Collagen scaffolds derived from fresh water fish origin...

Collagen scaffolds derived from fresh water fish originand their biocompatibility

Falguni Pati,1 Pallab Datta,1 Basudam Adhikari,2 Santanu Dhara,1 Kuntal Ghosh,3

Pradeep Kumar Das Mohapatra3

1School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, India2Materials Science Centre, Indian Institute of Technology, Kharagpur 721302, India3Department of Microbiology, Vidyasagar University, Midnapore 721102, India

Received 10 May 2011; revised 12 August 2011; accepted 15 August 2011

Published online 9 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33280

Abstract: Collagen, a major component of native extracellular

matrix, has diverse biomedical applications. However, its

application is limited due to lack of cost-effective production

and risk of disease transmission from bovine sources cur-

rently utilized. This study describes fabrication and character-

ization of nano/micro fibrous scaffolds utilizing collagen

extracted from fresh water fish origin. This is the first time

collagen extracted from fresh water fish origin was studied

for their biocompatibility and immunogenicity. The nano/

micro fibrous collagen scaffolds were fabricated through self-

assembly owing to its amphiphilic nature and were subse-

quently cross-linked. In vitro degradation study revealed

higher stability of the cross-linked scaffolds with only �50%

reduction of mass in 30 days, while the uncross-linked one

degraded completely in 4 days. Further, minimal inflamma-

tory response was observed when collagen solution was

injected in mice with or without adjuvant, without significant

dilution of sera. The fish collagen scaffolds exhibited consid-

erable cell viability and were comparable with that of bovine

collagen. SEM and fluorescence microscopic analysis

revealed significant proliferation rate of cells on the scaffolds

and within 5 days the cells were fully confluent. These find-

ings indicated that fish collagen scaffolds derived from fresh

water origin were highly biocompatible in nature. VC 2012 Wiley

Periodicals, Inc. J Biomed Mater Res Part A: 100A: 1068–1079, 2012.

Key Words: collagen, fresh water fish scale, nano/micro fi-

brous scaffold, immunological response, biocompatibility

How to cite this article: Pati F, Datta P, Adhikari B, Dhara S, Ghosh K, Mohapatra PKD. 2012. Collagen scaffolds derived from freshwater fish origin and their biocompatibility. J Biomed Mater Res Part A 2012:100A:1068–1079.

INTRODUCTION

Collagen, the principal structural protein of extracellularmatrix (ECM), plays a dominant role in maintaining biologi-cal and structural integrity of ECM and undergoes constantremodeling for physiological functions.1 It is also consideredto be an important morphogenetic factor in embryonicdevelopment and in regenerative processes.2 For these rea-sons, use of collagen is rapidly expanding in biomedical andpharmaceutical field as tissue engineering scaffold, wounddressing, and drug delivery vehicles either alone or in com-bination with other biomaterials.3–5 Amongst various typesof collagens, type I collagen has been extensively used forbiomedical application owing to its low antigenicity andhigh direct cell adhesion properties.5 Moreover, type I colla-gen accounts for 90% of the total collagen present in thebody and is abundant in skin, bone, and tendon.6

Commonly, type I collagen extracted from bovine sourceis used for medical applications.7 However, there has been agrowing concern of transmissible diseases, especially bovinespongiform encephalopathy (mad cow disease), ovine and

caprine scrapie, and other zoonoses for collagen products ofbovine origin and other animal sources as well.8 Conse-quently, there is an urgent need for easily available andsafer sources with no or less risks of disease transmission.Recombinant human collagens are now being produced inmammalian,9,10 bacterial,11 yeast,12,13 insect,14 and plant15

systems. Although, collagen produced using recombinantapproach may be useful in the longer term, but, their pro-duction is limited and not yet optimized for large scaleapplication.

A variety of alternative sources for collagen have beenproposed in recent times including fresh water and marinefishes,16,17 chicken skin,18 and different marine animals likesquid,19 octopus,20 jellyfish,21 starfish,22 and so forth. How-ever, recent outbreak of swine flu and avian flu furtherposes risk of disease transmission from pig and chicken,respectively. Fish source is comparatively safer and potentialalternate for extraction of collagen. Fish processing wastes,which are rich in collagen, may serve as a potential andsafer alternative for its extraction.23

Correspondence to: S. Dhara; e-mail: [email protected]

Contract grant sponsor: DBT, Govt. of India

1068 VC 2012 WILEY PERIODICALS, INC.

In spite of such advantages, fish collagens have not beenextensively studied for medical uses mainly due to their lowdenaturation temperature (Td). Interestingly, the living envi-ronment has direct influence on Td of collagen from anyspecies.24 Further, Td has also direct correlation with iminoacids (hydroxyproline and proline) content in collagen.25

The Td of fish collagens of marine sources reported in litera-ture is below 30�C,26 which limits their application in nativeform at physiological temperature of human body.27 Inter-estingly, Td of collagen from fresh water fish origin is rela-tively higher than that of marine sources.26 In fact, collagenfrom fresh water fish origin was isolated by our group andits denaturation temperature was found to be close to thatof mammalian one.28 While these abundantly availableresources can serve as economical, viable, and safer alterna-tive to bovine source, the environmental issue related topollution from fish wastes can also be addressed. However,the application of collagen from these sources will be real-ized upon immunological response, biocompatibility, andother safety concern. For application in tissue engineering,collagen can be formed into nano/micro fibrous scaffoldsthrough self-assembly at pH close to the isoelectric pointowing to its amphiphilic nature29 and can be subsequentlycross-linked for improved mechanical properties and toenhance their stability in vivo.30

This study describes fabrication and characterization ofnano/micro fibrous scaffolds through self-assembly byutilizing collagen extracted from fish scales of fresh waterorigin like Labeo rohita (Rohu) and Catla catla (Catla). Thescaffolds were stabilized by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)and N-hydroxysuccinimide (NHS). Enzymatic degradation ofcollagen scaffolds was assessed in PBS containing collage-nase. Immunological response of the isolated collagen wasevaluated by injecting the purified protein to mouse, bothwith and without adjuvant. Biocompatibility of the fish col-lagen scaffold was examined by evaluating the cellularresponses of both 3T3 and MG63 cells.

MATERIALS AND METHODS

Isolation of collagenIsolation of acid-soluble collagen. Collagen was isolatedfollowing the procedure described elsewhere by ourgroup.28 Briefly, fish scales of Rohu and Catla were washedthoroughly with distilled water. The scales were washed ina neutral solvent system for a period of 48 h to removeunwanted proteins from the surface. Demineralization of thescales was achieved by treating for 48 h with 0.5M EDTA(Merck, Mumbai) solution (pH 7.4). The demineralized fishscales were washed thrice with distilled water and used fur-ther. These scales were treated with 0.5M acetic acid(Merck, Mumbai) at pH 2.5 over a period of 48 h and insol-uble part of the scales was filtered out. NaCl was added tothe filtrate to a final concentration of 0.9M to induce saltingout of collagen and kept undisturbed for 24 h. The suspen-sion was centrifuged at 8000 rpm for 1 h and the precipi-tate was resolubilized in 0.5M acetic acid. The final solutionwas then dialyzed against 0.1M acetic acid and deionized

water for 24 h each and freeze-dried subsequently.Collagens extracted using acetic acid from Rohu and Catlaare referred as RA and CA, respectively.

Isolation of pepsin-solubilized collagen. The residue offish scales after acetic acid treatment was digested with1 mg/mL pepsin (from porcine gastric mucosa, 800–2500units/mg protein, Sigma) solution in 0.5M acetic acid at 4�C.After 24 h of incubation, mixtures were filtered and thesupernatant was dialyzed against 0.02M Na2HPO4 (Merck,Mumbai, India) in order to inactivate the pepsin. The pre-cipitate was salted out by addition of NaCl to a final concen-tration of 0.9M. Finally, the precipitate was dissolved in0.5M acetic acid, dialyzed against 0.1M acetic acid and dis-tilled water successively and freeze dried. Collagensextracted using pepsin from Rohu and Catla are referred toas RP and CP, respectively.

Preparation of collagen scaffoldsScaffolds were prepared by self-assembling of collagen intofibers following freeze drying. The isolated and purified fishcollagen was dissolved in 0.5M acetic acid to the desiredconcentration at 4�C with gentle stirring. Dialysis and incu-bation methods were used for in vitro fibrillogenesis ofmonomeric collagen. The collagen solution was dialyzedagainst 0.1M acetic acid following deionized water for 24 heach. After dialysis, the pH of obtained collagen solutionwas �6. Actually, the isoelectric point of collagen type I isat pH 9.3.31 Here it may be noted that, Jiang et al.31 haveshown that collagen molecules self-assembled into fibrillarstructures at pH 5.5–9.5. Dialyzed collagen solution was fur-ther poured into petri dish and was frozen at �80�C. After24 h of pre-freezing, the frozen mass was freeze dried for24 h. Collagen scaffolds were cross-linked by a mixture ofwater soluble carbodiimide, EDC (SRL Pvt. Ltd., Mumbai,India) and NHS (SRL Pvt. Ltd., Mumbai, India) following theprocedure described elsewhere.32 Briefly, cross-linking ofcollagen with EDC and NHS was performed by immersingsamples weighing l g (1.2 mM carboxylic acid groups) in 50mL water containing 6.0 mM EDC and 2.4 mM NHS for 2 hat room temperature. During cross-linking, solution pH wasmaintained at 5.5. The cross-linked samples were washedthoroughly with 0.1M Na2HPO4 solution for 2 h to hydrolyzeany remaining NHS activated carboxylic acid groups. Subse-quently, the samples were washed four times with distilledwater and lyophilized. Collagen samples with differentdegrees of cross-linking were obtained using three differentmolar ratios of EDC to NHS to COOH of 1:0.4:1, 3:1.2:1, and5:2:1, respectively. As control materials, cross-linked porousscaffolds were also prepared with bovine collagen (Fluka,Sigma Aldrich Chemie Gmbh, Buchs, Switzerland), using thesame method as described for fish scale collagen scaffolds.Scaffolds cross-linked with molar ratios of EDC to NHS toCOOH of 1:0.4:1, 3:1.2:1, and 5:2:1 are referred to as RA-1,RA-3, and RA-5 of acid extracted collagen from Rohu andCA-1, CA-3, and CA-5 of acid extracted collagen from Catla,respectively.

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | APR 2012 VOL 100A, ISSUE 4 1069

Microstructure analysis by SEMThe microstructure of the scaffolds were examined usingscanning electron microscopy (SEM) (JSM, Jeol, Japan). Priorto observation, samples were arranged on metal grids, usingdouble-sided adhesive carbon tape, and coated with goldunder vacuum.

Degree of cross-linkingThe degree of cross-linking of collagen scaffolds was deter-mined by ninhydrin assay,33 which was defined as the per-centage of free amino groups present in cross-linked sample.34

In this assay, the scaffolds (� 20 mg) were boiled at 100�Cwith a ninhydrin solution for 20 min. After boiling, optical ab-sorbance of the solution was recorded at 590 nm with a UV-visible spectrophotometer (Model- UV-1601, Shimadzu, Japan)using glycine at various known concentrations as standard.The amount of free amino groups present in the test sample,after boiling with ninhydrin, is proportional to the optical ab-sorbance of the solution.34,35 Each experiment was performedin triplicate. The degree of cross-linking of the samples wasdetermined following the method established previously.35

FTIR analysisCross-linked and uncross-linked collagen scaffolds were indi-vidually mixed with �5 times of vacuum dried KBr andpressed into pellets by hydraulic press. Infrared spectra wereobtained in the range between 4000 and 1000 cm�1 with KBrdisc method using an infrared spectrophotometer (Model–NEXUS-870, Thermo Nicolet Corporation, Madison, WI).

Mechanical testingMechanical properties of the scaffolds were evaluated undertensile mode by mechanical testing machine (Model-H25KS,Hounsfield, UK). The dimensions of scaffolds were 30 mm� 10 mm � 2 mm. The scaffolds were clamped to sampleholder and tested with the crosshead speed of 10 mm/minunder tension.

In vitro enzymatic biodegradabilityEnzymatic degradation of fish scale collagen scaffolds wasinvestigated by monitoring the mass loss of samples as afunction of exposure time to collagenase solution.21 Eachscaffold (15 mm � 15 mm � 2 mm) was weighed (W0) andsuspended in PBS containing 0.6 lg/mL collagenase (pH7.4) at 37�C with gentle shaking. Enzyme solution wasrefreshed every week to retain the activity. At scheduledintervals, collagen samples were washed with deionizedwater and lyophilized. Mass loss of the samples was trackedover time by weighing the samples after lyophilization (Wt).Five specimens were tested for each type. The percentweight remaining of collagen scaffolds was calculatedaccording to the following equation:

Residual mass ð%Þ ¼ ðWt=W0Þ � 100

where W0 is the initial weight of the collagen scaffold andWt is the weight of the scaffold after lyophilization at eachtime point.

Immunological studyMice were used for assessment of antibody responses to fishscale collagen. All animal experimentations were approved bythe Vidyasagar University Animal Care and Ethics Committee.

Mouse strains. Swiss albino mice (BALB/c strain) of eithersex were used, which has been shown previously to be re-sponsive to collagens.36 All animals were �8–9 weeks ofage before initial immunization. Mice were divided into10 groups (PBS, RA, RAþadjuvant, CA, CAþadjuvant, RP,RPþadjuvant, CP, CPþadjuvant, and Ovalbuminþadjuvant)with each group containing four mice (Table I).

Mouse immunization. Mice were immunized without adju-vant as well as with Incomplete Freund’s Adjuvant (IFA)(Santa Cruz Biotechnology Inc.) to assess the maximum im-munogenic potential of the injected immunogens (Table I).Mice treated without adjuvant were injected subcutaneously

TABLE I. Immunological Testing of Collagen Solution in Swiss Albino Mice

Group Sample Dose Route Immunization

Negativecontrol

PBS 100 lL of PBS Subcutaneous 1, 14, and35 days

RA Acid extracted collagen from Rohu 50 lL of 0.4 mg/mL fish collagenin PBS mixed with 50 lL of PBS

Subcutaneous -do-

CA Acid extracted collagen from Catla -do- Subcutaneous -do-RP Pepsin extracted collagen from Rohu -do- Subcutaneous -do-CP Pepsin extracted collagen from Catla -do- Subcutaneous -do-RAþIFAa Acid extracted collagen from Rohu 50 lL of 0.4 mg/mL collagen in PBS

mixed with 50 lL of IFAIntraperitoneal -do-

CAþIFAa Acid extracted collagen from Catla -do- Intraperitoneal -do-RPþIFAa Pepsin extracted collagen from Rohu -do- Intraperitoneal -do-CPþIFAa Pepsin extracted collagen from Catla -do- Intraperitoneal -do-Positive

controlOvalbumin 50 lL of 0.4 mg/mL ovalbumin in PBS

mixed with 50 lL of IFAIntraperitoneal -do-

a IFA, Incomplete Freund’s Adjuvant.

1070 PATI ET AL. COLLAGEN SCAFFOLDS DERIVED FROM FRESH WATER FISH ORIGIN

on day 1, 14, and 35 with 100 lL of protein solution (50 lLof 0.4 mg/mL fish collagen in PBS mixed with 50 lL ofPBS) (Table I). Mice treated with adjuvant were injectedintraperitoneally on day 1, 14, and 35 with 100 lL of pro-tein (50 lL of 0.4 mg/mL fish collagen in PBS mixed with50 lL of IFA) (Table I). A pre-bleed from the saphenousvein was taken prior to the first injection and then testsaphenous vein bleeds were taken 7 day after each immuni-zation. Approximately 20–40 lL of blood was drawn at eachbleed. At 42 day, animals were sacrificed and a terminalbleed was taken. All blood samples were allowed to clot,ringed, and centrifuged to separate and collect sera. Sam-ples were stored at �80�C prior to analysis.

Antibody analysis. Mouse sera were analyzed by a stand-ard enzyme linked immuno-sorbent assay (ELISA) for anti-bodies against the collagen preparation. For all sera, a 1/50dilution was examined against the protein. The secondaryantibody used was a 1/500 dilution of an affinity purifiedgoat anti-mouse IgG coupled to horseradish peroxidase(Assay Design), using 3,30, 5,50-tetramethylbenzidine (TMB)(Assay Design) as substrate.

Cell culture study on collagen scaffoldsFibroblasts (3T3) cells, obtained from the National Centrefor Cell Science (NCCS, Pune, India), were cultured in com-plete Dulbecco’s Modified Eagle’s Medium (DMEM) (Hime-dia, Mumbai, India) containing 10% fetal bovine serum(Himedia, Mumbai, India), and supplemented with 2 mM L-glutamine (Fluka, Sigma Aldrich, Japan), 1% penicillin–streptomycin (A002A, Himedia, Mumbai, India). Cells weremaintained at 37�C in a humidified incubator in an atmos-phere of 5% CO2.

MG63 cells are adherent human osteosarcoma cell line,obtained from the National Centre for Cell Science (NCCS,Pune, India), and relatively immature osteoblasts, whichexhibit a number of osteoblast-like phenotypic markers. Thecells were cultured in complete DMEM, (Himedia, Mumbai,India), which was supplemented with 10% fetal bovineserum (Himedia, Mumbai, India), 4 mM L-glutamine, 2 mMNa-pyruvate (Loba Chemie, Mumbai, India), and 1% penicil-lin–streptomycin (A002A, Himedia, Mumbai, India) in tissueculture flasks at 37�C with 5% CO2. The culture mediumwas changed every alternate day. Sub-culturing was doneon every 4th day by detaching cells from the flasks usingtrypsin–EDTA solution followed by splitting at 1:4 ratio.

The scaffolds were sterilized by immersion in 70% etha-nol solution for 1 h, washed three times with PBS, andsoaked in complete media for 12 h. For evaluation of bio-compatibility, both types of cells were seeded on the cross-linked fish collagen and bovine collagen scaffolds placed in12 well tissue culture plate (Tarson Products Ltd, Kolkata,India) separately at a density of 1 � 105 cells/cm2. Afterincubation for 3 h of the cell seeded scaffolds at 37�C, 2 mLfresh medium was added to each well containing collagenscaffolds. For the period of 7 day, culture medium wasrefreshed every alternate day.

Cell viability and morphology assay. The viability of cellson the scaffolds was evaluated after 1, 3, 5, and 7 day byMTT assay.37,38 Briefly, the scaffolds attached with cellswere incubated in a mixture of 360 lL of medium and40 lL MTT solution (5 mg/mL) in PBS for 4 h at 37�C and5% CO2. The intense red-colored formazan derivatives formedwere dissolved with 400 lL dimethyl sulfoxide for 15 minand the absorbance was measured at 590 nm with a micro-plate reader (Recorders and Medicare Systems, India).

The morphology of the adherent cells proliferating onthe scaffolds was examined using SEM. For SEM analysis,the samples were soaked in 2.5% glutaraldehyde PBS solu-tion for 4 h for cell fixing and dehydrated in an ascendingseries of ethanol aqueous solutions (50–100%) at roomtemperature and dried under vacuum. The samples weresputtered with gold and observed under SEM microscope(EVO 60, Carl ZEISS SMT, Germany).

Evaluation of cell proliferation on collagen scaffolds. Theproliferation of both types of cells on fish scale collagenscaffolds was observed under fluorescence microscopy afterstaining with the nucleic acid staining dye, Hoechst 33342(bisbenzimide trihydrochloride, Invitrogen, Eugene, OR),at specified intervals using manufacturers protocol. The cell-scaffold constructs were washed twice with PBS andincubated with dye (5 lg/mL of PBS) for 20 min at roomtemperature. After incubation, they were washed again withPBS to remove the excess dye and viewed under a fluores-cencet microscope (Zeiss Axio Observer Z1, Carl Zeiss,Germany) with UV excitation and emission of 346/442 nm.The cell nuclei appeared as blue fluorescencet after stainingwith Hoechst 33342. To ensure a representative count, eachscaffold sample was divided into quarters, and two fieldsper quarter were photographed at a magnification of 100�.The cells in each image were counted with Image J softwarefor all types of scaffolds and were expressed as averagenumber of cells per square mm2.

Fluorescence microscopy. Attachment of MG63 cells toscaffolds was evidenced using fluorescence microscopy. Thescaffolds were seeded with osteoblast cells (105) and cul-tured for 5 day in complete medium. The scaffolds werestained with rhodamine–phalloidin (Invitrogen, Eugene, OR)and Hoechst 33342 using manufacturer’s protocol. Scaffoldswere washed three times with PBS (pH 7.4), followed byincubation in 4% formaldehyde in PBS for 10 min. The sam-ples were further washed with PBS and the constructs werethen permeabilized using 0.1% Triton X-100 for 5 min. Thesamples were preincubated with 1% BSA for 30 min.Incubation with rhodamine–phalloidin for 20 min at roomtemperature, followed by washing with PBS and stainingwith 5 lg/mL Hoechst 33342 for 30 min were done. Fluo-rescence images from stained constructs were obtainedusing a fluorescence microscope (Zeiss Axio Observer Z1,Carl Zeiss, Germany) with ApoTome attachment.

Statistical analysisThe cell adhesion and proliferation experiments were per-formed in triplicate and the results were expressed as mean

ORIGINAL ARTICLE


6 standard deviation. Student’s t-test was used to assessstatistical significant difference of the results. Differencewas considered statistically significantly at p < 0.05.

RESULTS AND DISCUSSION

Fish scale is a mineralized tissue, which is composed oftype I collagen fibrils reinforced by calcium-deficienthydroxyapatite with highly ordered three-dimensional struc-ture. Thus, extraction of collagen from fish scale is achievedthrough primary decalcification step which exposes collagenfibrils to facilitate dissolution through direct solute–solventinteraction. The fibrous collagens are generally present inthe tissue in the form of covalent cross-linking between theindividual protein subunits.39 The cross-linking occursthrough lysyl oxidase mediated enzymatic reactions of alde-hydes generated enzymatically from lysine or hydroxylysineside-chains.40 Collagen is easier to isolate from those cross-linked through lysine aldehyde pathway as the initiallyformed aldimines cleave at low pH allowing the collagenmonomers to be solubilized in acidic medium. However, incase of hydroxylysine derived cross-linking neither the ini-tial cross-links nor their maturation products are labile tocleave,41 thus yield of acetic acid extraction is low. Whilepepsin has been reported to cleave peptides at acidic pH inthe telopeptide region, the extraction of collagen by pepsinrenders a higher yield.8 The yield of collagen extracted byacetic acid and pepsin digestion were �5% and �15%,respectively, from fish scales of both the species.

Fish collagen of fresh water origin generally has higherdenaturation temperature (Td) as reported elsewhere andhas a direct correlation with imino acids content.26 The Tdof collagen also has correlation to the habitat of the species.Collagen extracted from Rohu and Catla, fresh water origin,has Td of 36.5�C, which is close to mammalian collagen andis relatively higher than that from the marine source.28

Thus, collagens extracted from fresh water fish origin likeRohu and Catla may have advantages over marine sourcesfor biomedical application.

Collagen nano/micro fibrous scaffolds with highly openporous structure were successfully prepared by self-assem-bly through dialysis and incubation methods for the in vitrofibrillogenesis of monomeric collagen at pH �6. Collagenconcentration was varied to change fiber diameter, architec-ture, and pore interconnections as well. A 5 mg/mL of colla-gen solution was used in this study for maintaining similarporosity and scaffolds architecture during their preparation.Pore size, pore interconnectivity, and surface area arewidely recognized as important parameters for scaffolds tobe used in tissue engineering.42 SEM microscopy revealednano/micro fibrous architecture of the fabricated scaffoldsas shown in Figure 1. Further, these scaffolds had insignifi-cant change in microstructure after cross-linking by EDC/NHS. Three different molar ratios of EDC/NHS were used toassess their strength and biodegradation rate against varia-tion in cross-linked density of the collagen scaffolds. Mixtureof EDC and NHS were also effectively used for cross-linkingof collagen scaffolds derived from bovine and marine

FIGURE 1. Representative scanning electron microscopy images of porous fish cross-linked collagen scaffolds derived from Rohu (RA) and

Catla (CA).


origin.21,43 Further, the EDC/NHS cross-linked collagen wasreported to be non-cytotoxic in vitro,44 and biocompatibilitywas also demonstrated in animal models45 as the unreactedEDC/NHS can be washed off in aqueous media. The devel-oped cross-linked scaffolds containing nano/micro architec-ture may have resemblance to the intricate fibrillar struc-ture of native tissue, where collagen directly supports celladhesion by providing the chemical cues to cell surfacereceptors.5,7

Degree of cross-linkingThe degree of cross-linking of collagen scaffolds after treat-ment with three different concentrations of EDC and NHSratios with carboxylic group of collagen was determined byninhydrin assay. As shown in Table II, the degree of cross-linking of the scaffolds were 21.3%, 59.4%, and 68.3% forRA-1, RA-3, and RA-5, and 23.5%, 59.9%, and 69.5% forCA-1, CA-3, and CA-5, respectively. So, the cross-linkingdegree was increased with increasing cross-linker ratioowing to higher number of amide linkage formation. Fur-ther, RA-5 and CA-5 reveals the highest degree of cross-link-ing amongst them due to highest cross-linker ratio. Thecross-linking by EDC/NHS is facilitated by formation of o-isoacylurea from coupling of carbodiimides to carboxylicgroup. The resulting activated intermediate is attacked by anucleophilic primary amine to form an amide cross-link andthe isourea derivative of the applied carbodiimide is elimi-nated and can be washed out.44–46 Thus, the cross-linkersare not integrated into the samples and can easily beremoved with insignificant toxicity. Actually in EDC/NHScross-linking of collagen, peptide bonds are formed betweenglutamic- or aspartic acid residues, and lysine- or hydroxyly-sine residues.32,47 The use of NHS reduces side reactions ofthe EDC-activated groups such as hydrolysis or N-acyl shiftto form stable N-acylisourea.48

FTIR analysisThe FTIR spectra of collagens of RA and RP before and aftercross-linking are shown in Figure 2. The amide I band, withcharacteristic frequencies in the range from 1600 to 1700cm�1, was mainly associated with stretching vibrations ofthe carbonyl groups (C¼¼O bond) along the polypeptidebackbone,49 which is a sensitive marker of the peptide sec-

ondary structure.50 The absorption intensity of 1240 cm�1

(amide III) and 1454 cm�1 (amide II) band was approxi-mately equal, which confirms the triple helical structure ofRA and RP.51 The amide A band is associated with the NAHstretching frequency.52 A free NAH stretching vibrationoccurs in the range 3400–3440 cm�1, and when NH groupof a peptide is involved in hydrogen bonding, the position isshifted to lower frequencies. The amide A band of RA andRP were at 3440 cm�1 for both cases. When comparingwith the normal absorption range of the amide II bandsposition (1550–1600 cm�1), the position is found to beshifted to lower frequency, 1540 cm�1, which also indicatesthe existence of hydrogen bonding in each collagen.

After cross-linking with EDC/NHS, there is a slight varia-tion in the intensities of these bands (RA-5 and RP-5 in Fig.2). The intensity of amide II bands decreased in both RA-5and RP-5 as the intensity of ANH2 band in collagen mole-cules is stronger than that of N–H. Further, the intensity ofC¼O band was not changed significantly as amide bondformation has no effect on it. So, the change in intensity ofamide II bands corresponds to change in the free ANH2

groups in collagen molecules to NAH groups.53 Theseresults were in agreement with similar findings reportedelsewhere.53,54 Thus, intermolecular or intramolecularamide linkages were formed after cross-linking of collagenscaffolds with EDC/NHS.

TABLE II. Correlation of Mechanical Properties with Degree of Cross-Linking of Collagen Scaffolds

Sample nameaEDC:NHS:COOH(molar ratio)

Degree ofcross-linking (%)

Avg. Maxstress (MPa) % Elongation

RA – – 0.55 5.7RA-1 1:0.4:1 21.3 0.73 3.5RA-3 3:1.2:1 59.4 0.95 2.2RA-5 5:2:1 68.3 1.59 2.6CA – – 0.61 5.9CA-1 1:0.4:1 23.5 0.81 3.3CA-3 3:1.2:1 59.9 0.99 2.5CA-5 5:2:1 69.5 1.61 2.4

a RA and CA correspond to acid extracted collagens from Rohu and Catla.

FIGURE 2. FTIR spectra of collagen derived from Rohu and Catla

before (RA and CA) and after (RA-5 and CA-5) cross-linking.

ORIGINAL ARTICLE


Mechanical testingThe mechanical properties of the collagen scaffolds wereenhanced by EDC/NHS cross-linking (Table II). At the cross-linker concentration of 1:0.4:1 ratio, the tensile strengthincreased by more than 5% over the uncross-linked collagen[Fig. 3(a)]. At concentrations of 3:1.2:1 and 5:2:1 ratio, thetensile strength increased significantly to 74% and 192%,respectively, of the mean value of uncross-linked collagenscaffolds [Fig. 3(a)]. The tensile strength of the uncross-linked collagen scaffolds was 0.54 and 0.61 MPa for RA andCA, respectively. In fact, the tensile strength of cross-linkedscaffolds increased appreciably with 1.59 and 1.61 MPa forRA-5 and CA-5, respectively. The difference in tensilestrength between the uncross-linked (RA and CA) andcross-linked scaffolds (RA-5 and CA-5) was statistically sig-nificant (p < 0.05). In contrast, strain at break scaled inver-sely with EDC concentration. A maximum percent elongationwas evidenced in the control samples (5.7 and 5.9% for RAand CA, respectively) while the cross-linked at 3:1.2:1 ratiothe strain at break was least (2.3% for RA-3), 60% decreasein strain value was observed [Fig. 3(b)]. So, EDC/NHS couldbe effectively used to improve the mechanical property offish scale collagen scaffolds. Further, the tensile strength ofthe scaffolds can be tailored accordingly by varying thecross-link density.

In vitro enzymatic biodegradabilityStabilization of collagen-based matrix by cross-linking is anindispensable part for development of tissue engineeringscaffold to retard the rate of biodegradation and conse-quently the mechanical strength can also be improved.5,55

The cross-linking of collagen by EDC/NHS involves activa-tion of carboxylic groups of glutamic and aspartic acid resi-dues, as well as the formation of amide bonds in presenceof lysine or hydroxylysine residues.

The enzymatic degradation of fish collagen scaffolds wasinvestigated by monitoring the residual mass percent of thescaffolds as a function of time. The uncross-linked fish colla-gen scaffolds (RA) degraded rapidly as shown in Figure 3(c)and within 24 h the residual mass reduced to 18.4% andthese scaffolds were completely degraded within 4 d whentreated with 0.6 g/ml of collagenase solution. However, the

residual mass decreased to approximately 45%, 35%, and27%, respectively, for RA-5, RA-3, and RA-1 when they weretreated with 0.6 g/mL of collagenase solution for 30 d [Fig.3(c)]. This rate of degradation has reverse correlation withdegree of cross-linking of collagen scaffolds as rate of degra-dation gradually decreased with increasing cross-linked den-sity. So, the cross-linking of collagen scaffolds with EDC/NHS led to a significant improvement in biostability. There-fore, the degradation kinetics of fish collagen scaffolds couldbe controlled by varying the cross-linked density.

Immunological analysis of fish scale collagenAntibody responses against fish collagen were assessed inSwiss albino mice. While BALB/c mice are normally usedfor generating antibodies against globular proteins and theyare also shown to be responsive to collagens.56 In thisstudy, the total IgG response against the fish collagen pro-tein after 3 immunizations without adjuvant with sera col-lection over 42 days was minimal by ELISA, even withoutany significant dilution of the sera (Fig. 4). The fish collagenof both the species was found to be non-immunogenic inthe absence of adjuvant. Even in the presence of adjuvant, anegligible response was observed (Fig. 4). The test serawere comparable with the PBS control. Ovalbumin (Sigma-Aldrich, Germany), known as potent immunogen, was usedas positive control18 in this study. As the total IgG responsefrom the test sera was negligible, differentiation to specificantibody isotypes was not examined. Additionally, therewere no adverse reactions even after three repeat injectionsinto the mice over the 42 days time period indicating ab-sence of acute toxicity and sub-chronic toxicity. These datawere comparable to the commercial bovine collagen(Zyderm), which was also non-responsive in these micestrains.18

Cell culture studyIn vitro biocompatibility of fish scale collagen scaffoldsderived from both the species was investigated using 3T3and MG63 cells individually by MTT assay, which relies onthe mitochondrial activity of viable cells and represents aparameter for their metabolic activity.37 The morphology ofthe attached cells on the collagen scaffolds was also

FIGURE 3. (a) Tensile strength, (b) % elongation, and (c) in vitro degradation behavior of collagen scaffolds (RA) without and with cross-linking

(*p < 0.05).


evaluated under SEM and fluorescence microscope to assessthe cytocompatibility of the developed scaffolds. The bio-compatibility of collagen scaffolds derived from marinesources has also been demonstrated in similar study.21

Cell viability and morphology assay. The results of adirect-contact cytotoxicity assay using cells cultured on scaf-folds of fish scale are shown in Figure 5. Cell viability isexpressed as the absorbance at 590 nm. Interestingly, cross-linked fish scale collagen scaffolds of Rohu and Catla (RA-5,RP-5, CA-5, and CP-5) exhibited higher cell viability in com-parison with tissue culture plates (TCP) regardless of celltypes. Figure 5 shows the viability of 3T3 and MG63 cells,respectively, as a function of time. The viability of both typeof cells cultured on fish scale collagen, bovine collagen scaf-folds, and TCP at day 1 was similar and there was minutedifference in cell viability between samples at day 3. Fishscale collagens from both the species did not induce signifi-cant cytotoxic effect, but rather, exhibited higher cell viabil-ity than the TCP. In particular, the viability of 3T3 andMG63 cells in contact with fish scale collagen scaffolds wasmuch higher at day 7 than that of TCP. The viability of 3T3and MG63 cells increased within the 7 day observationperiod. Cell viability difference in case of 3T3 in betweencollagen scaffolds and TCP was statistically significant (p >

0.05) for RP-5, CA-5, CP-5 with TCP at day 7 and the differ-ence in cell viability with time was statistically significant (p< 0.05). Although the cell viability of fish scale collagenscaffolds was little higher than that of bovine collagen, butthey were not significant (p > 0.05). This may be due to thefact that collagen scaffolds provide higher surface area incomparison with TCP, thus, cells were able to proliferateand migrate well on the scaffolds. In case of TCP, there maybe contact inhibition of cells growth after 5 day. Thedifference in viability at day 5 and 7 for MG63 cell betweencollagen scaffolds and TCP was statistically significant(p < 0.05). Further, there was no significant difference in vi-ability of MG63 cells in between fish scale collagen scaffoldswith that of bovine one (p > 0.05).

The morphologies of 3T3 cultured on cross-linked fishcollagen scaffolds (RA-5 and CA-5) after 5 day culture areshown in Figure 6. As fish scaffolds had an interconnectedand highly porous structure, fibroblasts were distributedwell on the scaffolds (Fig. 6). This finding may be explained

FIGURE 4. Assessment of the immunological response before immu-

nization (Pre-Im) and at various time points after immunization of col-

lagen extracted from L. rohita (RA and RP are acetic acid and pepsin

extracted, respectively) and C. catla (CA and CP are acetic acid and

pepsin extracted, respectively). ELISA absorbance of 1/50 dilution of

sera for measurement of IgG developed in mice immunized (a) with-

out IF adjuvant and (b) with IF adjuvant. PBS and Ova (ovalbumin)

controls are indicated.

FIGURE 5. Viability of cells cultured in direct contact with cross-linked fish scale collagen (RA-5, RP-5, CA-5, and CP-5) and bovine collagen (BC-

5) scaffolds at 1, 3, 5, and 7 days, as determined by MTT assay. Viability is expressed as absorbance value at 590 nm. At 5 and 7 days, differen-

ces between samples with control are significant in viability for both types of cells (p < 0.05).

ORIGINAL ARTICLE


by the cells’ high affinity to collagen and their migrationaround the scaffold. The morphologies of MG63 seeded onfish collagen scaffolds (RA and CA) after 5 day culture areshown in Figure 6. The MG63 cells were also well distrib-uted on the scaffolds and became confluent in 5 day time.The cells were able to contact with each other with cellularextensions and protrusions. In fact, collagen was reported topromote cell attachment owing to the presence of domains(RGDS, YIGSR, etc.) in their molecules, which are recognizedby cell membrane receptors like integrin,57 and thus caneffectively enhance cell attachment and migration.58 Thebinding mediated by cell-surface integrins activates, often insynergy with soluble factors, the intracellular signaling cas-cades and transcription events that regulate cell cycling anddifferentiation.59 Particularly, it has been proposed thatnano-scale collagen fibers could provide the most favorablyspaced binding sites and geometrical signals because oftheir structural similarity to the natural collagen supramo-lecular arrangement in the ECM.60–62

Evaluation of cell proliferation on collagen scaffolds. Therepresentative images of the 3T3 cells attached to the scaf-folds and proliferation rate of 3T3 cells cultured on cross-linked fish collagen scaffolds (RA-5 and RP-5) during 5 dayculture are shown in Figure 7. Cells were stained withnucleic acid staining dye, Hoechst 33342, which can cross-interact with lipophilic cell membranes and label DNA withblue stains. The cells nuclei became visible after stainingwith Hoechst 33342.63 The 3T3 cells were found to attachto the cross-linked fish scale collagen scaffolds and prolifer-ated well as shown in Figure 7. At day 1, 183 6 17 and

168 6 10 cells/mm2 were found attached to the RA-5 andRP-5 scaffolds, respectively. Whereas, at day 3, cell numberincreases and there were 454 6 13 and 449 6 25 cells/mm2 present on RA-5 and RP-5 scaffolds, respectively. Cellnumber increases further and the scaffolds were confluentwith 596 6 17 and 585 6 22 cells/mm2 on RA-5 and RP-5scaffolds, respectively, at day 5. The difference in cells num-ber on both type of scaffolds between day 1, 3, and 5 werestatistically significant (p < 0.01). However, the differencein number of cells attached to RA-5 and RP-5 on each daywas insignificant (p > 0.05). Thus, both type of scaffoldssupported considerable cell attachment and proliferationand the scaffolds were highly biocompatible in nature. Thisfinding may be explained by high affinity of cells towards tocollagen, resulting in high cell proliferation and migration toall areas of the scaffolds. Song et al.21 also reported highnumber of cell attachment and proliferation on collagenscaffolds derived from marine origin.

Fluorescence microscopy. Attachment of MG63 cells on thefish collagen scaffolds were evaluated through fluorescencemicroscopy as shown in Figure 8. Cells proliferated rapidlyand became confluent at day 5. Further, Cells were observedto attach firmly on the nano/micro fibrous collagen scaf-folds and they congregated and oriented towards the fiberdirection with well spread-out morphology. The cells werefound to anchor to the nano/micro fibrous architecture ofthe scaffolds. Cell-cell contact with cellular extensionsand protrusions was also evident in Figure 8. Fluorescencemicroscopic results further complemented the SEM micro-scopic study.

FIGURE 6. Attachment of 3T3 and MG63 cells on collagen scaffolds of RA-5 and CA-5 in 5 day culture period.


FIGURE 8. Attachment of MG63 cells on cross-linked collagen scaffolds derived from (a) RA-5, (b) CA-5, (c) RP-5, and (d) CP-5 after 5 day culture

period. Red elongated portions represent actin filaments and blue dots represent the cells nuclei (scale bar, 100 lm). [Color figure can be viewed

in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 7. Proliferation of 3T3 cells on cross-linked collagen scaffolds derived from RA-5 and RP-5 during 5 day culture period. Blue dots repre-

sent the cells nuclei (scale bar, 200 lm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


CONCLUSION

Collagen isolated from abundantly available fresh water fishscales as an alternate to bovine collagen was utilized to de-velop scaffolds for tissue engineering application. Collagenextracted from these sources may be further considered assafer and economical alternative and by this way the envi-ronmental issue can also be addressed. The porous fishscale collagen scaffolds with nano/micro architecture wereprepared by self-assembly and was stabilized by cross-link-ing using EDC/NHS for tissue engineering application. More-over, the biostability and mechanical properties of fish colla-gen scaffolds can be tailored by varying the degree of cross-linking. In vitro cytotoxicity studies revealed that fish scalecollagen did not induce significant cytotoxic effect and hadconsiderable cell viability. The total IgG response against thefish collagen after three immunizations without adjuvantwith sera collection over 42 day was minimal as evidencedin ELISA assay. Even in the presence of adjuvant, a negligi-ble response was observed. Altogether, these findings indi-cate that fish scale collagen scaffolds derived from freshwater origin are biocompatible in nature and may havepotential tissue engineering applications.

ACKNOWLEDGMENTS

The authors thank IIT Kharagpur for providing infrastructuralfacility, and all lab members of Tissue Engineering laboratoryat SMST, IIT Kharagpur are acknowledged for their support.

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