International Journal of Biological Macromolecules 48 (2011) 571–576Contents lists available at ScienceDirectInternational Journal of BiologicalMacromoleculesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /i j b i o m acFabrication of chitosan/poly(caprolactone)nanofibrous scaffold for bone and skin tissue engineeringK.T.Shalumon a ,K.H.Anulekha a ,K.P.Chennazhi a ,H.Tamura b ,S.V.Nair a ,∗,R.Jayakumar a ,∗a Amrita Center for Nanosciences and Molecular Medicine,Amrita Institute of Medical Sciences and Research Centre,Amrita Viswa Vidyapeetham,Kochi 682041,India bFaculty of Chemistry,Materials and Bioengineering and High Technology Research Centre,Kansai University,Osaka 564-8680,Japana r t i c l e i n f o Article history:Received 15December 2010Received in revised form 18January 2011Accepted 24January 2011Available online 1 February 2011Key words:ChitosanTissue engineering Poly(caprolactone)Nanofibers Contact anglea b s t r a c tChitosan/poly(caprolactone)(CS/PCL)nanofibrous scaffold was prepared by a single step electrospinning technique.The presence of CS in CS/PCL scaffold aided a significant improvement in the hydrophilicity of the scaffold as confirmed by a decrease in contact angle,which thereby enhanced bioactivity and protein adsorption on the scaffold.The cyto-compatibility of the CS/PCL scaffold was examined using human osteoscarcoma cells (MG63)and found to be non toxic.Moreover,CS/PCL scaffold was found to support the attachment and proliferation of various cell lines such as mouse embryo fibroblasts (NIH3T3),murine aneuploid fibro sarcoma (L929),and MG63cells.Cell attachment and proliferation was further confirmed by nuclear staining using 4 ,6-diamidino-2-phenylindole (DAPI).All these results indicate that CS/PCL nanofibrous scaffold would be an excellent system for bone and skin tissue engineering.© 2011 Elsevier B.V. All rights reserved.1.IntroductionTissue engineering,a combination of principles of engineering and life sciences to improve tissue function has evolved decades ago [1].The main aspect of tissue engineering is the develop-ment of a suitable scaffold which can mimic the extra cellular matrix.Natural extra cellular matrix is a combination of proteo-glycans (glycosaminoglycans)and fibrous proteins.Certain specific requirements of the scaffolds for tissue reconstruction are ade-quate pore size for cell seeding,diffusability throughout the matrix,and biodegradability.The design of a scaffold involves the selec-tion of a suitable material which is biodegradable,biocompatible as well as non toxic to the cells,selection of a suitable method/type of scaffold which can provide better surface for cell attachment,proliferation and differentiation.The extra cellular environment formed on nanofibers compared to that on solid-walled surfaces has led to the report of increased cellular attachment with several cell lines including osteoblastic cells [2,3],fibroblasts [4],normal rat kidney cells,smooth muscle cells [5],neural stem cells [6],and embryonic stem cells [7].This increased attachment across various cell types provides tissue engineers,a potential tool to generate functional tissues in shorter time frames than would be possible on more traditional scaffolds.As of now so many nat-∗Corresponding authors.Tel.:+914842801234;fax:+914842802020.E-mail addresses:nairshanti@ (S.V.Nair),rjayakumar@ ,jayakumar77@ (R.Jayakumar).ural and synthetic polymers as well as their blends have been tried in this case.A wide variety of polymers are used in fabri-cating scaffolds viz-poly(lactic acid)[6],poly(glycolic acid)[8,9],poly(lactic-co-glycolic acid)[10],poly(caprolactone)[11],or natu-ral ones such as collagen [12],gelatin [13],silk [14]and chitosan [15,16].Recently there is seen a growing interest in the produc-tion of scaffolds by using natural polymers like chitin [17,18],chitosan [19,20],alginate [21],collagen [22],gelatin [23–25]etc.,due to their non-toxicity,enhanced biocompatibility,cell adhe-sion and proliferation.Since the use of natural polymers have certain disadvantages like low stability,toxic degradation products which can be harmful to the cells,the natural polymers are often blended with synthetic polymers [26,27].Also this have enhanced mechanical properties,degradation stability and enhanced affin-ity to the cellular components.Chitin and chitosan have been used as scaffolds due to their biodegradability,hydrophilicity,non-antigenicity,non-toxicity,antimicrobial activity,bio adherence and cell affinity,which make chitosan the ideal candidate for uses in a wide range of applications [28–31].The scaffold material in our study is a blend of chitosan and polycaprolactone nanofibers obtained by single step electrospinning.In this technique the poly-mer solution is pumped through a syringe,forms fibers when high electric field is applied.When the applied field overcomes the sur-face tension of the polymer solution,the polymer forms continuous filaments and can be collected in a collector which is grounded.Many properties of PCL such as thermal degradation,hydrophilic-ity,biodegradability and mechanical properties can be improved by incorporation of CS in PCL [32].The blend of the both polymers0141-8130/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.ijbiomac.2011.01.020572K.T.Shalumon et al./International Journal of Biological Macromolecules48 (2011) 571–576can render good environment for cell attachment,proliferation and differentiation.In the present work,nanofibrous scaffold was prepared by a single step electrospinning technique using a new solvent mixture of formic acid and acetone as reported in our pre-vious study[32].Bioactivity and cytocompatibility of the CS/PCL nanofibrous scaffold was evaluated by in vitro biomineralisation, contact angle measurements,cell adhesion,cytotoxicity and pro-tein adsorption studies.2.Materials and methods2.1.MaterialsChitosan(CS)with degree of deacetylation80–85%and MW: 100–150kDa was purchased from Koyo chemicals Co.Ltd.,Japan. Poly(caprolactone)(PCL)(M W43,000–50,000)was purchased from Polysciences Inc.,USA.Formic acid and acetone purchased from RFCL Ltd.and Qualigens Fine Chemicals Ltd.,India,respectively. Syringes and needles were purchased from BD Sciences Ltd.,Spain.2.2.Preparation of polymer solutionsThe polymer solution for electrospinning was prepared by opti-mizing the CS and PCL concentrations in the solvent mixture,formic acid/acetone(70:30).CS and PCL solutions were then blended in dif-ferent compositions and concentrations to achieve the optimum range for obtaining nanofibers as reported in our previous study [32].From the combination,CS1%and PCL8%in the blending ratio 1:3were selected for further studies.2.3.Electrospinning of polymer solutionCS/PCL nanofibrous scaffold was prepared by elecrospinning with selected combination of CS/PCL solution to a static target using a high voltage DC power supply connected to a multi terminal dis-tribution box(Model RR30P,0–30kV,Gamma High Voltage Inc., and USA).An infusion syringe pump(KDS220,kdScientific Inc., USA)with10ml syringe having blunt end needles of21gauge was used to deliver the polymer solutions.A distance of4cm was main-tained between the aluminium target and spinneret.All the sample solutions were spun at a rate of0.5ml/h to a grounded aluminium target.The aluminium targets werefixed on a wooden stand from the tip of the needle by applying a voltage of10kV.2.4.CharacterizationThe SEM images of the CS/PCL nanofibrous scaffold were taken using JEOL JSM6490LS operating at15kV.The samples werefixed on aluminium stubs and the stubs were coated with platinum using JEOL JFC1600for2min at10mA before imaging.The hydrophilicity of the CS/PCL scaffold was qualitatively determined by measuring the contact angle of the material with different media;distilled water and MEM using KRUSS drop shape analyzer(DSA100).The CS/PCL scaffold was cut into12mm2size pieces andfixed into the custom made sample holder of the drop shape analyzer.The analyzing media were taken in a2ml leur lock syringefitted with blunt edge needle.A single drop of volume∼2␮l was poured on the scaffold.The drop shape on the scaffold surface was recorded using the camera attached with the system.Initial contact angle values on the scaffolds were measured from each frame of the recorded videofiles.The contact angle was measured by the sessile drop approximation of the inbuilt software in the machine.2.5.Biomineralisation studiesCS/PCL nanofibrous scaffold with identical weight and shape were immersed in1.5×simulated bodyfluid(SBF)solution and were then incubated at37◦C in a closed Falcon tube for7and 14days.The SBF solution was prepared according to the litera-ture reported by Kukubo and Takadama[33].After prescribed time, samples were taken out of the medium and washed three times with deionised water to remove the impurities.CS/PCL nanofibrous scaffold was then lyophilized and examined in SEM for mineraliza-tion.The adsorbed minerals in the scaffold were also quantified using EDS attachment of the SEM.2.6.Protein absorption studiesThe protein absorption studies were carried out for the nanofi-brous scaffold for1,4and10h in MEM.The samples were cut in equal weight and size and kept immersed in MEM with serum proteins and growth factors for the respective time peri-ods.The samples were then given PBS washing once and were kept immersed in the elution buffer(1ml)at37◦C in the incu-bator for1h.200␮l of elution buffer was then mixed with25␮l of BCA/CuSO4solution in a96well plate;incubated at37◦C for 30min.The protein absorption was then quantified by measuring the absorbance of the eluted buffer/BCA/CuSO4at562nm in a plate reader(BioTEK2000).2.7.Cell culture studiesThree different cell lines such as NIH3T3,MG63and L929were obtained from NCCS,Pune and used for cell attachment studies on the CS/PCL nanofibrous scaffold.The cells were routinely cul-tured in MEM,supplemented with10%Fetal Bovine Serum(Sigma) and antibiotics(Penicillin,Streptomycin,Amphotericin and Gen-tamycin)(Invitrogen,USA)under standard conditions of37◦C and 5%CO2.The culture medium was changed twice a week and the cells were harvested prior to culture on the scaffold,by treating with0.25%trypsin-EDTA.2.8.Cytotoxicity studiesThe cell viability was measured using Alamar Blue Assay(Invit-rogen,USA).Alamar blue(Resazurin)is a nonfluorescent dye, which works based on the reduction of mitochondria of the living cells which converts Rezasurin to bright red-fluorescent resorufin of metabolically active cells.The amount offluorescence produced is proportional to the number of living cells.Initially,fibrous scaf-fold was incubated in culture medium for10min to wet the surface of the scaffold,prior to cell seeding.MG63cells were then seeded on the scaffold surface as per ISO standard10993-5for three time intervals viz;24,48and96h.After each specified culture period, culture medium with10%alamar blue was added to each well and incubated for5h.The absorbance of the solution was measured using the plate reader.The cell viability was measured in triplicate and plotted in terms of percentage optical density.2.9.Cell attachment and proliferation studiesCS/PCL scaffold was cut into12mm2size samples and steril-ized by immersion in70%ethanol for30min.Then the scaffold was washed thrice with sterilized PBS and transferred to24-well non-treated polystyrene cell culture plates to increase cell-matrix interactions and to reduce cell attachment on the plates.The scaf-fold was incubated for30min in1ml of the cell culture medium at37◦C before cell seeding to saturate the scaffold with growth medium and improve cell attachment.Three different cell linesK.T.Shalumon et al./International Journal of Biological Macromolecules 48 (2011) 571–576573Fig.1.SEM image of electrospun CS/PCL fiber in 1:3composition.such as NIH3T3,MG63and L929were suspended in MEM and seeded with a density of 5×103cells per each well.The cell seeded scaffold sections were then maintained at 37◦C with humidified 5%CO 2atmosphere for cell adhesion in the CO 2incubator for 48and 96h.The culture medium was changed every alternate day for the entire culture period.The cell attachment was then observed by taking the SEM image of the scaffold after 48and 96h.The attached cells on the scaf-fold were treated with 2.5%glutyraldehyde followed by thorough washing in PBS for two times.Then the cells adhered to the scaf-fold was dehydrated in an ethanol-graded series (50,60,70,80,90and 100%)for 10min each and allowed to dry on a clean petridish at room temperature.The surface of cell-adhered scaffold sections was observed by SEM after platinum coating.After culturing the cells on the scaffold for 48and 96h,the culture media of samples were washed with PBS twice and the cells on the scaffolds were then fixed in 4%phosphate-buffered paraformaldehyde.The samples were washed with PBS and per-meablized with 0.1%Triton-X100in PBS at room temperature for 5min.In order to reduce nonspecific background staining,samples were blocked with 1%Fetal Bovine Serum (FBS)for about 20min,followed by PBS washing.The cells were then mounted using DAPInuclear stain for 5min and stored at 4◦C.All incubations were con-ducted at room temperature.Nuclear staining images of the cells on scaffolds were recorded using fluorescent microscope (Olympus BX 51).3.Results and discussion3.1.Preparation and characterization of electrospun CS/PCL scaffoldsCS (1%)/PCL (8%)solution with 1:3composition was selected and electrospun to very fine nanofibers [32].The SEM images show that the fibers had approximately 102±24nm diameter and smooth morphology.As reported in our early study,the characteristic of the solvent system which used for the fabrication of fibers is the reason for obtaining fibers in very smaller diameter.Representative SEM image of electrospun fiber is shown in Fig.1.3.2.Contact angle measurementsIn contact angle measurements,the effect of incorporation of CS in PCL scaffolds was examined using two media viz;distilled water and MEM.Generally,hydrophilic surfaces would give a contact angle between 0◦and 30◦and less hydrophilic surfaces show a contact angle up to 90◦.Materials which show a con-tact angle more than 90◦is usually termed as hydrophobic.PCL scaffolds without CS show a Âvalue of 130.1±2and 129.8±3,respectively,for water and MEM,which in turn points towards the hydrophobic behavior of PCL scaffolds.When CS was incor-porated,a decrease in the contact angle values was observed both for water and MEM.CS/PCL scaffold show a contact angle 95.3±6for water and 94.9±5for MEM.The video contact angle images of CS/PCL scaffolds are shown in Fig.2A.In the figures,(a)&(b)represent the contact angle values of PCL scaffold alone and (c)&(d)represent contact angle values of CS/PCL scaffold for water and MEM.Blending of CS in PCL not only increases the hydrophilicity but also enhances the degradation possibility of thescaffold.Fig.2.(A)The contact angle images on PCL (a and b)and CS/PCL (c and d)scaffolds using distilled water and MEM,respectively,and (B)and (C)represents the SEM images of mineralized CS/PCL scaffolds after 7and 14days of SBF immersion.(D)Represents the Ca/P ratio of in biomineralized scaffold.574K.T.Shalumon et al./International Journal of Biological Macromolecules48 (2011) 571–576Fig.3.Protein adsorption studies with CS/PCL nanofibrousscaffolds.Fig.4.Cytotoxicity studies with MG63cells.3.3.Biomineralization studiesThe SEM images of the biomineralisation studies showed the mineral deposition over CS/PCL nanofibrous scaffolds.From the SEM images,it was found that the calcium phosphate deposition is increasing with time.The amount of minerals deposited on the sur-face of the scaffolds after 7and 14days are shown in Fig.2B and C.After 14days,scaffold shows a higher mineral deposition with sur-face breakage.As the calcium phosphate formation is important for the bone regeneration in the body,a scaffold which can provide immediate mineralization of hydroxyapatite would support for the formation of natural bone [34].The nanofibrous nature of the scaf-folds also increases the surface bioactivity of the scaffolds leading to the faster mineral deposition due to this reason.The EDS spectra of the apatite formation in scaffold having Ca/P ratio 1.69is given in Fig.2D,which is slightly higher than the original value.3.4.Protein adsorption studiesProtein adsorption was done using Bi cinchoninic Acid (BCA)assay.The principle of BCA assay relies on the reduction of Cu 2+to Cu 1+.The amount of reduction is proportional to the protein present.It has been shown that amino acids like cisterns,cystine,tryptophan,tyrosine and the peptide bond are able to reduce Cu 2+to Cu 1+.BCA forms a purple–blue complex with Cu 1+in alkaline environments,thus the reduction of alkaline Cu 2+by proteins can be determined.Protein absorption studies were carried out with the CS/PCL nanofibrous scaffolds for a period of 1,4,and 10h,respectively.As the incubation period increases,an increase in adsorption of proteins on the nanofibrous scaffolds was observed (Fig.3).The increases in protein deposition on the scaffolds are essential for the cell growth and differentiation.Also it has been already proved that the protein deposition on the fibrous scaffolds increases as the fiber diameter decreases.Also,in our previous study,PCL nanofibers exhibited higher protein adsorption rate on it compared to microfibers [35].So it is clear that CS/PCL nanofibers with much smaller diameter are expected to adsorb more amounts of proteins as a function of time.More studies are underway to eval-uate the long term protein adsorption as well as specific protein adsorption.3.5.Cytotoxicity studiesCytotoxicity of the prepared CS/PCL scaffold was analyzed using Alamar Blue Assay.Cell viability on the CS/PCL nanofibrous scaffold was examined for 24,48and 96h using MG63cell line (Fig.4).A time depended increase in optical density was observed for CS/PCL scaffold,when it was analyzed for three different time points.After 96h,the OD values show comparatively higher value with respect to negative control,which clearly points towards the nontoxicFig.5.SEM images of cell attachment on CS/PCL scaffold using MG63cells after 48and 96h (1A and 1B)and nuclear staining images on the same scaffold for same time using MG63(2A and 2B).Index image in 2B is the DAPI stained fibers without cells (2C).K.T.Shalumon et al./International Journal of Biological Macromolecules 48 (2011) 571–576575Fig.6.Represents the SEM images of cell attachment on CS/PCL scaffold using L929cells afetr 48and 96h (1A and 1B)and nuclear staining images on the same scaffold for same time using L929(2A and 2B).behavior of CS/PCL scaffold.Since the increase in OD values is the direct measure of number of living cells,our results clearly suggest that cell proliferation is taking place.3.6.Cell attachment studiesCell attachment studies were carried out using MG63,L929and NIH3T3cells.MG63cells show good attachment and spread-ing to CS/PCL fibers after 46and 96h,respectively,and it is shown in Fig.5(1A and 1B).The cells showed spread morphol-ogy at higher incubation periods (96h).The result was again confirmed with nuclear staining method using DAPI for 48and96h.The corresponding microscopic images for both the time points are given in Fig.5(2A and 2B).In the case of L929and NIH3T3,the scaffold showed spreading and proliferation similar to MG63(Figs.6and 7).The SEM image of morphology of L929and NIH3T3at 48and 96h are shown in Figs.6and 7(1A and 1B)and the corresponding nuclear staining images are shown in Figs.6and 7(2A and 2B).In the case of fibers without cells,no nuclear images were observed in DAPI staining and the correspond-ing image is shown as 2C in Fig.5.Since all the cells showed good attachment and spreading on the nanofibrous scaffold of CS/PCL,it can be used for bone and skin tissue engineering appli-cations.Fig.7.Represents the SEM images of cell attachment on CS/PCL scaffold using NIH3T3cells afetr 48and 96h (1A and 1B)and nuclear staining images on the same scaffold for same time using NIH3T3(2A and 2B).576K.T.Shalumon et al./International Journal of Biological Macromolecules48 (2011) 571–5764.ConclusionsThe CS/PCL nanofibrous scaffold was prepared andfiber diam-eter was confirmed by SEM.The contact angle measurements showed that the hydrophobic character of poly(caprolactone)has been reduced by CS blending.Bioactivity of the CS/PCL scaffold was confirmed by enhanced biomineralization and protein adsorption. 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