Materials Science and Engineering C 60 (2016) 143–150

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Materials Science and Engineering C

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Fabrication of novel high performance ductile poly(lactic acid) nanofiberscaffold coated with poly(vinyl alcohol) for tissueengineering applications

Abdalla Abdal-hay a,⁎, Kamal Hany Hussein b, Luca Casettari c,Khalil Abdelrazek Khalil d,e,⁎⁎, Abdel Salam Hamdy f

a Dept of Engineering Materials and Mechanical Design, Faculty of Engineering, South Valley of University, Qena 83523, Egyptb Stem Cell Institute and College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Koreac Department of Biomolecular Sciences, University of Urbino, Piazza Rinascimento, 6, Urbino, PU 61029, Italyd Dept. of Mechanical Engineering, College of Engineering, King Saud University, 800, Riyadh 11421, Saudi Arabiae Dept. of Mechanical Engineering, Faculty of Energy Engineering, Aswan University, Aswan, Egyptf Dept. of Manufacturing and Industrial Engineering, College of Engineering and Computer Science, University of Texas Rio Grande Valley, 1201West University Dr., Edinburg, TX 78541-2999, USA

⁎ Corresponding author.⁎⁎ Correspondence to: K.A. Khali, Dept. of MechanEngineering, King Saud University, P.O. Box 800, Riyadh 1

E-mail address: abda_55@jbnu.ac.kr (A. Abdal-hay).

http://dx.doi.org/10.1016/j.msec.2015.11.0240928-4931/© 2015 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 May 2015Received in revised form 3 October 2015Accepted 8 November 2015Available online 10 November 2015

Poly(lactic acid) (PLA) nanofiber scaffold has received increasing interest as a promising material for potentialapplication in the field of regenerative medicine. However, the low hydrophilicity and poor ductility restrict itspractical application. Integration of hydrophilic elastic polymer onto the surface of the nanofiber scaffold mayhelp to overcome the drawbacks of PLAmaterial. Herein, we successfully optimized the parameters for in situ de-position of poly(vinyl alcohol), (PVA) onto post-electrospun PLA nanofibers using a simple hydrothermal ap-proach. Our results showed that the average fiber diameter of coated nanofiber mat is about 1265 ± 222 nm,which is remarkably higher than its pristine counterpart (650 ± 180 nm). The hydrophilicity of PLA nanofiberscaffold coated with a PVA thin layer improved dramatically (36.11 ± 1.5°) compared to that of pristine PLA(119.7 ± 1.5°) scaffold. The mechanical testing showed that the PLA nanofiber scaffold could be convertedfrom rigid to ductile with enhanced tensile strength, due to maximizing the hydrogen bond interaction duringthe heat treatment and in the presence of PVA. Cytocompatibility performance of the pristine and coated PLA fi-bers with PVA was observed through an in vitro experiment based on cell attachment and the MTT assay byEA.hy926 human endothelial cells. The cytocompatibility results showed that human cells induced more favor-able attachment and proliferation behavior on hydrophilic PLA composite scaffold than that of pristine PLA.Hence, PVA coating resulted in an increase in initial human cell attachment and proliferation. We believe thatthe novel PVA-coated PLA nanofiber scaffold developed in this study, could be a promising high performance bio-material in regeneration medicine.

© 2015 Published by Elsevier B.V.

Keywords:Poly lactic acidPoly vinyl alcoholTissue regenerationHydrothermal depositionNanofiber scaffoldsCytocompatibilityBiodegradable synthetic polymers

1. Introduction

Biodegradable synthetic polymer fibers have attractedmassive inter-est as effective substitute materials in regeneration medicine applica-tions [1,2]. Specifically, poly(lactic acid) (PLA) is one of the mostwidely used synthetic polymers in this field due to the non-toxicity oflactic acid, which is naturally present in the human body, and FDA-approved [3–6]. However, there are some major limitations such as thehydrophobic nature and poor ductility of PLA which hinder its practical

ical Engineering, College of1421, Saudi Arabia.

use as substitute materials in tissue regeneration [3]. It is known thatthe surface wettability reflects the adhesion, growth of cells, and proteinabsorption on the surface of thematerial [3,6]. Some researchers noticedthat the porous scaffolds fabricated from PLA are floating in cell culturemedium [7]. Thus, the hydrophobic nature of PLA is a serious problemin a predominantly hydrophilic bioenvironment where the cells fail tohave initial attachment to the implanted scaffolds.

Mechanical properties play a crucial role in determining the in vivoperformance of the scaffolds in the tissue engineering field, such as vas-cular graft system [4], bone implants, and wound dressing [8]. The scaf-fold has to be composed of a durable biomaterial capable ofwithstanding physiological hemodynamic forces while maintainingstructural integrity until mature tissue forms in vivo.

Electrospun nanofibers represent an emerging class of biomimeticnanostructures that can act as proxies of the native tissue, while

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providing topographical and biochemical cues that promote tissuehealing. Development of advanced nanofiber scaffold in the secondhalf of the 20th century resulted in a marked revolution in advancedstructural materials [9] mainly because of the brittleness characteristicsof PLA nanofibers as a potential scaffold material [4,10,11].

Many efforts have been invested to address the aforementioned lim-itations of PLA nanofibers. For instance, poly blend nanofibers preparedby simple pre-mixing solution or melt-blending of PLA with organicpolymers [4,11–15] such as thermoplastic starch (TPS) were reported[14]. However, microscopic observations revealed non-uniform dis-persed PLA inclusions within the TPS matrix. Pitarresi et al. [16], pro-duced electrospun nanofiber scaffolds by employing PHEA-g-PLAcopolymer as a starting material. However, these scaffolds didn'tprovide significant biocompatible properties. Pi et al. [15], noticed theoccurrence of microphase separation in the coating layer through thefilm drying because of the dissimilarity of PLA and polyethylene oxide(PEO). This is because PEO is water soluble, while PLA is water-insoluble. Therefore, the preparation of wettable and ductile PLAscaffold using a facile approach for biomedical applications remains achallenge. As a result, changing the mechanical properties of post-electrospun PLA nanofibers from brittle/rigid to ductile using a simple,cost-effective approach is one of the main challenges in the presentstudy. Successful development of a hydrophilic and ductile PLA nano-fiber scaffold will open a new era towards the designing of highperformance advanced biomaterials in the field of tissue engineering.In this paper, we successfully developed a facile strategy that is basedon a surface modification of the post-electrospun (PE) PLA nanofibersthrough deposition of a hydrophilic and elastic layer on each singlePLA nanofiber using a simple hydrothermal route. The novelties of thispaper are: a) optimizing of a simple and cost-effective hydrothermalprocess for fabrication of a novel PLA based-scaffold with unique mor-phology and mechanical and biological properties; b) overcoming thelacks of pristine PLA; and c) introducing the amine groups in the fibersfor improving the cell adhesion and proliferation in tissue engineering.

Recently, our research group has successfully exploited a simple andinexpensive hydrothermal strategy for in situ deposition of inorganiccompounds onto PE nanofibers and creating advanced high perfor-mance 3D composite scaffolds formedical implants [17,18]. Interesting-ly, our process has no side effect on the properties of the polymer fibers.Furthermore, it provides sufficient interfacial bonding between thepolymer fibers and deposited compounds, and subsequently improvesthe mechanical properties of the scaffold. This novel process has beensuccessfully used by our group to apply an in situ deposition of a wetta-ble and elastic polymer thin layer on PE PLA nanofiber.

In this study, poly(vinyl alcohol) (PVA) was selected to conformalcoat each single PLA nanofiber because it is a water-soluble syntheticpolymer that possesses good biocompatibility, biodegradability, and ex-cellent mechanical properties [4]. PVA, in general, has a highwater con-tent and tissue-like elasticity. The abundant hydroxyl groups on PVAcan be readily modified to attach growth factors and adhesion proteins.We speculate that the hydrophilic properties of PVAmolecules can cre-ate a good physical/chemical interaction with PLA nanofibers on themolecular level by forming strong hydrogen bonding throughout a hy-drothermal treatment, and thereby enhancing the surface andmechan-ical properties of the PLA nanofibers. It is worthmentioning thatexploiting our strategy can overcome the difficulty of mixing both PLAand PVA phases at the macromolecular level because PVA is more hy-drophilic than PLA. Hence, the composite PVA/PLA nanofibrous scaffoldthat integrates the favorable wettability properties of PVA and elasticityas well as FDA approval of PLA is expected to significantly improve thematerial properties for tissue regeneration applications. Additionally,the cytocompatibility performance of the PLA nanofiber scaffold coatedwith a hydrophilic PVA thin layer using hydrothermal strategy wasstudied using the MTT test. The molecular interactions between PLAfiber and PVA molecules during the hydrothermal process werediscussed in detail.

2. Experimental

2.1. Fabrication of nanofibers

The electrospinning setting used in the current research for fabrica-tion of nanofibers and PLA pellets is a type of Ingeo Biopolymer 2003D,(a Nature Works, LLC (USA) product supplied by Green Chemical Co.,Ltd., Korea) as described in detail in our previous report [19]. Briefly,the solution for electrospinning was prepared by dissolving PLAin dichloromethane (Junsei Chemical Co., Japan) at a concentration of10 wt.%. The injection rate and the applied voltage were 0.5 ml/h and18 kV, respectively. The collected nanofibermatwas placed in a vacuumoven for 24 h at 40 °C to remove any potential residual solvents.

A PVA-coated PLA electrospun mat was prepared following theseprocedures: (1) the PLA as-electrospun mat was cut into rectangularspecimens (30 × 20 mm2). (2) These specimens were immersed intooptimized freshly prepared 1 wt.% PVA aqueous solution (it was ob-served that increasing the PVA solution concentration N1 wt.%, affectsnegatively the surface morphology of the nanofiber mats as shown inFig. S1 of the supplementary materials). PVA solution viscosity at1 wt.% was measured by a Brookfield, DV-III ultra programmable Rhe-ometer at room temperature and the result was about 17 cP.(3) 40 ml of PVA solution containing the PLA electrospun nanofibermat was placed in a Teflon-lined autoclave container and heat-treatedat 150 °C for 30 min. Previous studies reported that PVA and PLA poly-mers have good thermal resistivity up to this temperature [20,21].(4) After the reaction process is complete, the PVA-coated PLA matswere gently rinsed in distilled water to remove unattached PVA mole-cules from PLA nanofiber mats. (5) The resultant coated samples wereleft to dry at room temperature for four days until steady weight andthen introduced into a vacuum oven (10 mbar) at 35 °C for 72 h.(6) The amount of PVA adhering to the surface was evaluated fromthe dry weight of the substrates.

2.2. Surface/material characterizations

The surface micrographs of the pristine and composite mats werecharacterized by a field emission scanning electron microscope(FESEM; Hitachi S-7400, Japan). Themicrographs of the coated sampleswere taken at an accelerating voltage of 5 kV andwithmagnifications of5 and 25 K. To measure the fiber diameters, the FESEM images wereprocessed and analyzed by means of ImageJ software (National Insti-tutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/).FTIR (MB100 spectrometer, Bomen, Canada) analysis in a transmissionmode was used to identify the functional groups thereby reflectingphase structure of pristine and hydrothermally treated samples. Ther-mogravimetric analysis (TGA) was performed by a TGA-DSC, Q-20Perkin-Elmer Inc., USA, at a heating rate of 20 °C/min with a constantpurge of N2. Differential scanning calorimetry (DSC) datawere obtainedfrom a Perkin-Elmer Pyris Diamond DSC. Samples were scanned at aheating rate of 10 °C/min in N2 environment. The Tg values weremeasured as the change of the specific heat in the heat flow curves.

2.3. Surface wettability measurements

Flat mats were used to evaluate the hydrophilicity of pristine PLAand PVA/PLA composite nanofiber (treated) mats, using the water con-tact angle (WCA) measurements. 3 μl of purified water (ultrapuregrade) was pipetted out on top of the shiny side of 30 × 30 mm2 matspositioned on the stage of a bench-type contact angle goniometer(GBX; Digidrop, France), ensuring that the membrane mat wascompletely flat. The micrographs were taken after 1 s and WCA mea-surement was recorded. To confirm the coating homogeneity and coat-ing distribution of a PVA layer on the PLAmembranemat, theWCAwasmeasured at five different positions on each flat mat surface.

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The PVA/PLA composite scaffold was compared to the pristine PLAscaffold for their wettability or absorbability of purifiedwater. The puri-fied water was dropped on top of flat mats and the time required forcomplete absorption of the water into the scaffold was estimated tosee whether it is absorbable or un-absorbable. The flat mat was gentlystretched and fixed on a standard microscope glass slide (dimensionsapprox. 76 × 26 mm for glass slide). Each prepared sample wasmeasured three times, and the average value was recorded.

2.4. Mechanical properties

Pristine and treated PLAmats were subjected to stress–strain analy-sis using an Instron universal tester model: LLOYD instruments, LR5Kplus, UK. The samples were trimmed into a “dogbone” (see inset ofFig. 3) with offset ends via die cutting from the as-obtained mats to re-duce grip effects according to ASTMD-638. Testingwas conducted withthe tissue grips moving at a rate of 10 mm/min. The applied load wasconducted until the specimen experienced complete failure. The speci-men thicknesses were measured using a digital micrometer with a pre-cision of 1 μm (coating thickness gauge OMEGA instrument, OM179-745). The tensilemoduluswas calculated as the slope of the initial linearportion of the stress–strain curve. The data acquisition rate was set to20.0 Hz. Four membrane mat samples of each group were subjected totensile testing at room temperature. The data presentedwere expressedas the mean ± standard deviation. Statistical analysis was performedusing Student's t-test, and a p-value less than 0.05 was consideredsignificant.

2.5. Cytocompatibility studies (cell attachment and MTT assay)

To investigate the cell adhesion on the surfaces of as-prepared pris-tine PLA and treated PLA with PVA thin coated layer membrane sam-ples, the pristine PLA and PLA/PVA hybrid scaffolds were placed andfixed in the bottom of each well of a 24-well plate and then sterilizedwith ethylene oxide (ETO) gas for 24 h at room temperature. Subse-quently, EA.hy926 endothelial cells (passages 4–5) from AmericanType Culture Collection (ATCC) were then seeded onto the surfaces ofthe membrane samples at a density of 30 × 103 (300 μl of cell suspen-sion) and cultured with Dulbecco's Modified Eagle's Medium (DMEM;Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovineserum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin(P/S; Gibco, Grand Island, NY, USA) in a humidified incubator at 37 °Cand 5% CO2. Prior to cell culture on material surface, at 70% confluency,the cells were harvested by trypsinization and used for experiments.Samples containing cells were taken out after incubating the plates for3 and7days, rinsed twicewith phosphate-buffered solution (PBS) to re-move the non-attached cells, and subsequently fixedwith 2.5% glutaral-dehyde for 2 h at 4 °C overnight. Then, the sampleswere rinsed in 0.1MPBS and transferred to the critical-point dryer and dried with CO2. Thedried samples were sputtered with a thin layer of gold for observationof cell morphology using low voltage Bio-SEM (Hitachi, S-3000N,Japan) at an acceleration voltage of 10 kV.

To investigate cell proliferation, the attached cells viability (celldensity) on the pristine and hybridmembrane scaffold sampleswas de-termined by theMTT (3-[4,-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-lium bromide) assay. The MTT was prepared in phosphate bufferedsaline (PBS) at a final concentration of 5 mg/ml. For the assay, the scaf-folds were fixed in a 24-well cell culture plate and were sterilized withethylene oxide (ETO) steam for 24 h, using DMEM supplemented with10% FBS and 1% P/S, then medium was aspirated, and replaced by500 μl conditioned or control medium after adding 10% FBS. TheEA.hy926 endothelial cell response was evaluated after incubating theplate for 3, 5 and 7 days. At the end of the incubation, 50 μl of MTT solu-tion was added to each well followed by a 4-h incubation at 37 °C. Themedium was aspirated, and 350 μl dimethyl sulfoxide (DMSO) wasadded to each well to dissolve the blue formazan crystal. Then the

absorbance was recorded at a test wavelength of 570 nm and a refer-ence wavelength of 630 nm. A mean value was obtained from themea-surement of four test runs.

2.6. Statistical analysis

All the quantitative data were statistically analyzed to express asthemean (standard deviation (SD)). Statistical analysis was determinedby single factor ANOVA. p-Values less than 0.05 were consideredsignificant.

3. Results and discussion

The degrees of hydrolysis affect the solubility of PVA in water,whereas PVA with high degree of hydrolysis has low solubility inwater. Therefore, we used PVA (MW, 146,000–186,000) with 99% de-gree of hydrolysis. The selected PVA did not show solubility in waterat room temperature, but showed solubility at N80 °C under stirringcondition for 12 h. Once it dissolved, its solubility was maintainedwhen the temperature goes down to room temperature.

Fig. 1A–C (FESEM images) displays the fiber morphology of as-electrospun (pristine) PLA and PLA fibers coated with PVA. The pristinenanofibers exhibited a smooth surface and moderate uniform diameteralong their lengths as well as a non-interconnected (linear) fiber struc-ture (panel A). FESEM analysis of a pristine mat nanofiber verified thediameter of tissue scaffold to be 650 ± 180 nm. The PVA-coated PLAcomposite fiber mat showed cylindrical morphology with tiny defectsalong the fiber axis after the hydrothermal process (panels B and C).Moreover, PVA polymer is completelymasking the individual PLAnano-fiber along the fiber direction, and exhibits extremely extended nano-branches, of shooting the main nanofibers (Fig. 1B and C). No porousstructure was observed on the fiber surface, indicating that PVA playsa significant role to protect PLA fibers from any breakdown or fragmentformation during the deposition process (data are not shown). Ribeiroet al. [22] found that the nanofiber matrix of PLA changed into chunksconsisting of short fiber fragments with porous structure on their sur-face during a simple immersion in aqueous solution. They attributedsuch behavior to the simple hydrolysis of the ester backbone of aliphaticpolyester under aqueous conditions [23]. The imageJ software was usedto determine the diameter of different fibers. Results indicated that thefiber diameter increased after PVA incorporation onto the fiber surface(panel C). The average fiber diameter of the composite nanofiber matwas about 1265± 222 nm, which is remarkably higher than its pristinecounterpart (i.e., 650±180nm). PVA coating on the PLA fibers providesinterconnected and pseudo coagglomeration of nanofibers in additionto sufficient amount of joint-welding of the fibers at their neighboringpoints (panels B) which is absent in the pristine one (panel A). Highmagnification (panel C) reveals that PVA completely covered each indi-vidual PLA fiber and the PVA layer grew on the PLA main nanofiberswithout any phase separation. The mat coated with PVA showed a sig-nificant increase in its weight by about 0.38 mg PVA/1.0 cm2 of thePLA mat. These data demonstrate an increase in diameter size of thecomposite scaffold due to the deposition of a PVA layer on PLA fibers.We believe that during hydrothermal reaction at elevated temperature(150 °C) the PVA solution viscosity decreases resulting in higher solu-tion flow on the PLA single fibers causing the formation of a PVA layerwith a highly branched bridge web-like structure (panels B and C).We noticed that the PVAweb-like structure forms a kind of network be-tween the coated fibers (panels B and C) while maintaining the initial3D structure of the membrane mat. However, this behavior has notbeen detected at a temperature lower than 150 °C (data are notshown here). It seems that the web-like fibers act as bonding joints be-tween the main fibers. It's likely that decreasing viscosity at elevatedtemperature might cause higher PVA molecule mobility which can ex-plain the formation of branched PVA bridge fibers.

Fig. 1. SEM images of; (A) pristine PLA as-electrospun and PVA/PLA compositemat at low (B) and high (C)magnification. Dashed selected circular area in panel B is the respective positionfor panel C.

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The reaction temperature of composite sample, 150 °C,was preciselycontrolled based on our previous study on thermal analysis of PLAfibers[3] whereas the melting temperature of PLA is about 156 °C (Fig. 2A). Itwas reported that the thermal bonding of semi-crystalline polymer fi-bers, such as PLA, can occur near the melting temperature [24,25].Therefore, we hypothesize that some thermal interfiber-layer bondingwas effectively achieved between the PLA fiber and the PVA coatedlayer during the hydrothermal treatment. To verify our hypothesis,FTIRwas used to study the effect of hydrothermal treatment in the pres-ence of PVA solution on the interfacial bonding of the composite poly-mers (Fig. 2B). From the IR spectra in Fig. 2B, the characteristic bandsof pristine PVA occur at 838 cm−1 (rocking of CH), 919 cm−1 (bending

Fig. 2. (A) DSC; (B) FTIR spectra and (C) TGA curves of the pristine and composite mats. Dashnanofibers coated with PVA molecules.

of CH2), 1088 (stretching of CO and bending of OH from amorphous se-quence of PVA), 1143 cm−1 (stretching of CO from crystalline sequenceof PVA), 1417 cm−1 (wagging of CH2 and bending of OH), 2906 cm−1

(symmetric stretching of CH2), 2935 cm−1 (asymmetric stretching ofCH2) and 3278 cm−1 (stretching of OH). The comparison between theFTIR spectra of pristine and treated mats clearly shows that sufficientamount of PVA was deposited on the surface of PLA fibers as the inten-sity of different bands of PVA/PLAmats was significantly decreased andbecame narrower (Fig. 2B). It was reported that the bands of carbonylgroup of PLA and the bands of hydroxyl group of PVA occur at 1758and 3333 cm−1, respectively [26]. Accordingly, our results clearly iden-tified both bands in the hydrothermally treated PVA-coatedmats, while

ed arrows show the difference in absorption band of the pristine PLA nanofibers and PLA

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the 3333 cm−1 peak of pristine PLA disappeared (Fig. 2B). This providesanother evidence for depression of this peak on PLA due to the deposi-tion of PVA [26].

In the compositemat, the IR functional groups involved in strong in-termolecular hydrogen bonding often exhibit obvious shifts in their vi-bration frequencies (Fig. 2B). The characteristic peaks of the carbonyland hydroxyl groups of the composite mat were shifted towards higherfrequencies due to the intermolecular hydrogen bonding interactionbetween the hydroxyl groups of PVA and carbonyl group of PLA. Theseresults are in a good agreement with the FESEM morphology (Fig. 1B,C) where the interactions between the two polymers in the compositemat occurred without any phase-separation. Likewise, the crystalline-related peaks at 1417 and 1326 cm−1 in pure PVA disappeared in thebands of composite fibers, which can be also attributed to intermolecu-lar interaction between the polymers at the molecular level [26]. Fur-thermore, the band intensity of the composite mat was monotonicallydecreased after PVA deposition supporting themechanism of formationof an outer PVA layer on the surface of PLA fibers. The hydrothermaltreatment enhances the extent of shift of the carbonyl and hydroxylgroups indicating the formation of stronger hydrogen bonding and sug-gesting that such favorable interactions between the two polymers canlead to a miscible mat [26,27]. Additionally, it can also be identified thatthe intensity of the C–H stretching groups definitely varied (Fig. 2B)[28]. Taking the pristine mat as a base, the peak intensity of around2944 and 2995 cm−1 significantly decreased after hydrothermaldeposition of PVA. Indeed, the deposition of PVA using the hydrother-mal approach can assure strong intermolecular hydrogen-bondinginteractions [26,28].

Ribeiro et al. [22], reported that poly(L-lactide) electrospunfibers areamorphous but contain numerous crystal nuclei that could grow rapidlywhen the sample is heated up to 140 °C. Similarly, we expect that thedegree of crystallinity of the fibers can be tailored and controlled bythe hydrothermal treatment. The melting peaks of PLA showed no sig-nificant differences after PVA loading as revealed by DSCmeasurements(Fig. 2A). On the other hand, the Tm of pristine PVA could clearly be de-tected at 220 °C. After the hydrothermal process and formation of a PVAthin layer on PLA fibers, the PVAmelting temperature slightly shifted toa lower value (218 °C). The typical TGA thermogram curves of pristinePLA, PVA and PLA coated with PVA samples are demonstrated inFig. 2C. Notably, the TGA of pristine PVA polymer (prepared by sol–gelroute at a concentration of 1 wt.%) exhibited three distinct weightloss stages at 30–210 °C (5 wt.% loss of weakly physisorbed water),210–350 °C (decomposition of side chain of PVA) and 350–540 °C(decomposition of main chain of PVA), as shown in Fig. 2C. Sub-sequently, the pristine PLAnanofibers showed a single-step degradationat 305 °C, whereas the counterpart PVA/PLA composite nanofibersshowed degradation in two steps; first step occurs at 329 °C dueto the presence of PVA, and the second step occurs at 359 °C due toPLA (Fig. 2C). The onset temperatures of nanofibers were calculated tobe 335 °C and 285 °C for pristine and composite nanofibers, respectively.This data provides further evidence for the successful incorporationof PVA onto the PLA nanofibers. For the thermal decomposition of PLAmaterials, the thermal decomposition temperature (Td) was reducedby 15% for PLA nanofibers modified with PVA, as shown in Fig. 2C. Thedecrease in Td during the hydrothermal treatment can be attributedto the thermal degradation of PLA at 150 °C. Also, it is likely that thePLAfibers are partiallymelted (Tm=156 °C) during the treatment pro-cess to form an interfiber-layer bonding with PVAmolecules and createa miscible system. Otherwise, it could be also due to the thermal insta-bility of PLA [29]. Based on our experimental results, miscible polymersystems can produce new materials with designated properties super-seding those of their constituents. Collectively, it is worthmentioningthat the reduction in degradation temperature does not affect theproperties of the scaffold when it is used in biological temperature(i.e., 37 °C) [30] and was therefore deemed to be insignificant in practi-cal terms.

The glass transition temperature value of PLA slightly decreases afterthe treatment process and can be attributed to the presence of PVAphase. These observations provide another evidence that PLA and PVA,prepared by the hydrothermal process, can provide a good compatibil-ity [26]. Tsuji andMuramatsu [31] illustrated that PLA and PVA fabricat-ed at room temperature showed phase-separation in their blend filmsafter solvent evaporation. Other studies confirmed that PLA is partiallymiscible with PVA [32]. Leiggener et al. [33] reported through anin vivo study that the higher degree of crystallinity results in a higherchemical strength and loading capacity which promises advantagesfor long-term implantation. Our results showed that the incorporationof PVAmolecules at 150 °C induces the crystallization of composite sam-ples (Fig. 2A) due to the enhanced chain mobility [12], where the mea-sured heat of fusion (melting enthalpy) of the composite sample is40.61 J/g compared to 31.12 J/g for the pristine sample. Increasing theenthalpy of fusion suggests that the crystallinity and perfection of thecrystal structure are increased by PVA loading. The improvement incrystallinity after incorporation of a PVA thin layer onto the post-electrospun fibers indicates that there are interactions between PLAand PVA layer, i.e., formation of hydrogen bonding. In other words, thedeposition of a PVA layer onto the PLA fibers readily induces a chainconformation without defects in the crystalline phase of PLA. In addi-tion, it is known that the melting enthalpy reflects the crystallinity ofpolymer [40]. This can be explained by the decrease in the content ofthe amorphous domains due to crystalline growth [3]. This numerousgrowth (also known as nucleation) can be attributed to nonequilibriumchain conformations imposed by the electrospinning process that canbe frozen upon the evaporation of the solvent as noted by Zong et al.[34]. The crystallization behavior of PLA has been discussed elsewhere[35] and results showed that the crystallinity can be obtained at morethan 100 °C. The degree of crystallinity increases the treatment temper-ature below Tm. The temperature required for transition from a glassystate to a rubbery state will be higher. These results qualitatively dis-agree with the results presented previously [34] in which a relativelylow crystallinity was observed for electrospun polymer fibers from thesolution state. The chain conformation has also been noticed fromFTIR results (Fig. 2B). The 921 cm−1 absorption band which is charac-teristic of the α-crystal in PLA [20] is clear in the pristine mat and be-comes less intense in the composite mat (Fig. 2B). Fig. 2B shows thatthe increase in the crystallization degree is accompanied by significantnarrows and change in the shape of the absorption band between 830and 890 cm−1 for the composite mat [22]. The interfacial bonding andchain conformation can improve the mechanical properties of the com-posite mat. Collectively, hydrothermal treatment on PLA electrospunnanofibers in the presence of PVA molecules could create physicaljoint and chemical bonding between PLA fibers and PVA depositedlayer.

The substitute nanofiber materials used in tissue treatment shouldmaintain adequate mechanical strength over critical phases of thetissue-healing process to withstand the physiological bioenvironmentand tearing resistant during implantations. To investigate the mechani-cal properties of the pristine and composite mats, load displacementcurveswere obtainedwhereas the tensile strengths and percent of elon-gation (ductility)were calculated. The load displacement curves (Fig. 3)show that the tensile strength of the composite mat (12.9 MPa) washigher than that of the pristinemat (8.1± 1.5MPa). Interestingly, com-posite fabric induced excellent ductility compared to pristine fabric(over than 2.5 times). This indicates that the PVA coating has a strongaffinity on ductility improvement due to the increased elastic character-istic of the composite mat [21]. Ma and co-workers [36], found that theaddition of PVA to the blendfilms improves the extensibility of the com-posite blend films as a potential tissue engineeringmatrix. Norton et al.[37]were able to tune the ductile properties of gellan through the incor-poration of PVA as a secondary polymer network, for use in cartilageand skin as complex structures. The possible reasons could be due tothat formation of a PVA layer during the hydrothermal treatment

Fig. 3. Selected stress–strain curves of pristine and composite nanofibermats under tensileloading. Inset illustrates dog bone samples and their fixation at machine grip. Also, insetsare their respective water contact angle images.

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process resulting in an increase the stretching of the fiber compositemat. Our results are in good agreement with the previous study byYang et al. on PVA/gelatin composite polymers [38]. It was also reportedthat the heat treatment further improves the mechanical properties ofthe membranes [39]. In contrast, Martin and Avérous [14], found thata simple blending of PLGAwith PVA has a detrimental effect on theme-chanical properties, with a loss of 31% in Young's modulus and morethan 60% in tensile strength compared to the PLGA scaffold. The deposi-tion of PVA on PLA fibers sharply improves the tensile strength andelongation at break for composite polymers, because of the gooddeformability and flexibility of PVA.

We suggest that the improved tensile strength of the compositematcompared to pristine one can be attributed to increased tear resistanceof the branched PVA fibers (web-like fibers, parallel PLA fibers boundedto each other by PVA fibers of different diameters) compared to linear(non-branched) membrane rather than the presence of physical/chemical bonding between the polymers. The interconnected, networkstructure of the coated fibers resulted in higher tensile strength. It wasalready reported that the mechanical properties of nonwoven mats in-crease with increasing fiber junctions [40]. Thus, it is possible that theenhanced mechanical properties for the PVA-coated scaffold are alsodue to the fiber junction rather than the crystallinity improvement ofthe treated scaffold. These results describe the fact that the mechanicalbehavior of nonwoven mats depends mainly on the chemical composi-tion, treatment condition, morphology, and bonding structure of fibers.Hence, the relative improvement in the mechanical properties of thecomposite mat may be explained in terms of the increased affinitybetween the two macromolecules. The miscibility resulted in difficultslippage of chains under loading because of more entanglements andstrong physical/chemical interactions among the chains of compositepolymers, such as hydrogen bond. Therefore, the addition of PVA inthe presence of heat energy was quite helpful to improve the mechani-cal properties of PLA nanofiber scaffolds, overcome the brittleness na-ture of PLA and, to sharply convert it to ductile biomaterial.

Contact angles, which depend on topographic pattern and chemicalcomposition, reflect the hydrophilicity of scaffolds due to proteinabsorption and cell attachment [3,6,9]. The inset of Fig. 3 shows thatthe incorporation of a PVA thin layer sharply decreases the contactangle of PLA nanofibers and consequently, improves its hydrophilicity.We found out that the hydrophilicity of the coated PLA scaffold dramat-ically improved (36.11 ± 1.5°) compared to the pristine PLA (119.7 ±1.5°) scaffold which is similar to the author's previous report [3] onthe pristine PLA mat. The previous study also showed that the pristinePLA membrane surface possesses hydrophobic nature. The position-

dependent water contact angle changes for each PVA-coated PLA fibermat surface were not significant, indicating the homogeneity of PVAcoating on PLA fibers during the hydrothermal process. However, itwas reported that the water contact angle measurements of nanofiberscannot exactly reflect the degree of wettability of the polymers and theresults are purely qualitative, because the liquid drops on nanofiberscannot provide a full contact with the fiber surface [41]. Accordingly,the wettability was further confirmed in our study by the rateof water absorption to the prepared scaffolds. We noticed that thepristine PLA mat (Fig. S2A of supplementary materials) doesn't showwettability/absorbability until 80 min when water droplet has fallenon the surface, as expected, showing their hydrophobic characteristics.Conversely, the water absorption rate on the composite nanofiber matwas very fast within few seconds (Fig. S2B), which is qualitatively indi-cating that they aremore hydrophilic.We confirmed these results usinga video clip in the supporting information (Video clip S3 A and B). Ac-cording to British Standard 4554:1970 (Method of Test for Wettabilityof 3D Fabrics), fabrics giving times greater than 200 s with water areconsidered to be unwettable. The fast absorption and wetting of thePVA/PLA composite scaffolds are highly desirable for tissue engineeringapplications because the cells can be seeded directly and cultured in thishydrophilic scaffold without any further modification. Therefore,deposition of a PVA thin layer on the surface of PLAfibers during the hy-drothermal process could easily increase the hydrophilicity of as-spunfibers and increase its potential application in biological system.

To prove this hypothesis together with investigating the effects of aPVA thin layer formation on PLA, we studied the influence of EA.hy926endothelial cell attachments as well as proliferation on the pristine andtreated scaffolds at different culture times. The cell attachmentwas con-firmed using a bio-scanning electronmicroscope as shown in Fig. 4A–D.From the graphs, it can be seen that the endothelial cell has a good affin-ity to attach and growon both pristine and composite scaffolds at differ-ent culture times. However, the cell adhering on the hydrophilic scaffoldsurface was evidently better than that of the pristine PLA scaffold. Thecells have spread and the pseudopodia grew and extended along thecomposite scaffold (Fig. 4B and D) compared to that of the pristinePLA fibers (Fig. 4A and C), particularly after five days of culture, indicat-ing confluence growth of EA.hy926 cell. Nuttelman et al. [42] reportedthat the inability of cells to attach to tissue scaffold material, in general,relates to their hydrophilicity, leading to minimal adsorption of cell ad-hesion proteins on the scaffold surface. In addition, the abundant hy-droxyl groups on PVA (see FTIR data, Fig. 2B) during the hydrothermalprocess can be readily modified to attach growth factors, adhesion pro-teins, or other molecules of biological importance.

Previous in vitro and in vivo studies documented that hydrophilicityis an important factor when considering permeation of nutrients acrossthemembrane and cell compatibility [3,43]. These studies reported thathydrophobic materials are poorly wetted by cell culture medium,resulting in limited cell attachment to the scaffold and poor transfer ofnutrients and waste products across the membrane. From the aboveanalysis, the hydroxyl functional groups of PVA are present on the sur-face of the composite scaffolds and are absent on the PLA scaffold. Theappearance of PVA functional groups increased the hydrophilicity ofelectrospun PLA scaffolds. Indeed, coating of PVA to PLA nanofibers tofabricate a PVA/PLA composite scaffold using hydrothermal strategyproved to be an effective approach for improving the hydrophilicityand attachment properties of the scaffold and thus, overcome themain constrain of using PLA nanofibers in tissue engineeringapplications.

The cell proliferation (cell density or number of cells) was tested byMTT-assay and the results are shown in Fig. 5. In general, the cells on allscaffolds proliferated with increasing culture time points, indicating agood cytocompatibility of both pristine and composite scaffolds. Never-theless, at each time point interval there is a remarkable difference incell proliferation among the composite PVA/PLA scaffold (p less than0.005). The composite scaffold contributed the best proliferation result

Fig. 4. SEM observations of human endothelial cell adhesion and growth on pristine PLAmembrane scaffold (A and C) and composite membrane scaffold containing 1 wt.% PVA (B and D)after culture for 3 (A and B) and 5 (C and D) days.

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after culturing for 5 and 7 days, while the pristine PLA scaffold yieldedrelatively lowproliferation particularly after 7 days of culture, indicatingthat the PLA nanofiber scaffold coated with PVAmight have acceleratedthe proliferation and differentiation of EA.hy926 human cells. It can beobserved that EA.hy926 cells proliferated on the scaffold displays atime-dependent behavior, and the composite scaffold possesses highercell proliferation than that of the pristine PLA scaffold. The betterbiochemical interaction between cells and the hydrophilic membranesurface of the composite scaffold might provide good interaction ofEA.hy926 cells with PLA fibers coated with a PVA thin layer comparedto the pristine PLA fibers.

The durability of the repeated use of the composite fibrous mat wasevaluated using different strategies which were mentioned above. Our

Fig. 5.MTT cytotoxicity test on differentmats after 3, 5, and 7 days of culture. The viabilityof control cells was set at 100%, and the viability relative to the control was expressed. Thedata is reported as the mean ± standard deviation (n = 4 and p b 0.05).

results showed that the as-prepared scaffolds can be used repeatedlywithout a significant decrease in its efficiency. Therefore, we areexpecting that these 3D scaffold materials will have promising applica-tions in tissue engineering. We performed some tests for at least threetimes to confirm our data. We followed very systematic procedures toachieve and optimize our results by using several evaluation techniquessupported by several characterization tools to confirm our findings.Overall, based on our results, the PVA/PLA composite coating fabricatedby the hydrothermal approach can be considered as a promising, sim-ple, cost-effective and smart approach for fabrication of hydrophilizedscaffolds.

4. Conclusion

A novel polymer composite nanofiber scaffold was designedby exploiting a simple hydrothermal approach without using any sur-face modifier. The designed nanofibers demonstrated improvedhydrophilicity, mechanical properties, and EA.hy926 cell attachmentbehavior. The new scaffold promises new advancement in tissue engi-neering to meet the current clinical challenges and needs in this field.Our results confirmed that a PVA thin layer was successfully in situ de-posited and coated onto each single pre-electrospun PLA nanofiber.The deposition of PVA throughout the hydrothermal technique couldimprove the hydrogen bonding interaction and induce the crystallineconformation of PLA, resulting in sharp improvement in the PLA ductil-ity with significant enhancement in the tensile strength aswell. The de-signed PLA scaffold showed elongation over 2.5 times higher than thatof neat PLA. The conventional blending of PLA-based materials withPVA causes severe degradation of the fibrous scaffold, which can com-promise scaffold strength and affect the biodegradation rate beforethe healing process occurs completely. Using our novel coatingapproach, we were able to avoid degradation of the fibrous scaffold.However, further studies are needed to investigate the in vivobiocompatibility and biodegradability of the modified PLA scaffoldunder physiological conditions.

150 A. Abdal-hay et al. / Materials Science and Engineering C 60 (2016) 143–150

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.msec.2015.11.024.

Acknowledgments

The authors would like to extend their sincere appreciation to theDeanship of Scientific Research at King Saud University for fundingthis Research Group NO. (RG 1435-001).

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