Electrospun Composite Scaffolds Containing Poly(octanediol-co-citrate) Electrospun Scaffolds Containing Poly(octanediol-co-citrate) for CardiacComposite Tissue Engineering for Cardiac Tissue Engineering Molamma P. Prabhakaran,1 A. Sreekumaran Nair,1 Dan Kai,2 Seeram Ramakrishna1,3 1
Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore 117576
2
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
3
Department of Mechanical Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore 117576
Received 10 August 2011; revised 15 January 2012; accepted 17 January 2012 Published online 10 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22035
ABSTRACT:
increasing concentration of POC in the composite. The
A biocompatible and elastomeric nanofibrous scaffold is
morphology and cytoskeletal observation of the cells also
electrospun from a blend of poly(1,8-octanediol-co-
demonstrated the biocompatibility of the POC containing
citrate) [POC] and poly(L-lactic acid) -co-poly-
scaffolds. Electrospun POC/PLCL4060 nanofibers are
(3-caprolactone) [PLCL] for application as a
promising elastomeric substrates that might provide the
bioengineered patch for cardiac tissue engineering. The
necessary mechanical cues to cardiac muscle cells for
characterization of the scaffolds was carried out by
regeneration of the heart. # 2012 Wiley Periodicals, Inc.
Fourier transform infra red spectroscopy, scanning
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electron microscopy (SEM), and tensile measurement.
Keywords: poly(1,8-octanediol-co-citrate); elastomer;
The mechanical properties of the scaffolds are studied
cardiac; tensile strength; myoblasts
with regard to the percentage of POC incorporated with PLCL and the results of the study showed that the mechanical property and degradation behavior of the composites can be tuned with respect to the concentration of POC blended with PLCL. The composite scaffolds with POC: PLCL weight ratio of 40:60 [POC/PLCL4060] was found to have a tensile strength of 1.04 6 0.11 MPa and Young’s Modulus of 0.51 6 0.10 MPa, comparable to the native cardiac tissue. The proliferation of cardiac myoblast cells on the electrospun POC/PLCL scaffolds was found to increase from Days 2 to 8, with the
Correspondence to: Molamma P. Prabhakaran; e-mail:
[email protected] Contract grant sponsor: Singapore National Research Foundation under CREATE programme: The Regenerative Medicine Initiative in Cardiac Restoration Therapy Contract grant number: R-398-001-065-592 Contract grant sponsors: Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore C 2012 V
Wiley Periodicals, Inc.
Biopolymers Volume 97 / Number 7
This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com
INTRODUCTION
M
yocardial damage represents a major cause of morbidity and mortality to a huge population in both developed and developing countries. Myocardial infarction leads to the loss of cardiomyocytes and dilation of the left ventricle, thus imparing the cardiac function. Regenerating the cardiomyocytes once it is lost by heart failure is difficult to achieve and other than heart transplantation, there are very few procedures that can sufficiently prevent the heart disease progression. Strategies including cellular myoplasty are being explored for the regeneration of the infarct myocardium. Moreover myoblasts have the advantages of ease of expansion in vitro and high resistance to ischemia, with properties to be exploited for clinical use.1 But cardio-myoblasts are known
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to undergo a massive cell death when injected alone to the ischemic heart.2,3 In this respective, bio-engineered cardiac graft using a combination of cells and biomaterials could be a promising strategy for regeneration of the heart. Scaffolds for cardiac tissue engineering (TE) should be porous, elastic, biocompatible, and biodegradable.4,5 Among the various methods utilized for the fabrication of matrix mimicking scaffolds for cardiac TE, electrospinning is of great interest, since it is possible to make nonwoven membranes with fibers in nanometer scales that are architecturally similar to the native extracellular matrix (ECM) of the heart.6 Electrospun nanofibers provide high surface-to-volume ratios with interconnected pores, provide high versatility in material selection with ease of processing, and provide favorable surfaces for cell attachment and proliferation.7 Electrospinning has been applied for the fabrication of nanofibrous scaffolds from numerous biodegradable polymers, such as poly(e-caprolactone) (PCL), poly(lactic acid) (PLA), poly (glycolic acid) (PGA), etc.8–10 Aliphatic polyesters are biodegradable and produce nontoxic metabolites upon biological attack in the human body and they are also considered as clinically approved biomaterials.11,12 However, these biocompatible synthetic polymers are relatively rigid with higher elastic moduli (as high as 1000 MPa) that limits its application in soft TE. On the other hand, natural polymers like collagen cause large batch-to-batch variations during isolations, have low mechanical strength and involves high cost.13 Elasticity is an important criterion required of a heart patch, where by the scaffold should be flexible enough to accommodate the contractility of the cells, especially because the myocardial tissue undergoes constant cyclic deformations.14 Elastomers such as polyurethane (PU) and polycarbonate have been attempted for the engineering of cardiac tissues and PU scaffolds are demonstrated to withstand high stress, required of a cardiac patch.15 A scaffold for TE should have mechanical properties matching the host tissue at the site of implantation and POC is a polymer with elastomeric properties. Poly(1,8-octanediol-co-citrate) [POC] is a biodegradable, inexpensive, and hydrophilic polymer, which can be synthesized without the need of any toxic chemicals and solvents. Poly(L-lactic acidco-caprolactone) or PLCL is a biodegradable copolymer of poly-L-lactic acid and polycaprolactone, studied as a substrate for culture of endothelial cells and smooth muscle cells.16,17 The objective of this study was to prepare an elastomeric scaffold with ECM like morphology and a substrate that can provide the necessary mechanical cues essential for the regeneration of the heart. Here we report for the first time, the electrospinning of the elastomeric polymer POC, by blending it with another biocompatible polymer PLCL.
FIGURE 1 Schematics demonstrating the reaction of citric acid with octanediol to obtain POC.
The biocompatibility of the electrospun nanofibrous scaffolds was studied using cardiac myoblasts to realize its application in cardiac TE.
MATERIALS AND METHODS Materials The synthesis of poly(1,8-octanediol-co-citric acid) or POC was carried out according to literature reports and as briefly outlined below.18 Equimolar amounts of citric acid (ACS reagent, 99.5%, Sigma-Aldrich, Steinheim, Germany) and 1,8-Octanediol (98%, Aldrich, Steinheim, Germany) were taken in a round bottom flask and heated at 1608C in an oil bath under magnetic stirring and in an inert atmosphere of nitrogen for approximately 30 min. Citric acid, which is the multifunctional monomer, reacted with the diol under these conditions via polycondenzation reaction resulting in a cross-linked polymer. The temperature was subsequently lowered to 1408C and it was kept at the same temperature for an hour to further increase the degree of cross-linking. The structure of the polymer obtained via polycondenzation is shown in Figure 1. The viscous polymer thus formed was used for further experiments. Poly-L-lactic acid-co-caprolactone or [PLCL, 70:30] copolymer was bought from Boehringer Ingelheim (Ingelheim, Germany), and it had a molecular weight of 150 kDa. Solvents such as 1,1,1,3,3,3Hexafluoro-2-propanol (HFP) and ethanol were purchased from Sigma-Aldrich (St. Louis, USA). CellTiter 96 AQueous One Solution assay was purchased from Promega (Madison, USA) and hexamethyldisilazane (HMDS) was purchased from Fluka, Singapore. Dulbecco’s Modified Eagle’s media (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin–ethylenediaminetetraacetic acid (EDTA), pencillin-streptomycin were purchased from Gibco, Singapore.
Scaffold Fabrication POC and PLCL solutions were prepared by mixing definite proportions of both the components overnight at room temperature in HFP. Blended solutions of different amounts of POC: PLCL with a total polymer concentration of 9% (w/v) were prepared with ratios of 10:90, 25:75 and 40:60, during this study. Electrospinning was carried out, by placing the prepared solution in a 5-ml plastic syringe and the positive lead from the power supply was attached to a blunted 27 G stainless steel needle. Further a syringe pump (KD
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Electrospun Composite Scaffolds Scientific, USA) was used to feed the polymer solution into the needle tip and the solution was dispersed at a feed rate of 1.0 ml/h. The voltage applied to the needle tip was about 12 kV DC voltage, from a high-voltage power supplier (Gamma High Voltage Research, Ormond Beach, FL). When a high voltage was applied, the solutions in the syringe were driven by electrostatic force and ejected from the tip of the needle to generate nanofibers. The nanofibers were collected onto grounded 15 mm diameter coverslips or on aluminium sheets, located at a fixed distance of 12 cm from the needle tip. As a control, PLCL solution (9% w/v) was dissolved in HFP and electrospun to obtain PLCL nanofibers.
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After the incubation period, the solution was aliquoted in 96 well plates and read at 490 nm using a spectrophotometric plate reader (FLUOstar OPTIMA BMG Lab Technologies, Germany). The metabolically active cells will react with the tetrazolium salt in MTS reagent to form a soluble formazan dye (absorbance measured at 490 nm). Cells were also seeded on tissue cultured polystyrene (TCP) coverslips as a positive control. All the scaffolds and TCP were used in triplicate, and the experiments were repeated at least three times.
Cell Morphology on the Scaffolds
To determine the morphology and diameter of the fibrous scaffolds, they were viewed under a scanning electron microscope (S-3400N, Hitachi, Japan). Images were acquired at an accelerating voltage of 10-15 kV after gold coating of the scaffolds. The average diameters were determined by analyzing the SEM images using ImageJ software (National Institutes of Health, USA). Means and standard deviations of fiber dimensions were determined from 30 measurements per micrograph and calibration was made with the scale bar on each micrograph. Attenuated total reflectance Fourier transform infrared (ATRFTIR) spectra were obtained with AVATAR 380 FTIR machine (Thermo Electron, Waltham, MA), over a range of 700–3500 cm21 at a resolution of 2 cm21 after averaging 64 scans. The in vitro degradation of the scaffolds was assessed in phosphate buffered saline (PBS), pH 7.4 at 378C after a 4-week soaking period. Prior to taking the SEM, the scaffolds were rinsed in deionized water, dried, and coated with gold.
SEM micrographs were taken for detailed cell morphology studies on different substrates. Cells cultured on the different scaffolds were washed with phosphate buffered saline (PBS) and fixed with 3% glutaraldehyde for 3 h at room temperature. Thereafter, the samples were dehydrated using 50, 75, 90, and 100% ethanol solutions sequentially and dried with HMDS. The samples were sputter coated with gold (JFC-1200 fine coater; JEOL) and observed under SEM at an accelerating voltage of 10–15 kV. Cytoskeletal phenotype of the cells was observed using a confocal laser scanning microscope (CLSM, Olympus 1000V). For this, the cells were cultured for 4 days on the electrospun scaffolds, washed with PBS and fixed with 2% paraformaldehyde. Further the cellscaffold construct was permeabilized with 0.1% Triton X in PBS, stained with 5 lg/ml of phalloidin FITC labeled (Sigma Aldrich, Singapore) and visualized under laser scanning confocal microscope (LSCM). The cell nuclei were stained with 40 ,60 -diamidino-2-phenylindole hydrochloride (DAPI). The migration of cells into the scaffold was further imaged using Imaris software in order to obtain a 3D view of the cell-scaffold construct after staining the cells with phalloidin and (with or without) DAPI.
Mechanical Properties
Statistical Analysis
Scaffold Characterization and Degradation
Tensile tests were carried out using a 3345 MicroTester (Instron, Norwood, MA) equipped with a 10N load cell and 30-mm gauge length. Nanofibers were of rectangular (10 3 20 mm2) in shape and the test was performed at room temperature. The scaffolds were tested at a rate of 5 mm/min. Young’s modulus was calculated from the linear section of the initial slope of the curve obtained for the scaffolds. For each composition, tests were performed for at least five times.
In Vitro Cell Culture H9c2 rat myoblast cells were purchased from ATCC (CRL-1446; American Type Culture Collection, MD), and they were cultured in DMEM containing 4.5 g/l glucose, 4 mM L-glutamine, 25 units/ml penicillin/streptomycin and 10% FBS. Tissue culture incubators were set for 5% CO2 and 378C. When the cells reached 80–90% confluence, they were trypsinized and subcultured at 1:3 ratios. Nanofibers deposited on 15-mm coverslips were washed with PBS and incubated with complete media overnight. Cells were further seeded on the nanofibers at a density of 1 3 104 cells/ cm2 and the cell proliferation on the electrospun fibers was studied by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt] colorimetric method. In brief, after different culturing periods, the cell seeded nanofibers were incubated in DMEM containing 20% cell titer reagent for 3 h.
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Data were analyzed using one way analysis of variance (ANOVA), where ever required, and the statistical significance was tested at P 0.05.
RESULTS Morphology of the Scaffolds Figure 2 shows the morphology of the electrospun PLCL and composite POC/PLCL fibers obtained during this study. The conditions used for the electrospinning process were optimized to obtain uniform bead free fibers, and the fiber diameters of 10:90, 25:75, and 40:60 compositional ratios of POC: PLCL were obtained as 412 6 119 nm [POCPLCL1090]; 451 6 120 nm [POCPLCL2575] and 490 6 88 nm [POCPLCL4060], respectively. On the other hand, electrospun PLCL scaffolds were found to have a fiber diameter of 620 6 97 nm. The total concentration of the polymeric blend used for electrospinning was the same for all the POC/PLCL composite scaffolds. However, we observed a decrease in the fiber diameters for POC/PLCL scaffolds compared to the electrospun PLCL scaffolds. A mixture of two synthetic
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FIGURE 2 Morphology of electrospun scaffolds by SEM (A) PLCL (B) POC/PLCL1090 (C) POC/PLCL2575 (D) POC/PLCL4060.
polymeric solutions produces composite fibers with smaller diameters on electrospinning. Such observation of reduced fiber diameters were also demonstrated by Zhang et al (2010), where they obtained poly(L-lactide)/poly(D-lactide) [PLLA/PDLA] nanofibers of 529 6 104 nm compared with pure PLLA fibers of 761 6 262 nm, and a similar trend was observed in our study also.19
Characterization of Electrospun Scaffolds Figure 3 shows the typical FTIR spectra obtained for electrospun PLCL and POC/PLCL scaffolds. Ester absorption peaks were seen at 1760 cm21 for PLCL scaffolds, whereas the COC stretching vibrations of PLCL were observed at 1097, 1133, and 1187 cm21. FTIR analysis of POC/PLCL scaffold showed broader peaks from 1610–1800 cm21, partic-
FIGURE 3 FTIR spectra of electrospun PLCL and POC/PLCL scaffold.
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FIGURE 4 Degradation of the electrospun scaffolds after 4 weeks period in PBS, evaluated by SEM (A) PLCL (B) POC/PLCL1090 (C) POC/PLCL2575 (D) POC/PLCL4060. *Intertwining of the fibers is indicated by arrows.
ular of the carbonyl (C¼ ¼O) groups. The composite scaffold showed a broader peak at 2916 cm21 in the spectra representative of the methylene groups of the POC polymer. The SEM images of the scaffold after four weeks degradation period is shown in Figure 4. Typically PLCL is viewed as a slow degrading polymer and due to its rigidity pure PLCL scaffolds cannot be applied solely for soft TE. With incorporation of POC in the composite scaffold, the results of the SEM image for 4 week degraded POC/PLCL4060 (Figure 4D) showed intertwining (and clinginess) of the fibers, which was not observed in the SEM images (Figure 4A–4C) of other degraded scaffolds. This could be inferred as the increased degradation behavior of the POC/PLCL4060 scaffold, which was more obvious from the SEM image of POC/PLCL4060 scaffolds after 4 weeks of biodegradation time. Figure 5 shows the results of stress–strain curves obtained for electrospun PLCL and POC/PLCL scaffolds. The tensile strength of the composite POC scaffold were as high as 2.62 6 0.23 MPa (POC/PLCL1090) and their Young’s modulus ranged from 0.51 6 0.10 for POC/PLCL4060 to 1.12 6 0.15 MPa for POC/PLCL1090, while the elongation at break Biopolymers
ranged from 156 to 283%. As shown in Figure 5, the tensile strength of the composite scaffolds decreased from 2.62 6 0.23 MPa (POC/PLCL 1090) to 1.04 6 0.11 MPa (POC/ PLCL 4060), with increasing concentration of POC in the
FIGURE 5 Stress–strain curve of electrospun POC/PLCL scaffolds evaluated by Instron 3345 tensile tester.
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Biocompatibility of POC/PLCL Scaffolds
FIGURE 6 Proliferation of H9c2 cells as determined by MTS assay. *Significant against cell proliferation on PLCL scaffolds at P 0.05.
composite scaffolds. The elongation at break of POC/ PLCL4060 (167%) was more than two times higher compared to virgin PLCL nanofibers (56%), thereby imparting the elastomeric characteristics of POC in composite POC/ PLCL4060 scaffold.
The potential application of POC/PLCL scaffolds with respect to in vitro cardiac TE was evaluated by carrying out cell proliferation on electrospun scaffolds by using H9c2 cardiac myoblast cells. An increase in cell proliferation was observed during the 8-day cell culture compared with cell proliferation on PLCL nanofibers. Results of the colorimetric MTS assay (Figure 6) suggested the nontoxicity of POC containing scaffolds, where by the cardiac myoblasts were found to proliferate significantly higher on the composite POC/ PLCL scaffolds compared to cell proliferation on pure PLCL scaffolds (P 0.05). The cell proliferation on POC/ PLCL2575 and POC/PLCL4060 scaffolds was found 26 and 33% higher, respectively after 8 days of cell culture compared to the cell proliferation on PLCL scaffolds. Such enhanced cell proliferations on POC containing scaffolds could be due to the mechanical cues provided by the elastomeric polymer within these composites, allowing better flexibility for cellscaffold integration. The morphology of the cardiac myoblast cells grown on TCP and POC scaffolds on Day 4 were observed by SEM. Figure 7A–7E shows that the cells attached and spread over
FIGURE 7 Interaction of H9c2 cells on (A) TCP as control; and electrospun scaffolds (B) PLCL (C) POC/PLCL1090 (D) POC/PLCL2575 (E) POC/PLCL4060 observed on Day 4 by SEM.
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FIGURE 8 Cytoskeletal images of H9c2 cells stained by phalloidin FITC on Day 4 on (A) TCP (B) PLCL (C) POC/PLCL1090 (D) POC/PLCL2575 (E) POC/PLCL4060 scaffolds.
the electrospun scaffolds, but higher cell proliferations was observed on POC/PLCL4060 scaffolds. Few numbers of cells were found attached on the POC/PLCL1090 scaffolds (Figure 7C) compared with the cells attached on POC/PLCL2575 scaffolds (Figure 7D). Moreover the cell phenotype was found more organized and aligned on the POC/PLCL4060 scaffolds than on pure PLCL scaffolds and even the TCP. The cell alignment found on POC/PLCL4060 scaffolds could be due to the result of the mechanical cues provided by these scaffolds due to its elastic properties (more suitable for soft tissue regeneration), compared with the stiffer PLCL scaffolds. Cells were found to completely cover the available surface area of the scaffolds that the underlying fibers were less visible on POC/PLCL4060 nanofibers compared to the cell spreading on pure PLCL fibers. The morphology and cytoskeletal architecture of the cells on different electrospun scaffolds were further assessed on Day 4 by staining with phalloidin-FITC and the results are shown in Figure 8A–8E. Cell spreading on the POC containing scaffolds were visible with more alignment of cellular features, with high cell density and organization of actin filaments were observed for POC/PLCL4060 nanofibers. However, this was less observed for pure PLCL and POC/ Biopolymers
PLCL1090 nanofibers. The cell-scaffold construct was also imaged for a three dimensional view of the cells within the scaffolds and Figure 9 shows the image obtained on Day 4 via Imaris software. The flexibility of the elastomeric scaffold allowed for deeper penetration of cells (20 lm) within the POC/PLCL4060 scaffold, compared to cell penetration in PLCL scaffolds (9 lm), calculated using the Imaris software. Biomaterials provide a suitable platform for cell attachment and proliferation, as well as guide the cells for tissue establishment. Designing an elastomeric scaffold with stress–strain responses comparable with the mechanical property of the heart tissue, could favor the best transmission of mechanical cues to the constituent cells and tissues during the process of regeneration. Results of the mechanical properties together with the biocompatibility evaluations showed that the electrospun POC/PLCL4060 nanofibers are promising substrates for regeneration of soft tissues.
DISCUSSION Research have been carried out in identifying the most complex mixture of ECM components required for the repair of any natural tissue, however the mechanical properties of the
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FIGURE 9 Confocal 3D imaging of phalloidin FITC on cell-scaffold constructs using Imaris software on Day 4 on (A) PLCL (B) POC/PLCL1090 (C) POC/PLCL2575 (D) POC/PLCL4060 scaffolds.
materials are always given little priority during tissue repair. Mechanical characteristics of heart tissue are of paramount importance due to the elasticity and beating phenomena of cardiac cells, which specifies the need for a less resistant material that can assist and support the beating cells. Considering the force generating demands of cardiac tissue, we focused on the fabrication of elastomeric scaffolds by electrospinning so as to mimic the nanofibrous architecture of ECM together with the mechanical properties of the cardiac tissue of regeneration. Bioengineered cardiac patches have been developed thus improving the cardiac function, and to support and repair the damaged sites by replacing damaged myocardial tissues.20 Mechanical cues such as scaffold elasticity (elastic modulus of \ 1 MPa) is very important for application of scaffolds in cardiac applications, as the cardiac cells get exposed to longterm mechanical cyclic strains.21–23 A TE scaffold should
allow for the contraction of the growing tissues and withstand the contractions of surrounding myocardium upon implantation, meaning the scaffold should be flexible and not stiff. Moreover, cells rely on mechanical stimuli from the supporting scaffold for feedback on its proliferation. Bioelastomers have great potential as promising biodegradable materials, provided their degradation products are nontoxic and can enter the metabolic cycles of bio-organisms.11,24 However, synthesis of elastomeric materials involved complex synthetic routes and procedures, utilize toxic chemicals, solvents, and involve high cost of production.25–27 In a recent study by Chen et al, low cost and nontoxic precursors such as glycerol and sebacic acid were utilized for the fabrication of new generation elastomers such as poly glycerol sebacate (PGS) and were applied for cardiac TE.28 In this study, we utilized two simple chemicals, namely citric acid and octanediol and performed the poly-condensation reaction to Biopolymers
Electrospun Composite Scaffolds
produce POC polymer networks. Another advantage is the use of citric acid, where it is one of products of the metabolic pathway of human body (through Kreb cycle), involved in the chemical conversion of fats, proteins and carbohydrates into carbondioxide and water. In this study we have chosen citric acid as a monomer to react with the bifunctional monomer to synthesize POC. Electrospinning was chosen as a technique to fabricate scaffolds, because of the ease of method adaptability and ability to produce nanoscale fibers from various natural and synthetic polymers. Moreover, the electrospun fibers can undergo large strains and rotations, and can induce complex behaviors at the macro-specimen scale which cannot be accomplished in other material fabrication methods.29 Electrospinning of pure POC solution was not possible, due to its high viscosity and hence we focused on the fabrication of composite scaffolds with different compositional ratios of POC with PLCL for cardiac TE. It was possible to incorporate as high as 40 wt% of POC with PLCL to electrospin POC/PLCL4060 scaffolds. Previous reports are available on the utilization of novel elastomeric polymer POC for soft TE applications, though the fabrication of electrospun POC polymeric composite scaffolds has not been reported yet.30,31 Among the different elastomeric scaffolds used for cardiac TE, poly glycerol sebacate (PGS) scaffolds are reported to have a tensile strength of 0.50 MPa and Young’s modulus of 0.04–0.28 MPa while poly ester urethane urea (PEUU) scaffolds are demonstrated to have a tensile strength of 0.97–1.64 MPa.28,32,33 POC is an elastomeric polymer that is crosslinked through covalent linkages, and it is creep-resistant and capable of undergoing repetitive mechanical loading too.34 The tensile strength of the POC/PLCL scaffolds decreased with increase in the concentration of POC (from 2.62 MPa for POC/PLCL1090 to 1.04 MPa for POC/PLCL4060) in the composite. The Young’s modulus of electrospun POC/ PLCL4060 scaffold was obtained as 0.51 MPa, much comparable to the Young’s modulus of human myocardium (0.2– 0.5 MPa at the end of diastole). Results suggest that the electrospun POC/PLCL4060 demonstrates the fulfillment of the elasticity requirement of a cardiac graft.28 Therefore, the electrospun POC/PLCL4060 scaffolds might have sufficient elasticity to provide mechanical stimulation for the engineered biografts and provide mechanical strength matching soft tissues.35,32 It is also known that a cardiac construct with suitable mechanical properties can promote the proliferation of cardiac cells by stimulating organized matrix formation.36 Cell-matrix interactions mimicking the native microenvironment with suitable structural and chemical cues are aimed in TE37–39 and hence we blended POC with PLCL to obtain a composite scaffold with suitable mechanical, degradation Biopolymers
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properties for cell attachment and proliferation. POC-based scaffolds prepared by particulate leaching and solvent casting methods has been studied in vitro by Kang et al. for the proliferation of chondrocytes.40 Studies have suggested that myoblast seeded grafts could improve the cardiac function by minimizing adverse ventricular remodeling or wall thinning.41,42 Results by Kutschka et al. (2006) suggested the superior performance of H9c2 cell seeded grafts compared to plain collagen implantations towards the improved function of left ventricle in rat models.2 Results of our cell proliferation together with cell morphology studies show the biocompatibility of POC containing scaffolds for application in cardiac TE. POC films were fabricated by Bastida et al. (2007), where they studied the attachment of HL-1 cardiomyocytes after coating the scaffolds with cell adhesive molecules such as laminin, fibronectin, collagen etc.43 However, such physical coatings with ECM molecules might only assist in the initial attachment of cells, and here we utilized a polymeric blend of POC with PLCL to electrospin and fabricate nanofibers suitable for cell proliferations. The biocompatibility studies on POC containing scaffolds using cardiac myoblasts was found nontoxic, whereby the POC/PLCL4060 scaffolds outperformed the POC/PLCL1090 and POC/PLCL2575 scaffolds in cell proliferation. It is also known that the proliferative myoblast population switches towards a bundle of terminally differentiated myofiber formation and it occurs with the formation of elongated phenotype of the myoblasts.44 Results of the cell morphology by SEM analysis and cytoskeletal organization of myoblasts on the POC/PLCL 2575 and POC/PLCL4060 showed that the cells were more aligned, elongated and probably getting organized towards the formation of myofibers. Our studies demonstrate the feasibility of electrospinning POC containing elastomeric scaffold, together with its application as a suitable substrate for cardiac TE.
CONCLUSIONS Bio-composite scaffolds of poly diol citrates are elastomeric substrates potentially beneficial for soft tissue engineering. The primary goal of this study was to explore the electrospinnability of different compositional ratios of POC and PLCL polymers, and to evaluate the mechanical properties of the electrospun scaffolds. The electrospun POC/PLCL4060 scaffolds was found to have good tensile strength and elastic modulus, comparable to human heart, and could be suitable for providing the necessary mechanical cues required for cardiac tissue regeneration. Moreover, the biocompatibility of the scaffolds was studied using cardiac myoblasts, where the scaffolds were found to attach, proliferate and interact well
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with the POC/PLCL4060 nanofibers. Our study shows that POC containing scaffolds can be an ideal cost-effective solution to fabricate scaffolds for soft TE and other novel biodegradable polydiol citrates can be expected in the future.
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Reviewing Editor: C. Allen Bush
Biopolymers
