Long-Term Stabilization of Polysaccharide Electrospun Fibres by In Situ Cross-Linking

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  • This article was downloaded by: [York University Libraries]On: 11 November 2014, At: 05:53Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

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    Long-Term Stabilization ofPolysaccharide Electrospun Fibresby In Situ Cross-LinkingLiya Shi a , Catherine Le Visage b & Sing Yian Chew ca School of Chemical and Biomedical Engineering, N1.2-B2-20, Nanyang Technological University, 62 NanyangDrive, 637459 Singaporeb Inserm, U698, Bio-ingnierie Cardiovasculaire, CHUX. Bichat, 46 Rue Henri Huchard, 75877 Paris Cedex 18,Francec School of Chemical and Biomedical Engineering, N1.2-B2-20, Nanyang Technological University, 62 NanyangDrive, 637459 SingaporePublished online: 02 Apr 2012.

    To cite this article: Liya Shi , Catherine Le Visage & Sing Yian Chew (2011) Long-TermStabilization of Polysaccharide Electrospun Fibres by In Situ Cross-Linking, Journal ofBiomaterials Science, Polymer Edition, 22:11, 1459-1472, DOI: 10.1163/092050610X512108

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    http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions

  • Journal of Biomaterials Science 22 (2011) 14591472brill.nl/jbs

    Long-Term Stabilization of Polysaccharide ElectrospunFibres by In Situ Cross-Linking

    Liya Shi a, Catherine Le Visage b and Sing Yian Chew a,

    a School of Chemical and Biomedical Engineering, N1.2-B2-20, Nanyang Technological University,62 Nanyang Drive, 637459 Singapore

    b Inserm, U698, Bio-ingnierie Cardiovasculaire, CHU X. Bichat, 46 Rue Henri Huchard,75877 Paris Cedex 18, France

    Received 21 April 2010; accepted 21 May 2010

    AbstractCross-linking of polysaccharide electrospun constructs using currently available techniques results in poorscaffold structural stability. In general, cross-linked substrates lose their nanofibrous architecture within ashort time under physiological conditions. In this study, we introduce an in situ cross-linking electrospin-ning technique to fabricate and stabilize pullulan/dextran fibres. Pullulan/dextran (4:1 weight ratio, 16.7 and20 wt%) solutions were preloaded with the chemical cross-linker, trisodium trimetaphosphate (STMP), toenable cross-linking during electrospinning. By increasing STMP from 4 to 16 wt%, the average diameter ofelectrospun fibres increased significantly from 26835 nm to 41674 nm (P < 0.05). Additionally, the en-hanced cross-linking effectively decreased the swelling extent of the scaffolds. In particular, in the presenceof 10 wt% gelatin, a significant decrease in scaffold swelling ratio was observed (208.5 31.3% at 4 wt%STMP vs 133.1 9.1% at 16 wt% STMP, P < 0.05). In vitro stability studies demonstrated the retentionof scaffold fibrous morphology and negligible weight loss in all samples after 28 days. Environmental SEManalysis revealed that at least 16 wt% STMP was required in order to retain the nanofibrous structure of thescaffolds under hydrated conditions. Compared with hydrogels of similar chemical content, the nanofibrousarchitecture of electrospun scaffolds significantly enhanced human dermal fibroblast (HDF) viability at days3 and 7 (P < 0.05). The incorporation of gelatin and the increase in scaffold cross-linking density favouredHDF cell attachment and spreading. In particular, 16 wt% STMP promoted actin stress fibre formation.Taken together, the results support the promise of using STMP in situ cross-linking for long-term stabiliza-tion of polysaccharide electrospun fibres and the advantage of polysaccharide nanofibrous constructs fortissue engineering. Koninklijke Brill NV, Leiden, 2011

    KeywordsPullulan, dextran, nanofibres, cytocompatibility, regenerative medicine

    * To whom correspondence should be addressed. Tel.: (65) 6316-8812; Fax: (65) 6794-7553; e-mail:SYChew@ntu.edu.sg

    Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050610X512108

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  • 1460 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472

    1. Introduction

    Among the variety of materials that have been electrospun, polysaccharides are oneof the most popular due to their abundance, stability and non-toxicity [1]. However,the instability of water-soluble polysaccharide scaffolds under hydrated conditionsremains a major hurdle to the widespread application of these materials in regener-ative medicine in the form of nanofibrous constructs. Without proper stabilizationvia efficient cross-linking, these water-soluble electrospun scaffolds typically losetheir unique fibrous architecture under aqueous conditions. Some commonly usedcross-linking methods adopted to stabilize polysaccharide scaffolds include UVirradiation [2, 3], immersion of scaffolds into cross-linker solutions [4, 5] and ex-posure of these scaffolds to cross-linker vapours [69]. However, these methods aretypically laborious and time-consuming and the structural stability of the result-ing constructs is often limited. An improved cross-linking approach for long-termstabilization of polysaccharide nanofibrous substrates is, therefore, required.

    Due to its ease of chemical modification and lack of immunogenicity [10], pul-lulan has been found applications in biomedicine [1113], tissue engineering [14]and drug/gene delivery [1518]. Dextran, a bacterial polysaccharide, has been fre-quently used as blood substitutes and drug-delivery carriers [19]. Hydrogels com-prising pullulan and dextran can support the culture and proliferation of smoothmuscle cells for vascular tissue engineering [14, 20]. However, the electrospinningof pullulan and dextran mixtures and the long-term stabilization of these materialsin the form of nanofibres remain unexplored.

    In this study, we propose an in situ cross-linking method to fabricate and stabilizepullulan/dextran nanofibres for at least 28 days under hydrated conditions. The ad-vantage of using pullulan/dextran in the form of nanofibrous cell-culture substratesover hydrogels is also demonstrated using human dermal fibroblasts.

    2. Materials and Methods

    2.1. Electrospinning of Pullulan/Dextran Fibres

    Pullulan (Mw 200 000, Hayashibara) and dextran (Mw 500 000, Pharmacia) weredissolved at a weight ratio of 4:1 in deionized water at room temperature at a con-centration of 20% (w/w). Trisodium trimetaphosphate (STMP, Sigma) was thenadded into the polysaccharide solution at concentrations of 4, 8, 12 and 16 wt%of pullulan/dextran and vortexed for 2 h. The resultant solution was then mixedwith dimethylformamide (DMF, Sigma) at a volume ratio of 10:1 (polysaccharidesolution/DMF). Before electrospinning, 10 wt% NaOH aqueous solution (NaOH,Sigma) was added at a volume ratio of 1:10 (NaOH/polysaccharide solution) toprovide an alkaline condition to activate cross-linking. The resulting mixture wasthen transferred into a syringe that is charged at +18 kV (Gamma High VoltageResearch). A rotating target (150 rpm) charged at 4 kV was placed 10 cm awayfrom the needle tip, resulting in an overall electric potential of 22 kV. By dispensing

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  • L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472 1461

    Table 1.Processing parameters for electrospun pullulan/dextran scaffolds

    Sample Polymer Gelatin STMPconcentration content content(wt%) (wt%) (wt%)

    Fibres without gelatinSTMP4GEL0 20 0 4STMP8GEL0 20 0 8STMP12GEL0 20 0 12STMP16GEL0 20 0 16

    Fibres with gelatinSTMP4GEL10 20 10 4STMP8GEL10 20 10 8STMP12GEL10 20 10 12STMP16GEL10 16.7 10 16

    All polysaccharide solutions were charged at 22 kV anddispensed at 3 ml/h during electrospinning. Nomenclature:STMPXGELY , where X and Y represent the weight percentageof STMP and gelatin (GEL), respectively.

    the polysaccharide solution at 3 ml/h through a 22 G needle, random electrospunfibres were obtained. Samples containing 10 wt% gelatin were fabricated by elec-trospinning pullulan/dextran/gelatin solution with different loading concentrationsof STMP under the same conditions. Table 1 summarizes the samples and theirchemical content. All samples are denoted as STMPXGELY , where X and Y rep-resent the weight percentage of STMP and gelatin added respectively. Followingelectrospinning, all scaffolds were dried at 37C for 7 days before rinsing with wa-ter to remove unreacted reagents and air-dried at 37C until consistent weight.

    2.2. Characterization of Pullulan/Dextran Fibrous Scaffolds

    2.2.1. Scaffold MorphologyThe surface morphology of cross-linked polysaccharide fibres was observed usinga field emission scanning electron microscope (JEOL JSM 6700F FESEM) at anaccelerating voltage of 10 kV after gold sputter coating. The average fibre diameterswere then computed by measuring at least 60 fibres using ImageJ software.

    2.2.2. Phosphorous Content of ScaffoldThe phosphorous content of fibrous scaffolds were determined according to a col-orimetric method based on the yellow complex produced by mixing ammoniumvanadate, molybdate and phosphate [21]. Phosphorous content was determinedfrom a calibration curve prepared with known amounts of phosphorus. Results wereexpressed as mmol/g dried scaffold, and three scaffolds were tested for each sample.

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  • 1462 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472

    2.2.3. Scaffold Swelling BehaviourThree separately electrospun scaffolds (diameter 12.5 mm) were tested for eachsample. All dried scaffolds were incubated in DMEM (Gibco) for 4 h at 37C.Thereafter, the cross-sectional areas of dried and swollen scaffolds were measuredusing light microscopy and ImageJ software as Ad and Aw, respectively. Scaffoldswelling ratio was finally computed as (Aw/Ad) 100%.2.2.4. Scaffold StabilityDried scaffolds (weight (Wo) approx. 0.3 g) were immersed in phosphate-bufferedsaline (PBS, Invitrogen) at 37C for 2, 4, 7, 14, 21 and 28 days. At each time pointthree scaffolds were retrieved, washed with deionized water and air-dried at 37Cfor two days before the recording of weight (Wr). The percentage weight remainingwas finally calculated as (Wr/Wo) 100%. For observation under the SEM, allscaffolds were washed 3 times in deionized water and dehydrated using gradientethanol treatment (30, 50, 70, 90% and pure ethanol for 15 min at each step) priorto critical point drying (CPD, BAL-TEC CPD 030). Thereafter, all samples weregold sputter-coated for SEM analysis.

    2.3. Evaluation of Cellular Interactions with Electrospun Pullulan/DextranScaffolds

    Prior to cell seeding, all scaffolds (diameter 12.5 mm) were sterilized under UVfor 20 min on each side and equilibrated in culture medium (DMEM, 10% fetalbovine serum (FBS, Hyclone) and 1% penicillinstreptomycin (Gibco)) for 1 h at37C. Thereafter, 1 ml human dermal fibroblasts (HDF, P14, 1 104 cells/ml) wasseeded onto each scaffold. Cells were cultured in a humidified CO2 incubator at37C with a change of fresh media every 3 days until further evaluation at days 3and 7. Pullulan/dextran hydrogels with the same chemical content were preparedaccording to previous protocols [14] in order to understand the effects of scaffoldtopography on HDF-substrate response.

    2.3.1. Confocal Fluorescent Microscopy ImagingAt days 3 and 7, all scaffolds were washed once with PBS to remove unattachedcells prior to fixation with 4% paraformaldehyde for 20 min. Next, cells werepermeabilized with 0.05% Triton-X and 50 mM glycine for 20 min. Cell actin cy-toskeleton was stained with Oregon green-phalloidin (1:500, Invitrogen) and nucleiwith propidium iodide (1:250, Invitrogen) for 30 min. All scaffolds were washed3 times with PBS in between each step prior to observation under a confocal micro-scope (Zeiss, LSM 510).

    2.3.2. SEM AnalysisAt days 3 and 7, all scaffolds were washed once with PBS and then were fixedwith 2.5% glutaraldehyde for 30 min and washed with PBS for three times. Theconstructs were then treated according to the drying process indicated above.

    To observe cell-seeded scaffolds under hydrated conditions, all scaffolds werefixed using 2.5% glutaraldehyde for 1 h prior to examination in PBS under an envi-

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  • L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472 1463

    ronmental SEM (Philips ESEM-FEG XL30) with an accelerating voltage of 15 kVat a pressure of 3.5 Torr.

    2.3.3. HDF Viability and ProliferationAt days 3 and 7, scaffolds were washed 3 times with PBS and transferred to new48-well plates. Cells seeded on tissue-culture polystyrene (TCPS) served as con-trols. WST-1 reagent (Roche, 20 l) was then added into each well and diluted with180 l culture medium. After incubation for 4 h at 37C, absorbance at 450 nmwas recorded using a microplate reader (Tecan, Infinite f200) against the referencewavelength of 620 nm. WST-1 reagent and plain culture medium served as back-ground control. All experiments were run in triplicate.

    2.4. Statistical Analysis

    All results are expressed as mean SD. Statistical analyses for fibre diameterswere carried out using the KruskalWallis method, followed by the MannWhitneyU -test. For WST-1 assay results, the independent-samples t-test was used. A valueof P < 0.05 was considered statistically significant.

    3. Results

    3.1. Characterization of Pullulan/Dextran Fibrous Scaffolds

    3.1.1. Scaffold MorphologyAs indicated in Fig. 1 and Table 2, scaffolds obtained under the optimized condi-tions listed in Table 1 possessed uniform fibre diameters, ranging from 260 nm to416 nm. For scaffolds without gelatin, there was a significant increase in fibre diam-eter as STMP content increased from 8 to 16 wt%. This trend was also observed inscaffolds comprising gelatin when STMP content increased from 4 to 12 wt%. Theincrease in fibre diameter is likely due to the increase in polysaccharide solution vis-cosity [22] as STMP concentration increased. In the case of STMP16GEL10, dueto high solution viscosity, a stable spinning process could only be obtained by de-creasing the polysaccharide concentration from 20 to 16.7 wt%. Correspondingly,a decrease in fibre diameter was observed.

    3.1.2. Scaffold Swelling BehaviourTable 2 highlights the swelling ratios of all scaffolds as monitored by the changein cross-sectional areas of the samples after immersion in DMEM. As STMP con-tent increased from 4 to 16 wt%, a decreasing trend in swelling ratio was observedfor plain pullulan/dextran scaffolds. In the presence of 10 wt% gelatin, a signifi-cant increase in swelling ratio was observed at 4 wt% STMP as compared to allother loading levels. The variation in swelling ratio as STMP concentration in-creased from 8 to 16 wt% was, however, insignificant with or without the inclusionof gelatin.

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  • 1464 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472

    Figure 1. FESEM micrographs of (ad) as-spun pullulan/dextran fibres and (eh) pullulan/dextran fi-bres after 28 days incubation under physiological conditions. Results indicate the retention of scaffoldnanofibrous architecture for 28 days, particularly at 16 wt% STMP.

    Table 2.Average diameters of pullulan/dextran fibres increased with increasing STMP content whilst theswelling ratios of pullulan/dextran scaffolds decreased with increasing STMP

    Sample Fibre diameter Swelling ratio Phosphorous content(nm) (%) (mmol/g scaffold)

    Fibres without gelatinSTMP4GEL0 268 35 192.7 34.7 0.22 0.04STMP8GEL0 261 40a 160.1 21.0 0.44 0.05STMP12GEL0 303 47a,b 160.3 45.2 0.54 0.10STMP16GEL0 416 74b 141.2 8.3 0.80 0.06

    Fibres with gelatinSTMP4GEL10 282 50c 208.5 31.3 0.23 0.02STMP8GEL10 326 71c,d 142.2 16.2 0.48 0.06STMP12GEL10 367 53d,e 130.5 11.0 0.58 0.05STMP16GEL10 323 54e 133.1 9.1 0.84 0.16The phosphorous contents indicate that cross-linking extent increased with increasing STMP. The

    same letters in the fibre diameter column indicate significant difference between the two samples(P < 0.05). P < 0.05, significant difference compared to STMP4GEL10. For phosphorous content,the STMP16 samples were significantly different from STMP4 samples.

    3.1.3. Phosphorous ContentBecause the cross-linking is realized through phosphate esters, the phosphorouscontent is a valuable estimate of the synthesis efficacy of phosphorus incorporationon the polymer chains. Table 2 shows that the amount of phosphorus in fibres in-creased from 0.22 0.04 mmol/g to 0.80 0.06 mmol/g with increase of addedSTMP from 4% to 16%. Higher STMP concentration leads to a higher phosphorouscontent, as already reported for plain hydrogels [23].

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    Figure 2. In vitro stability of electrospun pullulan/dextran scaffolds (a) without gelatin and (b) withgelatin, indicating structural stability over a period of 28 days under physiological conditions.

    3.1.4. Scaffold StabilityFigure 2 illustrates the in vitro stability of pullulan/dextran scaffolds over a pe-riod of 4 weeks under hydrated conditions. With the incorporation of gelatin, aslightly larger weight loss (8090%) was observed as compared to scaffolds withoutgelatin (9095%), although the difference was not statistically significant. These re-sults indicated that cross-linked pullulan/dextran scaffolds have excellent structuralstability and could serve as potential scaffolding materials capable of providingnanofibrous topographic cues to seeded cells over prolonged time periods.

    The FESEM images of scaffolds after incubation for 28 days are shown inFig. 1eh. All scaffolds maintained their porous structures after 28 days. However,fusion of fibres occurred at low STMP levels, particularly in the case of 4 wt%STMP. For scaffolds with 16 wt% STMP, there was negligible fibre fusion and thescaffold architecture was comparable to that observed at day 0. The increase incross-linker concentration appeared to improve the structural stability of the fibres

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  • 1466 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472

    under hydrated conditions over a prolonged time period, and at least 16 wt% STMPwas necessary to prevent obvious fibre fusion after 4 weeks.

    3.2. Morphology and Distribution of HDF on Pullulan/Dextran Fibrous Scaffolds

    HDF was used as the model cell type to evaluate the cytocompatibility of electro-spun pullulan/dextran scaffolds. As there appeared to be insignificant differences inphysical properties of scaffolds comprising 816 wt% STMP, only scaffolds with4 wt% and 16 wt% STMP with or without gelatin were selected for analyses oncellsubstrate interactions.

    On day 3, few cells were found on STMP4GEL0, STMP4GEL10 and all hydro-gels, indicating poor cell attachment on these substrates (data not shown). For theresults on Day 7, as indicated in Fig. 3, an increase in STMP content enhanced cellattachment. The inclusion of 10 wt% gelatin further promoted cell spreading. Stressfibre formation was observed only in STMP16GEL10 samples, indicating that cellattachment was most pronounced on these substrates and was comparable to that onTCPS. In contrast, cellular attachment was not observed on hydrogels with similarcontent of STMP16GEL10 even at day 7.

    Figure 4 illustrates cellular interactions with pullulan/dextran fibrous scaffolds asobserved under the SEM. The results appeared consistent with fluorescent imaging.Cell attachment was poor on STMP4GEL0 scaffolds with rounded cells appearingto detach. In contrast, HDF adhered well to STMP4GEL10, STMP16GEL0 andSTMP16GEL10 scaffolds. Examination of the cellsubstrate interface indicatedgood integration between HDF and scaffolds that comprised of gelatin (Fig. 4cand 4d inset). Additionally, the fibrous architecture of STMP16 scaffolds was re-tained after 7 days of culture, whilst fibre fusion occurred in scaffolds with only4 wt% STMP. Furthermore, a confluent layer of cells was observed on each of theSTMP16 scaffolds, the high cell density promoted cell alignment and led to cellsadopting a spindle-like morphology.

    Scaffold morphology and cellular attachment onto pullulan/dextran scaffolds un-der hydrated condition in PBS are shown in Fig. 4e and 4f. In the presence of 4 wt%STMP with gelatin, the scaffold fibrous structure was not observed (Fig. 4e). Incontrast, the fibrous architecture was retained in the presence of 16 wt% STMPwith gelatin, clearly demonstrating the effects of enhanced cross-linker contentin stabilizing the architecture of the electrospun scaffolds (Fig. 4f). Cellsubstrateinteractions under hydrated conditions also appeared in a similar way to those ob-served under ESEM (Fig. 4f, dotted circles).

    3.3. HDF Viability on Pullulan/Dextran Fibrous Scaffolds

    Figure 5 shows the metabolic activity of cells as indicated by WST-1 assay. Pullu-lan/dextran hydrogels and TCPS served as controls. On Day 3, higher cell viabilitywas observed on fibres with 16 wt% STMP, with no significant difference betweenscaffolds with or without gelatin. However, the incorporation of gelatin enhancedcell metabolic activity significantly on day 7 for both STMP4 and STMP16 samples

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    Figure 3. (a, b) Fluorescent microscopy images of cells cultured for 7 days on scaffolds withoutgelatin (a, STMP4GEL0; b, STMP16GEL0) and (c, d) on scaffolds with gelatin (c, STMP4GEL10;d, STMP16GEL10). (e) TCPS (control). Results indicate enhanced cellular attachment and spread-ing in the presence of gelatin and high STMP content (green, actin cytoskeleton; red, cell nuclei).This figure is published in colour in the online edition of this journal, that can be accessed viahttp://www.brill.nl/jbs

    (P < 0.05). In contrast, cell viability increased only slightly for scaffolds withoutgelatin. Cells cultured on STMP16GEL10 samples demonstrated the highest levelof metabolic activity and was similar to that on TCPS. At all time points, there wasnegligible cell viability in hydrogels.

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  • 1468 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472

    Figure 4. (a, b) FESEM images of cells cultured for 7 days on scaffolds without gelatin(a, STMP4GEL0; b, STMP16GEL0) and (c, d) on scaffolds with gelatin (c, STMP4GEL10;d, STMP16GEL10). Results demonstrate enhanced cell adhesion and spreading, and improvedcellsubstrate integration in the presence of gelatin and high STMP content. Inset: white arrows indi-cate cellsubstrate interface, the scale bar indicate 2 m. (e, f) ESEM images of (e) STMP4GEL10 and(f) STMP16GEL10 (dotted circles indicate cells on the scaffolds) on day 7. The fibrous architectureremains intact under hydrated conditions in the presence of 16 wt% STMP.

    4. Discussion

    In this study, we demonstrate a one-step approach to electrospin cross-linked pul-lulan/dextran fibres. Prior to this, the poor electrospinnability of polysaccharidesand the instability of electrospun polysaccharide scaffolds limited the applicationof these materials as nanofibrous constructs in regenerative medicine [19]. The poorelectrospinnability of polysaccharides is largely due to the fact that these materials

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    Figure 5. Metabolic activity of HDF on pullulan/dextran scaffolds as evaluated using WST-1 assay.,#P < 0.05, significant difference compared to STMP16GEL10 and TCPS, respectively, at the sametime point; @P < 0.05, significant difference due to the addition of gelatin. Results indicate that cellviability was enhanced on electrospun scaffolds as compared to hydrogels, and in the presence of highSTMP content and gelatin.

    typically have poor solubility (e.g., pullulan can dissolve in water or DMSO only[24]); result in polymer solutions that lack polymer chain entanglements that arenecessary for electrospinning; or possess high solution viscosity or surface tension[19]. Furthermore, since pullulan and dextran are water-soluble polysaccharides,there is the additional challenge of preserving the structural integrity of these fibrousconstructs under physiological conditions. Although the electrospinning and pho-tochemical cross-linking of dextran have been reported, such an approach involvesmultiple processing steps such as polymer modification and UV-irradiation [2]. Tocircumvent these laborious fabrication steps, Schiffman et al. [25] introduced aone-step approach by adding glutaraldehyde directly into chitosan solutions priorto electrospinning. Unfortunately, the stability of the cross-linked chitosan scaf-folds remains limited within 3 days under aqueous conditions. In contrast, our studyclearly demonstrates the prolonged scaffold structural stability of at least 28 days.This indicates that cells seeded on these scaffolds will continuously be subjected tonanofibrous topographical cues throughout the period of cell culture. Furthermore,STMP cross-linking is also applicable to other polysaccharides such as starch [26]and guar gum [27].

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    STMP chemically cross-links pullulan and dextran under alkaline condition[28]. Therefore, higher concentrations of STMP or NaOH led to a faster rate ofcross-linking and gelation of the solutions, resulting in unstable electrospinningprocesses; shorter electrospinning duration prior to complete solution gelation; andan increase in fibre diameter distribution. On the other hand, if the STMP concen-tration was below 4 wt%, the electrospun constructs lose their fibrous structure im-mediately upon exposure to aqueous environment due to incomplete cross-linking.The conditions shown in Table 1 ensured a stable electrospinning process for atleast 20 min at a flow rate of 3 ml/h before solution gelation occurred.

    Scaffolds prepared with different cross-linker concentrations are anticipated topossess different physical properties such as swelling ratio and degradation rate.However, except for scaffolds prepared with 4 wt% STMP, the swelling ratio ofall other substrates did not vary significantly. Swelling degree is influenced by twoantagonistic phenomena: the cross-linking which reduces swelling by stiffening themacromolecular matrix and the phosphate anionic charges which generate elec-trostatic repulsions between the neighbouring chains of the polysaccharides, thushaving a positive effect over swelling [23]. Here, an STMP concentration of 8 wt%or higher might have led to a balance in swelling. In the case of scaffold degradationrate, no significant difference was observed amongst all samples considered in thisstudy. Over the course of 7 days cell culture, STMP4 scaffolds adopted a film-likestructure (Figure 4a, 4c and 4e), likely due to the swelling and fusion of electrospunfibres. On the other hand, scaffolds prepared with 16 wt% STMP, retained their fi-brous morphology. It appears that, although 4 wt% STMP (with or without gelatin)was sufficient in preventing the complete dissolution of pullulandextran scaffoldsunder aqueous conditions, this concentration of STMP was insufficient in retaininga stable fibrous architecture. On the other hand, 16 wt% STMP (with or withoutgelatin) is sufficient in stabilizing the nanofibrous structures for at least 28 dayswhile remaining cytocompatible.

    Cytocompatibility of pullulan/dextran fibres was analyzed by culturing HDFsfor 3 and 7 days. As pullulan and dextran are hydrophilic, gelatin was included topromote cell adhesion [29, 30]. In particular, gelatin appeared to have significant ef-fects on enhancing cell proliferation after 7 days of culture. In order to elucidate theeffects of scaffold topography on cellular behaviour, hydrogels of similar chemicalcontent were analyzed in parallel. From microscopy analyses and WST-1 assay, itappears that regardless of STMP concentration, electrospun scaffolds enhanced celladhesion and viability more significantly as compared to hydrogels. This is likelydue to the increase in surface area provided by nanofibres for cell attachment andgrowth [31]. Although cell adhesion and spreading were improved, no cellular pen-etration was observed in the fibrous constructs. This lack of cell penetration intorandomly-oriented electrospun fibres is consistent with previous in vitro and in vivostudies where macrophage invasion and in vivo cell penetration were observed onlyon scaffolds comprising of aligned electrospun fibres [31]. Comparing cellular be-haviour on STMP16 samples versus STMP4 samples, an increase in cross-linking

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    density favoured HDF cell adhesion and proliferation. The synergistic effects ofgelatin and higher cross-linking density in STMP16GEL10 samples further pro-moted similar extent of cell metabolic activity and actin stress fibre formation ascompared to TCPS after 7 days of culture. Altogether, these results suggested that ahigher cross-linking density and the fibrous surface of pullulan/dextran electrospunscaffolds promote more positive cellsubstrate interactions in HDF than hydrogels.

    5. Conclusions

    In this study the long-term structural stability of cross-linked pullulan/dextrannanofibres was demonstrated. By varying the cross-linker concentration from 4 to16 wt%, nanofibrous architecture could be retained for at least 28 days under hy-drated conditions. The advantage of presenting pullulan/dextran as nanofibrous con-structs over hydrogels was also shown with enhanced cell attachment and metabolicactivity being observed in cells cultured on nanofibres. Taken together, electrospunpullulan/dextran fibrous scaffolds have the potential to serve as biomimetic con-structs capable of providing topographic signals to seeded cells over prolonged timeperiods for tissue engineering.

    Acknowledgements

    Partial funding support by the National Medical Research Council (NMRC) Ex-ploratory/Development Grant (NMRC/EDG/0027/2008) and the MOE Tier 1 Re-search Grant (RG36/07) is acknowledged. We thank Mr. Frederic Nadaud (UTCCompiegne, France) for performing the ESEM analysis and Dr. J. M. Anderson(Case Western Reserve University, Cleveland, OH, USA) for vetting through themanuscript.

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