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Macromolecular Nanotechnology

Synthesis and characterisation of gelatin/zeolite porous scaffold

Neethu Ninan a,b,d,, Yves Grohens a, Anne Elain a, Nandakumar Kalarikkal b,c, Sabu Thomas b,d

a Universit de Bretagne Sud, Laboratoire Ingnierie des Matriaux de Bretagne, BP 92116, 56321 Lorient Cedex, Franceb Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Priyadarsini Hills PO, Kottayam 686 560, Kerala, Indiac School of Pure and Applied Physics, Mahatma Gandhi University, Priyadarsini Hills PO, Kottayam 686 560, Kerala, Indiad School of Chemical Sciences, Mahatma Gandhi University, Priyadarsini Hills PO, Kottayam 686 560, Kerala, India

a r t i c l e i n f o

Article history:

Received 2 August 2012Received in revised form 11 February 2013Accepted 19 February 2013Available online 6 March 2013

Keywords:

GelatinScaffoldZeoliteLyophilisationPorosityCrosslinking

a b s t r a c t

Exploring the possibility of using zeolites in tissue engineering scaffolds is a potentialapproach in regenerative medicine because of their biocompatibility and cation exchangeability. A novel method to synthesize formaldehyde crosslinked gelatin/zeolite scaffolds bylyophilisation technique is reported in this paper. AFM images of gelatin solutions obtainedbefore and after the addition of formaldehyde, revealed the coil to helix transformation ofgelatin after crosslinking. The pore size of gelatin control scaffold was in the range of50750lm while it was greatly reduced to 10350lm after incorporation of 0.5% zeolitesin gelatin matrix, G(0.5%). Micro-CT analysis showed that porosity of G(0.5%) was 81% andthe pores were well interconnected. The elemental analysis and crystallinity studies con-firmed the presence of zeolites in G(0.5%). Interestingly, contact angle was found toincrease from 88.6 to 108 with the increase in concentration of zeolites in gelatin.G(0.5%) showed the highest glass transition temperature (37 C) as well as dynamic com-pression modulus (737 kPa). Swelling and degradation of scaffolds were tuned by adjust-

ing concentration of zeolites in the composite scaffolds. All these results suggest that theycan be further investigated for their application in tissue engineering.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Tissue engineering scaffolds are porous structures fabri-cated from synthetic and naturally derived biodegradable

polymers which serve as transitory artificial extracellularmatrix (ECM) that promotes cell attachment and 3-dimensional (3D) tissue formation [1]. There are varioustechniques to fabricate 3D porous scaffolds namely, fiberbonding[2], lyophilisation[3], supercritical fluid technol-ogy[4], compression moulding and salt leaching[5], gasfoaming [6], rapid prototyping [7] and electrospinning[8]. Lyophilisation or freeze drying is a dehydration tech-

nique in which liquid samples are frozen below its glasstransition temperature (Tg) or melting point and frozensolvents are removed by sublimation process, therebyobtaining porous, interconnected structures[9]. Comparedto other techniques, the distinct benefits of lyophilisationare that no toxic organic solvents are being used and thelow temperature helps to maintain the activity of biomac-romolecules and pharmaceutical products for long period.Unlike normal drying process, this technique involveslow surface tension which can maintain the pore structure.Hence, scaffolds with wealth of pore morphologies and

0014-3057/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.eurpolymj.2013.02.014

Abbreviations: CAF, copper activated faujasites; G(0%), gelatin withoutCAF; G(0.25%), gelatin with 0.25% CAF; G(0.5%), gelatin with 0.5% CAF;G(2.5%), gelatin with 2.5% CAF; G(5%), gelatin with 5% CAF; 3D, 3dimensional; ECM, extra cellular matrix; Tg, glass transition temperature;Vd, defect volume; Vs, volume of scaffold material; Wi, initial weight ofsample before immersing in water;Wt, final weight of sample after wateruptake; W0, initial weight of scaffold before degradation studies; W1, finalweight of scaffold after degradation studies; mV, milli volt; lm,micrometer; SD, standard deviation; PBS, phosphate buffered saline. Corresponding author. Address: Universit de Bretagne Sud, Labora-

toire dIngnierie des Matriaux de Bretagne (LIMatB), Centre de Recher-che Christiaan Huygens, Rue de St. Maud, BP 92116, Bureau 32 bis,56321 Lorient Cedex, France. Tel.: +33 751464109/+91 484 2557031; fax:+33 02 97 87 45 19.

E-mail addresses: [email protected] (N. Ninan), [email protected] (Y. Grohens), [email protected] (A. Elain), [email protected] (N. Kalarikkal), [email protected](S. Thomas).

European Polymer Journal 49 (2013) 24332445

Contents lists available at SciVerse ScienceDirect

European Polymer Journal

j 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 / e u r o p o l j

MACROMOLECULARNANOTECHNOLOGY
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nanostructures can be synthesised by adjusting variousparameters in freeze drying[10].

Natural or synthetic biopolymers are used for fabricat-ing lyophilised scaffolds. Natural polymers have severaladvantages over synthetic polymers as they could be easilyrecognised by surrounding biological microenvironmentand are metabolically processed through established path-

ways. On the other hand, synthetic polymers may generatetoxicity and chronic inflammation due to lack of cell recog-nition signals [11]. Among the various natural polymers,gelatin is an ECM protein with extensive pharmaceuticaland medical applications. It is obtained by the partialhydrolysis of collagen, which is extracted from bone, skin,tendon and cartilage[12]. It gels and melts below normalbody temperature. Gelatin polypeptide chains exist as flex-ible, unfolded coils at elevated temperatures and undergocoil to helix transformation at lower temperatures[13]. Itis pro-angiogenic [14], non-immunogenic [15], biocompat-ible and biodegradable [16]. Literature reports a wideapplication range for gelatin based scaffolds in bone tissueengineering [17], gene transfection[18], wound dressing[19], drug delivery [20], corneal endothelial cell therapy[21], and innumerable other fields. Due to the differentfunctional groups in gelatin like ANH2, ASH, ACOOH, dou-ble bonds, gelatin can be modified with bio-molecules andeven nanoparticles[22]. Zhang et al. has utilised this prop-erty to synthesis microspheres of gelatin and zeolite forcontrolled drug delivery[23]. Our attempt was to innovatea method by which we could incorporate inorganic carrierslike zeolites in 3D porous scaffolds that allow high cellseeding density for dermal tissue growth and diffusion ofnutrients and waste products from the cells.

Zeolites are microporous molecular sieves made up ofcrystalline aluminosilicates. They are biocompatible and

areused as safeoral magnetic resonanceimaging(MRI)con-trast agents[24],drug carriers[23], skin whitening agents[25], anticancer agents[26], reduce TiO2 induced reactiveoxygen species in fibroblast cell lines [27] andenhanceoxy-gen delivery to cells under hypoxia [28]. In the presentstudy, copper activated faujasite (CAF), a mineral group inthe zeolite family, was used. According to the InternationalZeolite Association, faujasites (Framework type code FAU)have cubo-octahedral sodalite cages, connected by hexago-nal prisms. Copper ions balance the negative charge of fauj-asite lattice.Thesehaveexcellention exchangecapacity andlatticestability. Very fewworks are reportedon theapplica-tion of CAF for tissue engineering applications.

Owing to the merits and versatility of freeze drying, itsapplication was exploited to synthesize a novel 3D porousscaffold based on gelatin/CAF composite. The structural,mechanical, thermal, water uptake andin vitrodegradationstudiesof thepreparedcompositescaffoldswereinvestigatedin this paper. The antibacterial activity, cytotoxicity andin vivo studies are discussed in detail in forthcoming paper.

2. Experimental

2.1. Materials

Gelatin powder (type B) was purchased from MerckChemicals, India. Glycerol (purity 99%) and formaldehyde

(36.538%) solutions were supplied by Sigma Aldrich(France). CAF was a kind gift from IRMA (France). Phos-phate buffered saline (PBS) powder was purchased fromSigma Aldrich. All chemicals used for synthesis were ofanalytical grade and were not further purified.

2.2. Preparation of porous gelatin/CAF scaffold

A 2% (w/v) gelatin solution was prepared by dissolvinggelatin powder in distilled water by magnetic stirring at60 C. To this solution, 5% (v/v) glycerol was added as plas-ticiser and again stirred. Then, 0.5% (w/v) of CAF was dis-persed in distilled water and sonicated for half an hour,before being added to gelatin solution by syringe addition.After complete interdispersion of the solutions, the tem-perature was decreased to 37 C. 0.38% (v/v) formaldehydewas added to this solution under thorough and continuousmixing to crosslink gelatin containing CAF. The solutionwas then poured in petri plates, pre-frozen at 20 C indeep freezer, followed by lyophilisation (Christ Alpha 12LD Plus Freeze Dryer) at 50 C to prepare porous gela-tin/CAF composite scaffolds. By keeping the concentrationof gelatin and formaldehyde constant, composite scaffoldswith 0.25%, 0.5%, 2.5% and 5% (w/w) of CAF namely,G(0.25%), G(0.5%), G(2.5%), G(5%), were prepared alongwith a control scaffold, G(0%), without any CAF.

2.3. Characterisation

The hydrodynamic diameter and distribution of CAFparticles were determined by a light scattering techniqueusing Malvern Zeta sizer Nano ZS. Surface charge and sta-bility of CAF suspension were also analysed.

A commercial multimode Nanoscope IIIa atomic force

microscope from Veeco (Santa Barbara, CA) equipped withtypical Silicon tips (LTESP, Veeco) was used to analyse themorphology of gelatin coils before and after the addition ofcrosslinker. 5 lL of sample was dispersed in 100 ml of Mil-lipore water. The solution was filtered and drop depositedon freshly cleaved mica and dried at 25 C in vacuum oven.Tapping mode was used to record the images. The springconstant was 0.57 N/m and imaging was done at a scanspeed of 3.5 Hz. Obtained images were analysed.

Micro-CT experiments were performed at the technicalresource centre, Morlaix (France) using monochromaticbeam of X-rays. The system allowed scanning objects withvoxel sizes of 4 lm, using its 240 kV/320 W directional

V(TOMEX) 240D X-ray tube and precision manipulator.The sample with thickness of 2.5 mm was rotated step bystep through a full 360rotation at increments of less than1 per step and a series of 2-dimensional (2D) X-ray imageswere acquired, which were then reconstructed to obtain3D images. The reconstruction process involved severalsteps like recording of projections, logarithm, ramp filter-ing and back projection. The object was rotated such thatit was always inside cone X-ray. In case of manipulator,the maximum size of object diameter was 500 mm andlength was 600 mm. The maximum distance between sam-ple and detector was 1500 mm. The high resolution detec-tor, Type XRD 1640 had output type directional angle at60. During reconstruction of 3D images, the first 25 back

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projections were done slowly and the other 475, in speed.The linear attenuation coefficient was distributed in65,536 levels of gray scale and acquisition speed was 15frames/s. Defect analysis of the scaffolds were done andporosity was estimated by the following equation,

Porosity Vd=Vd Vs 1

where (Vd) is defect volume and (Vs) is volume of scaffoldmaterial.

The morphology of the scaffolds was investigated byusing Scanning Electron Microscope (SEM) from JEOL,JSM 6031, Kyoto, Japan. The scaffold samples were pre-pared by taking thin sections with a razor blade. These sec-tions were gold sputtered in vacuum by means of Polaronsputtering apparatus and then analysed. The pore size wasevaluated from geometrical measurements of SEM images.

Energy dispersive X-ray spectroscopy (EDX) system Ox-ford INCA Energy 200 Premium was used to analyse thematerial composition of the prepared scaffolds.

To study the wettability and surface energy of the pre-pared scaffolds, contact angle measurements were carriedout usingGBX DigidropContact Angle meterequipped withhigh resolution CCD 2/3 inch camera. Sessile drop methodwas used to determine drop orientation. Rectangular sec-tions of thecompositescaffolds (5 2 1 cm3) wereplacedon silicate glass microscope slides. They were then posi-tioned flat using special sample holder. The liquid dropletvolume was 6 lL and temperature was maintained at25 C. The droplets were released at 1 cm above the surfaceandvideo capturing system measured theimages of dropletonthe surfaceafter30 s. For each sample, sixmeasurementswere taken on different regions and average of the contactangle values wasthentabulated. Thesurface energywascal-culated using Owens and Wendt equation [29].

Fourier Transform Infrared (FTIR) spectra of the scaf-folds and CAF powder were obtained using Perkin ElmerSpectrum D400, by spanning along a frequency range from4000 to 400 cm1.

X-Ray diffraction (XRD) patterns of CAF powder and thecomposite scaffolds were taken using Philips PW3710 dif-fractometer operating with Cu Karadiation with a wave-length of 1.5418 . The patterns were recorded, analysedand phase matched by XPERT software, data collectorand graphics.

The mechanical properties of the scaffold were deter-mined using dynamic mechanical analyser, DMA 2980 TAinstrument. The compression modulus was measured for

rectangular samples with dimensions 10 10 5 mm3

.The tests were performed at 5 C/min from 20 to 75 C, ata frequency of 1 Hz and displacement of 0.05 mm.

Differential scanning calorimetric (DSC) data of gelatinand gelatin/CAF scaffolds were obtained with Mettler-To-ledo DSC 882 instrument. 10 mg samples were placed inaluminium pans and heated at 5 C/min in nitrogen atmo-sphere over a temperature range from 10 to 80 C.

The water uptake ability of the scaffolds in deionisedwater was analysed for 3 weeks. The composite scaffoldswere cutinto small pieces withequalweights andimmersedin deionised water. Thesoaked sampleswere removed after1, 7 and 14 days, gentlyblotted on filter paper and weighed.The initial and final weights were found to be (Wi)and(Wt),

respectively. The percentage of water uptake was deter-mined from Eq. (2) and wasexpressedas mean SD(n= 3).

Percentage of water uptake Wi Wt=Wi 100 2

The degradation of gelatin based scaffolds was studiedusing PBS (pH = 7.4) at 37 C. Three samples of each scaf-fold were taken and their initial weight was noted (W0).

They were then immersed in PBS and incubated at 37 Cfor 1, 7 and 14 days. After soaking them, they were re-moved from PBS, washed in deionised water and freezedried. The dry weight of the scaffold was recorded as(W1). The percentage of degradation was calculated usingthe Eq.(3)and was expressed as mean SD (n= 3).

Percentage of degradation %

W0 W1=W1 100 3

3. Results and discussion

In the present study, 3D porous scaffolds were synthes-

ised by lyophilisation of chemically crosslinked gelatin/CAF hydrogel. A mechanism was proposed for the forma-tion of the composite scaffold. As shown in Scheme 1,when gelatin was heated at 60 C, it adopted coil configu-ration and at a pH below its isoelectric point (pH 4.5),these coils attained positive charge[30]. When CAF sus-pension was added to gelatin solution, the negativelycharged CAF particles might have interacted with posi-tively charged segments of gelatin. Finally, the gelatin/CAF was crosslinked by formaldehyde to maintain struc-tural integrity. During formaldehyde crosslinking, lysineand arginine residues present in gelatin, got converted totheir respective methylols. These methylols reacted to

form lysinearginine crosslinks as reported by Salsa andco-workers [31]. As a result, gelatin coils were convertedto triple helical structures. The gelatin/CAF solution wasthen poured into petri plates and frozen at20 C. As tem-perature was lowered, gelatin underwent complete trans-formation from coil to helical configuration[32]. Duringfreezing step, ice crystals grew and were phase separatedfrom the concentrated gelatin solution. The frozen solventwas then removed by sublimation under vacuum, resultingin the formation of porous scaffolds [16].

3.1. Particle size and zeta potential of CAF

Dynamic light scattering principle was used to analysethehydrodynamic diameterof zeolites[33,34]. The sizedis-tribution of aqueous suspensions of 0.5% (w/v) CAF wasfound in the range of 13 lm(Fig. 1). Its zeta potentialwas38 mV (lesser than30 mV) atpH 6.4 and thereby,had sufficient charge to keep particles from interacting andwas hence a stable system [35].

3.2. AFM image analysis

Prior to lyophilisation, the nanostructures of gelatincoils were observed using AFM to study their transforma-tion from coil to helix configuration during formaldehydecrosslinking. Several factors were taken into account while

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Scheme 1. Synthesis of porous gelatin/CAF scaffolds by lyophilisation. (A) Gelatin in its coil configuration at 600 C, (B) CAF particles, and (C) CAF incrosslinked gelatin matrix.

Fig. 1. Particle size distribution profile of CAF.




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imaging. Tapping mode was adopted because it decreasedforces between sample and tip and prevented damage toeach, unlike contact and non-contact modes[36]. Triangu-lar cantilever was used since its baseline was wider and itwas easier to find the tip while handling the sample[37].Air was chosen as the environment for imaging becausein water, only the global surface structure of gelatin can

be visualised at a very low resolution [38].At elevated temperatures, gelatin was found in its

coiled state. To confirm this, we took aqueous samples ofgelatin and glycerol, dissolved at higher temperaturesand conducted AFM analysis.Fig. 2a showed the sphericalcoacervates of gelatin that are formed through sequentialself-charge neutralisation of intermolecular gelatin mole-cules. Gelatin, being a polyampholyte has positively andnegatively charged amino acid residues at a given pH.When glycerol was added to the gelatin aqueous solution,the chains collapsed and the positively charged segmentsinteracted with negatively charged segments through cou-lomb interaction, thereby forming spherical structures[39]. Cavities were observed due to swelling of gelatin asreported by Yang and co-workers[37].

Fig. 2b and c depicted the AFM images of crosslinkedgelatin/CAF solution before freezing. The fibrillar structurewith average diameter of 10 nm, revealed that gelatin hasstarted undergoing coil to helix transformation due tothe addition of formaldehyde. Besides, the fibrillar struc-ture, gelatin coils were also found, as it was the onset ofcrosslinking. Once they are frozen, complete transforma-tion from coil to helix takes place[32].

3.3. SEM and EDX analysis

Several samples were prepared by varying the concen-tration of CAF. Out of these, G(0.5%) with highest mechan-ical strength and Tg was chosen as the ideal scaffold forfurther investigation.

SEM analysis yielded the 2D images of the preparedscaffolds. Fig. 3a showed well interconnected pores ofG(0.5%) while Fig. 3c indicated poorly interconnected poresof G(0.0%).Fig. 3b displayed CAF particles dispersed in gel-atin matrix of G(0.5%). Using ImageJ software, we esti-mated the pore size distribution of G(0.5%) and G(0.0%)by analysing around 50 pores. The pore size of G(0.5%)was in the range of 10350 lm (Fig. 3d) while that of

G(0%) (Fig. 3e) was in the range of 50750lm. The averagepore size of G(0.5%) was 161 lm and that of G(0%) was272lm. The highest number of pores was found in therange of 1050 lm in case of G(0.5%) and 250300 lmfor G(0%). These analysis confirmed that sizes of poreswere reduced by the incorporation of CAF into the gelatinmatrix. This might be due to the interaction of negatively

charged CAF particles with positively charged gelatin asexplained inScheme 1.

Previous reports revealed that pore size of lyophilisedgelatin scaffolds were 189415 lm for gelatin/hyaluronicacid[40]; 300500 lm for salt leached gelatin [41]; 50100lm for glutaraldehyde crosslinked gelatin[42]; 100200lm for gelatin scaffolds formed by cryogenic treat-ment[16]; 250400 lm for gelatin/apatite scaffolds[15];10100 lm for gelatin fibrinogen cryogel [43]; 180 lmfor gelatin/chondroitin/hyaluronan tri polymer scaffolds[44]; 461 lm for ethylene diamine carbodiimide cross-linked gelatin hydrogels[45]. Comparing with the litera-ture, the obtained size range of pores for gelatin/CAF wasideal for promoting nutrient supply to cells and for en-abling their migration and proliferation[46].

In the EDX spectra of CAF (Fig. 3f), the peaks corre-sponding to Si, Al and O were contributed by aluminosili-cates present in it. The peaks representing the energylevels of copper confirmed that it was a copper activatedfaujasite. The prominent peak for carbon was mainly fromgelatin. The peaks represented by CAF were found in theEDX spectra of G(0.5%) with reduced intensity (Fig. 3g).According to previous reports, if Si/Al ratio was 3 or higher,it would be a Y type faujasite[47]. From EDX analysis, theSi/Al ratio was 3.0 for both CAF and G(0.5%). All these dataconfirmed the presence of CAF in gelatin matrix.

3.4. Micro-CT analysis

Micro-CT is a non-destructive technique which enablesreconstruction of 3D images of the prepared scaffolds[48].Unlike SEM, the internal features of the scaffolds can bevisualised using micro-CT. Recently, it was used in the 3Devaluation of scaffolds for bone augmentation [49], tocharacterise internal structure of drug delivery devices[50], quantitative analysis of tumor induced brain destruc-tion[51], evaluation of angiogenesis[52]and neovascular-isation[53].

Fig. 2. AFM images of: (a) G(0%), (b) G(0.5%) solution, and (c) magnified image of G(0.5%).




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Micro-CT was used to determine the architectural fea-tures like pore size, surface area to volume ratio and poros-ity of G(0.5%), which affect its mechanical properties andbiological functionality.Fig. 4presented the top and bot-tom images of G(0.5%), along with their cross sectionalimages. These indicated that pores were well intercon-nected and distributed throughout the scaffold. A scaffoldwith good pore interconnectivity will have higher diffusionefficiency of oxygen, nutrients and metabolic wastes[54].

Around 5480 pores were analysed and the pore sizewas found in the range from 40 to 450 lm. The highestnumber of pores was found in the range from 20 to50 lm. This was in agreement with SEM analysis. How-ever, pore size measured by SEM was found to be lowercompared to that of micro-CT. This was because of thefact that pore diameters were calculated on the basis ofamount of pixels present in the pore [16]. But, the distri-bution of pores was not uniform as there were meso andmicropores. Peter et al. has mentioned about two factorsthat were responsible for the formation of larger pores.Nucleation rate was the first factor which depended onthe diffusion of atoms in the solution. The second factorwas the direction of high heat and protein transfer inwhich ice crystals were formed [16]. From the micro-CTimages, the larger pores were found in those regions

where atoms diffused to form a cluster and the rate ofheat transfer was high.

Mueller has reported that surface area to volume ra-tio should be of the order of 100 cm2/cm3 for porousscaffolds [55]. G(0.5%) showed a surface area to volumeratio of 114 cm2/cm3, which was comparatively higherto assist cellular adhesion. From the defect volume anal-ysis (Fig. 3e), porosity of G(0.5%) was estimated to be81%. Porosity is an important factor that determinesthe cell seeding efficiency, diffusion and mechanicalstrength of scaffolds. Freed et al. has reported that scaf-folds require a minimum porosity of 90% but the disad-vantage of very high porosity was that the mechanicalproperty will be lowered [56]. So, we designed G(0.5%)so that there was a compromise between scaffold poros-ity and mechanical properties. Thus, G(0.5%) was ob-served to have good pore interconnectivity, surface tovolume ratio and porosity, which would enable molecu-lar transport.

3.5. Contact angle measurements

The surface wettability of the polymer scaffolds werestudied in detail (Fig. 5). It was found that the contact an-gle of scaffolds increased in the range of 88.6108 for

Fig. 3. SEMimages showing: (a) G(0.5%), (b) CAF incorporated in G(0.5%) and (c) G(0%); pore size distribution profile of (d) G(0.5%) and (e) G(0%); EDXof (f)

CAF (g) G(0.5%).




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water, 6279 for formamide and 4960 for tricresylphosphate (Table 1), on increasing the concentration ofCAF. Earlier reports have observed high contact angle val-ues for gelatin despite its hydrophilicity[5759]. Amongthe different samples of gelatin/CAF scaffold, the parame-ters like concentration of polymer and crosslinker werekept constant. The only variable parameter was concentra-tion of CAF. So, the increase in contact angle can be due tothe interaction between the positively charged amino acidsof gelatin with negatively charged CAF particles, resultingin the reorientation of the hydrophobic amino acid chains(including, leucine, valine, isoleucine, phenylalanine andmethionine), which were then exposed at the solid/air

interface [60]. After 60 s, the contact angle was reduceddue to absorption of water, which then slowly percolatedthrough the pores of scaffold. The surface energy was low-

ered due to electrostatic interactions between gelatin andCAF. If the surface was extremely hydrophilic, the scaffoldwould lose its integrity when it is in the culture medium.On the other hand, a highly hydrophobic surface preventedthe adhesion of cells[61]. G(0.5%) was the optimised scaf-fold with a slightly hydrophobic nature that may preventagglomeration of cells.

3.6. FTIR analysis

The characteristicFTIR peaks of pure gelatin were amideA peakat 3258 cm1 due toANH stretching vibrations [62],amide-I band at 1630 cm1 due to AC@O stretching vibra-

tions, amide-II band at 1524 cm1

due to ANH bend cou-pled with ACH stretch, amide III band at 1234 cm1 dueto ANH bend[63]. The distinctive FTIR peaks of CAF arosemainly due to their framework vibrations at mid and farinfrared spectra [64]. The regions corresponding to1250900 cm1 were assigned for asymmetrical TAOATstretching, where T represented Si or Al[65]. Samples con-taining copper or zinc showed a characteristic peak around400 cm1 [65]. So, the prominent peak at 447 cm1 wastypical for CAF. The peaks corresponding to gelatin andCAF were found in FTIR spectra of composite scaffolds withminor shifts, showing that CAF particles were incorporatedin the polymer matrix (Fig. 6). A visible difference betweenG(0%) and composite scaffolds was that the peak at

Fig. 4. Micro-CT images of G(0.5%) (a) and (b) side view and its cross section, (c) and (d) top view and its cross section, (e) defect volume analysis.

Fig. 5. Images of water drops on: (a) G(0%), (b) G(2.5%) after 30 s.




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3280 cm1 has become more prominent in case of compos-ite scaffolds due to stretching vibrations of AlAOH andSiAOH present in CAF. The peaks of G(0%) at 1630 wasdownshifted to 1646 cm1 and the peak at 1040 wasshifted to 1020 cm1 in case of composite scaffolds, sug-gesting electrostatic interactions between positivelycharged amino acids of gelatin and negatively chargedCAF particles[66].

The FTIR data was used to confirm whether crosslinkinghas taken place. During formaldehyde crosslinking, lysine

and arginine present in gelatin were converted to theirrespective methylols. Both the methylols reacted to formargininelysine crosslinks[31]. As a result, the amide IIband was downshifted from 1524 to 1548 cm1, whichcan be observed in all gelatin/CAF scaffolds. This shift arosedue to increased hydrogen bonding in aqueous environ-ment. The peaks at 29502850 cm1 in the compositescaffolds corresponding to stretching modes of hydrocar-bon and the broad modes at 30003600 cm1 due toAOH and ANH stretching vibrations were also the impactsof formaldehyde crosslinking[67]. Thus, FTIR spectra sug-gested that electrostatic interactions have taken place be-tween gelatin and CAF besides the formaldehydecrosslinking.

3.7. XRD analysis

Fig. 6a represented the XRD spectra of pure gelatinwhich confirmed that it was generally amorphous. It hadno XRD peaks except a broad peak at 21.Fig. 6f displayedthe XRD spectra of CAF which showed that it was highlycrystalline. 2h of CAF was observed at 6 due to (111)plane. The other prominent diffraction peaks were

observed at 15, 23, 27 and 31, attributed by (33 1),(533), (731) and (555) planes[68]. When low concentra-tion of CAF was incorporated in the gelatin matrix as inG(0.25%), the composite scaffold (Fig. 7b) was found tobe amorphous. When higher concentrations of CAF wereintegrated into gelatin matrix, the peaks corresponding tocrystalline CAF were observed in the composite scaffolds(Fig. 7bd), whose intensity increased with increase inthe concentration of CAF.

3.8. Thermal properties

InFig. 8, the DSC curves of G(0%) and composite scaf-folds were reported. The polymeric chains of G(0%) under-went segmental mobility at low temperatures and hencehad a low Tg at 24 C. The presence of CAF in the gelatinmatrix reduced the segmental mobility of polymeric chainsand thereby increased Tg.

Now, concentration of CAF played an important role indetermining the Tg value. At low concentration of CAF(Fig. 8b and c), there was good interaction between thepolymer and CAF and hence the Tg was found to increasetill 37 C. At high concentration of CAF (Fig. 8d and e),

the CAFCAF interaction dominated the polymerCAFinteraction and hence the Tg was found to decrease to23 C. So, G(0.5%) was found to have the highest Tg(37 C) among other samples. This was attributed dueto electrostatic interaction between gelatin and CAF be-sides formaldehyde crosslinking of gelatin. However, incase of G(2.5%) and G(5%), there was aggregation of CAFparticles and Tg dropped drastically. Jeffrey et al. hasdiscussed the relation between Tg and polymerfillerinteraction in support of these results[69].

Table 1

List of contact angle and surface energy.

Samples Contact anglewith water ()

Contact angle withformamide ()

Contact angle withtricresyl phosphate ()

Surface energy(mJ/m2)

G(0%) 88.6 62 49 31G(0.25%) 93.3 65 53 29.2G(0.5%) 96 68 57 27.2G(2.5%) 101 69 59 26.9G(5%) 108 71 60 27.7

Fig. 6. FTIR spectra of: (a) gelatin, (b) CAF, (c) G(0.25%), (d) G(0.5%), (e) G(2.5%), and (f) G(5%).




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3.9. Mechanical properties

Tissue engineering scaffolds are subjected to variousstresses and need to maintain their integrity and preventdamage of cultured cells[70].Fig. 9compiled changes indynamic compression modulus when the concentration

of CAF was increased. The modulus was found to increasefrom 7 kPa to 737 kPa with increasing fraction of CAFupto 0.5%. But as the concentration of CAF was further in-creased, the interaction between CAF particles has over-come the interaction between polymer and CAF and themodulus was decreased drastically. The results obtainedby DMA were in support with DSC results. From these data,a direct relation was observed between Tg and mechanicalproperties. As the Tg increased, the dynamic compressionmodulus of samples were found to increase and vice versa.Tsou et al. has prepared composites with higher Tg andmechanical properties [71]. Among the four samples,G(0.5%) was found to have the highest Tg and dynamiccompression modulus.

3.10. Water uptake studies

The main concern about gelatin scaffold was its highlyhygroscopic nature due to which it swells easily in water[72]. We observed that water uptake can be controlled byincorporating CAF in the polymer matrix. This was attrib-

uted due to reduction in pore size of scaffold due to thepresence of CAF, as observed in case of G(0.5%). The controlscaffold G(0%) showed 82% water uptake in 24 h whereasG(5%) showed 50% uptake (Fig. 10). G(0%) acquired maxi-mum swelling capacity on seventh day and thereafter per-centage of water uptake was found to decline. This wasmainly due to disruption of polymer chains at the maxi-mum stage of swelling. On the other hand, the compositescaffolds showed increase in the swelling capacity till14th day. Uncontrolled swelling can badly affect themechanical property of scaffolds. So, it was advantageousto tune the swelling capacity of scaffolds so that therewas easy passage of nutrients to the cells and betterabsorption of culture medium[73].

Fig. 7. XRD spectra of: (a) G(0%) (b) G(0.25%) (c) G(0.5%) (d) G(2.5%) (e) G(5%) (f) CAF.

Fig. 8. DSC curves of: (a) G(0%), (b) G(0.25%), (c) G(0.5%), (d) G(2.5%), and (e) G(5%).




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3.11. In vitro degradation studies

The prepared scaffolds should be biodegradable andthereby provide enormous space for cell growth and tis-sue development[74]but the degradation rate should behighly controlled. Fig. 11represented the in vitro degra-dation studies of the control and composite scaffolds.On the seventh day, 87% of G(0%) was degraded whereasonly 45% of G(5%) underwent degradation. On the 14thday, nearly 92% of G(0%) was degraded whereas only58% of G(5%) got degraded. From the studies conducted,

it was obvious that percentage of degradation was re-duced greatly after incorporation of CAF into gelatin ma-trix. With the passage of time, degradation rate was

found to increase in case of all the scaffolds. These re-sults further support that along with formaldehydecrosslinking, there was strong electrostatic interactionbetween gelatin and CAF, which aided in the loweringof degradability of the scaffolds. Thus, degradability ofthe scaffolds could be adjusted by varying the concentra-tion of CAF.

4. Conclusion

3D porous scaffolds were prepared by lyophilisation ofgelatin/CAF hydrogel. The coil to helix transformation ofgelatin coils was studied using AFM. The CAF suspension

Fig. 9. Dynamic compression modulus of G(0%), G(0.25%), G(0.5%), G(2.5%) and G(5%).

Fig. 10. Percentage of water uptake of: (a) G(0%), (b) G(0.25%), (c) G(0.5%), (d) G(2.5%), and (e) G(5%) for 14 days.




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was characterised using DLS and Zeta sizer. Highly porousand well interconnected pores were observed using SEM.The architectural features like pore size distribution, sur-face area to volume ratio and porosity were estimatedusing micro-CT. EDX and XRD analysis confirmed the pres-ence of CAF in gelatin matrix. FTIR studies suggested thatelectrostatic interactions have taken place between gelatinand CAF. Among the different samples, G(0.5%) was foundto have highest Tg and dynamic compression modulus. Thedirect relation between Tg and mechanical properties werediscussed. G(0.5%) showed controlled water uptake andbiodegradation rate compared to G(0%). These results sup-

ported the efficacy of composite scaffolds for their applica-tion in tissue engineering. The antibacterial studies,cytotoxicity and animal studies of the prepared scaffoldswill be conveyed through forthcoming paper.

Acknowledgements

We deeply acknowledge financial support from TheBrittany Region, the European Union (FEDER) and theFrench Ministry for Research.

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