated by centrifuging at 12 000 rpm at 4 °C for 20 min. The isolated RNA was stabilized by 70% ethanol/nuclease-free water. The absorbance of the solution was measured at 260 and 280 nm using the Multiskan microplate reader to obtain concentration and purity of the isolated RNA. The first-strand cDNA was synthesized using oligo primer (25081; Intron Biotech) according to the manufacturer’s instruction. Gene expression was detected using qRT-PCR. The PCR was performed using Mastermix kit, d.d. H2O, forward or reverse primer and cDNA on Real-Time PCR instrument (a Rotor-Gene6000, Corbett Life Science, U.S.A.). The data were normalized to GAPDH and expressed as fold change for PEEUU−PCL 1/1 and PEEUU relative to PCL (ΔΔCt). Table S1 represents the used primer sequences. Subcutaneous Implantation of Films. The animal study was carried out according to the guidelines approved by Animal Research Care Committee at University Tehran, Iran. Adult male Wistar rats with a weight of 200−250 g were selected and anesthesia was induced with ketamine/xylazine. 1.0 cm transverse incision was made on rat skin in the left dorsal region and films (6 mm diameter ×160 μm thickness) were sutured in this region on muscle by a 6−0 nonabsorbable polypropylene. The skin was closed using 4−0 polyglactin absorbable suture and bandaged. For prevention of the infection, cefazoline antibiotic (100 mg/kg, 1 dose in a day up to 3d) was injected into animals. After 8 weeks postimplantation, the explants were frozen in 2-methylbutane, which was precooled in liquid nitrogen. The sections were then stained with hematoxylin and eosin (H&E, Sigma-Aldrich, U.S.A.) for histological assessment. Statistical Analysis. All the obtained results were expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed by SPSS 16.0 software to assess differences between the measured groups followed by Tukey’s HSD post hoc test. P < 0.05 was considered significant.

Figure 2. (A) SEM micrographs of the fabricated films. (B) Macroscopic images and (C and D) SEM images of the scaffolds fabricated by the solvent casting and salt leaching method. (C) and (D) micrographs with low and high magnification, respectively. Scale bars: 200 μm.

Table 1. Morphological Characteristics of the Porous Scaffolds scaffold PEEUU PEEUU− PCL 3/1 PEEUU− PCL 1/1 PEEUU− PCL 1/3 PCL

average pore size (μm)

range of pores (μm)

pore density (×104 /cm3)

porosity (%)

77 ± 18 142 ± 40

65−110 71−242

133 ± 8 286 ± 10.5

79 ± 2 91 ± 2

139 ± 21

125−155

497 ± 14.3

94 ± 1

170 ± 52

114−355

226 ± 12.4

89 ± 4

190 ± 74

130−320

189 ± 15

87 ± 3

investigation, a polymer consisting of hydrophobic PDEGA and hydrophilic PEG as soft segments in the structure of PEEUU was successfully synthesized (Figure. 1). We then generated the films and 3D macroporous scaffolds from the synthesized PEEUU, PEEUU−PCL blends, and PCL with the hope of attaining better physicochemical properties and suitable cellular responses from blend patches. SEM micrographs of the films were shown in Figure 2A. After blending two polymers, micropores appeared onto the surface of PEEUU−PCL blends, but these micropores were not observed on the surface of PEEUU and PCL films. The blend films had micropore sizes in the range of 4−9 μm. The PEEUU−PCL 1/1 blend had the highest porosity and pore size (51% and 9 ± 0.4 μm, respectively). These micropores can be attributed to the presence of moisture during the film casting and solvent evaporation. In blends with two distinct hydrophilic (PU) and hydrophobic (PCL) phases, it is possible that hydrophilic polymer such as synthesized polyurethane migrates to the surface of the films during solvent evaporation,

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RESULTS AND DISCUSSION Physico-Chemical Characterization of the Scaffolds. To promote repair and regeneration of infarcted myocardium, it is important to provide constructs with appropriate cell− scaffold interactions. Cells respond to the situations such as structure and chemical composition of scaffolds. Owing to the elasticity of polyurethane elastomers, they are applied as a scaffold in cardiovascular tissues including the cardiac tissue, heart valves, and blood vessels.34 In this D


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Figure 3. Physicochemical characterization of the films. (A) DSC curve of the synthesized PEEUU. (B) Surface wettability of films. (C) The swelling ratio of specimens. (D) Mass remaining of the films; To evaluate in vitro degradation rate, the samples were immersed in DPBS and incubated at 37 °C for 13 weeks. ***p < 0.001.

Table 2. Wettability and Degradation Rate of the Films¶ wettability films PEEUU PEEUU−PCL 1/3 PEEUU−PCL 1/1 PEEUU−PCL 3/1 PCL

mass remaining (%)

contact angle (deg)

swelling ratio (%) (after 72 h)

60.5 ± 2a 87.5 ± 2.5b

30.05 ± 3.85 7.34 ± 0.7

90 ± 1.14d 97 ± 1.52d

19.7 ± 1.18

86.08 ± 1.46d

67.9 ± 4.1a

25.27 ± 3.35

88 ± 1.14d

90.2 ± 4.2b

4.69 ± 0.45

75.5 ± 4c

density with closed pores while blend scaffolds had larger pore sizes, containing mainly interconnected and opened pores. The range of mean pore sizes was 77−190 μm. Previous studies suggested that a pore size of 100−150 μm in cardiac tissue engineering allows appropriate cell survival and vascularization in scaffolds.39 In this study, the composite scaffolds had pore sizes in the range of 142−170 μm, which seems to be suitable for cardiac tissue engineering. The scaffold with optimal interconnected pore networks (made from PEEUU−PCL 1/1) had a pore density of 494 × 104/cm3. The results showed that the increase in porosity of scaffolds results in the elevated pore density in samples (Table 1). The porosity of the scaffolds was approximately similar for all specimens except in PEEUU which was 79 ± 2%. The PEEUU−PCL 1/1 scaffold had a better porosity (94 ± 1%). In the macroporous scaffolds, it was found that the average pore size, pore density, and overall porosity increase after adding PCL in blends compared to the PEEUU scaffold. The PCL led to the improved porosity of the blend scaffolds. The superior mechanical strength and high molecular weight of PCL might lead to the improvement of porosity in blend scaffolds. After adding porogen such as NaCl, the mechanical properties of the scaffolds were severely reduced, which is mainly due to the porous structures of the scaffolds. The SEM images also showed obvious interconnected pores in blends. The result of the DSC analysis of the synthesized polymer was plotted in Figure 3A. The PEEUU presented low glass transition temperatures (−44.7 °C) associated with the soft segment that indicates a rubbery state of the polymer at room temperature. Hard segment transition was not observed on the DSC curve that demonstrates a low degree of phase separation. The PEEUU with an amorphous PDEGA and PEG soft segments had not detectable melting temperature.

in vitro test

101 ± 1.6

¶

a, b, and c imply significantly different groups. d denotes that mean difference is significant in each group after 13 weeks, (p < 0.001 versus initial time).

where this polymer can be exposed to the moisture and undergo degradation, resulting in micropores formation.35 Here, synthesized polyurethane and polycaprolactone were hydrophilic and hydrophobic polymers, respectively. These micropores in blend films can accelerate cell attachment and proliferation compared with flat films.36 Cellular behaviors such as migration, organization, proliferation, and differentiation could be affected by 3D scaffolds.37 It is required to optimize scaffolds in terms of pore-related features and swelling behavior to improve diffusion of nutrients, cell infiltration, and cellular behavior.38 The microstructure and macrostructure of porous scaffolds are shown in Figure 2B−D. The SEM analysis carried out to assess the structure and morphology of scaffolds such as pore size, pore density, and interconnectivity between the pores. SEM images were obtained from cross sections of macroporous scaffolds. As shown in Table 1 as well as Figure 2C,D, the scaffolds made from PEEUU had the least pore sizes and pore E


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Figure 4. Mechanical properties of the films. (A) Representative tensile stress−strain curves for the polymer films. (B) Details of tensile stress at a low strain of figure (A). (C) Ultimate and yield strength for the samples. (D) Maximum strain and energy at the break for the films. (E) Young’s modulus and maximum force of constructs. The PEEUU shows the lowest modulus. n = 3, (*P < 0.05, ***P < 0.001).

hydrophilic nature of this polymer as expected while the PCL had the highest contact angles (90 °C, p < 0.001) and hydrophobicity among the groups. The presence of PEG segment in the backbone of PEEUU with the ability to establish hydrogen bonding interaction with water molecules was the reason for the observed hydrophilic character of the PEEUU. The water contact angle of blends changed with enhancement of the PEEUU in blends so that with an increased ratio of polyurethane elastomer, low water contact angle was measured.

Wettability is an important feature of the scaffold in order to support the early stages of cell adhesion onto the scaffold surface. Biomaterials are expected to provide a highly hydrated substrate, similar to soft tissues having high water content. Thus, tuning the surface properties of the biomaterial in terms of hydrophilicity influences its biological efficiency. To determine the surface hydrophilicity of the films, water contact angle was measured (Table 2 and Figure 3B). The results of surface wettability showed that the PEEUU had the lowest contact angles (60.5 °C, p < 0.001), indicating highly F


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Figure 5. Biocompatibility test for the film constructs. (A) H9c2 cells cultured on films (TCPS as a control group) after 48, 72 and 96 h of cell seeding (n = 3). The results indicate that the scaffolds are biocompatible, and the cell proliferation is higher on the surface of the blend films as compared with the PEEUU and TCPS. (B) Fluorescently stained H9c2 cells on the surface of the PEEUU−PCL 1/1 film after 48 and 96 h of cell culture. DAPI staining. Scale bar: 100 μm. (C) SEM images of H9c2 cells seeded into the macroporous PEEUU-PCL 1/1 patches.

remarkably affected the hydrolytic degradation of the scaffolds. Because of the highest hydrophobicity of the PCL and PEEUU−PCL 1/3 compared with other samples, these polymers had the lowest degradation rates after 13 weeks (p < 0.001).40 PEEUU showed the highest surface and bulk hydrophilicity, but this polymer was degraded slower than the PEEUU−PCL 1/1 and 3/1 blends. The reason for the faster degradation rate observed in these blends could be attributed to either the higher concentration of water molecules in the vicinity of ester bonds due to higher hydrophilicity of this samples (compared to PCL and PEEUU−PCL 1/3) or the existence of micropores on their surface compared with the PEEUU film, which consequently increase the surface area for interaction and penetration of water molecules. The degradation of the PEEUU and blend scaffolds took over 8

The swelling properties were determined by water absorption rate as they are related to bulk hydrophilicity. Swelling behavior of the scaffolds affects degradation properties, the nutrition, and waste transfer rate within the scaffolds.40 Swelling results of scaffolds were shown in Figure 3C. The PEEUU−PCL blends presented lower water absorption compared with the PEEUU. In samples, the increase in the amount of PCL decreased the water content that occurred because of hydrophobicity of PCL. Polyesters are often blended in other polymers to control degradation rate. To avoid fibrous capsule formation and inflammatory response in the heart, the cardiac patch should biodegrade over period regeneration after myocardial infarction, which often takes 6−8 weeks.8 The hydrolytic degradation of dry films was measured in DPBS at 37 °C for 13 weeks (Table 2 and Figure 3D). The ratio of water absorption G


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Figure 6. Immunofluorescence staining of the cardiomyoblasts cultured onto the film constructs. (A) Staining results for Ki67. (D) Statically analysis of Ki67 (at least p < 0.05). (B) Immunofluorescent of cardiac α-actin expression on day 4. (E) Quantitative analysis of α-actin. (C) Immunofluorescent images of GATA4 expression. (F) GATA4 statically analysis. (G) Gene expression profiling of H9c2 cardiomyoblast for the cardiac-specific proteins on the films after 7 days of cell seeding (n = 3). Scale bar: 50 μm. *p < 0.05, **p < 0.01 and ***p < 0.001.

blend films enhanced along with the increase in the PEEUU content (Figure 4F). The elastic modulus of these films was a magnitude similar to that of native heart tissue: the PEEUU (0.62 ± 16 MPa), the PEEUU−PCL 3/1 (12.62 ± 3.1 MPa), and the PEEUU−PCL 1/1 (63.3 ± 4.07 MPa). Low stiffness may be an appropriate feature with respect to integration with native tissue because a sorely stiff matrix inhibits the growth and function of cardiac cells. We achieved an improved mechanical strength and good biocompatibility utilizing the blend of polyurethane with PCL. Because of increased

weeks. Hence, the degradation rate blends and PEEUU was appropriate for cardiac tissue. Mechanical Characterization of the Scaffolds. The mechanical tests were accomplished on films at room temperature in air. The stress−strain curves and related mechanical properties are shown in Figure 4 and Table S2. While tensile stress−strain curves of the PEEUU−PCL blends and PCL films became nonlinear at about 13% strain, resulting from collapsing and densification structure, stress−strain curve of the PEEUU was linear (Figure 4A). The elasticity of the H


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Figure 7. H&E staining of the implanted scaffolds. Large pores are seen in the blend scaffolds, resulting in the blend scaffolds degraded faster than other groups. (A) PEEUU−PCL 1/3, (B) PEEUU−PCL 3/1, (C) PEEUU−PCL 1/1, (D) PEEUU, and (E) PCL films.

Metabolic Activity and Cell Morphology. The development of cardiac patches requires appropriate scaffolds seeded by necessary cells in order to represent the characteristics of the cardiac tissue. Therefore, we evaluated the effect of scaffolds on viability and metabolic activity of H9c2 cardiomyoblasts through MTT assay at 48, 72, and 96 h. The results illustrated that the highest rates of viability are significantly related to the blend films compared with the PEEUU film and TCPS (Figure 5A,B, P < 0.05). The surface topography of the composite films might be responsible for the highest rates of cell viability compared to PEEUU film and TCPS. The PEEUU and PCL had no micropores on their surfaces, while the surface of PEEUU−PCL blends exhibited microporous features. The increase of metabolic activity on the surface of the blend scaffolds was confirmed using fluorescence microscope following nucleus staining at 48 and 96 h after cell seeding. As shown in Figure 5B, there was an increase in the growth of cells on the surface of PEEUU−PCL 1/1 at 96 h compared with 48 h. SEM images of cardiomyoblasts cultured into the macroporous PEEUU-PCL 1/1 scaffold also displayed a good cell adhesion into patches after 5 days (Figure 5C). These results demonstrated the usability of the scaffolds as a suitable substrate for the adhesion and growth of cells. Expression of Cardiac-Specific Proteins on Scaffolds. The scaffolds influence cells with respect to not only their morphology but also their function. To analyze the cellular responses of the cardiomyoblasts seeded onto the films, we investigated cellular proliferation by immunofluorescence staining and the expression of cardiac-specific markers using immunofluorescence staining and quantitative real-time PCR. The Ki67 protein is a cellular marker to determine cell proliferation. We assayed the proliferation of H9c2 cells on the PEEUU and PEEUU−PCL 1/1 films by expression of the Ki67 protein (Figure 6A,D). While the significant population

Figure 8. Representative images of the tissue responses to the grafts, (A) PEEUU−PCL 3/1, (B) PEEUU−PCL1/1, (C) PEEUU−PCL1/ 3, and (D) PEEUU. (E) and (F) Microvessels were observed in the connective tissue area between the grafted (E) PEEUU or (F) the PEEUU−PCL 1/1 constructs and the native muscle of rat (black arrows show microvessels). Scale bar: 100 μm.

intermolecular forces associated with increased PCL crystallinity at a higher amount of PCL, the PEEUU−PCL blends exhibited higher tensile strengths than PEEUU. I


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ACS Biomaterials Science & Engineering of Ki67-positive H9c2 cells (at least P < 0.05) were found on the PEEUU−PCL 1/1 blend (60.5 ± 1.7%), there was a lower level of Ki67 expression on the PEEUU film (14.3 ± 1.2%) (p < 0.001) (Figure 6D). The previous studies have suggested that the cellular response to microporous films (e.g., cell adhesion and proliferation) arises from the adsorption of FBS’s fibronectin in the microporous structure of the film, which leads to increased cell adhesion and proliferation. The blend scaffold had these micropores while there were no microporous structures in the PEEUU and PCL films. The cells were then assessed by immunofluorescence staining for the α-actin (muscle-specific intermediate filament protein) and GATA4 (a transcription factor that regulates myocardial function). The expression of α-actin is shown in Figure 6B,E. The elongation of the H9c2 cells onto the PEEUU−PCL 1/1 film was more than the control groups (Figure 6B). As plotted in Figure 6E, statistical analysis of αactin expression showed that cells on the PEEUU−PCL 1/1 film have the highest expression (92.2 ± 1.94%) (p < 0.001). The GATA4 is a transcription factor which involves the cardiac commitment from the precursor cells. More expression of GATA4 in cytoplasm indicates normal physiological activity, while nuclear localization of GATA4 indicates the pathological hypertrophic condition of cardiomyoblasts.41 Expression of GATA4 in the H9c2 cardiomyoblasts is displayed in Figure 6C,F. There were the lowest expressions of GATA4 in the seeded cells on PEEUU (9.9 ± 0.8%) and PCL (10.7 ± 0.96%) films that these expressions were further localized in the cell nucleus. However, the highest levels of the expression were found in the group of cardiomyoblasts cultured on the TCPS (61 ± 1.25%) and PEEUU−PCL 1/1 film (59 ± 1.47%) (p < 0.001) that distributed majorly in the cytoplasm (Figure 6F). Quantitative real-time PCR was carried out for GATA4, NKX 2.5, MEF2C, cardiac troponin I (cTNI), connexin 43, and cardiac α-actin genes. Cardiomyoblasts were seeded on PEEUU, PEEUU−PCL 1/1, and PCL films for a week, and results are shown in Figure 6G. In the H9c2 cardiomyoblasts cultured on the PEEUU−PCL 1/1 and PEEUU films, the expression of NKX 2.5 (PEEUU: 3.1 ± 0.16, PEEUU−PCL 1/ 1:3.6 ± 0.3 Fold), MEF (PEEUU: 0.9 ± 0.07, PEEUU−PCL 1/1: 1.16 ± 0.1 Fold), and cardiac α-actin (PEEUU: 2.1 ± 0.31, PEEUU−PCL 1/1: 3.5 ± 0.4 Fold) genes was upregulated in comparison with the PCL film (at least p < 0.05). In addition, there was an increase in the expression of GATA4, cTNI and connexin 43 genes on the PEEUU−PCL 1/1 blend (2.3 ± 0.08, 1.28 ± 0.2 and 1.1 ± 0.2 Fold) compared with the PEEUU (0.7 ± 0.2, 0.6 ± 0.2 and 0.5 ± 0.1 fold) films (at least p < 0.05). The expression of early cardiac markers was significantly upregulated on the blend film relative to the PEEUU and PCL films. This illustrated the tendency of H9c2 cells to become cardiomyoblast and maintain their morphology and function on the blend film at a week after cell seeding. Therefore, our results indicated that the blend film provided an appropriate substrate for the cardiomyoblasts to adhere, proliferate and present better function. In Vivo Subcutaneous Implantation of Scaffolds. To assess in vivo biodegradation rate and tissue response to the PEEUU, blends, and PCL films, subcutaneous implantation of the scaffolds was accomplished in male Wistar rats. Figure S2 shows macroscopic images of the implanted samples before (Figure S2A) and after (Figure S2B,C) follow-up period. After dissection, in all animals, samples were observed to be covered

with connective tissue (Figure S2C). As shown in Figure S2B, the PEEUU−PCL blends degraded faster than other groups. The entire PCL and the majority of the PEEUU film were remained almost intact and preserved their circular form. These results were further confirmed by H&E staining of constructs 8 weeks after the implantation. Micropores also appeared on the surface of blend patches (Figure 7). These findings indicate that blend patches did not completely degrade after 2 months, matching the degradation rate with the desired period. The results of in vivo degradation also indicated that degradation of the samples implanted into the animal body was slightly higher than that of in vitro assessments. This can be attributed to the intricate aqueous environment (such as interstitial body fluid) compared with the DPBS used in vitro. The biological medium leads to the degradation of the PEEUU and PEEUU−PCL and PCL by means of the oxidation and hydrolytic-enzyme processes (mediated by the cells specifically neutrophil and monocytederived macrophages; enzymatic agents related to the acute foreign body response). Hydrolytic degradation of polyester urethane occurs in aliphatic ester bond.42,43 Existing inflammatory-cell-derived enzymes in the physiological environment also accelerate the degradation of PEEUU. Santerre et al. carried out an investigation on enzymes and confirmed that monocyte-derived macrophages secreted enzymes including cholesterol esterase and monocyte specific esterase which increased the degradation of polycarbonate-based polyurethanes.44 Macroscopically, the scaffolds were surrounded by a connective tissue and blood vessels were formed in the surrounding tissue of each scaffold. The tissue response to the constructs of the PEEUU and PEEUU−PCL blends in the rats are shown in Figure 8. H&E staining shows that the constructs were surrounded by connective tissue. Microvessels were formed in the connective tissue region between the constructs and the adjacent muscle of the host animal (Figure 8E,F). The scaffold integration into tissue was increased by enhanced vascularization in the surrounding tissue of grafts.45

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CONCLUSIONS The new polyurethane synthesized using a novel “one-pot method” resulted in PEEUU−PCL scaffolds with improved mechanical and physicochemical properties. The PEEUU− PCL blends revealed appropriate cell−scaffold interactions, resulting in enhanced functional activities of the cardiomyoblasts by expressing the cardiac-specific markers. These blend patches revealed mechanical features similar to those of the native heart tissue. Therefore, our findings suggested that the PEEUU−PCL patches could be an attractive substrate for the cardiac regeneration that can potentially be utilized in cellbased therapies for myocardial regeneration.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00848. Additional information about ATR-FTIR analysis, mechanical properties, primer sequences used for RTPCR analysis, and macroscopic images of implanted scaffolds (PDF) J


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AUTHOR INFORMATION

Corresponding Author

*Tel: +98-21-4305 2200. Fax: +98-21-8899 1117. E-mail: [email protected]. ORCID

Shiva Asadpour: 0000-0001-5574-7142 Hamid Yeganeh: 0000-0002-1293-0737 Jafar Ai: 0000-0001-8417-5913 Saeid Kargozar: 0000-0002-3785-1322 Alexander Seifalian: 0000-0002-1180-3322 Hossein Ghanbari: 0000-0001-7639-995X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Tehran University of Medical Sciences and Health Services (Grant No: 93-01-8724919) for financial support.

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ABBREVIATIONS (PEEUU), poly(ester-ether urethane urea); (PCL), polycaprolactone; (MEF2C), myocyte enhancer factor 2C; (cTNI), cardiac troponin I; (CONNX43), connexin 43; (GAPDH), glyceraldehyde 3-phosphate dehydrogenase

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