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Electrospun Biopolyesters as Drug Screening Platformsfor Corneal KeratocytesPedram Azaria, Ng Sook Luanb, Seng Neon Gana, Rosiyah Yahyaa, Chiow San Wongd, Kien HuiChuab & Belinda Pingguan-Murphyc

a Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysiab Department of Physiology, Faculty of Medicine, University Kebangsaan Malaysia, Jalan RajaMuda Abdul Aziz, Kuala Lumpur, Malaysiac Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, KualaLumpur, Malaysiad Plasma Technology Research Center, Physics Department, Faculty of Science, University ofMalaya, Kuala Lumpur, MalaysiaPublished online: 21 May 2015.

To cite this article: Pedram Azari, Ng Sook Luan, Seng Neon Gan, Rosiyah Yahya, Chiow San Wong, Kien Hui Chua & BelindaPingguan-Murphy (2015) Electrospun Biopolyesters as Drug Screening Platforms for Corneal Keratocytes, International Journalof Polymeric Materials and Polymeric Biomaterials, 64:15, 785-791, DOI: 10.1080/00914037.2015.1030648

To link to this article: http://dx.doi.org/10.1080/00914037.2015.1030648

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Electrospun Biopolyesters as Drug Screening Platforms forCorneal Keratocytes

PEDRAM AZARI1, NG SOOK LUAN2, SENG NEON GAN1, ROSIYAH YAHYA1, CHIOW SAN WONG4,KIEN HUI CHUA2, and BELINDA PINGGUAN-MURPHY3

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia2Department of Physiology, Faculty of Medicine, University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia3Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia4Plasma Technology Research Center, Physics Department, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

Received 3 October 2014, Accepted 14 March 2015

In vitro drug screening techniques provide rapid and easy to analyze data, while saving a lot of animals from being sacrificed. Animportant part of any in vitro drug screening platform is a biomaterial which promotes cell growth and proliferation. The potentialof electrospun scaffolds made of polyhydroxybutyrate (PHB), poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV), andpolycaprolactone (PCL) were studied to serve as drug screening platform for corneal keratocyte tissues. The results showed that theproliferation rate was slightly higher for PCL and PHBV on day 7. Gene expression results showed that PCL was the best inmaintaining keratocyte genes.

Keywords: Biopolyesters, corneal keratocytes, electrospinning, polycaprolactone, polyhydroxybutyrate, polyhydroxybutyrate-co-hydroxyvalerate

1. Introduction

Infection, trauma, viral, and atopic disease as well as geneticdeficiencies are just some of the causes for corneal functiondisorders [1,2] that lead to corneal blindness of more than45 million individuals all around the world annually [3].Although mild injuries can be cured by the natural immune

system response or via clinically available techniques, severecases still await the development of novel drugs [4]. In orderto assess the toxicity of any novel treatment, animal exper-imentation is needed. Besides being costly, animal experimen-tation requires a large number of animals to be sacrificed [5].As an alternative to animal tests, in vitro drug screening couldbe considered, a method that has the benefits of ease of appli-cation, simplicity of testing, and straightforward datainterpretation as well as not involving animals. It providesrapid and cost-effective screening with good sensitivity [6].Although accurate extrapolation of the in vitro results toin vivo conditions remains challenging, it can still be very use-ful for preliminary stages of studies and saves a lot of animals.

Address correspondence to: Belinda Pingguan-Murphy,Department of Biomedical Engineering, Faculty of Engineer-ing, University of Malaya, 50603 Kuala Lumpur, Malaysia.E-mail: bpingguan@gmail.com

Color versions of one or more of the figures in the articlecan be found online at www.tandfonline.com/gpom.

International Journal of Polymeric Materials and Polymeric Biomaterials, 64: 785–791

Copyright # 2015 Taylor & Francis Group, LLC

ISSN: 0091-4037 print/1563-535X online

DOI: 10.1080/00914037.2015.1030648

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In order to undertake such in vitro drug testing forapplication to the cornea it is necessary to have a reliablescaffold material capable of promoting and maintainingthe growth of keratocytes, the cells which make up about90% of the volume of corneal stroma, being bonded to anextracellular matrix of highly regular collageneous lamella.

In this research, we have developed electrospun scaffoldsfor this purpose from three commercially available biopoly-esters and studied their potential to promote corneal kerato-cyte growth and characteristics.

Notably, keratocytes usually stay in the quiescent stateand maintain noncrystalline structures to make the cornealtransparent and have optimal refraction. However, theyare able to undergo transition into repair phenotypes,namely activated fibroblasts or myofibroblasts in respondto specific environmental signals, example injury and woundhealing [7]. It is important for keratocyte to differentiatebecause it helps in retaining corneal transparency and deter-mining the corneal response to injury [8]. This clearly estab-lished that keratocytes are sensitive to the changes inenvironmental condition, and therefore understanding thebehaviors of keratocytes in different testing conditions willgreatly increase our knowledge in corneal wound healingand regeneration.

Concerning the use of biopolyesters, it is notable thatthese have been used previously as cell carriers for biomedi-cal applications, suggesting a possible application here aswell [9–12]. Advantageously, these polymers have a low costof synthesis, degrade easily through hydrolysis of esterbonds, and produce nontoxic degradation products [13,14].Production of scaffold is via electrospinning technology,which allows fabrication of a controllable fibrous scaffoldsimilar to the natural extracellular matrix (ECM) [15,16],in a simple, versatile, and cost-effective manner. In the elec-trospinning process micro and nano fibers are produced bystretching a polymeric solution by high electric field[17,18]. There are a number of processing parameters thatcan affect the morphology of the fibers, which include thechemical nature of the polymer, solvent, applied voltage,spinneret to collector distance, feeding rate, and capillarydiameter. Based on the applied parameters the morphologycan be varied to produce uniform fibers, beaded fibers, andfibers with spindles on string [19]. In this comparative studywe have subjected three different biopolyesters to the sameelectrospinning parameters and obtained different morphol-ogies, presumably due to the different chemical natures ofthe polymers. The potential of these materials in terms ofcorneal keratocyte proliferation and differentiation werethen investigated.

2. Experimental

2.1 Chemicals

Poly[(R)-3-hydroxybutyric acid] (PHB), poly(3-hydroxy-butyric acid-co-3-hydroxyvaleric acid) (PHBV), andpolycaprolactone (Mn¼ 80000 g=mol) (PCL) were pur-chased from Aldrich. F12: DMEM (FD), antibiotic-antimycotic, trypsin-EDTA glutamax, and fetal bovine

serum were purchased from Gibco Invitrogen, USA. 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromidesolution was purchased from Sigma Aldrich USA. VitaminC, dimethyl sulfoxide, paraformaldehyde, chloroform,dimethylformamide (DMF), and phosphate buffer salinewere purchased from Merck Millipore, Germany. Alamarblue solution was purchased from Life Technologies, USA.TRI Reagent and polyacryl carrier were purchased fromMolecular Research Center, USA. DNAse- and RNAse-free distilled water and SuperScript III First-StrandSynthesis SuperMix kit were purchased from Invitrogen,USA.

2.2 Electrospinning

For ease of comparison the same parameters were applied toPHB, PHBV, and PCL. A 10% (wt=v) solution of polymerwas prepared in a co-solvent mixture comprising of chloro-form and DMF with a ratio of 9:1. The solution was stirredat 60�C in a round bottom flask fitted with a condenser for5 h to reach homogeneity. The solution was loaded into a20 mL syringe (Terumo) placed 18 cm away from the collec-tor horizontally. The feeding rate of the syringe pump wasset at 3 mL=h. A semi rectified DC voltage of 12 kV was gen-erated from a custom-made high voltage power supply toconduct electrospinning.

2.3 Field Emission Scanning Electron Microscopy

The morphology of the electrospun fibers and cells werestudied by using field emission scanning electron microscopy(FEI: QUANTA FEG 250). Average diameter of the fibersfor each sample was measured using ImageJ software. Foreach sample 25 measurements was carried out at differentrandom spots. Fiber intersections and beads on fibers wereexcluded from measurements. For samples seeded with cells,cell fixation was carried out prior to observation. Cells werefixed with a 4% paraformaldehyde after removal of the cul-ture medium followed by washing in PBS. Field emissionscanning electron microscopy was conducted without apply-ing any coating on the samples. The samples were studied ondays 1, 3, 5, and 7.

2.4 Cell Culture

Six New Zealand white rabbits’ corneal tissues wereobtained from the local animal slaughterhouse. The cornealtissues were processed using the techniques reported byGhafar et al. [20]. The use of corneal cells for research pur-pose was approved by the Universiti Kebangsaan Malaysiaanimal ethic committee with approval number of: FF-092-2012. Keratocytes were harvested from the cornealstroma with 0.3% collagenase type I digestion as mentionedin a recent paper [4]. Cell cultures were maintained in F12:DMEM (FD) media supplemented with 10% fetal bovineserum (Gibco Invitrogen, USA), 1% antibiotic-antimycotic,1% glutamax, and 1% vitamin C under standard incubationat 37�C, 5% CO2, and 95% humidity. Primary culture (P0)was passaged at a split ratio of 1:4 when cells reached

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approximately 90% confluence using 0.125% trypsin-EDTA.Passage 1 culture was used in the study.

2.5 Leachate Preparation

Leachate was prepared at a surface-area-to-extractant vol-ume ratio based on ISO 10993–12. The ratio was 120 cm2

per 20 mL extraction vehicle when the material thickness isless than or equal to 0.5 mm. The scaffolds (PCL, PHB,and PHBV) with size 2.27 cm2 were prepared and disinfectedwith 70% ethanol. Twenty scaffolds were rinsed with PBS1X thrice and incubated in FD media at 37�C and 5%CO2 for 3 days.

2.6 Cell Viability and Cytotoxicity Test

Cytotoxicity of scaffold leachate was assessed via 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)assay. Passage 1 keratocytes with the seeding density of50,000 cells=mL were seeded onto 96-well culture plateovernight. The keratocytes were treated with 10 leachate con-centrations using a dilution factor of 10 in FD media for72 h. MTT solution was added and incubated for 4 h at 37�Cand 5% CO2. Dimethylsulfoxide (DMSO) was added to dis-solve the purple formazan before reading its absorbance atwavelength 570 nm.

2.7 Cell Proliferation Assay

The Alamar Blue assay was used to measure the cell prolifer-ation continuously since the cells remained viable afterperforming the cytotoxicity test. Passage 1 keratocytes witha seeding density of 3.5� 104 cells were seeded onto thescaffolds (PCL, PHB and PHBV) respectively for 4 hours.Scaffolds were rinsed with PBS X1 in order to remove theunattached cells, then the scaffolds were transferred intonew culture wells with 2 mL FDþ 10%FBS medium. Onday 1 Alamar Blue assay, the culture media were removedand substituted with 550 lL medium containing 10% Ala-mar blue. After 4 h incubation at 37�C and 5% CO2, 200lL of medium was aspirated and read using ELISA micro-plate reader with wavelength 570 nm. The Alamar Blue testwas repeated for cultures for days 3, 5, and 7. On day 7, thekeratocytes cultured on the scaffolds were harvested usingTrypsin-EDTA 0.125% after the Alamar Blue assay. Kerato-cytes were centrifuged and the pellet was preserved usingTRI Reagent (Molecular Research Center, Cincinnati,USA) for total RNA extraction. A standard curve wasplotted based on the Alamar blue absorbance reading inserial dilutions of cell number to obtain an equation, andto calculate the number of viable cells.

2.8 Total RNA Extraction and Two-Step ReverseTranscriptase Polymerase Chain Reaction (RT-PCR)

Total RNA extraction was carried out according to the man-ufacturer’s protocol. Polyacryl carrier (Molecular ResearchCenter, USA) was used for total RNA precipitation. Thetotal RNA pellet was washed with 75% ethanol and air driedbefore dissolving in DNAse- and RNAse- free distilled water

(Invitrogen, Carlsbad, CA, USA). The purity and concen-tration of extracted total RNA was determined by Nano-drop ND-100 spectrophotometer (Wilmington, DE, USA)and stored at� 80�C for further use. The synthesis of comp-lementary DNA was performed using SuperScript IIIFirst-Strand Synthesis SuperMix kit (Invitrogen) accordingto the manufacturer’s instruction. The reverse transcriptionwas carried out at 50�C for 30 min. The forward and reverseprimers used in the quantitative RT-PCR were designed fromthe sequences obtained in NIH Genbank database usingPrimer-3 software as published in [4]. Glycerylaldehyde-3--phospate dehydrogenase (GAPDH) was used as housekeep-ing gene for the data normalisation. Two-step RT-PCR wascarried out using iQSYBR Supermix in Bio-Rad iCycler(Bio-Rad, USA). The protocol conditions were initiatedwith the activation of Taq DNA polymerase at 94�C for3 min, followed by 45 cycles of PCR amplification at 94�Cfor 10 s and 60�C for 30 s and then melting curve analysis.The specificity and size of PCR products were confirmedwith 2% agarose gel electrophoresis.

2.9 Statistical Analysis

Quantitative data were obtained for six samples and testedfor statistical significance using Statistical package for SocialSciences (SPSS) version 20. The results were presented ingroups as mean �standard error of mean (SEM). One-wayanalysis of variance (ANOVA) was performed in normallydistributed data, while Kruskal-Wallis test was used as non-parametric test. Post hoc tests were Tukey HSD andMann-Whitney. P< 0.05 was considered to be statisticallysignificant.

3. Results and Discussion

3.1 FESEM

Figure 1 shows a microscopic view of blank electrospun scaf-folds. As the image suggests, PCL leads to the formation offibers with a smaller diameter than those of PHB and PHBVunder the same conditions. PCL, due to its lower molecularweight, resulted in less viscose solution with less chain entan-glement (Figure 1a). Therefore, during electrospinning itcould be stretched to a much lower diameter. However, alower viscosity could result in less stable jet, with formationof more random fibers, and formation of beads within theelectrospun fibers [21]. In agreement with this, fiber diameterdistribution patterns of the fibers (Figure 1) confirm PCLexhibits the lowest diameter, at 300 nm, followed by PHBat 1.12 mm and PHBV at 1.35 mm. PHB, comparatively, ismore rigid and stiff because of its high crystallinity, formedmore straight fibers [22] (Figure 1b). As a result of the incor-poration of hydroxyvalerate into PHBV, the polymer is alsomore elastic. PHBV fibers show good uniformity as well asporosity, and have a looped shape rather than just beingstraight.

Figure 2 shows cell morphology and cell to scaffold inter-action at four different days: 1, 3, 5, and 7. The SEM imagesof cells show cells growing on the electrospun scaffolds.

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These cells can spread and penetrate into the fibers. The filo-pedia extend along the fibers on all the scaffolds, as guided bythe orientation of the fibers. In PHB scaffolds, the micro-graphs showed the cells were larger, flatter and stretched.

This latter observation may indicate higher expression of a-SMA2 on PHB. During corneal wound healing, keratocytesare activated and transformed into fibroblasts and eventuallybecome alpha-smooth muscle actin (a -SMA)-expressed

Fig. 2. FESEM images showing the morphology of keratocytes cultured on PCL (a–d) days 1–7, PHB (e–h) days 1–7, and PHBV(j–n) days 1-7 with magnifications of 2000�.

Fig. 1. FESEM microscopy of the blank scaffolds (a) PCL, (b) PHB, (c) PHBV, and their relevant fiber diameter distributionpattern.

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myofibroblasts and contribute to the wound contraction [23].The micrographs demonstrated that the diameter and align-ment of fibers in scaffolds could guide cytoskeleton extensionof cells and led to different levels of gene expression, asshown in q-PCR results. These findings also showed thatPCL fiber of a smaller diameter has produced a hierarchicallyordered scaffold closely imitating that of native collagenfibers. The micrographs clearly showed that the changes incell morphology depended on the type of scaffold on whichit was seeded. The cells on the PCL fiber tend to be bulkierand more on the surface mainly due to the smaller pore size.The cells on PHBV show a planar morphology. Cell pen-etration inside the scaffold also could be observed on PHBV.

3.2 Cell Viability and Cytotoxicity

Figure 3 shows the average MTT activity of the scaffolds(n¼ 5) at 10 different concentrations of leachate solution.

As can be seen, none of the scaffolds show signs of inhibitionof cell proliferation, and the MTT activity for all scaffolds atdifferent concentrations of leachate solution remains posi-tive. The concerns over solvent residue in the electrospunfibers can be safely dismissed.

3.3 Cell Proliferation Assay

Keratocyte interactions between different scaffolds wereassessed in vitro by seeding cells in PCL, PHB, and PHBVat various time points. Figure 4 shows average keratocyteproliferation measurements on different days and scaffolds(n¼ 6), based on the Alamar Blue proliferation assay. Thiscell population shows an ascending trend on PCL, PHBand PHBV, and all the scaffolds support keratocyte prolifer-ation. The increase in the number of cells on PCL was signifi-cant for all days. However, for PHB and PHBV this increase

Fig. 3. Keratocytes’ proliferation in several dilutions of scaffoldleachate.

Fig. 4. Alamar Blue proliferation assay results for keratocytesseeded in scaffold (PCL, PHB, and PHBV) and time point.The number of keratocytes increased on PCL, PHB, and PHBVas a function of time, however, there is no significant differencefor days 5 and 7 for PHB and PHBV (P> 0.05). No significancedifference in cell numbers was observed among the differentscaffolds, except on day 5, PHB and PHBV exhibit higher cellnumbers compare to PCL and it is statistically significant(P< 0.05). �Significant difference.

Fig. 5. Alamar Blue proliferation assay plotted separately foreach individual scaffold (a) PCL, (b) PHB, and (c) PHBV) ondifferent days. The number of keratocytes shows that it signifi-cantly increased on PCL over the time. For PHB and PHBVexcept for days 5 and 7 the increase in the number of kerato-cytes is significant for the rest of the days (P< 0.05).#Significant difference.

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was significant until day 5 and there was no significantdifference between day 5 and day 7 (Figure 5). The results sug-gest that the cells may have reached confluency after day 5 onPHB and PHBV. The keratocytes have reached confluence byday 7 in all scaffolds, although the comparison betweengroups showed some differences on day 5 (Figure 4). Overall,the results suggested that the cell proliferation rates on PCL,PHB, and PHBV were almost the same.

3.4 Real-Time PCR

Figure 5 shows gene expression for five targeted genes;lumican (LUM), aldehyde dehyrogenase ALDH), vimentin(VIM), collagen type 1 (COL.1), and a -SMA2. PCL showsgood potential for maintaining cell phenotype with highexpression of LUM, ALDH, VIM, COL.1, and a -SMA2.All these genes were significantly higher in expression onPCL, compared to PHBV. Among the three scaffolds,PCL expressed the highest LUM. LUM, a small leucine-richrepeat proteoglycan (SLRP) is a major keratan sulfate pro-teoglycan in maintaining corneal transparency [24]. LUMalso regulates collagen fibrillogenesis and modulates cornealepithelial cell migration [25]. Meanwhile, ALDH is abun-dantly expressed by corneal stromal keratocytes in PCL aswell. ALDH is responsible for the production of cornealcrystallins, which contributes to cellular transparency [26].The high level of expression of a -SMA2 in PHB suggeststhat this scaffold is good for wound healing. However, overexpression of a -SMA2 may result in corneal scar formationand affects optical transparency [27]. It is postulated that thehigher expression of a -SMA2 is the result of cell stretchingover the PHB sample. Expression of vimentin (VIM), amesenchymal cell marker characterized as a fibroblast, is

important to show the transition of keratocytes to activatedfibroblasts, providing structural properties of cell andenhancing the cell migration in the corneal wound healing[28]. COL.1 is the main extracellular matrix in cornealstroma, while the well-organized collagen 1 fibrils are neces-sary to regulate corneal transparency [29]. Overall, PCL wasshown to be the best option as a scaffold from those tested,as it is able to maintain corneal stromal cell functionalitybetter than PHB or PHBV

4. Conclusion

The electro-spinning technique can produce fibers that areable to support keratocyte growth and proliferation. All ofthe scaffolds are capable of promoting corneal keratocytesattachment and growth. There is a preference for up-regulation of gene expression when PCL scaffolds are used,including an up-regulation of LUM, ALDH, VIM, andCOL.1. We believe this is due to the similarity of PCL fiberorientation to native collagen in extracellular matrix interms of fiber morphology and diameter. PHB showed a bet-ter a -SMA2 expression, which makes it a suitable material forcorneal wound healing applications. This study shows that theselection of biopolymer for scaffolding material can have animportant influence upon gene expression, and requires care-ful consideration in the design of drug-screening technologies.

Funding

This study was supported by High Impact Research Grant(UM.C=HIR=MOHE=ENG=44) from the Ministry of Edu-cation Malaysia and Postgraduate Research Grant (PPP)(PV-107-2012A), University of Malaya.

Fig. 6. Quantitative gene expression of cultured corneal keratocytes. The expression values for lumican (LUM), a -SMA2, ALDH,collagen type 1 (COL.1), and vimentin (VIM) relative to the expression values of GAPDH as the internal control. Higherexpression of LUM in PCL scaffold compare to PHB and PHBV. �Significant difference among PCL, PHB, and PHBV (P< 0.05).

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