Polycaprolactone/oligomer compound scaffolds for cardiac tissue engineering Chaganti Srinivasa Reddy,1,2 Jayarama Reddy Venugopal,3 Seeram Ramakrishna,3 Eyal Zussman1 1
Department of Mechanical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel Institute of Biomaterial Science, Helmholtz-Zentrum Geesthacht, Kantstraße 55, Teltow 14513, Germany 3 Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore 2
Received 9 May 2013; revised 8 November 2013; accepted 18 November 2013 Published online 12 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35045 Abstract: Polycaprolactone (PCL), a synthetic biocompatible and biodegradable polymer generally used as a scaffold material for tissue engineering applications. The high stiffness and hydrophobicity of the PCL fiber mesh does not provide significant cell attachment and proliferation in cardiac tissue engineering. Towards this goal, the study focused on a compound of PCL and oligomer hydrogel [Bisphenol A ethoxylated dimethacrylate (BPAEDMA)] processed into electrospun nanofibrous scaffolds. The composition, morphology and mechanical properties of the compound scaffolds, composed of varying ratios of PCL and hydrogel were characterized by scanning electron microscopy, infrared spectroscopy and dynamic mechanical analyzer. The elastic modulus of PCL/BPAEDMA nanofibrous scaffolds was shown to be varying the BPAEDMA weight fraction and was decreased by increasing the BPAEDMA weight fraction. Compound fiber meshes containing 75 wt % BPAEDMA oligomer
hydrogel exhibited lower modulus (3.55 MPa) and contact angle of 25o. Rabbit cardiac cells cultured for 10 days on these PCL/ BPAEDMA compound nanofibrous scaffolds remained viable and expressed cardiac troponin and alpha-actinin proteins for the normal functioning of myocardium. Cell adhesion and proliferations were significantly increased on compound fiber meshes containing 75 wt % BPAEDMA, when compared with other nanofibrous scaffolds. The results observed that the produced PCL/BPAEDMA compound nanofibrous scaffolds promote cell adhesion, proliferation and normal functioning of cardiac cells to clinically beneficial levels, relevant for cardiac tissue engineering. C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3713– V
3725, 2014.
Key Words: elastomer, nanofibers, hydrogel, cardiac cells, troponin, actinin
How to cite this article: Reddy CS, Venugopal JR, Ramakrishna S, Zussman E. 2014. Polycaprolactone/oligomer compound scaffolds for cardiac tissue engineering. J Biomed Mater Res Part A 2014:102A:3713–3725.
INTRODUCTION
Polycaprolactone (PCL) is a semicrystalline thermoplastic polymer widely used in tissue engineering applications due to its non-immunogenicity, slow biodegradability and good drug permeability.1 Electrospun nanofiber mesh of PCL resembles the morphology of extracellular matrix (ECM), and has been used as a scaffold material for cell adhesion and proliferation in tissue regeneration. Mimicking the fibrillar structure of ECM, engineered scaffold provides essential guidance for cell organization, survival, and function.2–6 By ensuring such support, the scaffolds set the foundation for efficient and compact tissue formation and an expedited recovery process. The ester and keto group functionalities of PCL render it highly hydrophobic, leaving it with no physiologically active sites. Thus, while epithelial cell growth was enhanced on a PCL membrane, cell affinity towards PCL is generally poor.7 Biomimicking of ECM, the poor hydrophilicity of electrospun PCL mats led to an over-
all reduction in cell adhesion, migration, proliferation and differentiation.8,9 The scaffold chemistry plays an important role in cell adhesion and differentiation following implantation. Studies have reported that adhesion and organization of transplanted cardiac cells into functional contractile tissue are far superior on natural ECM compared with synthetic scaffolds.10,11 The substrate environment ideal for promoting cell proliferation has been a persistent area of debate in tissue engineering. Candidate substrate materials must exhibit biocompatibility, while supporting cell proliferation, which is hypothesized to be a direct function of its stiffness. Bioengineered scaffold stiffness must be closely resemble that of native ECM and flexible to allow the contraction of growing cells.12 In the case of PCL membranes, 3T3 fibroblast cell proliferation was most significant on low stiffness samples.13 In addition, high scaffold surface area and porosity are essential to allow for intercellular transport of nutrients,
Correspondence to: J. R. Venugopal; e-mail: [email protected] or E. Zussman; e-mail: [email protected] Contract grant sponsor: National Research Foundation of Singapore in the framework of the Regenerative Medicine Initiative in Cardiac Restoration Therapy research program and Technion-Israel Institute of Technology, Haifa, Israel; contract grant number: R-398-001-065-592
C 2013 WILEY PERIODICALS, INC. V
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TABLE I. Electrospinning Parameters for As-Spun and Post-Cured Scaffolds Chemical Composition and Physical Properties Scaffold Propertiesa
Scaffold Composition [wt.%]
As-Spun
Electrospinning Parameters
Scaffold
PCL
BPAEDMA
Voltage [kV]
PCL PCL/BPAEDMA (75/25) PCL/BPAEDMA (50/50) PCL/BPAEDMA (25/75)
100 75 50 25
0 25 50 75
12 12 12 12
Post-Cured
Storage Modulus [MPa]
Storage Modulus [MPa]
Working Distance [cm]
Flow Rate [mL/h]
Wetting Angle [ ]
26 C
37 C
26 C
37 C
11 11 11 11
1 1 1 1
82 31 15 25
39 17 12 9
32 12 9 7
39 31 12 29
32 23 10 26
a
Mean value of n 5 5 samples were presented.
metabolites and regulatory molecules. Furthermore, as mentioned earlier, scaffold chemistry highly influences cell adhesion and growth potentials. After observing reduced relocation of transplanted labeled mesenchymal stem cells to remote organs and non-infarcted myocardium when implanted along with a collagen matrix, Dai et al. concluded that matrices of this nature can enhance clinical outcomes of implanted cells.14 Numerous studies have been reported to create fibrous electrospun nanofibrous scaffolds composed of polymer blend solutions with emphasis on application of biocompatible and biodegradable polymers.15–17 Shin et al.18 reported considerable cardiomyocyte attachment to PCL based meshes, along with the expression of cardiac-specific proteins, such as myosin heavy chain, connexin 43 and cardiac troponin. They hypothesized that such 3D structures may induce cardiomyocyte-based vascularization upon provision of angiogenic factors. Schnell et al.19 observed glial cell migration and axonal growth on electrospun fibrous scaffolds of PCL versus collagen/PCL scaffolds. They demonstrated that Schwann cells, fibroblasts, and olfactory ensheathing cells were significantly increased on collagen/ PCL fibers, when compared with pure PCL fibers. Nguyen et al.20 demonstrated that the increasing ratio of PLGA (poly lactic acid/glycolic acid) in electrospun PCL/PLGA blend scaffolds, the altered mechanical properties of electrospun membranes resulted in increased fibrous mat biocompatibility. In addition, the elastomeric polyglycerol sebacate yields porous, elastomeric three-dimensional (3D) scaffolds with controllable stiffness and anisotropy. Such scaffolds have been reported effective in myocardial tissue engineering applications,21,22 as illustrated by their potential to promote the formation of cardiac grafts comprised of aligned heart cells with mechanical properties closely resembling those of the native myocardium. Landis et al.23 fabricated a saltleached tissue engineering scaffolds based on photo-crosslinkable bisphenol A ethoxylated dimethacrylate (BPAEDMA) monomers pertinent for hard tissue engineering. These monomers are extensively used in the formulation of matrices for restorative dental composites.24–28 This study focused on fabricating bioengineered scaffold suitable for cardiac tissue engineering, with low stiffness and balanced hydrophobicity. To achieve this goal, PCL was
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blended with a photocurable BPAEDMA hydrogel precursor, with a degree of ethoxylation equivalent to 30. BPAEDMA with high degree of ethoxylation lead to obtain highly flexible elastomeric hydrogel when treated by UV radiation. The blended oligomer contained soft ethylene oxide segments, providing flexibility and allow bioadhesion of cells, as well as polymerizable methacrylate end groups, for mechanical strength tunability. In addition, these elements are both non-toxic and non-immunogenic, which are two essential requirements for biocompatible implantable materials. Photopolymerization of this oligomer leads to the formation of flexible elastomeric hydrogel films and nanofibers.29–32 Assessment of the tissue engineering capacities of the described compound fiber scaffolds embedded with rabbit cardiac cells, demonstrated their low stiffness and balanced hydrophobic properties were suitable for cell adhesion and proliferation of cardiac cells. The newly developed PCL/ BPAEDMA compound nanofibrous scaffolds may bear considerable potential in cardiac tissue engineering applications. MATERIALS AND METHODS
Materials PCL (Mw: 80,000), PEG-derived oligomer hydrogel precursor BPAEDMA (Mw: 1700), 2, 2-dimethoxy, 2-phenyl acetophenone and chloroform were obtained from Sigma Aldrich. PCL was dissolved in chloroform to obtain 10% (wt/wt) solution. BPAEDMA and 2,2-Dimethoxy-2-phenylacetophenone photo-initiator (PI) (2 wt/wt % to BPAEDMA) were then added to PCL solution at the desired ratios. The compositions are presented in Table I and the chemical formulas of the materials and graphical representation of electrospun compound fibers are shown in Scheme 1. Electrospinning and polymerization The flow controlled by a syringe pump of the PCL/ BPAEDMA blended solution was set at a constant rate of 1 mL/h with an electrostatic field of 12 kV (Table I). An aluminum foil sheet was placed 11 cm beneath the spinneret exit for the collection of non-woven fibrous scaffolds. PCL/ BPAEDMA electrospun fibrous compound scaffolds were placed on aluminum foil sheets supported on a metal disc at a distance of 15 cm from two horizontal 20W high-pressure
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SCHEME 1. (a) Chemical formula of reagents, and (b) Schematic representation of different steps involved in the process of scaffold preparation.
mercury lamps and then BPAEDMA in the compound fibers was cured by UV radiation for 10 min as reported earlier.29,31 Characterization of the fibrous scaffolds Microscopic characterization. Electrospun PCL/BPAEDMA nanofibrous compound scaffolds collected on carbon tapecovered metal stub and sputter-coated with gold were analyzed using Phenom desktop scanning electron microscope (SEM) (5 kV accelerating voltage, FEI Company, Hillsboro, OR). The surface morphology of PCL and PCL/BPAEDMA naofibrous scaffolds were analyzed by stained with eosin solution for about 30 s and extensive washed with water to remove excess stain and vacuum dried at 40 C. Eosinstained PCL and PCL/BPAEDMA scaffolds were sputtercoated with gold and analyzed by SEM. Wettability of the nanofibrous scaffolds Sessile droplet of double distilled water (DDW) was poured at the center of each nanofibrous scaffolds. Droplet evolution in time was imaged with an electronic camera (MotionScope-Redlake Imaging Corporation). The water contact angle (hw) was determined at time 15 s after placing of the water droplet. The camera speed was 2000 f.p.s. and the shutter speed 0.05 ms. The camera was equipped with a 70–180 mm, f/4.5 zoom lens. The light source (v) (400W HMI lamp with Dedolight DEB400D electronic ballast) was placed against the camera along its line of sight. Five different PCL and PCL/BPAEDMA nanofibrous compound scaffolds were evaluated for water contact angle measurements. Swelling experiments The swelling characteristics of as-spun and post-cured PCL/ BPAEDMA nanofibrous compound scaffolds were assessed
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in aqueous medium. PCL/BPAEDMA nanofibrous compound scaffolds were soaked in water for 78 h at 37 C and swollen to an equilibrium state. Swelling percentages were calculated as: Water fraction 5 ½ðwsm 2wdm Þ=wdm 3100 where, wsm 5 weight of swollen compound fiber mat and wdm 5 weight of dry compound fiber mats. Differential scanning calorimetry Differential scanning calorimetric experiments were carried out using a Differential scanning calorimeter (METTLERDSC 1). All DSC measurements were repeated on five samples for each PCL/BPAEDMA nanofibrous scaffold under nitrogen atmosphere and at temperatures ranging from 10 to 180 C at a heating rate of 10 C/min on sample sizes that ranged between 0.5 and 3 mg. The DSC was calibrated with an indium and zinc-melting standard. Dynamic mechanical analysis Dynamic mechanical analyzer (TA, DMA Q 800) in the tension film mode was used to measure the mechanical properties of as-spun and post cured nanofibrous scaffolds. The samples were subjected to a sinusoidal displacement at 10-mm amplitude and at a fixed frequency of 1 Hz from 280 to 40 C with a heating rate of 2 C/min. The storage moduli of as-spun and post cured nanofibrous scaffolds were measured in the temperature interval from 280 to 40 C. Stress–strain measurements at 26 C were conducted on 5 3 3 3 0.2mm rectangular samples at a strain rate of 0.5 mm/min. Wide angle X-ray scattering Wide-angle X-ray diffraction (WAXD) patterns of as-spun and post cured nanofibrous scaffolds were recorded using a
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Philips PW3710 diffractometer and PW3020 goniometer (Philips, MBLE, Brussels, Belgium) operating at 10 kV and 10 mA with Cu Ka radiation (k 5 0.154 nm). The dried PCL/BPAEDMA nanofibrous scaffolds were placed on Vaseline and diiffractograms were acquired at an angular range of 2h from 5 to 60 with a step size of 0.05 , and 4 s counting time for each step. Infrared spectroscopic measurements To confirm the existence of cured BPAEDMA in PCL/ BPAEDMA nanofibrous compound scaffolds after swollen in water/phosphate buffered saline (PBS) for 7 days, samples were removed from water and vacuum dried at 40 C. Fourier transform infrared spectral analysis was performed on dried PCL/BPAEDMA nanofibrous scaffolds using a NEXUS 870 FTIR (Thermo Nicolet) in a humidity-free atmosphere at room temperature with 4 cm21 resolution and 32 scans signal average and spectra were recorded from 400 to 4600 cm21 interval in the transmission mode using a DTG TEC detector. The spectral data of as-spun nanofibrous scaffolds are presented in this paper without any correction or modification. Isolation and culture of cardiac cells Rabbit cardiac cells were isolated using a collagenasebased digestion method. The rabbit aorta was cannulated and perfused using a bicarbonate solution (118.5 mM NaCl, 25.0 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.0 mM glucose, and 2.5 mM CaCl2). The rabbit heart was then thoroughly washed thrice, for 20 min each time, with antibiotic solutions diluted in PBS to concentrations of 33 and 23, and then fragmented the ventricles into fine pieces. Tissue fragments were subsequently treated with 1% collagenase (Type I) diluted in PBS, for 30 min at 37 C. The dispersed cells were harvested by decantation after each 5 min of incubation. The fine cardiac pieces were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (GIBCO Invitrogen) and 1% antibiotic and antimycotic solutions (Invitrogen Corporation) in 75 cm2 cell culture flasks, to allow for cardiac cell isolation. Trypsinized cells were centrifuged, counted following trypan blue staining and then seeded on the nanofibrous scaffolds. Cell seeding on scaffolds Tissue culture plates (TCP), PCL, PCL/BPAEDMA (75/25), PCL/BPAEDMA (50/50), and PCL/BPAEDMA (25/75) elastomeric nanofibrous scaffolds were sterilized with 70% ethanol, washed three times with PBS, and subsequently immersed in complete DMEM overnight before cell seeding. Electrospun compound nanofibers were collected on 15 mM round glass cover slips and then placed in 24-well plates along with a stainless steel ring to prevent lifting of fibers. The trypsinized cells collected from culture flasks, were counted and seeded onto the TCP (control) or on the scaffolds at a density of 1 3 104 cells/well. Cells were allowed to adhere for 15 min before adding medium, which was replaced every 3 days.
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Cell proliferation Cell adhesion and proliferation on the nanofibrous scaffolds were determined using the colorimetric MTS assay (CellTiter 96 AQueous One solution, Promega, Madison, WI), in which the yellow tetrazolium salt [3-(4, 5-dimethylthiazol-2yl)25-(3-carboxymethoxyphenyl)22(4sulfophenyl)22H tetrazolium] is reduced by the mitochondrial dehydrogenase enzymes secreted in metabolically active cells, to form purple formazan crystals. The formazan dye absorbs light at 492 nm and the number of crystals formed is said to be directly proportional to the number of viable cells. Cells were rinsed with PBS, to remove non-adherent cells, and incubated with 20% MTS reagent in a serum-free medium for 3 h at 37 C. Samples of culture medium were then pipetted out into fresh 96-well plates. Absorbance was measured at 490 nm using a microplate reader (Fluostar Optima, BMG Lab Technologies, Germany). Cell morphology After 10 days in culture, cardiac cells cultured on the various scaffolds were analyzed with a SEM. The scaffolds were rinsed twice with PBS and fixed in 3% glutaraldehyde (Sigma Aldrich) for 3 h. Thereafter, scaffolds were rinsed in deionized water and dehydrated with increasing concentrations of ethanol (50%, 70% 90%, 100%) for 10 min each. Samples were then treated with hexamethyldisilazane (Fluka Chemical Corporation, Milwaukee, WI) and then airdried in a fume hood. Finally, the scaffolds were sputtercoated with gold (JEOL JFC-1200 fine coater, Japan) and observed by SEM (FEI-QUANTA 200F, Netherland) at an accelerating voltage of 10 kV. Labeling cells with fluorescent dyes. Cardiac cells (1 3 104cells/cm2) seeded on nanofibrous scaffolds were washed with PBS after 10 days in culture, and then stained with 5chloromethyl fluorescein diacetate (25 lM CMFDA at 37 C for 60 min, CellTracker green, Promega USA) which is cleaved by cytosolic esterases to form a highly fluorescent and membrane impermeable CMFDA derivative. The CMFDA-supplemented medium was then replaced by complete medium and cardiac cells were incubated overnight. Cells were then washed with PBS and after addition of serum-free medium observed under an inverted Leica DM IRB laser scanning microscope (Leica DC 300F) at 488 nm. Expression of cardiac-specific protein markers Cardiac cells grown on nanofibrous scaffolds were fixed using 100% chilled methanol, then washed once with PBS for 15 min and incubated in a 0.5% Triton-X solution for 5 min to permeabilize the cell membrane. Non-specific binding sites were blocked by incubating the cells in 3% BSA (Sigma Aldrich) for 1 h. Thereafter, they were stained with anti-sarcomeric alpha-actinin antibodies and anti-troponin antibody (1:100 dilution, Sigma Aldrich), followed by treatment with an FITC-labeled (1:250 dilution, Sigma Aldrich) secondary antibody. Nuclei were stained with 40 , 6diamidino-2-phenylindole (1:5000 dilutions, Invitrogen). Finally, samples were extensively washed and then mounted
PCL/OLIGOMER COMPOUND SCAFFOLDS FOR CARDIAC TISSUE ENGINEERING
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FIGURE 1. SEM images of nanofibrous scaffolds. PCL (a), PCL/BPAEDMA (75/25) (b), PCL/BPAEDMA (50/50) (c), and PCL/BPAEDMA (25/75) (d).
using Vectashield (Vector Laboratories), before being analyzed under a confocal laser scanning microscope (Olympus FV1000). Statistical analysis All the data presented in the manuscript are expressed as mean 6 standard deviation (SD). Significance level was determined using Student’s t-test. Differences were considered statistically significant at p 0.05. RESULTS AND DISCUSSION
Influence of BPAEDMA on morphology of compound fibers Cell-scaffold interactions are determined by the physical and chemical properties of constituent materials, with particular weight on their chemical composition, particle size and surface properties which include topography, roughness, surface energy and wettability.29 Morphologies of representative, nonpolymerized PCL/BPAEDMA compound fibers are shown in Figure 1. Fiber diameters were within the range of 0.5–1 lm and a
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fiber mat thickness was 200 lm. SEM images of eosin-stained PCL fiber mats [Fig. 2(a,b)] showed fibers with many craters on the surface. Whereas, PCL/BPAEDMA nanofibrous scaffold [Fig. 2(c,d)] with 50 wt % BPAEDMA showed fibers with spherical and elliptical microdomains on the surface, and they were distributed along the fiber axis. PCL/BPAEDMA nanofibrous scaffolds with >50 wt % BPAEDMA, surface of PCL/BPAEDMA nanofibers exhibited biphasic morphology being PCL as the continuous phase and BPAEDMA as dispersed phase along the fiber axis. These surface microdomains are expected to render the compound fibers more pertinent to cell adhesion applications. As-spun and post cured PCL/BPAEDMA (50/50) nanofibrous compound scaffolds were placed in water for one week at 37 C and they were removed from water and vacuum dried overnight at 40 C. The surface morphology of as-spun and post cured PCL/BPAEDMA compound fibers (Fig. 3) resembled the surface morphology of the non-swollen compound fibers, their diameters increased by an average of 15% than PCL fibers, confirming the existence of hydrophilic BPAEDMA. The dried swollen PCL/BPAEDMA nanofibrous scaffolds were analyzed by
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FIGURE 2. SEM images of eosin-stained as-spun nanofibrous scaffolds. PCL (a, b), and PCL/BPAEDMA (50/50) (c, d) at two different magnifications.
FTIR spectroscopy (Fig. 4). The dried preswollen compound scaffolds exhibited an absorbance bands at 1610 cm21 and 1637 cm21, attributed to an aromatic double bond (AC6H4A) and aliphatic double bond (AC@CA) of BPAEDMA, respectively. BPAEDMA was partially cured during spinning of PCL/ BPAEDMA compound solution in chloroform and post curing of PCL/BPAEDMA nanofibrous scaffolds by UV radiation lead to decreased intensity of aliphatic double bond (AC@CA) absorbance band at 1637 cm21 of BPAEDMA, as reported earlier.29 Mechanical properties of compound nanofibrous scaffolds Mechanical properties of as-spun and post cured nanofibrous scaffolds were measured [Fig. 5(a–d), and Table I]. Storage modulus and loss modulus curves depend on the temperature [Fig. 5(a,b)] of PCL/BPAEDMA nanofibrous scaffolds clearly indicate that PCL and BPAEDMA were phase separated. The storage modulus curves of PCL and PCL/BPAEDMA nanofibrous scaffolds displayed the typical behavior of semicrystalline polymers with four distinctive zones. At < 260 C, the storage modulus slightly decreased with temperatures. This observed limited temperature effect is due to the fact that the polymer was in glassy state,
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where molecular motions were largely restricted to vibrations and short-range rotations. Around 260 to 220 C, a broad transition showed as a sharp drop in storage modulus was observed. At this temperature range, the main relaxation process corresponded to amorphous PCL domains associated with the glass transition of PCL. At higher temperatures range, 220 to 40 C, the storage modulus decreased due to the progressive softening of the PCL matrix. Here amorphous, rubbery and crystalline domains coexisted. At higher temperatures between 45 C and 70 C, the storage modulus dropped sharply because complete melting of the PCL crystalline domains [not shown in Fig. 5(a,b)], as observed from DSC melting thermograms. Whereas, at higher temperatures between 10 C and 30 C, PCL/BPAEDMA nanofibrous scaffolds storage modulus decreased abruptly due to melting of BPAEDMA crystalline domains as observed by DSC measurements. PCL scaffold loss modulus as a function of temperature showed a peak maximum at 254 C, and this peak maximum temperature was defined as PCL glass transition temperature. PCL/ BPAEDMA nanofibrous compound scaffolds loss modulus as function of temperature typically represented a mixed amorphous phase with broad glass transition temperature.
PCL/OLIGOMER COMPOUND SCAFFOLDS FOR CARDIAC TISSUE ENGINEERING
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FIGURE 3. SEM images of dried preswollen nanofibrous scaffolds. As-spun (a, b) and post cured (c, d) PCL/BPAEDMA (50/50) shown at two different magnifications.
PCL/BPAEDMA nanofibrous compound scaffolds glass transition temperature was shifted by 2 C to 10 C toward higher temperature side compared with PCL with increasing
FIGURE 4. FTIR spectra of dried preswollen scaffolds and BPAEDMA. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), PCL/BPAEDMA (25/75) (dark gray), and BPAEDMA (dot black).
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BPAEDMA weight fraction. This increased glass transition temperature might be attributed to the existence partially cured BPAEDMA sub-micrometer domains in the PCL/ BPAEDMA compound nanofibers as observed from morphology of eosin-stained compound scaffolds. When PCL/ BPAEDMA compound scaffolds with partially cured BPAEDMA were post cured by UV radiation about 10 min an increase in tensile storage modulus was observed, when compared with as-spun counterparts likely associated with the fraction of the cross-linked BPAEDMA in the compound fibers. Stress–strain behavior of PCL and post cured PCL/ BPAEDMA compound nanofibrous scaffolds at 26 C are presented in Figure 5(c,d). PCL scaffold showed an ultimate tensile strength of 2.8 MPa, elastic modulus of 30 MPa and strain at break of 300 6 18 (%). PCL/BPAEDMA (75/25) exhibited an ultimate tensile strength of 2.5 MPa, elastic modulus of 10 MPa, and strain at break of 178 6 30 (%). PCL/BPAEDMA (50/50) compound nanofibrous scaffold showed an ultimate tensile strength of 1.5 MPa, elastic modulus of 2 MPa and strain at break of 106 6 30 (%). PCL/BPAEDMA (25/75) compound scaffold exhibited an
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FIGURE 5. Storage modulus and loss modulus of as-spun (a), post cured (b), stress–strain curves until break (c), linear region of stress–strain curves, (d) of post cured scaffolds. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
ultimate tensile strength of 2.3 MPa, elastic modulus of 7 MPa and strain at break of 111 6 44 (%). Post cured PCL/BPAEDMA compound nanofibrous scaffolds showed reduced elastic modulus and strain at break compared with PCL scaffolds. This reduced strain at break can be attributed to increased crosslink density of PCL/ BPAEDMA compound nanofibrous scaffolds and reduced elastic modulus might be due to the reduced crystallinity of PCL in the compound scaffolds. However, the PCL/ BPAEDMA 50/50 nanofibrous compound scaffolds exhibited higher crystallinity than PCL scaffold might be due to the coexistence of mixed PCL and BPAEDMA crystalline phase. Wettability and swelling properties PCL and PCL/BPAEDMA compound nanofibrous scaffolds surface wettability with water, measured by sessile water drop contact angle measurements (Table I), were 60% < comparison with PCL scaffold indicating increased hydrophilicity of the compound fiber mats. The swelling characteristics of as-spun and post cured scaffolds were assessed in an aqueous medium (Fig. 6). Fiber swelling capacities decreased significantly with increasing hydrogel weight fraction in correlation with the increased crosslink density of PCL/BPAEDMA compound nanofibrous scaffolds. The difference between swelling capacities as-spun and post
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cured nanofibrous scaffolds was only 20%, likely due to increased crosslink density of PCL/BPAEDMA compound nanofibrous scaffolds upon post curing.
FIGURE 6. Swelling characteristics of as-spun and post cured scaffolds. Compound fibers comprised of varying ratios of PCL and BPAEDMA were soaked in water for 78 h before calculation of swelling percentages [5100 3 (wsm 2 wdm)/wdm)]. Mean values (6SD) are presented (n 5 5).
PCL/OLIGOMER COMPOUND SCAFFOLDS FOR CARDIAC TISSUE ENGINEERING
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FIGURE 7. WAXD patterns of as-spun (a) and post cured (b) scaffolds. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
Wide-angle X-ray diffraction Structural characteristics of as-spun and post cured nanofibrous scaffolds were analyzed by WAXD to determine the impact of BPAEDMA weight fraction, on the crystallinity of PCL (Fig. 7). The WAXD pattern of as-spun naofibrous scaffolds [Fig. 7(a)] was comprised of two main planes [(110) at 2u 5 21.35 and (200) at 2u 5 23.65], which can be attributed to the orthorhombic crystalline structure of semicrystalline PCL.33 A decrease in intensity of the peak at 2h 5 21.35, for the PCL/BPAEDMA compound nanofibrous scaffolds was observed and can be attributed to the decreased weight fraction of PCL in these scaffolds. The full width at half maximum intensity of peak (b) at 2h 5 21.35, for the PCL/BPAEDMA nanofibrous scaffolds also decreased in comparison with that recorded for PCL scaffold, suggesting an increase in crystallite size of PCL in the compound fibers. This trend was further intensified in post cured PCL/ BPAEDMA compound nanofibrous scaffolds [Fig. 7(b)].
Thermal stability of PCL and PCL/BPAEDMA compound fibers Determination of thermal stability by means of differential scanning calorimetery (DSC) is the most common and simplest method for evaluating melting temperature, melting
enthalpy and crystallinity of a polymer with respect to the melting enthalpy of its fully crystalline state. The melting temperature, melting enthalpy and degree of crystallinity of as-spun and post cured nanofibrous scaffolds are presented in Table II and the DSC thermograms are shown in Figure 8. The melting enthalpies of as-spun nanofibrous compound scaffolds (except PCL/BPAEDMA 50/50) were lower in comparison with the as-spun PCL scaffolds. The melting enthalpy of PCL/BPAEDMA (50/50) compound nanofibrous scaffold was higher than the PCL scaffold, which suggests that coexistence of mixed PCL and BPAEDMA crystalline phase. However, the BPAEDMA phase of compound nanofibrous scaffolds inhibited PCL crystallization, as demonstrated by the drop in PCL melting enthalpy in these scaffolds compared with PCL scaffold. The melting enthalpy and degree of crystallinity of PCL in post cured PCL/BPAEDMA compound nanofibrous scaffolds (except PCL/BPAEDMA 50/50) were further increased, suggested to be the result of structural modification of PCL by low intensity UV radiation.
Cardiac cell proliferation and morphology on PCL/BPAEDMA scaffolds This study fabricated elastomeric PCL/BPAEDMA compound fibers for the culture of cardiac cells as designated for
TABLE II. Thermal Properties of As-Spun and Post-Cured Scaffolds Scaffold Composition [wt %]
Melting Temperature [ C]
Crystallinitya [%]
Melting Enthalpy [J/g]
As-Spun
Post-Cured
Scaffold
PCL
BPAEDMA
AS-SPUN
Post-Cured
As-Spun
Post-Cured
PCL
PCL
PCL PCL/BPAEDMA (75/25) PCL/BPAEDMA (50/50) PCL/BPAEDMA (25/75)
100 75 50 25
0 25 50 75
61 59 60 60
60 59 60 59
71 46 118 55
84 54 93 59
52 34 86 40
61 39 68 43
a
The melting enthalpy of fully crystalline PCL 5 136 J/g,42 and the melting enthalpy of fully crystalline PEO 5 197 J/g.43
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FIGURE 8. DSC melting curves of as-spun and inset is BPAEDMA heating cooling thermogram (a) and post cured (b) scaffolds. PCL (black), PCL/ BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
myocardial infarction (MI). Cell proliferation on PCL/ BPAEDMA (25/75) meshes showed significantly (p 0.05) higher than PCL and PCL/BPAEDMA (75/25) nanofibrous scaffolds on days 10 and 15, respectively (Fig. 9). These observations suggested that the PCL/BPAEDMA fibrous material provides a suitable microenvironment to support cell growth and expression of cardiac proteins may boost tissue reorganization in the myocardium. Increased cell adhesion on the elastomeric nanofiber matrix can also be attributed to adhesion proteins such as fibronectin and vitronectin present in the serum used for cell culture.34,35. In this study, the balanced hydrophilicity and lower stiffness of the designed compound scaffolds promotes cell adhesion and proliferation than that of PCL fiber mesh alone. Normal cell morphology was observed in cells grown for 10 days on elastomeric PCL/BPAEDMA (25/75) nanofibrous scaffolds, in contrast to that of cells cultured on PCL/BPAEDMA (75/ 25) nanofibrous scaffolds (Fig. 10). These results confirm that the porous, hydrophilic compound nanofibrous scaf-
FIGURE 9. Proliferation of rabbit cardiac cells on various electrospun nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (Control), PCL, PCL/BPAEDMA (75/25), PCL/BPAEDMA (50/50), and PCL/ BPAEDMA (25/75) elastomeric nanofibrous scaffolds for up to 15 days. Cell proliferation was determined using the MTS assay. Values (6SD) represent mean optical density as measured at 490 nm (n 5 6). (HG-BPEDMA hydrogel). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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folds provide cell adhesion sites and support both proliferation and transport of nutrients for cardiac tissue engineering. Cell viability and expression of cardiac proteins CMFDA dye belongs to a group of chloromethyl derivatives developed for in vitro labeling of live cells.36,37 The CMFDA compound is metabolized intracellularly in viable cells to a cell-impermeant and fluorescent state within 1 h. Addition of CMFDA to the cell culture medium causes the compound to freely permeate the cell membrane and be acted upon by cytosolic esterases, producing a CMFDA derivative that is brightly fluorescent. To determine the viability of cells embedded on PCL and PCL/BPAEDMA compound scaffolds, samples were treated with CMFDA after 10 days in culture. While CMFDA fluorescence was observed in cardiomyocyte cultures grown on control and elastomeric nanofibrous scaffolds, greater cell density and normal morphology were observed among those cultured on elastomeric PCL/ BPAEDMA (25/75) nanofibrous scaffolds (Fig. 11). As scaffold biomaterials have been reported to influence cellular activities, the expression of alpha-actinin and troponin T, two markers specific to cardiac lineage and both essential for cardiomyocyte functioning was evaluated in cells grown on the various elastomeric nanofiber scaffolds. Alpha-actinin is an actin cross-linking protein38 critical to cardiac contractility, while troponin T regulates the force and velocity of myocardial contraction.39 Troponin T is also an indicator of cardiomyocytes differentiation.40,41 Figure 12 shows the expression of Troponin and alpha-actinin in the cardiac cells grown on PCL and PCL/BPAEDMA (25/75) elastomeric scaffolds compared with the TCP. The expression of alphaactinin and Troponin by the cardiac cells cultured on PCL/ BPAEDMA (25/75) compound nanofibrous scaffolds indicates that the cells retains their normal functionality. The observed results showed that the designed PCL/BPAEDMA (25/75) elastomeric nanofibrous scaffolds properties suitable for the application in the repair of MI in cardiac tissue engineering.
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FIGURE 10. Morphology of cardiac cells cultured on elastomeric PCL/BPAEDMA nanofibers. Representative SEM images of rabbit cardiac cells grown on nanofibrous scaffolds for 10 days. Rabbit cardiac cells were cultured on TCP (Control) (a), PCL (b), PCL/BPAEDMA (75/25) (c), PCL/ BPAEDMA (50/50) (d), and PCL/BPAEDMA (25/75) fibers (e).
FIGURE 11. Rabbit cardiac cell viability when cultured on electrospun PCL/BPAEDMA compound nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (Control) (a), PCL (b), PCL/BPAEDMA (75/25) (c), PCL/BPAEDMA (50/50) (d), and PCL/BPAEDMA (25/75) (e) elastomeric nanofibrous scaffolds for 10 days and then treated with CMFDA to determine cell viability (scale bar 100 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 12. Expression of cardiac proteins in cardiac cells grown on PCL, PCL/BPAEDMA compound nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (a, d), PCL (b, e) and PCL/BPAEDMA (25/75) scaffolds (c, f) for 10 days. Samples were fixed and stained for cardiac proteins Toponin (a–c) and actinin (d–f), both detected with an FITC filter (scale bar 10 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CONCLUSIONS
The site of MI is a poor environment for cell growth; to increase cell viability, some factors to improve such an infertile environment are desirable for cardiac tissue engineering. The described electrospun neat nano/micro PCL fiber meshes resemble the fibrillar structural organization of ECM and also favor the proliferation of rabbit cardiac cells in vitro. However, cardiac cells proliferation increased upon addition of BPAEDMA to PCL fibrous scaffolds, seemingly due to the balanced hydrophilicity of the resulting scaffold and decreased modulus of PCL from 39.68 MPa to 3.55 MPa. Compound fiber mesh comprised of 25 wt % PCL and 75 wt % BPAEDMA, allowed for enhanced cell adhesion and proliferation of cultured cardiac cells and expression of key cardiac-specific proteins. The PCL/BPAEDMA (25/75) nanofibrous scaffolds provided a fiber mesh support with low stiffness, balanced hydrophilicity, along with wettable properties relevant for cardiac tissue engineering.
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12. Mukherjee S, Venugopal JR, Ravichandran R, Ramakrishna S, Raghunath M. Multimodal biomaterial strategies for regeneration of infarcted myocardium. J Mater Chem 2010;20:8819–8831. 13. Tan PS, Teoh SH. Effect of stiffness of polycaprolactone (PCL) membrane on cell proliferation. Mater Sci Eng C 2007;27:304–308. 14. Dai WD, Hale SL, Kay GL, Jyrala AJ, Kloner RA. Delivering stem cells to the heart in a collagen matrix reduces relocation of cells to other organs as assessed by nanoparticle technology. Regen Med 2009;4:387–395. 15. Chen ZG, Mo XM, Qing FL. Electrospinning of collagen-chitosan complex. Mater Lett 2007;61:3490–3494. 16. Xu XL, Chen XS, Wang ZF, Jing XB. Ultrafine PEG-PLA fibers loaded with both paclitaxel and doxorubicin hydrochloride and their in vitro cytotoxicity. Eur J Pharm Biopharm 2009;72:18–25. 17. Yang DZ, Li YN, Nie J. Preparation of gelatin/PVA nanofibers and their potential application in controlled release of drugs. Carbohydr Polym 2007;69:538–543. 18. Shin M, Ishii O, Sueda T, Vacanti JP. Contractile cardiac grafts using a novel nanofibrous mesh. Biomaterials 2004;25:3717–3723. 19. Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, Mey J. Guidance of glial cell. Migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials 2007;28:3012– 3025. 20. Nguyen TH, Lee BT. Electro-spinning of PLGA/PCL blends for tissue engineering and their biocompatibility. J Mater Sci Mater Med 2010;21:1969–1978. 21. Engelmayr GC, Cheng MY, Bettinger CJ, Borenstein JT, Langer R, Freed LE. Accordion-like honeycombs for tissue engineering of cardiac anisotropy. Nat Mater 2008;7:1003–1010. 22. Radisic M, Park H, Martens TP, Salazar-Lazaro JE, Geng WL, Wang YD, Langer R, Freed LE, Vunjak-Novakovic G. Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue. J Biomed Mater Res Part A 2008;86A: 713–724. 23. Landis FA, Stephens JS, Cooper JA, Cicerone MT, Lin-Gibson S. Tissue engineering scaffolds based on photocured dimethacrylate polymers for in vitro optical imaging. Biomacromolecules 2006;7: 1751–1757. 24. Moszner N, Salz U. New developments of polymeric dental composites. Prog Polym Sci 2001;26:535–576. 25. Skrtic D, Antonucci JM, Liu DW. Ethoxylated bisphenol dimethacrylate-based amorphous calcium phosphate composites. Acta Biomater 2006;2:85–94. 26. Skrtic D, Antonucci JM, Eanes ED, Eldelman N. Dental composites based on hybrid and surface-modified amorphous calcium phosphates. Biomaterials 2004;25:1141–1150. 27. Skrtic D, Antonucci JM, Eanes ED. Effect of the monomer and filler systems on the remineralizing potential of bioactive dental composites based on amorphous calcium phosphate. Polym Adv Technol 2001;12:369–379.
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28. Skrtic D, Antonucci JM. Dental composites based on amorphous calcium phosphate—Resin composition/physicochemical properties study. J Biomater Appl 2007;21:375–393. 29. Reddy CS, Arinstein A, Avrahami R, Zussman E. Fabrication of thermoset polymer nanofibers by co-electrospinning of uniform core-shell structures. J Mater Chem 2009;19:7198–7201. 30. Amerio E, Sangermano M, Colucci G, Malucelli G, Messori M, Taurino R, Fabbri P. UV curing of organic-inorganic hybrid coatings containing polyhedral oligomeric silsesquioxane blocks. Macromol Mater Eng 2008;293:700–707. 31. Reddy CS, Arinstein A, Zussman E. Polymerization kinetics under confinement. Polym Chem 2011;2:835–839. 32. Guo XY, Gough JE, Xiao P, Liu J, Shen ZJ. Fabrication of nanostructured hydroxyapatite and analysis of human osteoblastic cellular response. J Biomed Mater Res Part A 2007;82A:1022–1032. 33. Bittiger H, Marchess Rh, Niegisch WD. Crystal structure of polyeta-caprolactone. Acta Crystallogr Section B 1970;B 26:1923–1927. 34. Anselme K. Osteoblast adhesion on biomaterials. Biomaterials 2000;21:667–681. 35. Grinnell F. Focal adhesion sites and the removal of substratumbound fibronectin. J Cell Biol 1986;103:2697–2706. 36. Poot M, Kavanagh TJ, Kang HC, Haugland RP, Rabinovitch PS. Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser-dye with Hoechst-33342. Cytometry 1991;12:184–187. 37. Zhang YZ, Olson N, Mao F, Roth B, Haugland RP. New fluorescent-probes for long-term tracing of living cells. FASEB J 1992;6:A1835–A1835. 38. Cabello N, Remelli R, Canela L, Soriguera A, Mallol J, Canela EI, Robbins MJ, Lluis C, Franco R, McIlhinney RAJ, et al. Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor. J Biol Chem 2007;282:12143–12153. 39. Xing Y, Huang PJ, Zhang KM. Cardiac troponin T and I: Application in myocardial injury and forensic medicine. Fa Yi Xue Za Zhi 2003;19:242–244. 40. Parmacek MS, Solaro RJ. Biology of the troponin complex in cardiac myocytes. Prog Cardiovasc Dis 2004;47:159–176. 41. Tan MY, Zhi W, Wei RQ, Huang YC, Zhou KP, Tan B, Deng L, Luo JC, Li XQ, Xie HQ and others. Repair of infarcted myocardium using mesenchymal stem cell seeded small intestinal submucosa in rabbits. Biomaterials 2009;30:3234–3240. 42. Hartman O, Zhang C, Adams EL, Farach-Carson MC, Petrelli NJ, Chase BD, Rabolt JE. Biofunctionalization of electrospun PCLbased scaffolds with perlecan domain IV peptide to create a 3-D pharmacokinetic cancer model. Biomaterials 2010;31:5700–5718. 43. Campbell C, Viras K, Richardson MJ, Masters AJ, Booth C. Crystallinity of uniform triblock oxyethylene/methylene/oxyethylene (EmCnEm) oligomers studied by X-ray diffraction, differential scanning calorimetry and Raman spectroscopy. Die Makromol Chem 1993;194:799–816.
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Department of Mechanical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel Institute of Biomaterial Science, Helmholtz-Zentrum Geesthacht, Kantstraße 55, Teltow 14513, Germany 3 Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore 2
Received 9 May 2013; revised 8 November 2013; accepted 18 November 2013 Published online 12 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35045 Abstract: Polycaprolactone (PCL), a synthetic biocompatible and biodegradable polymer generally used as a scaffold material for tissue engineering applications. The high stiffness and hydrophobicity of the PCL fiber mesh does not provide significant cell attachment and proliferation in cardiac tissue engineering. Towards this goal, the study focused on a compound of PCL and oligomer hydrogel [Bisphenol A ethoxylated dimethacrylate (BPAEDMA)] processed into electrospun nanofibrous scaffolds. The composition, morphology and mechanical properties of the compound scaffolds, composed of varying ratios of PCL and hydrogel were characterized by scanning electron microscopy, infrared spectroscopy and dynamic mechanical analyzer. The elastic modulus of PCL/BPAEDMA nanofibrous scaffolds was shown to be varying the BPAEDMA weight fraction and was decreased by increasing the BPAEDMA weight fraction. Compound fiber meshes containing 75 wt % BPAEDMA oligomer
hydrogel exhibited lower modulus (3.55 MPa) and contact angle of 25o. Rabbit cardiac cells cultured for 10 days on these PCL/ BPAEDMA compound nanofibrous scaffolds remained viable and expressed cardiac troponin and alpha-actinin proteins for the normal functioning of myocardium. Cell adhesion and proliferations were significantly increased on compound fiber meshes containing 75 wt % BPAEDMA, when compared with other nanofibrous scaffolds. The results observed that the produced PCL/BPAEDMA compound nanofibrous scaffolds promote cell adhesion, proliferation and normal functioning of cardiac cells to clinically beneficial levels, relevant for cardiac tissue engineering. C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3713– V
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Key Words: elastomer, nanofibers, hydrogel, cardiac cells, troponin, actinin
How to cite this article: Reddy CS, Venugopal JR, Ramakrishna S, Zussman E. 2014. Polycaprolactone/oligomer compound scaffolds for cardiac tissue engineering. J Biomed Mater Res Part A 2014:102A:3713–3725.
INTRODUCTION
Polycaprolactone (PCL) is a semicrystalline thermoplastic polymer widely used in tissue engineering applications due to its non-immunogenicity, slow biodegradability and good drug permeability.1 Electrospun nanofiber mesh of PCL resembles the morphology of extracellular matrix (ECM), and has been used as a scaffold material for cell adhesion and proliferation in tissue regeneration. Mimicking the fibrillar structure of ECM, engineered scaffold provides essential guidance for cell organization, survival, and function.2–6 By ensuring such support, the scaffolds set the foundation for efficient and compact tissue formation and an expedited recovery process. The ester and keto group functionalities of PCL render it highly hydrophobic, leaving it with no physiologically active sites. Thus, while epithelial cell growth was enhanced on a PCL membrane, cell affinity towards PCL is generally poor.7 Biomimicking of ECM, the poor hydrophilicity of electrospun PCL mats led to an over-
all reduction in cell adhesion, migration, proliferation and differentiation.8,9 The scaffold chemistry plays an important role in cell adhesion and differentiation following implantation. Studies have reported that adhesion and organization of transplanted cardiac cells into functional contractile tissue are far superior on natural ECM compared with synthetic scaffolds.10,11 The substrate environment ideal for promoting cell proliferation has been a persistent area of debate in tissue engineering. Candidate substrate materials must exhibit biocompatibility, while supporting cell proliferation, which is hypothesized to be a direct function of its stiffness. Bioengineered scaffold stiffness must be closely resemble that of native ECM and flexible to allow the contraction of growing cells.12 In the case of PCL membranes, 3T3 fibroblast cell proliferation was most significant on low stiffness samples.13 In addition, high scaffold surface area and porosity are essential to allow for intercellular transport of nutrients,
Correspondence to: J. R. Venugopal; e-mail: [email protected] or E. Zussman; e-mail: [email protected] Contract grant sponsor: National Research Foundation of Singapore in the framework of the Regenerative Medicine Initiative in Cardiac Restoration Therapy research program and Technion-Israel Institute of Technology, Haifa, Israel; contract grant number: R-398-001-065-592
C 2013 WILEY PERIODICALS, INC. V
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TABLE I. Electrospinning Parameters for As-Spun and Post-Cured Scaffolds Chemical Composition and Physical Properties Scaffold Propertiesa
Scaffold Composition [wt.%]
As-Spun
Electrospinning Parameters
Scaffold
PCL
BPAEDMA
Voltage [kV]
PCL PCL/BPAEDMA (75/25) PCL/BPAEDMA (50/50) PCL/BPAEDMA (25/75)
100 75 50 25
0 25 50 75
12 12 12 12
Post-Cured
Storage Modulus [MPa]
Storage Modulus [MPa]
Working Distance [cm]
Flow Rate [mL/h]
Wetting Angle [ ]
26 C
37 C
26 C
37 C
11 11 11 11
1 1 1 1
82 31 15 25
39 17 12 9
32 12 9 7
39 31 12 29
32 23 10 26
a
Mean value of n 5 5 samples were presented.
metabolites and regulatory molecules. Furthermore, as mentioned earlier, scaffold chemistry highly influences cell adhesion and growth potentials. After observing reduced relocation of transplanted labeled mesenchymal stem cells to remote organs and non-infarcted myocardium when implanted along with a collagen matrix, Dai et al. concluded that matrices of this nature can enhance clinical outcomes of implanted cells.14 Numerous studies have been reported to create fibrous electrospun nanofibrous scaffolds composed of polymer blend solutions with emphasis on application of biocompatible and biodegradable polymers.15–17 Shin et al.18 reported considerable cardiomyocyte attachment to PCL based meshes, along with the expression of cardiac-specific proteins, such as myosin heavy chain, connexin 43 and cardiac troponin. They hypothesized that such 3D structures may induce cardiomyocyte-based vascularization upon provision of angiogenic factors. Schnell et al.19 observed glial cell migration and axonal growth on electrospun fibrous scaffolds of PCL versus collagen/PCL scaffolds. They demonstrated that Schwann cells, fibroblasts, and olfactory ensheathing cells were significantly increased on collagen/ PCL fibers, when compared with pure PCL fibers. Nguyen et al.20 demonstrated that the increasing ratio of PLGA (poly lactic acid/glycolic acid) in electrospun PCL/PLGA blend scaffolds, the altered mechanical properties of electrospun membranes resulted in increased fibrous mat biocompatibility. In addition, the elastomeric polyglycerol sebacate yields porous, elastomeric three-dimensional (3D) scaffolds with controllable stiffness and anisotropy. Such scaffolds have been reported effective in myocardial tissue engineering applications,21,22 as illustrated by their potential to promote the formation of cardiac grafts comprised of aligned heart cells with mechanical properties closely resembling those of the native myocardium. Landis et al.23 fabricated a saltleached tissue engineering scaffolds based on photo-crosslinkable bisphenol A ethoxylated dimethacrylate (BPAEDMA) monomers pertinent for hard tissue engineering. These monomers are extensively used in the formulation of matrices for restorative dental composites.24–28 This study focused on fabricating bioengineered scaffold suitable for cardiac tissue engineering, with low stiffness and balanced hydrophobicity. To achieve this goal, PCL was
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blended with a photocurable BPAEDMA hydrogel precursor, with a degree of ethoxylation equivalent to 30. BPAEDMA with high degree of ethoxylation lead to obtain highly flexible elastomeric hydrogel when treated by UV radiation. The blended oligomer contained soft ethylene oxide segments, providing flexibility and allow bioadhesion of cells, as well as polymerizable methacrylate end groups, for mechanical strength tunability. In addition, these elements are both non-toxic and non-immunogenic, which are two essential requirements for biocompatible implantable materials. Photopolymerization of this oligomer leads to the formation of flexible elastomeric hydrogel films and nanofibers.29–32 Assessment of the tissue engineering capacities of the described compound fiber scaffolds embedded with rabbit cardiac cells, demonstrated their low stiffness and balanced hydrophobic properties were suitable for cell adhesion and proliferation of cardiac cells. The newly developed PCL/ BPAEDMA compound nanofibrous scaffolds may bear considerable potential in cardiac tissue engineering applications. MATERIALS AND METHODS
Materials PCL (Mw: 80,000), PEG-derived oligomer hydrogel precursor BPAEDMA (Mw: 1700), 2, 2-dimethoxy, 2-phenyl acetophenone and chloroform were obtained from Sigma Aldrich. PCL was dissolved in chloroform to obtain 10% (wt/wt) solution. BPAEDMA and 2,2-Dimethoxy-2-phenylacetophenone photo-initiator (PI) (2 wt/wt % to BPAEDMA) were then added to PCL solution at the desired ratios. The compositions are presented in Table I and the chemical formulas of the materials and graphical representation of electrospun compound fibers are shown in Scheme 1. Electrospinning and polymerization The flow controlled by a syringe pump of the PCL/ BPAEDMA blended solution was set at a constant rate of 1 mL/h with an electrostatic field of 12 kV (Table I). An aluminum foil sheet was placed 11 cm beneath the spinneret exit for the collection of non-woven fibrous scaffolds. PCL/ BPAEDMA electrospun fibrous compound scaffolds were placed on aluminum foil sheets supported on a metal disc at a distance of 15 cm from two horizontal 20W high-pressure
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SCHEME 1. (a) Chemical formula of reagents, and (b) Schematic representation of different steps involved in the process of scaffold preparation.
mercury lamps and then BPAEDMA in the compound fibers was cured by UV radiation for 10 min as reported earlier.29,31 Characterization of the fibrous scaffolds Microscopic characterization. Electrospun PCL/BPAEDMA nanofibrous compound scaffolds collected on carbon tapecovered metal stub and sputter-coated with gold were analyzed using Phenom desktop scanning electron microscope (SEM) (5 kV accelerating voltage, FEI Company, Hillsboro, OR). The surface morphology of PCL and PCL/BPAEDMA naofibrous scaffolds were analyzed by stained with eosin solution for about 30 s and extensive washed with water to remove excess stain and vacuum dried at 40 C. Eosinstained PCL and PCL/BPAEDMA scaffolds were sputtercoated with gold and analyzed by SEM. Wettability of the nanofibrous scaffolds Sessile droplet of double distilled water (DDW) was poured at the center of each nanofibrous scaffolds. Droplet evolution in time was imaged with an electronic camera (MotionScope-Redlake Imaging Corporation). The water contact angle (hw) was determined at time 15 s after placing of the water droplet. The camera speed was 2000 f.p.s. and the shutter speed 0.05 ms. The camera was equipped with a 70–180 mm, f/4.5 zoom lens. The light source (v) (400W HMI lamp with Dedolight DEB400D electronic ballast) was placed against the camera along its line of sight. Five different PCL and PCL/BPAEDMA nanofibrous compound scaffolds were evaluated for water contact angle measurements. Swelling experiments The swelling characteristics of as-spun and post-cured PCL/ BPAEDMA nanofibrous compound scaffolds were assessed
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in aqueous medium. PCL/BPAEDMA nanofibrous compound scaffolds were soaked in water for 78 h at 37 C and swollen to an equilibrium state. Swelling percentages were calculated as: Water fraction 5 ½ðwsm 2wdm Þ=wdm 3100 where, wsm 5 weight of swollen compound fiber mat and wdm 5 weight of dry compound fiber mats. Differential scanning calorimetry Differential scanning calorimetric experiments were carried out using a Differential scanning calorimeter (METTLERDSC 1). All DSC measurements were repeated on five samples for each PCL/BPAEDMA nanofibrous scaffold under nitrogen atmosphere and at temperatures ranging from 10 to 180 C at a heating rate of 10 C/min on sample sizes that ranged between 0.5 and 3 mg. The DSC was calibrated with an indium and zinc-melting standard. Dynamic mechanical analysis Dynamic mechanical analyzer (TA, DMA Q 800) in the tension film mode was used to measure the mechanical properties of as-spun and post cured nanofibrous scaffolds. The samples were subjected to a sinusoidal displacement at 10-mm amplitude and at a fixed frequency of 1 Hz from 280 to 40 C with a heating rate of 2 C/min. The storage moduli of as-spun and post cured nanofibrous scaffolds were measured in the temperature interval from 280 to 40 C. Stress–strain measurements at 26 C were conducted on 5 3 3 3 0.2mm rectangular samples at a strain rate of 0.5 mm/min. Wide angle X-ray scattering Wide-angle X-ray diffraction (WAXD) patterns of as-spun and post cured nanofibrous scaffolds were recorded using a
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Philips PW3710 diffractometer and PW3020 goniometer (Philips, MBLE, Brussels, Belgium) operating at 10 kV and 10 mA with Cu Ka radiation (k 5 0.154 nm). The dried PCL/BPAEDMA nanofibrous scaffolds were placed on Vaseline and diiffractograms were acquired at an angular range of 2h from 5 to 60 with a step size of 0.05 , and 4 s counting time for each step. Infrared spectroscopic measurements To confirm the existence of cured BPAEDMA in PCL/ BPAEDMA nanofibrous compound scaffolds after swollen in water/phosphate buffered saline (PBS) for 7 days, samples were removed from water and vacuum dried at 40 C. Fourier transform infrared spectral analysis was performed on dried PCL/BPAEDMA nanofibrous scaffolds using a NEXUS 870 FTIR (Thermo Nicolet) in a humidity-free atmosphere at room temperature with 4 cm21 resolution and 32 scans signal average and spectra were recorded from 400 to 4600 cm21 interval in the transmission mode using a DTG TEC detector. The spectral data of as-spun nanofibrous scaffolds are presented in this paper without any correction or modification. Isolation and culture of cardiac cells Rabbit cardiac cells were isolated using a collagenasebased digestion method. The rabbit aorta was cannulated and perfused using a bicarbonate solution (118.5 mM NaCl, 25.0 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.0 mM glucose, and 2.5 mM CaCl2). The rabbit heart was then thoroughly washed thrice, for 20 min each time, with antibiotic solutions diluted in PBS to concentrations of 33 and 23, and then fragmented the ventricles into fine pieces. Tissue fragments were subsequently treated with 1% collagenase (Type I) diluted in PBS, for 30 min at 37 C. The dispersed cells were harvested by decantation after each 5 min of incubation. The fine cardiac pieces were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (GIBCO Invitrogen) and 1% antibiotic and antimycotic solutions (Invitrogen Corporation) in 75 cm2 cell culture flasks, to allow for cardiac cell isolation. Trypsinized cells were centrifuged, counted following trypan blue staining and then seeded on the nanofibrous scaffolds. Cell seeding on scaffolds Tissue culture plates (TCP), PCL, PCL/BPAEDMA (75/25), PCL/BPAEDMA (50/50), and PCL/BPAEDMA (25/75) elastomeric nanofibrous scaffolds were sterilized with 70% ethanol, washed three times with PBS, and subsequently immersed in complete DMEM overnight before cell seeding. Electrospun compound nanofibers were collected on 15 mM round glass cover slips and then placed in 24-well plates along with a stainless steel ring to prevent lifting of fibers. The trypsinized cells collected from culture flasks, were counted and seeded onto the TCP (control) or on the scaffolds at a density of 1 3 104 cells/well. Cells were allowed to adhere for 15 min before adding medium, which was replaced every 3 days.
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Cell proliferation Cell adhesion and proliferation on the nanofibrous scaffolds were determined using the colorimetric MTS assay (CellTiter 96 AQueous One solution, Promega, Madison, WI), in which the yellow tetrazolium salt [3-(4, 5-dimethylthiazol-2yl)25-(3-carboxymethoxyphenyl)22(4sulfophenyl)22H tetrazolium] is reduced by the mitochondrial dehydrogenase enzymes secreted in metabolically active cells, to form purple formazan crystals. The formazan dye absorbs light at 492 nm and the number of crystals formed is said to be directly proportional to the number of viable cells. Cells were rinsed with PBS, to remove non-adherent cells, and incubated with 20% MTS reagent in a serum-free medium for 3 h at 37 C. Samples of culture medium were then pipetted out into fresh 96-well plates. Absorbance was measured at 490 nm using a microplate reader (Fluostar Optima, BMG Lab Technologies, Germany). Cell morphology After 10 days in culture, cardiac cells cultured on the various scaffolds were analyzed with a SEM. The scaffolds were rinsed twice with PBS and fixed in 3% glutaraldehyde (Sigma Aldrich) for 3 h. Thereafter, scaffolds were rinsed in deionized water and dehydrated with increasing concentrations of ethanol (50%, 70% 90%, 100%) for 10 min each. Samples were then treated with hexamethyldisilazane (Fluka Chemical Corporation, Milwaukee, WI) and then airdried in a fume hood. Finally, the scaffolds were sputtercoated with gold (JEOL JFC-1200 fine coater, Japan) and observed by SEM (FEI-QUANTA 200F, Netherland) at an accelerating voltage of 10 kV. Labeling cells with fluorescent dyes. Cardiac cells (1 3 104cells/cm2) seeded on nanofibrous scaffolds were washed with PBS after 10 days in culture, and then stained with 5chloromethyl fluorescein diacetate (25 lM CMFDA at 37 C for 60 min, CellTracker green, Promega USA) which is cleaved by cytosolic esterases to form a highly fluorescent and membrane impermeable CMFDA derivative. The CMFDA-supplemented medium was then replaced by complete medium and cardiac cells were incubated overnight. Cells were then washed with PBS and after addition of serum-free medium observed under an inverted Leica DM IRB laser scanning microscope (Leica DC 300F) at 488 nm. Expression of cardiac-specific protein markers Cardiac cells grown on nanofibrous scaffolds were fixed using 100% chilled methanol, then washed once with PBS for 15 min and incubated in a 0.5% Triton-X solution for 5 min to permeabilize the cell membrane. Non-specific binding sites were blocked by incubating the cells in 3% BSA (Sigma Aldrich) for 1 h. Thereafter, they were stained with anti-sarcomeric alpha-actinin antibodies and anti-troponin antibody (1:100 dilution, Sigma Aldrich), followed by treatment with an FITC-labeled (1:250 dilution, Sigma Aldrich) secondary antibody. Nuclei were stained with 40 , 6diamidino-2-phenylindole (1:5000 dilutions, Invitrogen). Finally, samples were extensively washed and then mounted
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FIGURE 1. SEM images of nanofibrous scaffolds. PCL (a), PCL/BPAEDMA (75/25) (b), PCL/BPAEDMA (50/50) (c), and PCL/BPAEDMA (25/75) (d).
using Vectashield (Vector Laboratories), before being analyzed under a confocal laser scanning microscope (Olympus FV1000). Statistical analysis All the data presented in the manuscript are expressed as mean 6 standard deviation (SD). Significance level was determined using Student’s t-test. Differences were considered statistically significant at p 0.05. RESULTS AND DISCUSSION
Influence of BPAEDMA on morphology of compound fibers Cell-scaffold interactions are determined by the physical and chemical properties of constituent materials, with particular weight on their chemical composition, particle size and surface properties which include topography, roughness, surface energy and wettability.29 Morphologies of representative, nonpolymerized PCL/BPAEDMA compound fibers are shown in Figure 1. Fiber diameters were within the range of 0.5–1 lm and a
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fiber mat thickness was 200 lm. SEM images of eosin-stained PCL fiber mats [Fig. 2(a,b)] showed fibers with many craters on the surface. Whereas, PCL/BPAEDMA nanofibrous scaffold [Fig. 2(c,d)] with 50 wt % BPAEDMA showed fibers with spherical and elliptical microdomains on the surface, and they were distributed along the fiber axis. PCL/BPAEDMA nanofibrous scaffolds with >50 wt % BPAEDMA, surface of PCL/BPAEDMA nanofibers exhibited biphasic morphology being PCL as the continuous phase and BPAEDMA as dispersed phase along the fiber axis. These surface microdomains are expected to render the compound fibers more pertinent to cell adhesion applications. As-spun and post cured PCL/BPAEDMA (50/50) nanofibrous compound scaffolds were placed in water for one week at 37 C and they were removed from water and vacuum dried overnight at 40 C. The surface morphology of as-spun and post cured PCL/BPAEDMA compound fibers (Fig. 3) resembled the surface morphology of the non-swollen compound fibers, their diameters increased by an average of 15% than PCL fibers, confirming the existence of hydrophilic BPAEDMA. The dried swollen PCL/BPAEDMA nanofibrous scaffolds were analyzed by
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FIGURE 2. SEM images of eosin-stained as-spun nanofibrous scaffolds. PCL (a, b), and PCL/BPAEDMA (50/50) (c, d) at two different magnifications.
FTIR spectroscopy (Fig. 4). The dried preswollen compound scaffolds exhibited an absorbance bands at 1610 cm21 and 1637 cm21, attributed to an aromatic double bond (AC6H4A) and aliphatic double bond (AC@CA) of BPAEDMA, respectively. BPAEDMA was partially cured during spinning of PCL/ BPAEDMA compound solution in chloroform and post curing of PCL/BPAEDMA nanofibrous scaffolds by UV radiation lead to decreased intensity of aliphatic double bond (AC@CA) absorbance band at 1637 cm21 of BPAEDMA, as reported earlier.29 Mechanical properties of compound nanofibrous scaffolds Mechanical properties of as-spun and post cured nanofibrous scaffolds were measured [Fig. 5(a–d), and Table I]. Storage modulus and loss modulus curves depend on the temperature [Fig. 5(a,b)] of PCL/BPAEDMA nanofibrous scaffolds clearly indicate that PCL and BPAEDMA were phase separated. The storage modulus curves of PCL and PCL/BPAEDMA nanofibrous scaffolds displayed the typical behavior of semicrystalline polymers with four distinctive zones. At < 260 C, the storage modulus slightly decreased with temperatures. This observed limited temperature effect is due to the fact that the polymer was in glassy state,
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where molecular motions were largely restricted to vibrations and short-range rotations. Around 260 to 220 C, a broad transition showed as a sharp drop in storage modulus was observed. At this temperature range, the main relaxation process corresponded to amorphous PCL domains associated with the glass transition of PCL. At higher temperatures range, 220 to 40 C, the storage modulus decreased due to the progressive softening of the PCL matrix. Here amorphous, rubbery and crystalline domains coexisted. At higher temperatures between 45 C and 70 C, the storage modulus dropped sharply because complete melting of the PCL crystalline domains [not shown in Fig. 5(a,b)], as observed from DSC melting thermograms. Whereas, at higher temperatures between 10 C and 30 C, PCL/BPAEDMA nanofibrous scaffolds storage modulus decreased abruptly due to melting of BPAEDMA crystalline domains as observed by DSC measurements. PCL scaffold loss modulus as a function of temperature showed a peak maximum at 254 C, and this peak maximum temperature was defined as PCL glass transition temperature. PCL/ BPAEDMA nanofibrous compound scaffolds loss modulus as function of temperature typically represented a mixed amorphous phase with broad glass transition temperature.
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FIGURE 3. SEM images of dried preswollen nanofibrous scaffolds. As-spun (a, b) and post cured (c, d) PCL/BPAEDMA (50/50) shown at two different magnifications.
PCL/BPAEDMA nanofibrous compound scaffolds glass transition temperature was shifted by 2 C to 10 C toward higher temperature side compared with PCL with increasing
FIGURE 4. FTIR spectra of dried preswollen scaffolds and BPAEDMA. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), PCL/BPAEDMA (25/75) (dark gray), and BPAEDMA (dot black).
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BPAEDMA weight fraction. This increased glass transition temperature might be attributed to the existence partially cured BPAEDMA sub-micrometer domains in the PCL/ BPAEDMA compound nanofibers as observed from morphology of eosin-stained compound scaffolds. When PCL/ BPAEDMA compound scaffolds with partially cured BPAEDMA were post cured by UV radiation about 10 min an increase in tensile storage modulus was observed, when compared with as-spun counterparts likely associated with the fraction of the cross-linked BPAEDMA in the compound fibers. Stress–strain behavior of PCL and post cured PCL/ BPAEDMA compound nanofibrous scaffolds at 26 C are presented in Figure 5(c,d). PCL scaffold showed an ultimate tensile strength of 2.8 MPa, elastic modulus of 30 MPa and strain at break of 300 6 18 (%). PCL/BPAEDMA (75/25) exhibited an ultimate tensile strength of 2.5 MPa, elastic modulus of 10 MPa, and strain at break of 178 6 30 (%). PCL/BPAEDMA (50/50) compound nanofibrous scaffold showed an ultimate tensile strength of 1.5 MPa, elastic modulus of 2 MPa and strain at break of 106 6 30 (%). PCL/BPAEDMA (25/75) compound scaffold exhibited an
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FIGURE 5. Storage modulus and loss modulus of as-spun (a), post cured (b), stress–strain curves until break (c), linear region of stress–strain curves, (d) of post cured scaffolds. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
ultimate tensile strength of 2.3 MPa, elastic modulus of 7 MPa and strain at break of 111 6 44 (%). Post cured PCL/BPAEDMA compound nanofibrous scaffolds showed reduced elastic modulus and strain at break compared with PCL scaffolds. This reduced strain at break can be attributed to increased crosslink density of PCL/ BPAEDMA compound nanofibrous scaffolds and reduced elastic modulus might be due to the reduced crystallinity of PCL in the compound scaffolds. However, the PCL/ BPAEDMA 50/50 nanofibrous compound scaffolds exhibited higher crystallinity than PCL scaffold might be due to the coexistence of mixed PCL and BPAEDMA crystalline phase. Wettability and swelling properties PCL and PCL/BPAEDMA compound nanofibrous scaffolds surface wettability with water, measured by sessile water drop contact angle measurements (Table I), were 60% < comparison with PCL scaffold indicating increased hydrophilicity of the compound fiber mats. The swelling characteristics of as-spun and post cured scaffolds were assessed in an aqueous medium (Fig. 6). Fiber swelling capacities decreased significantly with increasing hydrogel weight fraction in correlation with the increased crosslink density of PCL/BPAEDMA compound nanofibrous scaffolds. The difference between swelling capacities as-spun and post
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cured nanofibrous scaffolds was only 20%, likely due to increased crosslink density of PCL/BPAEDMA compound nanofibrous scaffolds upon post curing.
FIGURE 6. Swelling characteristics of as-spun and post cured scaffolds. Compound fibers comprised of varying ratios of PCL and BPAEDMA were soaked in water for 78 h before calculation of swelling percentages [5100 3 (wsm 2 wdm)/wdm)]. Mean values (6SD) are presented (n 5 5).
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FIGURE 7. WAXD patterns of as-spun (a) and post cured (b) scaffolds. PCL (black), PCL/BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
Wide-angle X-ray diffraction Structural characteristics of as-spun and post cured nanofibrous scaffolds were analyzed by WAXD to determine the impact of BPAEDMA weight fraction, on the crystallinity of PCL (Fig. 7). The WAXD pattern of as-spun naofibrous scaffolds [Fig. 7(a)] was comprised of two main planes [(110) at 2u 5 21.35 and (200) at 2u 5 23.65], which can be attributed to the orthorhombic crystalline structure of semicrystalline PCL.33 A decrease in intensity of the peak at 2h 5 21.35, for the PCL/BPAEDMA compound nanofibrous scaffolds was observed and can be attributed to the decreased weight fraction of PCL in these scaffolds. The full width at half maximum intensity of peak (b) at 2h 5 21.35, for the PCL/BPAEDMA nanofibrous scaffolds also decreased in comparison with that recorded for PCL scaffold, suggesting an increase in crystallite size of PCL in the compound fibers. This trend was further intensified in post cured PCL/ BPAEDMA compound nanofibrous scaffolds [Fig. 7(b)].
Thermal stability of PCL and PCL/BPAEDMA compound fibers Determination of thermal stability by means of differential scanning calorimetery (DSC) is the most common and simplest method for evaluating melting temperature, melting
enthalpy and crystallinity of a polymer with respect to the melting enthalpy of its fully crystalline state. The melting temperature, melting enthalpy and degree of crystallinity of as-spun and post cured nanofibrous scaffolds are presented in Table II and the DSC thermograms are shown in Figure 8. The melting enthalpies of as-spun nanofibrous compound scaffolds (except PCL/BPAEDMA 50/50) were lower in comparison with the as-spun PCL scaffolds. The melting enthalpy of PCL/BPAEDMA (50/50) compound nanofibrous scaffold was higher than the PCL scaffold, which suggests that coexistence of mixed PCL and BPAEDMA crystalline phase. However, the BPAEDMA phase of compound nanofibrous scaffolds inhibited PCL crystallization, as demonstrated by the drop in PCL melting enthalpy in these scaffolds compared with PCL scaffold. The melting enthalpy and degree of crystallinity of PCL in post cured PCL/BPAEDMA compound nanofibrous scaffolds (except PCL/BPAEDMA 50/50) were further increased, suggested to be the result of structural modification of PCL by low intensity UV radiation.
Cardiac cell proliferation and morphology on PCL/BPAEDMA scaffolds This study fabricated elastomeric PCL/BPAEDMA compound fibers for the culture of cardiac cells as designated for
TABLE II. Thermal Properties of As-Spun and Post-Cured Scaffolds Scaffold Composition [wt %]
Melting Temperature [ C]
Crystallinitya [%]
Melting Enthalpy [J/g]
As-Spun
Post-Cured
Scaffold
PCL
BPAEDMA
AS-SPUN
Post-Cured
As-Spun
Post-Cured
PCL
PCL
PCL PCL/BPAEDMA (75/25) PCL/BPAEDMA (50/50) PCL/BPAEDMA (25/75)
100 75 50 25
0 25 50 75
61 59 60 60
60 59 60 59
71 46 118 55
84 54 93 59
52 34 86 40
61 39 68 43
a
The melting enthalpy of fully crystalline PCL 5 136 J/g,42 and the melting enthalpy of fully crystalline PEO 5 197 J/g.43
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FIGURE 8. DSC melting curves of as-spun and inset is BPAEDMA heating cooling thermogram (a) and post cured (b) scaffolds. PCL (black), PCL/ BPAEDMA (75/25) (light gray), PCL/BPAEDMA (50/50) (gray), and PCL/BPAEDMA (25/75) (dark gray).
myocardial infarction (MI). Cell proliferation on PCL/ BPAEDMA (25/75) meshes showed significantly (p 0.05) higher than PCL and PCL/BPAEDMA (75/25) nanofibrous scaffolds on days 10 and 15, respectively (Fig. 9). These observations suggested that the PCL/BPAEDMA fibrous material provides a suitable microenvironment to support cell growth and expression of cardiac proteins may boost tissue reorganization in the myocardium. Increased cell adhesion on the elastomeric nanofiber matrix can also be attributed to adhesion proteins such as fibronectin and vitronectin present in the serum used for cell culture.34,35. In this study, the balanced hydrophilicity and lower stiffness of the designed compound scaffolds promotes cell adhesion and proliferation than that of PCL fiber mesh alone. Normal cell morphology was observed in cells grown for 10 days on elastomeric PCL/BPAEDMA (25/75) nanofibrous scaffolds, in contrast to that of cells cultured on PCL/BPAEDMA (75/ 25) nanofibrous scaffolds (Fig. 10). These results confirm that the porous, hydrophilic compound nanofibrous scaf-
FIGURE 9. Proliferation of rabbit cardiac cells on various electrospun nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (Control), PCL, PCL/BPAEDMA (75/25), PCL/BPAEDMA (50/50), and PCL/ BPAEDMA (25/75) elastomeric nanofibrous scaffolds for up to 15 days. Cell proliferation was determined using the MTS assay. Values (6SD) represent mean optical density as measured at 490 nm (n 5 6). (HG-BPEDMA hydrogel). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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folds provide cell adhesion sites and support both proliferation and transport of nutrients for cardiac tissue engineering. Cell viability and expression of cardiac proteins CMFDA dye belongs to a group of chloromethyl derivatives developed for in vitro labeling of live cells.36,37 The CMFDA compound is metabolized intracellularly in viable cells to a cell-impermeant and fluorescent state within 1 h. Addition of CMFDA to the cell culture medium causes the compound to freely permeate the cell membrane and be acted upon by cytosolic esterases, producing a CMFDA derivative that is brightly fluorescent. To determine the viability of cells embedded on PCL and PCL/BPAEDMA compound scaffolds, samples were treated with CMFDA after 10 days in culture. While CMFDA fluorescence was observed in cardiomyocyte cultures grown on control and elastomeric nanofibrous scaffolds, greater cell density and normal morphology were observed among those cultured on elastomeric PCL/ BPAEDMA (25/75) nanofibrous scaffolds (Fig. 11). As scaffold biomaterials have been reported to influence cellular activities, the expression of alpha-actinin and troponin T, two markers specific to cardiac lineage and both essential for cardiomyocyte functioning was evaluated in cells grown on the various elastomeric nanofiber scaffolds. Alpha-actinin is an actin cross-linking protein38 critical to cardiac contractility, while troponin T regulates the force and velocity of myocardial contraction.39 Troponin T is also an indicator of cardiomyocytes differentiation.40,41 Figure 12 shows the expression of Troponin and alpha-actinin in the cardiac cells grown on PCL and PCL/BPAEDMA (25/75) elastomeric scaffolds compared with the TCP. The expression of alphaactinin and Troponin by the cardiac cells cultured on PCL/ BPAEDMA (25/75) compound nanofibrous scaffolds indicates that the cells retains their normal functionality. The observed results showed that the designed PCL/BPAEDMA (25/75) elastomeric nanofibrous scaffolds properties suitable for the application in the repair of MI in cardiac tissue engineering.
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FIGURE 10. Morphology of cardiac cells cultured on elastomeric PCL/BPAEDMA nanofibers. Representative SEM images of rabbit cardiac cells grown on nanofibrous scaffolds for 10 days. Rabbit cardiac cells were cultured on TCP (Control) (a), PCL (b), PCL/BPAEDMA (75/25) (c), PCL/ BPAEDMA (50/50) (d), and PCL/BPAEDMA (25/75) fibers (e).
FIGURE 11. Rabbit cardiac cell viability when cultured on electrospun PCL/BPAEDMA compound nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (Control) (a), PCL (b), PCL/BPAEDMA (75/25) (c), PCL/BPAEDMA (50/50) (d), and PCL/BPAEDMA (25/75) (e) elastomeric nanofibrous scaffolds for 10 days and then treated with CMFDA to determine cell viability (scale bar 100 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 12. Expression of cardiac proteins in cardiac cells grown on PCL, PCL/BPAEDMA compound nanofibrous scaffolds. Rabbit cardiac cells were cultured on TCP (a, d), PCL (b, e) and PCL/BPAEDMA (25/75) scaffolds (c, f) for 10 days. Samples were fixed and stained for cardiac proteins Toponin (a–c) and actinin (d–f), both detected with an FITC filter (scale bar 10 mm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CONCLUSIONS
The site of MI is a poor environment for cell growth; to increase cell viability, some factors to improve such an infertile environment are desirable for cardiac tissue engineering. The described electrospun neat nano/micro PCL fiber meshes resemble the fibrillar structural organization of ECM and also favor the proliferation of rabbit cardiac cells in vitro. However, cardiac cells proliferation increased upon addition of BPAEDMA to PCL fibrous scaffolds, seemingly due to the balanced hydrophilicity of the resulting scaffold and decreased modulus of PCL from 39.68 MPa to 3.55 MPa. Compound fiber mesh comprised of 25 wt % PCL and 75 wt % BPAEDMA, allowed for enhanced cell adhesion and proliferation of cultured cardiac cells and expression of key cardiac-specific proteins. The PCL/BPAEDMA (25/75) nanofibrous scaffolds provided a fiber mesh support with low stiffness, balanced hydrophilicity, along with wettable properties relevant for cardiac tissue engineering.
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