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

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<ul><li><p>This article was downloaded by: [York University Libraries]On: 11 November 2014, At: 05:53Publisher: Taylor &amp; FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK</p><p>Journal of Biomaterials Science,Polymer EditionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tbsp20</p><p>Long-Term Stabilization ofPolysaccharide Electrospun Fibresby In Situ Cross-LinkingLiya Shi a , Catherine Le Visage b &amp; 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.</p><p>To cite this article: Liya Shi , Catherine Le Visage &amp; 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</p><p>To link to this article: http://dx.doi.org/10.1163/092050610X512108</p><p>PLEASE SCROLL DOWN FOR ARTICLE</p><p>Taylor &amp; Francis makes every effort to ensure the accuracy of all the information(the Content) contained in the publications on our platform. However, Taylor&amp; Francis, our agents, and our licensors make no representations or warrantieswhatsoever as to the accuracy, completeness, or suitability for any purposeof the Content. Any opinions and views expressed in this publication are theopinions and views of the authors, and are not the views of or endorsed byTaylor &amp; Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor andFrancis shall not be liable for any losses, actions, claims, proceedings, demands,costs, expenses, damages, and other liabilities whatsoever or howsoever caused</p><p>http://www.tandfonline.com/loi/tbsp20http://www.tandfonline.com/action/showCitFormats?doi=10.1163/092050610X512108http://dx.doi.org/10.1163/092050610X512108</p></li><li><p>arising directly or indirectly in connection with, in relation to or arising out of theuse of the Content.</p><p>This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expresslyforbidden. Terms &amp; Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Yor</p><p>k U</p><p>nive</p><p>rsity</p><p> Lib</p><p>rari</p><p>es] </p><p>at 0</p><p>5:53</p><p> 11 </p><p>Nov</p><p>embe</p><p>r 20</p><p>14 </p><p>http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions</p></li><li><p>Journal of Biomaterials Science 22 (2011) 14591472brill.nl/jbs</p><p>Long-Term Stabilization of Polysaccharide ElectrospunFibres by In Situ Cross-Linking</p><p>Liya Shi a, Catherine Le Visage b and Sing Yian Chew a,</p><p>a School of Chemical and Biomedical Engineering, N1.2-B2-20, Nanyang Technological University,62 Nanyang Drive, 637459 Singapore</p><p>b Inserm, U698, Bio-ingnierie Cardiovasculaire, CHU X. Bichat, 46 Rue Henri Huchard,75877 Paris Cedex 18, France</p><p>Received 21 April 2010; accepted 21 May 2010</p><p>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 &lt; 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 &lt; 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 &lt; 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</p><p>KeywordsPullulan, dextran, nanofibres, cytocompatibility, regenerative medicine</p><p>* To whom correspondence should be addressed. Tel.: (65) 6316-8812; Fax: (65) 6794-7553; e-mail:SYChew@ntu.edu.sg</p><p> Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050610X512108</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Yor</p><p>k U</p><p>nive</p><p>rsity</p><p> Lib</p><p>rari</p><p>es] </p><p>at 0</p><p>5:53</p><p> 11 </p><p>Nov</p><p>embe</p><p>r 20</p><p>14 </p></li><li><p>1460 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472</p><p>1. Introduction</p><p>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.</p><p>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.</p><p>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.</p><p>2. Materials and Methods</p><p>2.1. Electrospinning of Pullulan/Dextran Fibres</p><p>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</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Yor</p><p>k U</p><p>nive</p><p>rsity</p><p> Lib</p><p>rari</p><p>es] </p><p>at 0</p><p>5:53</p><p> 11 </p><p>Nov</p><p>embe</p><p>r 20</p><p>14 </p></li><li><p>L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472 1461</p><p>Table 1.Processing parameters for electrospun pullulan/dextran scaffolds</p><p>Sample Polymer Gelatin STMPconcentration content content(wt%) (wt%) (wt%)</p><p>Fibres without gelatinSTMP4GEL0 20 0 4STMP8GEL0 20 0 8STMP12GEL0 20 0 12STMP16GEL0 20 0 16</p><p>Fibres with gelatinSTMP4GEL10 20 10 4STMP8GEL10 20 10 8STMP12GEL10 20 10 12STMP16GEL10 16.7 10 16</p><p>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.</p><p>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.</p><p>2.2. Characterization of Pullulan/Dextran Fibrous Scaffolds</p><p>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.</p><p>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.</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Yor</p><p>k U</p><p>nive</p><p>rsity</p><p> Lib</p><p>rari</p><p>es] </p><p>at 0</p><p>5:53</p><p> 11 </p><p>Nov</p><p>embe</p><p>r 20</p><p>14 </p></li><li><p>1462 L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472</p><p>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.</p><p>2.3. Evaluation of Cellular Interactions with Electrospun Pullulan/DextranScaffolds</p><p>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.</p><p>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).</p><p>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.</p><p>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-</p><p>Dow</p><p>nloa</p><p>ded </p><p>by [</p><p>Yor</p><p>k U</p><p>nive</p><p>rsity</p><p> Lib</p><p>rari</p><p>es] </p><p>at 0</p><p>5:53</p><p> 11 </p><p>Nov</p><p>embe</p><p>r 20</p><p>14 </p></li><li><p>L. Shi et al. / Journal of Biomaterials Science 22 (2011) 14591472 1463</p><p>ronmental SEM (Philips ESEM-FEG XL30) with an accelerating voltage of 15 kVat a pressure of 3.5 Torr.</p><p>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.</p><p>2.4. Statistical Analysis</p><p>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-samp...</p></li></ul>

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