in vitro biocompatibility of electrospun and solvent-cast chitosan substrata towards schwann,...

European Polymer Journal 46 (2010) 428–440

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Contents lists available at ScienceDirect

European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Macromolecular Nanotechnology

In vitro biocompatibility of electrospun and solvent-cast chitosansubstrata towards Schwann, osteoblast, keratinocyte and fibroblast cells

Pakakrong Sangsanoh a,b, Orawan Suwantong a,b, Artphop Neamnark a,b,Poonlarp Cheepsunthorn c, Prasit Pavasant d, Pitt Supaphol a,b,*

a The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailandb The Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailandc Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailandd Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o

Article history:Received 5 August 2009Received in revised form 5 October 2009Accepted 24 October 2009Available online 4 November 2009

Keywords:ElectrospinningFibrous membranesChitosanBiocompatibility

0014-3057/$ - see front matter � 2009 Elsevier Ltddoi:10.1016/j.eurpolymj.2009.10.029

* Corresponding author.E-mail address: [email protected] (P. Supaphol).

a b s t r a c t

Chitosan or poly(N-acetyl-D-glucosamine-co-D-glucosamine) with a degree of deacetylationof about 85% was fabricated into nanofibrous membranes by electrospinning from 7% w/vchitosan solution in 70:30 v/v trifluoroacetic acid/dichloromethane solvent system. Theobtained fibers were smooth without the presence of beads. The diameters of the individualfiber segments were 126 ± 20 nm. The potential for use of the electrospun chitosan nanofi-brous membranes as substrates for cell/tissue culture was evaluated with four different celltypes, i.e., Schwann cells, osteoblast-like cells, keratinocytes and fibroblasts, in terms of theattachment and the proliferation of the cells as well as the morphology of the seeded andthe cultured cells. The results were compared with the corresponding solvent-cast films. Bothtypes of the chitosan substrates supported the attachment and, at the same time, promotedthe largest increase in the viability of the cultured keratinocytes. The viability of Schwanncells cultured on these substrates increased marginally well, but the attachment of the cellson the surfaces was relatively poor. Finally, both types of the chitosan substrates showedcytostatic property towards both osteoblast-like cells and fibroblasts, despite the convinc-ingly good attachment of osteoblast-like cells on the surfaces.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Tissue engineering is an interdisciplinary technologythat applies materials engineering, cellular biology and ge-netic engineering towards the development of biologicalsubstitutes for tissues, such as skin, cartilage for joints, boneand so on [1]. The primary objectives of these substitutes areto restore, maintain and/or improve tissue functions. Aneffectual scaffold for tissue engineering must support anddefine the three-dimensional (3D) organization of the tis-sue-engineered space and maintain the normal differenti-ated state of cells within the cellular compartment. Ideally,an effectual scaffold should mimic the structure and biolog-

. All rights reserved.

ical functions of native extracellular matrix (ECM) proteins[2], so as to provide mechanical support and regulate cellu-lar activities [3]. A wide variety of fabrication techniqueshave been used to generate 3D polymeric scaffolds for po-tential for use in tissue regeneration. Electrospinning is atechnique capable of producing ultra-fine fibers with diam-eters in sub-micrometer down to nanometer range throughthe action of a high electric field [4]. Under such a high elec-tric field, a stream of a polymer liquid (solution or melt) isejected towards a collector. It undergoes a significantstretching during its flight to the collector, resulting in thedeposition of solid fibers in the form of a non-woven fabricon the collector [4]. The 3D structure and topography ofthe electrospun polymeric fiber mats resemble those of thecollagen bundles in the natural ECM [2]. Other characteris-tics, such as high surface area, high porosity and high

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P. Sangsanoh et al. / European Polymer Journal 46 (2010) 428–440 429

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inter-pore connectivity, make electrospun fiber mats excel-lent candidates for tissue engineering [2,3,5–7].

Over the past decade, development of scaffolds for cell/tissue culture based on biodegradable and biocompatiblesynthetic or natural polymers has been heavily explored[8–12]. Among the various natural polymers, chitosan hasbeen regarded as one of the most suitable materials for bio-medical applications, owing to its biocompatibility, biode-gradability and non-toxicity [13]. Chitosan or poly(N-acetyl-D-glucosamine-co-D-glucosamine) is a partially N-deacetylated derivative of chitin or poly(N-acetyl-D-gluco-samine), one of the most abundant polysaccharides, derivedmainly from shells of arthropods as well as internal flexiblebackbone of cephalopods [13]. Direct electrospinning ofchitosan is quite difficult [14]. Consequently, electrospin-ning of chitosan has usually been carried out from its blendsolutions with another electrospinnable polymer, such aspoly(ethylene oxide) (PEO) in an aqueous solution of aceticacid [15–17], or acetic acid, hydrochloric acid, or trifluoro-acetic acid (TFA) [18]; ultra-high molecular weight PEO ina mixture of an aqueous solution of acetic acid and dimethylsulfoxide [19]; silk fibroin in an aqueous solution of formicacid [20]; poly(vinyl alcohol) (PVA) in an aqueous solutionof formic acid [14], acetic acid [14,21–26], or acrylic acid[27–29]; PVA and poly(vinyl pyrrolidone) (PVP) in an aque-ous solution of acetic acid [30]; poly(ethylene terephthal-ate) (PET) in TFA [31]; type I collagen in a mixed solventsystem of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and TFA[32–34]; type I collagen and PEO in an aqueous solution ofacetic acid [35]; polyacrylamide (PAAm) in an aqueous solu-tion of acetic acid [36]; poly(lactic acid) (PLA) in TFA [37]. Inaddition, electrospinning of certain derivatives of chitin orchitosan either in its native form or in its blend with anotherpolymer has been reported. Some of these are carboxy-methyl chitin [38] and carboxyethyl chitosan [39] in theirblends with PVA in distilled water; hexanoyl chitosan inchloroform [40]; and quaternary ammonium chitosan inits blend with either PVA [41] or PVP [42] in distilled water.

Notwithstanding, successful fabrication of pure chitosannanofibers has been reported from the electrospinning ofchitosan solutions in TFA or a mixture of TFA and dichloro-methane (DCM) [14,31,43–51], deacetylation of chitinnanofibers obtained from the electrospinning of chitin solu-tions in HFP [52], and the electrospinning of chitosan solu-tions in 90% aqueous acetic acid solution [53,54]. Purechitosan nanofibers could also be obtained by a two-step ap-proach: the preparation of bicomponent nanofibers of chito-san and another electrospinnable polymer (e.g., PVA) andthe subsequent removal of the polymer by solvent-extrac-tion [21,25]. Coaxial electrospinning, with chitosan formingthe core and PEO forming the sheath, can alternatively beused to prepare pure chitosan nanofibers by subsequentremoval of the PEO sheath [55]. Based on these previousreports [14,31,43–51], the fabrication of pure chitosannanofibers from chitosan solutions in TFA or TFA/DCM byelectrospinning has been most effective. However, furtherutilization of the chitosan nanofibrous membranes that areprepared from these chitosan solution systems is often lim-ited by the loss of the fibrous structure as soon as they are incontact with an aqueous medium [43,46,47]. The dissolu-tion of the electrospun chitosan fibers that are prepared

from the chitosan solutions in TFA or TFA/DCM occurs as aresult of the high solubility in the aqueous medium of the–NH3

+CF3COO- salt residues that are formed when chitosanis dissolved in TFA [43]. This, however, can be alleviated bycross-linking with glutaraldehyde [46,47]. The use of glutar-aldehyde as the cross-linking agent via either the one- [47]or the two-step [46] cross-linking process rendered thecross-linked fibers to be swellable, but insoluble, in neutraland acidic aqueous media. However, the biodegradabilityof the cross-linked chitosan fibers may be adversely af-fected, if the extent of cross-linking is too great.

In a previous work by some of us [43], the stability ofthe chitosan nanofibrous membranes in an aqueous med-ium was improved by immersing the membranes in 5 MNa2CO3 aqueous solution for 3 h at ambient condition. Itwas shown that the post-neutralized membranes couldmaintain their fibrous structure even after a continuoussubmersion in phosphate buffer saline (PBS, pH = 7.4) ordistilled water for 12 weeks. As an evidence for actual use-fulness of the proposed neutralization method, it wasshown that the post-neutralized chitosan nanofibrousmembranes could be used as substrates for in vitro bio-compatibility study with a schwannoma cell line, RT4-D6P2T [6]. Recently, Wan et al. [49] utilized the proposedneutralization procedure to ascertain the stability of boththe poly(chitosan-g-DL-lactic acid) (PCLA) and the chitosanscaffolds prepared by electro-wet-spinning process in thecell culturing condition using primary dermal fibroblastsharvested from a New Zealand rabbit as reference.

Despite the numerous reports on the in vitro responsesof difference cell lineages on blend and pure electrospunchitosan nanofibers [6,17,35,45,49], a similar report thatstudy the in vitro responses and cellular behavior of anytype of the cell lineages on pure chitosan nanofibrous scaf-folds is still lacking. Here, we report, the in vitro biologicalevaluation of the pure electrospun chitosan nanofibrousmembranes towards four different types of cells, i.e., Schw-ann cells, osteoblast-like cells, keratinocytes and fibro-blasts, in comparison with the corresponding solvent-castfilms and the tissue-culture polystyrene plate (TCPS).

2. Experimental

2.1. Materials

Chitosan [powder, degree of deacetylation (%DD) � 85%;see detailed characterization in supplementary data], triflu-oroacetic acid (TFA, CF3COOH; �98% purity) and dichloro-methane (DCM) were purchased from Sigma–Aldrich(USA). Na2CO3, used as the neutralizing agent, was pur-chased from Sigma–Aldrich (USA). Both the weight-averageand the number-average molecular weights of chitosanwere determined to be about 610 and 110 kg mol�1, respec-tively (see detailed characterization in supplementary data).

2.2. Fabrication of electrospun chitosan nanofibrousmembranes and corresponding solvent-cast films

Electrospun chitosan nanofibrous membranes were pre-pared in a manner similar to a previous report [43]. Briefly,7% w/v chitosan solution was prepared by dissolving a mea-

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sured amount of chitosan powder in 70:30 v/v TFA/DCMmixture. The as-prepared chitosan solution was stirred con-tinuously for 12 h at room temperature and later fed into a 5-mL glass syringe fitted with a blunt 20-gauge stainless steelneedle (OD = 0.91 mm), used as the nozzle. Both the syringeand the needle were tilted 45� from a horizontal baseline.The electrospun chitosan nanofibers were collected on analuminum sheet wrapped around a homemade rotating cyl-inder (width and diameter�15 cm; 40–50 rpm), placed at afixed distance of 20 cm from the needle tip. The needle wasconnected to the emitting electrode of positive polarity of aGamma High-Voltage Research ES30P-5 W power supply.Both the electrical potential and the collection time werefixed at 25 kV and 24 h, unless noted otherwise. The solutionfeed rate was driven mainly by the gravity and the electricalforces generated during electrospinning. The resultingchitosan nanofibrous membranes were dried in vacuo atroom temperature prior to further investigation.

To further stabilize the chitosan nanofibrous mem-branes in the cell culturing medium, the membranes weresubjected to neutralization by a method previously pro-posed by some of us [43]. Briefly, the membranes were im-mersed in 5 M Na2CO3 aqueous solution for 3 h at ambientcondition. After the immersion, the membranes wererepeatedly washed with distilled water until of neutralpH, dried at ambient condition for 1 d and further driedin an oven at 40 �C overnight prior to further studies.

For comparison purposes, chitosan was also fabricatedinto films by the solvent-casting technique. The castingchitosan solution (at 2.5% w/v) was prepared by dissolvinga measured amount of chitosan powder in 1 wt.% acetic acidaqueous solution. After having been stirred until a clearsolution was obtained, it was cast on glass Petri dishes anddried in vacuo at room temperature prior to furtherinvestigation.

2.3. Wettability of electrospun chitosan nanofibrousmembranes and corresponding solvent-cast films

Wettability of the chitosan nanofibrous membrane andthe corresponding film surfaces was assessed by watercontact angle measurements. The static water contact an-gles were measured by a sessile drop method using a Krüsscontact angle measurement system. A distilled water drop-let (about 40 lL) was gently plated on the surface of eachspecimen. At least 10 readings on different parts of thespecimen were averaged to attain a data point.

2.4. Cell culture and cell seeding

To evaluate the potential for use of the electrospunchitosan nanofibrous membranes as scaffolding materials,their biocompatibility in terms of attachment and prolifer-ation of four different cell lineages, including murineSchwann cells (RT4-D6P2T; a schwannoma cell line de-rived from a N-ethyl-N-nitrosourea (ENU)-induced ratperipheral neurotumor), murine osteoblast-like cells(MC3T3-E1; mouse calvaria-derived, preosteoblastic cells),human keratinocytes (HaCaT; an immortalized, but non-tumorigenic cell line) and human foreskin fibroblasts(HFF; a non-transformed cell line, originating from pooled

foreskins of children), was evaluated in comparison withthat of the corresponding films and TCPS.

RT4-D6P2T and MC3T3-E1 were first cultured as a mono-layer in Dulbecco’s modified Eagle’s medium (DMEM; Sig-ma–Aldrich, USA), supplemented by 5% fetal bovine serum(FBS; Biochrom AG, Germany), 1% L-glutamine (Invitrogen,USA) and 1% antibiotic and antimycotic formulation [con-taining penicillin G sodium, streptomycin sulfate andamphotericin B (Invitrogen, USA)]. The cells were incubatedat 37 �C in a humidified atmosphere containing 5% CO2 andthe culture medium was replaced once in every 2 d. Eachof the free-standing chitosan nanofibrous membrane andthe corresponding film samples was cut into circular disks(�15 mm in diameter) and the disk specimens were placedin wells of a 24-well tissue-culture polystyrene plate (TCPS;Biokom System, Poland), which were later sterilized in 70%ethanol for 30 min. The specimens were then washed withautoclaved deionized water and subsequently immersed inDMEM overnight. To ensure a complete contact betweenthe specimens and the wells, a stainless steel ring (stainlesssteel grade 304; �14 mm in diameter) was placed on top ofeach specimen as well as in empty wells of TCPS. The refer-ence cells from the cultures were trypsinized [0.25% trypsincontaining 1 mM ethylene diamine tetraaceticacid (EDTA;Invitrogen, USA)], counted by a haemocytometer (HausserScientific, USA) and seeded at a density of about40,000 cells/well on both the nanofibrous membrane andthe film specimens and empty wells of TCPS that were usedas control.

HaCaT and HFF were also cultured based on the sameprocedure but slightly different in the ingredients of thecell culturing medium supplement, i.e., 10% fetal bovineserum (FBS, BIOCHROM, Germany), 1% L-glutamine (Invit-rogen, USA), together with 100 U mL�1 penicillin (Invitro-gen, USA) and 100 lg mL�1 streptomycin (Invitrogen,USA).

2.5. Cell attachment and cell proliferation

For the cell attachment study, the reference cells wereseeded on both the nanofibrous membrane and the filmspecimens in wells of a 24-well TCPS at 40,000 cells/welland allowed to attach to the surfaces of the specimensfor 2, 8 and 24 h. The viability of the cells in bare wells ofthe TCPS was used as control. At each specified seedingtime, the viability of the attached cells was quantified by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay. Each specimen was rinsed withphosphate-buffered saline (PBS; Sigma–Aldrich) to removeunattached cells prior to the MTT assay. For the cell prolif-eration study, the cells were first seeded on both the nano-fibrous membrane and the film specimens in wells of a 24-well TCPS at 40,000 cells/well and a priori allowed to attachto the specimens for 24 h. The viability of the proliferatedcells on the specimens was quantified by the MTT assayafter the cells had been cultured for 1, 3 and 5 d. The meth-od was similar to the cell attachment study except for thefact that the culture medium was replaced with SFM ondays 1 and 3 in order to get rid of the effect of the nutrientsthat are present in the medium on the increase in the via-bility of the cultured cells.

Fig. 1. Representative SEM image of electrospun chitosan nanofibrousmembrane.


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2.6. Quantification of viable cells by MTT assay

The MTT assay is the method used to quantify the via-bility of cells on the basis of the reduction of the yellow tet-razolium salt to purple formazan crystals bydehydrogenase enzymes secreted from the mitochondriaof metabolically active cells. The amount of purple forma-zan crystals relates to the number of the viable cells in alinear manner. First, the culture medium of each culturedspecimen was removed and replaced with 500 lL/well ofMTT solution (Sigma–Aldrich, USA) and then the platewas incubated for 1 h. After incubation, the MTT solutionwas removed. Then, 500 lL/well of dimethyl sulfoxide(DMSO; Riedel-de Haën, Germany) was added to dissolvethe formazan crystals and the plate was left at room tem-perature in darkness for 1 h on a rotary shaker. Finally, theabsorbance at 570 nm, representing the proportion of theviable cells, was recorded by a Thermospectronic Gene-sis10 UV–visible spectrophotometer.

2.7. Statistical analysis

The data are presented as means ± standard errors ofthe means (n = 3). A one-way ANOVA was used to comparethe means of different data sets and a statistical signifi-cance was accepted at a 0.05 confidence level.

2.8. Morphological observation of cultured cells

After the culture medium had been removed, the cell-cultured nanofibrous membrane and film specimens wererinsed with PBS twice and the cells were fixed with 3% glu-taraldehyde solution [diluted from 50% glutaraldehydesolution (Electron Microscopy Science, USA) with PBS] at500 lL/well. After 30 min, they were rinsed again withPBS and kept in PBS at 4 �C. After cell fixation, the speci-mens were dehydrated in an ethanol solution of varyingconcentration (i.e., 30%, 50%, 70% and 90%) and, finally,with pure ethanol for about 2 min each. The specimenswere then dried in 100% hexamethyldisilazane (HMDS;Sigma–Aldrich, USA) for 5 min and later dried in air afterthe removal of HMDS. After being completely dried, thespecimens were mounted on copper stubs, coated withgold using a JEOL JFC-1100E sputtering device for 3 minand observed by a JEOL JSM-5200 scanning electron micro-scope. For comparison, the morphology of the cells thathad been seeded or cultured on a glass substrate (coverglass slide, 1 mm in diameter; Menzel, Germany) was alsoinvestigated.

3. Results and discussion

As mentioned, an attempt to mimic the structure andfunction of the natural ECM has been the most importantpoint for the development of an effectual scaffold thatcan restore, maintain and/or improve the functions of tis-sues. This can be achieved with the right choices of thematerials and the methods with which the materials arefabricated. Chitosan, due to the presence of the N-acetyl-D-glucosamine co-monomeric unit, is, at least partly, struc-

turally similar to hyaluronic acid or poly(D-glucuronicacid-co-N-acetyl-D-glucosamine), one of the major glycos-aminoglycans (GAGs) of the natural ECM in native tissues[56]. The fabrication of chitosan into fibrous form by elec-trospinning, with size equivalent to that of the collagenbundles found in the native tissues [2], could render thematerial a suitable candidate as an effectual scaffold forcell/tissue culture.

Here, electrospinning of chitosan was carried out in amanner similar to a previous report by some of us [43].Smooth fibers without the presence of beads were ob-tained (see Fig. 1) and the diameters of these fibers were126 ± 20 nm. After consecutive spinning for �2 d, thethickness of the chitosan nanofibrous membranes was35 ± 2 lm. As shown in supplementary data, predomi-nantly smooth fibers were only obtained from 7% and 8%w/v chitosan solutions in TFA/DCM, while a combinationof smooth and beaded fibers was obtained from 5% and6% w/v chitosan solutions. While the electrospinning ofchitosan from its solution in TFA could be achieved at alow concentration (i.e., 2.7% w/v) [46,47], the electrospin-ning of the polymer from its solutions in TFA/DCM, onthe other hand, was successfully performed at relativelygreater concentrations [14,43,45,48,50,51] (see summaryof the data obtained from the literature in supplementarydata). This is postulated to be a result of the reduction inthe viscosity of the chitosan solutions due to the presenceof DCM as the modifying co-solvent.

In the present work, the potential for use of the electro-spun chitosan nanofibrous membranes as substrates forcell/tissue culture was evaluated with four different typesof cells, e.g., murine Schwann cells (RT4-D6P2T; hereafter,Schwann cells), murine osteoblast-like cells (MC3T3-E1;hereafter, osteoblast-like cells), human keratinocytes (Ha-CaT; hereafter keratinocytes) and human fibroblasts(HFF; hereafter, fibroblasts), in terms of the attachmentand the proliferation of the cells as well as the morphologyof the seeded and the cultured cells. The corresponding sol-vent-cast films and TCPS were used as the internal and thepositive controls, respectively. The thickness of the sol-vent-cast chitosan films was 72 ± 5 lm. The reason forthe aqueous solution of acetic acid being used instead ofthe TFA/DCM mixture as the casting solvent for the fabrica-


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tion of chitosan films was due mainly to the cost of TFA.The choice of either solvent system, however, did not havea noticeable effect on the surface morphology of the ob-tained films (see additional experiment in supplementarydata).

It is a known fact that wettability of the surface of ascaffold plays an important role on the adhesion of ECMbiomolecules which promote seeded or cultured cells to at-tach, proliferate and differentiate. The static water contactangle assessment of the surfaces of the electrospun chito-san nanofibrous membranes and corresponding solvent-cast films indicated that the wettability of the membranesurfaces was complete as perfect spreading of water drop-lets was observed (data not shown).

3.1. Cell attachment and cell proliferation

The ability to support the attachment of cells is one ofthe foremost characteristics of an effectual scaffold. Toevaluate such a characteristic, the reference cells wereseeded on the chitosan nanofibrous substrates for variouscell seeding time intervals. The results were comparedwith those obtained with the corresponding film sub-strates and TCPS. Fig. 2 shows the viability of the four dif-

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Fig. 2. Attachment of (a) murine Schwann cells (RT4-D6P2T), (b) murine osteoblafibroblasts (HFF) that had been seeded on electrospun chitosan nanofibrous andand 24 h. The viability of the attached cells that had been seeded on TCPS for 2 h wshown in the figure. *,#Significantly different at p < 0.05.

ferent cell types after the cells had been seeded on thechitosan nanofibrous and the film substrates, in compari-son with that on TCPS, for 2, 8 and 24 h. The viability ofthe cells attached on TCPS at 2 h after cell seeding was usedas the reference value to calculate the relative viability ofthe attached cells shown in the figure. For any given celltype, the viability of the attached cells on these substratesgenerally increased significantly with an increase in thecell seeding time, with an exception to Schwann cells onthe nanofibrous substrates which showed a decrease, andfibroblasts on both the nanofibrous and the film substrateswhich showed a constancy, in their viability between 8 and24 h of cell seeding.

At any given cell seeding time point, the viability of thecells attached on TCPS was generally greater than that ofthe cells attached on both the nanofibrous and the filmsubstrates. However, equivalent values were observed forosteoblast-like cells that had been seeded on the nanofi-brous substrates for 2 h, keratinocytes that had beenseeded on the film substrates for 8 h, and keratinocytesthat had been seeded on both the fibrous and the film sub-strates for 24 h. Comparatively, between the nanofibrousand the film substrates, the viability of Schwann cells,osteoblast-like cells and keratinocytes that had been

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st-like cells (MC3T3-E1), (c) human keratinocytes (HaCaT), and (d) humancorresponding solvent-cast film substrates and TCPS (i.e., control) for 2, 8as used as reference to calculate the relative viability of the attached cells


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seeded on the nanofibrous substrates, in most cases, wasgreater than that of the cells on the film counterparts, withan exception to fibroblasts that had been seeded for 24 hwhich showed a comparable value. Additionally, the viabil-ity of keratinocytes that had been seeded on the nanofi-brous substrates for 2 and 8 h was inferior to that of thecells on the film counterparts. However, at 24 h of cellseeding, the viability of the cells on both types of sub-strates showed equivalent values.

The ability of the different substrates in promoting theattachment of the cells could be evaluated further byobserving the viability of the cells attached on a given typeof substrates whether it was either increased or decreasedbetween two adjacent seeding time points. Between 2 and8 h of cell seeding, the largest increase in the viability ofthe attached Schwann cells and osteoblast-like cells wasobserved when they were seeded on TCPS, while that ofthe attached keratinocytes and fibroblasts was observedwhen they were seeded on the nanofibrous and the filmsubstrates, respectively. Interestingly, osteoblast-like cellsthat had been seeded on the film substrates showed anequivalent increase in the viability of the attached cellsto that on TCPS. Between 8 and 24 h of cell seeding, thelargest increase in the viability of the attached osteo-blast-like cells and fibroblasts was observed when they


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Fig. 3. Proliferation of (a) murine Schwann cells (RT4-D6P2T), (b) murine ostehuman fibroblasts (HFF) that had been cultured on electrospun chitosan nanofibrfor 1, 3 and 5 d. The viability of the attached cells that had been cultured on TCproliferated cells shown in the figure. *,#Significantly different at p < 0.05.

were seeded on TCPS, while that of the attached keratino-cytes and osteoblasts-like cells was observed when theywere seeded on the nanofibrous and the film substrates,respectively.

The biocompatibility of the chitosan nanofibrous andthe corresponding film substrates was further evaluatedin terms of their ability to promote the proliferation ofthe cells that had been allowed to attach on their surfaces,in comparison with that of TCPS, for 24 h. Fig. 3 shows theviability of Schwann cells, osteoblast-like cells, keratino-cytes and fibroblasts that had been cultured on the varioussubstrates for 1, 3 and 5 d. It should be noted that the via-bility of the cells that had been allowed to attach on thevarious substrates for 24 h was taken as the viability ofthe proliferated cells on day 1 and that the viability ofthe cells that had been cultured on TCPS on day 1 was usedas the reference value to determine the relative viability ofthe proliferated cells shown in the figure. Similar to theattachment assay, the viability of the cells proliferated onthe surfaces of these substrates generally increased withan increase in the cell culturing time. However, fibroblaststhat had been cultured on the film substrates showed noincrease in their viability between days 1 and 3 of cell cul-turing. The similar behaviors were also observed on osteo-blast-like cells and fibroblasts that had been cultured on


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oblast-like cells (MC3T3-E1), (c) human keratinocytes (HaCaT), and (d)ous and corresponding solvent-cast film substrates and TCPS (i.e., control)PS for 1 d was used as reference to calculate the relative viability of the

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the film and the nanofibrous substrates between days 3and 5 of cell culturing, respectively.

Evidently, the viability of the cells that had been cul-tured on TCPS, at any given cell culturing time point, wasconsistently greater than that of the cells on both the nano-fibrous and the film substrates, with an exception to that ofkeratinocytes on both the nanofibrous and the film sub-strates on day 1 which showed equivalent values to thatof TCPS. In most cases, the viability of the cells that hadbeen cultured on the nanofibrous substrates, at any givencell culturing time point, was greater than that of the cellson the film counterparts. An exception to this was forSchwann cells that had been cultured on the film sub-strates for 3 d which showed a reversed trend. Moreover,no appreciable difference in the viability of keratinocytesthat had been cultured on the nanofibrous and the filmsubstrates at any given cell culturing time point was ob-served. Between days 1 and 3 of cell culturing, the largestincrease in the viability of all types of the proliferated cellswas observed when they were grown on TCPS. Between

Table 1Representative SEM images of murine Schwann cells (RT4-D6P2T) hat had been seesolvent-cast film substrates and TCPS (i.e., control) for 2 h, 8 h, 1 d, 3 d and 5 d.

Cell seeding/culturing time point Type of substrate

Glass

2 h

8 h

24 h or 1 d

3 d

5 d

days 3 and 5, a similar observation was obtained, with anexception to keratinocytes which showed the largest in-crease in the viability when they were grown on both typesof film and nanofibrous substrates.

3.2. Morphology of attached and proliferated cells

The assessment on the ability of a scaffold to supportthe attachment and to promote the proliferation of theseeded or the cultured cells should not rely solely on theresults obtained by the MTT assay. This is because themethod, despite its simplicity, only provides a biologicalsnapshot of the mitochondria’s activity that is taken to rep-resent the viability of the cells. Any factor that affects tothe mitochondria’s activity can also affect the MTT absor-bance, despite an equivalent number of cells under inves-tigation. To better assess the ability of a scaffold tosupport the attachment and to promote the proliferationof the cells, morphological observation of the seeded orthe cultured cells can give additional information about

ded or cultured on electrospun chitosan (CS) nanofibrous and corresponding

CS nanofibers CS film


the phenotypic nature of the cells, intercellular interaction,and possible interaction between the cells and the sub-strates onto which they adhere. Tables 1–4, respectively,show representative SEM images of Schwann cells, osteo-blast-like cells, keratinocytes and fibroblasts that had beenseeded or cultured on the surfaces of the chitosan nanofi-brous and the corresponding film substrates as well as thatof the glass substrates for 2 h, 8 h, 1 d, 3 d and 5 d. It shouldbe noted that the cell seeding/culturing period of 24 h or1 d was considered both as the late attachment periodand as the early proliferation period.

Studies related to the attachment and the proliferation ofSchwann cells on the chitosan nanofibrous and the film sub-strates stem from the facts that Schwann cells play a crucialrole during nerve regeneration through the production ofgrowth factors and the excretion of ECM [57]. Developmentof an effectual bioartificial nerve graft, comprised of a bio-material pre-seeded with Schwann cells, should be an inter-esting approach for effective nerve regeneration [58].According to Table 1, even at 2 h after cell seeding, the cells

Table 2Representative SEM images of murine osteoblast-like cells (MC3T3-E1) hat hadcorresponding solvent-cast film substrates and TCPS (i.e., control) for 2 h, 8 h, 1 d


Glass

2 h

8 h

24 h or 1 d

3 d

5 d

cultured on both the film and the glass substrates appearedin their typical spindle shape with bipolar or multipolarshape. On the contrary, those cultured on the nanofibroussubstrates were still round, suggesting that the cells mightnot be fully attached on the surface. At 8 h and 1 d after cellseeding and cell culturing, the cells on both the film and theglass substrates extended their cytoplasm in the form of thinand long fibrils, similar to lamellipodia, from the leadingedges. On the other hand, those cultured on the nanofibroussubstrates, despite an evidence of the cells extending theircytoplasm along the individual fibers, were still round. Thecytoplasmic expansion of the cells cultured on the nanofi-brous substrates was observed for the first time on day 3after cell culturing. On day 5 after cell culturing, intercon-nection of the cultured cells on any type of substrates wasdiscernible.

Chitosan was shown to be an effective substrate thatsupports the initial attachment and the spreading of oste-oblasts [59]. According to Table 2, osteoblast-like cells thathad been seeded on all types of the substrates were round,

been seeded or cultured on electrospun chitosan (CS) nanofibrous and, 3 d and 5 d.

CS nanofibers CS film

MA

CRO

MO

LECU

LAR

NA

NO

TECH

NO

LOG

Y

Table 3Representative SEM images of human keratinocytes (HaCaT) hat had been seeded or cultured on electrospun chitosan (CS) nanofibrous and correspondingsolvent-cast film substrates and TCPS (i.e., control) for 2 h, 8 h, 1 d, 3 d and 5 d.


Glass CS nanofibers CS film

2 h

8 h

24 h or 1 d

3 d

5 d


MA

CRO

MO

LECU

LAR

NA

NO

TECH

NO

LOG

Y

with the ones that had been seeded on both the film andthe glass substrates exhibiting the evidence of lamellipodiaon their surface. The round morphology of the cells indi-cated that the cells might not yet be fully attached onthe surfaces of the substrates. At 8 h after cell seeding,the cells that had been seeded on the glass substratesexhibited an evidence of lamellipodia extending from theedge of the cells, but the morphology of the cells was stillround. Similarly, the majority of the cells seeded on thenanofibrous substrates were also round, while those onthe film counterparts assumed the spindle shape. On day1 after cell seeding/culturing, the majority of the cellsgrown on all types of the substrates were spindle-like. Fur-ther increasing the culturing time to 3 and 5 d resulted inthe expansion of the cytoplasm of the cells grown on theglass substrates to assume a polygonal shape. On the otherhand, while the majority of the cells grown on both thenanofibrous and the film substrates still assumed the spin-dle morphology, the cells that had been cultured on thenanofibrous substrates expanded relatively better.

Studies related to the attachment and the proliferationof keratinocytes and fibroblasts on the chitosan nanofi-brous and the film substrates stem from the fact that chito-san has been heavily explored as a promising material forwound healing [60] and artificial skin applications [61–66], due to its haemostatic and antibacterial properties.According to Table 3, keratinocytes on all types of the sub-strates after 2 and 8 h of cell seeding were still round.Lamellipodia were, however, observed on the surface ofthese cells. Interestingly, even at the short attachmenttime point of 8 h, aggregation of adjacent cells was evident.Clearly, these cells exhibited the characteristic cobblestonemorphology after they had been seeded on these sub-strates for only 8 h. On day 1 after cell seeding/culturing,the cells attached on the glass substrates existed in twopopulations, i.e., round cells with evidence of lamellipodiaand expanded cells that formed intercellular tight junc-tions with adjacent cells. In a similar manner, the majorityof the cells grown on both types of the chitosan substrateswere well-expanded, with the cells forming intercellular

Table 4Representative SEM images of human fibroblasts (HFF) hat had been seeded or cultured on electrospun chitosan (CS) nanofibrous and corresponding solvent-cast film substrates and TCPS (i.e., control) for 2 h, 8 h, 1 d, 3 d and 5 d.


Glass CS nanofibers CS film

2 h

8 h

24 h or 1 d

3 d

5 d


MA

CRO

MO

LECU

LAR

NA

NO

TECH

NO

LOG

Y

tight junctions with adjacent cells. On days 3 and 5 aftercell culturing, not only the cultured cells were well-ex-panded, with the cells forming intercellular tight junctionswith adjacent cells, but they also appeared to grow on topof one another, forming into multilayers of a cellular con-struct. It should be noted that, after about 3 d of cell cultur-ing, the chitosan substrates were well covered with thecells as well as their secreted matrix.

For potential for uses as wound dressing and skin substi-tutes, all of the chitosan substrates were further evaluatedwith fibroblasts. According to Table 4, the cells that had beenseeded on all types of the substrates for 2 h were round. At8 h after cell seeding, lamellipodia were observed aroundthe edge of the cells seeded on the films, indicating thebeginning of cellular expansion. A similar behavior was ob-served for the majority of the cells seeded on the glass sub-strates, however with slightly greater extent of cellularexpansion. Evidently, only the cells seeded on the nanofi-brous substrates were well-expanded. At the cell culturingtimes greater than or equal to 1 d, the cells that had been

grown on the glass substrates assumed their typical spindlemorphology and readily spread over the surface. On days 3and 5 after cell culturing, interconnection of adjacent cellswas evident. A similar result was observed with the cellsgrown on the nanofibrous substrates. On the film substrateshowever, both round and spindle-like cells were observedon their surface on day 1 after cell seeding/culturing. Moreexpansion of the cells and the interconnection of adjacent,expanded cells were observed when they had been grownon the film surface for the minimum period of 3 d.

3.3. Further discussion

Studies related to the in vitro biocompatibility of chito-san with mammalian cells have usually been conducted onchitosan in the form of solvent-cast films [58–60,62–67]with various cell types, e.g., Schwann cells (native cellsfrom sciatic nerve fragments of 1–2-d-old neonatal Spra-gue–Dawley rats) [58] and (primary rat Schwann cellsfrom sciatic nerve) [59], osteoblast-like cells (MC3T3-E1)


MA

CRO

MO

LECU

LAR

NA

NO

TECH

NO

LOG

Y

[60], fibroblasts (e.g., 3T3 [60], L929 [62], native cells fromforeskins of children [63], native cells from specimens ofskins from healthy donors [64,67], and normal adult hu-man dermal fibroblasts (NAHDF) [67]), keratinocytes(e.g., native cells from foreskins of children [63], HaCaT[64], and native cells from specimens of skins from healthydonors [67]), and baby hamster kidney cells (BHK21(C13))[62]. On the other hand, related studies of chitosan in theform of electrospun nanofibrous membranes are quite lim-ited, with few known reports being on fibroblasts (e.g., hu-man embryo skin fibroblasts (hESFs) [45] and native cellsfrom the back skin of a 60-d-old male New Zealand rabbit[49]). Many of these reports showed that a good number offactors affected the in vitro biocompatibility of chitosan,i.e., the degree of deacetylation (%DD) [59,62–64,66],molecular weight [64], residual proteins [64], and sub-strate morphology [i.e., film versus microfibers (15 lm indiameter)] [58]. Yuan et al. [58] showed that morphologyof the substrates played a major role in mediating thebehavior of the cultured cells. Specifically, they showedthat despite the fact that the viability of the native ratSchwann cells after having been cultured on the surfaceof the chitosan membranes for various cell culturing times,ranging from 1 to 14 d, was greater than that observed onthe chitosan macrofibers, the cells migrated more readilyonto the stereoframe of the fibers than they did on the sur-face of the films [58].

The extracellular matrix (ECM) proteins have the capac-ity to regulate cell behaviors such as adhesion, spreading,growth, and migration [59]. In a cell culture experiment,the factors influencing the adsorption of proteins are surfacewettability and surface charge [68,69]. It is a known fact thatcells carry a negative surface charge at physiological pH andthe magnitude of the charge depends mainly on the compo-sition of the cell membranes [62,70]. Chatelet et al. [63]showed that, in addition to the effect of %DD that influencedthe behavior of the seeded/cultured native keratinocytesfrom foreskins of children, the adhesion of the cells was fa-vored on smoother surfaces of the substrates. While both%DD and surface topography play major roles in regulatingthe adhesion of the cells onto chitosan substrates, the sur-face charge is another influencing factor. For chitosan, thepKa value is in the range of 6.2–7.0 [71], hence, in the phys-iological pH, chitosan would largely be expressed as neutralentity (i.e., no surface charge). As a result, the surface chargeshould not be regarded as a factor influencing the attach-ment of cells on chitosan substrates. The difference in theattachment of various cell lineages on the chitosan sub-strates, as observed in the present work, could be collectivecontributions from the different degrees of negative chargesof the cell membranes, the difference in the mediating effectof adsorbed proteins which could be different for differentcell lineages, and the smoothness of the substrates thatmay facilitate the mobility of the cells which could vary fordifferent cell types [63,64].

In the present work, even though the chitosan nanofi-brous substrates were slightly better in supporting theattachment and promoting the proliferation of Schwanncells, the cells grown on the surface of the films developedinto their phenotypic morphology much faster than theydid on the surface of the nanofibrous substrates, with the

formation of thin and long fibrils radiating from the lead-ing edges of the cells being evident only after 8 h after cellseeding. A similar result was observed with the cells grownon the glass substrates. These results suggest that Schwanncells may prefer the flat over rough surfaces [6]. Here, thesurface of the chitosan nanofibrous membranes was con-sidered rough when the sizes of the pores or other irregu-larities on the surface of the membranes were muchsmaller than those of the cells. Notwithstanding, a recentstudy by Wang et al. [51] demonstrated that preferentialalignment of the underlying chitosan nanofibrous struc-tures could play a part in mediating the alignment ofSchwann cells that had been cultured on their surfacesthrough the contact guidance phenomenon.

Fakhry et al. [60] showed that, at 1 h after cell seeding,osteoblasts (MC3T3-E1) adhered much better than fibro-blasts (3T3) did on the surfaces of the chitosan-coatedglasses. However, at 24 h after cell seeding, the number ofthe attached fibroblasts increased significantly, with the lev-els being equivalent to those of the attached osteoblasts. At1 h after cell seeding, osteoblasts already began to spread,while fibroblasts were still round. At 24 h after cell seeding,a combination of expanded and round fibroblasts was evi-dent [60]. On the other hand, Chatelet et al. [63] showed thatwhile fibroblasts (native cells from foreskin of children) at-tached twice more than keratinocytes on the surfaces ofchitosan of different %DD’s (3–47%), they could not prolifer-ate on these surfaces. They attributed this phenomenon tothe very high adhesion of fibroblasts on these surfaces thatcould inhibit their growth (i.e., cytostatic property: this isnot cytotoxic but inhibits cell proliferation). [63]. Others[62,64–66], on the other hand, showed that film substratesfabricated from chitosan with a relatively high %DD (i.e.,>90) could support the growth of fibroblasts relatively well.In the present work, both the chitosan nanofibrous and thecorresponding film substrates exhibited cytostatic propertytowards osteoblast-like cells and fibroblasts, as suggestedby the relative constancy in the MTT viabilities (see Fig. 3band d) and the relatively low number of cells seen on theSEM images (see Tables 2 and 4). On the other hand, as indi-cated by the MTT and the SEM results, both types of thechitosan substrates supported the attachment and the pro-liferation of keratinocytes extremely well, but they ap-peared to support the proliferation of Schwann cells onlymarginally.

4. Conclusion

Electrospun nanofibrous membranes of chitosan orpoly(N-acetyl-D-glucosamine-co-D-glucosamine) with the%DD of about 85% and the weight- and the number-aver-age molecular weights of 610 and 110 kg mol�1, respec-tively, were prepared from 7% w/v chitosan solution in70:30 v/v TFA/DCM. Smooth fibers with the diameters ofthe individual fiber segments being 126 ± 20 nm were ob-tained. The membranes were evaluated for their potentialfor use as substrates for cell/tissue culture on four differentcell lineages, i.e., Schwann cells, osteoblast-like cells, kerat-inocytes and fibroblasts, against the corresponding sol-vent-cast films. Both types of the chitosan substratessupported the attachment and the proliferation of kerati-


MA

CRO

MO

LECU

LAR

NA

NO

TECH

NO

LOG

Y

nocytes very wells. Despite the poor attachment on thesubstrates, Schwann cells proliferated marginally well onthese substrates. Finally, despite the good attachment ofosteoblast-like cells, both osteoblast-like cells and fibro-blasts were not able to proliferate on these substrates.

Acknowledgements

This work was supported in part by (1) the Ratchadaph-isek Somphot Endowment Fund for Research and ResearchUnit, Chulalongkorn University and (2) the Center forPetroleum, Petrochemicals and Advanced Materials (C-PPAM). P. Sangsanoh thanks a doctoral scholarship (PHD/0191/2550) received from the Royal Golden Jubilee Ph.D.Program, the Thailand Research Fund (TRF).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.eurpolymj.2009.10.029.

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in vitro biocompatibility of electrospun and solvent-cast chitosan substrata towards schwann,...

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