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Biomaterials 34 (2013) 2855e2864

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Biomaterials

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The promotion of osseointegration of titanium surfaces by coatingwith silk protein sericin

Sunita Nayak, Tuli Dey, Deboki Naskar, Subhas C. Kundu*

Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India

a r t i c l e i n f o

Article history:Received 7 December 2012Accepted 4 January 2013Available online 26 January 2013

Keywords:SilkSericinOsseointegrationTitaniumCytokinesOsteoblast

* Corresponding author. Tel.: þ91 3222 283764; faxE-mail address: kundu@hijli.iitkgp.ernet.in (S.C. Ku

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.01.019

a b s t r a c t

A promising strategy to influence the osseointegration process around orthopaedic titanium implants isthe immobilization of bioactive molecules. This recruits appropriate interaction between the surface andthe tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In thisstudy, we aimed to investigate the functionalization of metallic implant titaniumwith silk protein sericin.Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde ascrosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugatedwith integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbo-diimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titaniumwas characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD func-tionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion,proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNAexpressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblastcells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significantamount of pro-inflammatory cytokines TNF-a, IL-1b and nitric oxide production were recorded whenmacrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized ti-tanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially usefulbioactive coated materials for titanium-based medical implants.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Titanium (Ti) and its alloys are progressively studied for bio-medical applications as a key material in orthopaedic, dentalimplant, hip-joint replacement devices and heart-valves [1]. Tiimplant is non toxic with excellent mechanical properties and alsoshows better response to bone. In addition, its surface is corrosionresistant and bio inert in vivo [2]. However, Ti based orthopaedicimplants encounter specific complications associated with itsincorporation and stability at host interface due to insufficient celladhesion and the implants susceptibility to bacterial infections [3].The implant surface should support osseointegration and con-ductiveness, regardless of implantation site, bone quality and bonequantity for appropriate bone healing and regeneration. Osseoin-tegration permits the direct anchorage of an implant by the for-mation of bony tissue at the boneeimplant interface; whereasosteoconductive property of implant material allows bone growth

: þ91 3222 278433.ndu).

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at the surface [4]. However the osseointegration and osteo-conductive properties of pristine Ti implants are essentially limited.One of the leading factors that govern the interactions between theimplants and their surrounding tissues in bone integration is thephysicochemical surface properties of the implants [5]. Severalmechanical and chemical treatments like blasting, plasma spraying[6] alkaline treatment [7], acid etching [8], hydroxyapatite coating[9] are established for the surface modifications of Ti implants forimproved bone response in healing process. However, the mostpromising and investigated strategy for enhancement of boneregeneration, integration at the interface with implant devices isimmobilization of biomolecules to Ti surfaces [2]. This biochemicalsurface modification can provide characteristics chemical andphysical signals that can direct the differentiation and assembly ofcells and to build conditions for adsorption of matrix proteins andpromote cell-extracellular matrix (ECM) interaction.

The immobilization of different biomolecules on biomaterialsurfaces has been proven to enhance cell attachment and prolif-eration in vitro [10]. There are many methods available for immo-bilization of biomolecules onto a surface, e.g. physical adsorption,encapsulation, entrapment and covalent or ionic binding, etc. The

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advantages of covalent immobilization are that the immobilizedbiomolecules are not easy to be removed by ware and tear androbust enough to withstand in vivo exposure [11]. Silane couplingreagents have been widely used for the immobilization of cell ad-hesive proteins [12]. Immobilization of bioactive molecules such asgrowth factors, peptides, proteins, cell adhesive proteins, poly-saccharides for enhancing cell attachment, cell spreading and cellactivity have been reported [2,13e26]. Other approach reported isthe use of functional parts of larger molecules and its immobili-zation to the surface to get suitable response. Examples of suchmolecules are the sequence arginine-glycine-aspartate (RGD) [27],laminin sequences [28], collagen-derived peptide [29], vitronectinpeptide [12], fibroblast growth factor-fibronectin (FGF-FN) fusionprotein [30] and others are peptidomimetics [31] and aptamers[32].

As a natural biomolecule, sericin exhibits various biologicalproperties that recognize it as a potential material in pharmaco-logical, biomedical and biotechnological field [33e36]. Sericina water soluble globular protein and envelops the fibroin fibre withsuccessive sticky layers that help in the formation of a cocoon. Itconstitutes about 20e30% of the total cocoonweight. In serum-freeculture it enhances proliferation and attachment of mammaliancell, insect lines and hybridoma cells [37]. Sericin peptides havenegligible immunogenicity in vivo [38] which may help its candi-dature as a potential biomaterial. Sericin of the cocoon protein ismostly discarded as waste in silk industries during degummingprocess of silk fibres [39]. The various biological activities of sericinmake this unutilized by-product sericin protein a potential materialfor pharmaceuticals and biomedical use.

Considering the upcoming challenges of acquiring a model or-thopaedic implant with osteoconductive surface to influence theosseointegration process, we aimed to develop Antheraea mylittacocoon derived sericin and sericin/RGD peptides functionalized Tisubstrates. The modification of Ti substrate was characterized byFourier transform infrared spectroscopy, X-ray photoelectronspectroscopy, atomic force microscopy and scanning electron mi-croscopy. The effect of surface-immobilized sericin and sericin/RGDpeptide on adherence, proliferation and function of osteoblast likecells were evaluated. Bio-functionality of the adhered bone cells interms of related gene expression; alkaline phosphatase activity andcalcium deposition were investigated.

2. Materials and methods

2.1. Chemicals

Fresh non-mulberry Indian tropical tasar cocoon of A. mylitta (Am), collectedfrom Taldangra Farm, Bankura District, West Bengal, India. Titanium foil (99.9%purity), APTES, glutaraldehyde, RGD peptide sequence (Gly-Arg-Gly-Asp-Ser) andother chemicals (Sigma Aldrich, St Louise, USA). Dulbecco’s Modified Eagle’s Me-dium (DMEM), trypsineEDTA, antibiotic solution and FBS (Gibco BRL (Grand Island,NY, USA), calcein AM and ethidium homodimer (LIVE/DEAD Viability/CytotoxicityKit - Molecular Probes - Invitrogen), Cellulose dialysis tubing with 3.5 and 12 kDacut-off ranges (Pierce, Rockford, IL, USA). Rhodamine phalloidin, Hoescht 33258,ELISA kit for TNF-a and IL-1b quantification (Invitrogen, USA) were obtained for thisstudy.

2.2. Titanium substrate preparation

Titanium foil of technical purity (cp, grade 2) obtained from sigma USA. The Tifoils of 0.5 mm thickness were cut into 1 cm � 1 cm pieces. The pieces were thensonicated for 15 min in acetone (70% by volume), ethanol, and deionized water,respectively to remove residual surface impurities and finally dried to use forchemical functionalization.

2.3. Chemical functionalization of titanium

The cleaned Ti foils were activated in 2.5 M NaOH solution at 80 �C for 24 h, andthen thoroughly rinsed with dH2O and blown dry. The NaOH-activated Ti substrateobtained is referred to as TiOH. This activation process produced a very hydrophilic

substrate, which was then silanized by immersing into a 2% v/v solution of APTES in99.8% anhydrous ethanol for 10 h to generate an amino-silane surface. The silanizedTiOH surface by APTES was denoted as TiOHA and was sonicated in ethanol toremove the excess physio-absorbed molecules. For further protein molecule adhe-sion, another linker molecule, gluteraldehyde was used. 2% v/v solution of gluter-aldehyde in deionized water was prepared and stirred for 1 h. The wateregluteraldehyde solution was poured over the Ti-APTES samples ensuring completecoverage of the metal surface. The containers were then sealed and left for 24 h.Afterwards the samples were rinsed thoroughly with deionized water and placed ina petri dish.

2.4. Extraction of silk sericin (SS) from A. mylitta silk cocoon

Silk sericin protein was extracted by the alkaline degumming method fromcocoons of the non-mulberry silk worm A. mylitta using a protocol described [35].Briefly cocoons were cut into finer pieces and were added to 0.02 M Na2CO3 (Merck)solution. The contents were boiled for 1 h. The solution was centrifuged and thesericin solution in supernatant was collected while the undissolved pellet wasdiscarded. The supernatant was then dialysed for 48 h against water using 12 kDadialysis membrane (Pierce, USA) to remove Na2CO3 and further concentrated againstPEG-8000 using 3.5 kDa dialysis membrane.

2.5. Surface coating with sericin

The samples were further treated with sericin solution. A solution of 1% (w/v)sericin in deionized water was prepared, and used to immerse the TiOHA samplesfor 2 h at 37 �C. The samples were further rinsed repeatedly with PBS to remove theunattached sericin protein. The sample was referred as Ti-SS.

2.6. RGD peptide immobilization

The sericin-functionalized Ti substrates were treated at 20 �C with an RGDpeptide solution (200 mg/ml) containing EDAC (20mM) and NHS (50mM) in 0.1 mMMES buffer for 4 h, followed by rinsing with phosphate buffered saline (PBS,pH ¼ 7.4). The sample was referred as Ti-SS-RGD.

2.7. Surface analysis by SEM

Surface morphology of Ti, Ti-SS, Ti-SS-RGD foils were observed using a scanningelectron microscope (JEOL, JSM-5800). SEM images were acquired after gold sput-tering at operating voltage of 20 kV.

2.8. FTIR study of the surface coating on titanium

The infrared absorption spectra of the SS, Ti-SS, and Ti-SS-RGD complex wereobtained from an FTIR spectrometer (NICOLET 5700, USA) in diffuse reflectancemode. For each spectrum obtained, a total of 64 scans were accumulated at 4 cm�1

resolution. Scanning was conducted in the range from 400 to 4000 cm�1.

2.9. X-ray photoelectron spectroscopy (XPS)

The elemental chemical composition of pristine Ti, Ti-SS and Ti-SS-RGD surfacewas determined by XPS (Phi5000 versaprobe II with Al (1486,6 eV) and Mg(1253,6 eV) ion source). The binding energy scale was referenced by setting Cls peakat 284.6 eV.

2.10. Atomic force microscopy (AFM)

The surface topography and roughness of the Ti substrates before and afterimmobilization were characterized using a Scanning Probe Microscope (SPM)[Model: Multiview- 1000�] in tapping mode. Atomic force microscopy was per-formed in non-contact mode with tips mounted on cantilevers at spring constant of40 N/m (as specified by manufacturer) for surface analysis. Measurements weredone with 10 � 10 mm2 scan area at room temperature. Quantitative AFM analysiscalculated using AFM WSxM4.0.

2.11. Cell culture

Mouse osteoblast cell line (MG-63) and mouse macrophage (RAW 264.7) cellswere cultured in DMEM (Invitrogen, USA) supplemented with 10% foetal bovineserum, 100 U/ml penicillin and 100 mg/ml streptomycin. All cell lines were incu-bated at 37 �C in a humidified atmosphere of 5% CO2 with the growthmedia changedevery 48 h. Cultured cells were detached by trypsinization (0.25% trypsine EDTA),suspended in fresh culture media and used for the experiments described below.

2.12. Cell viability by Alamar blue assay

MG-63 cells were seeded at a density of 1 �105 cells/ml on pristine Ti, Ti-SS andTi-SS-RGD surface. At 1, 3, 7, and 14 days of incubation time, each sample were

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washed in PBS, placed into fresh, sterile wells and a 1 in 10 dilution of Alamar Blue(AB) dye (Invitrogen, USA) in incomplete media was added. Cells cultured on tissueculture plastic were used as controls. The plates were incubated for 4 h and the dyereagents were carefully transferred to 96-well plates. The optical density wasmeasured at 570 and 600 nm using a microplate reader (Thermo Scientific Multis-kan Spectrum, Japan). The data were expressed as percentage of AB reduction thatcorrelates with the number of viable cells.

2.13. Cell viability by live/dead assay

The viability of cells on the surface of pristine Ti, Ti-SS and Ti-SS-RGD wasevaluated by live/dead staining. Briefly, MG-63 cells were carefully seeded ata density of 5 � 104 cells/ml on the surface of each sample in 24-well plates. After 1and 5 days of incubation, cells/specimens were rinsed three times with PBS and thenincubated with live/dead stain (2 mM calcein AM and 4 mM ethidium homodimer-1)for 30 min at room temperature. Viable cells (green) and dead cells (red) wereevaluated under a confocal laser scanning microscope (CLSM, Olympus FluoView1000).

2.14. Cell attachment and proliferation

The proliferation of the osteoblast cells on substrates was investigated bymeasuring the number of cells after 1 and 3 days of culture. Cells were seeded ontosubstrates at a density of 5000 cells/cm2. At each designated points in time, the cellson the substrates were rinsed with PBS, fixed with 4% paraformaldehyde (PFA,Merck) stained with rodhamine phalloidin for actin and Hoechst 33258 for nucleus.For cell attachment study, osteoblast cell suspensions at a density of 1�104 cells/cm2

were seeded onto the substrates and incubated in a 5% CO2 incubator for 24 h. Afterthe pre-specified incubation period, the substrates were washed twice with PBSbefore fixing with 3% glutaraldehyde for at least 30 min at room temperature. Thesubstrates were then subjected to step dehydration with a graded series of ethanol-water solutions (70%e100%) for 10 min each and sputter coated with gold, for im-aging using a scanning electron microscope (SEM) (JEOL, Model 5600LV).

2.15. Alizarin red staining for calcium deposition

Alizarin red staining was performed after day 7 and 14 days of culture in order tocorroborate osteogenic gene expression. Mineralization of the cell layer wasexamined through the use of alizarin red S. The cell layers were washed twice withphosphate buffered saline (PBS) fixed with 10% formalin solution for 30 min, andthen washed with pure water two times. For alizarin red S staining, the cells wereplaced in 1% alizarin red S (Sigma, USA) solution for 10 min, washed with pure waterthree times and then air-dried. Staining layers were visualized with phase micro-scopy using an inverted microscope (Nikon ECLIPSE TS100).

2.16. RT-PCR analysis

Oestoblast cells were cultured on pristine Ti, Ti-SS and Ti-SS-RGD surface for 7days. mRNA level of bone sialoprotein (BSP), osteocalcin (OC) and alkaline phos-phatase (ALP) were analysed by semi-quantitative RT-PCR reaction using custommade forward and reverse primers (Table 1) in respect to GAPDH level as controls.Total RNA was prepared using Trizol (Invitrogen, Carlsbad, CA, USA) followed by theRNA isolation kit (Qiagen) to isolate total RNA. First strand cDNA was synthesizedfrom 10 mg of total RNA using first strand cDNA synthesis kit (Fermentas).

Table 1Primers used for semi-quantitative RT-PCR.

Gene name Gene Sequence (5‘-3‘) Size Size (primer)

Bone sialoprotein Forward primer 198 bp 23 bp, 21 bpagggcacctcgaagacaacaaccReverse primerccccacgaggttccccgttct

Osteocalcin Forward primer 173 bp 20 bp, 21 bpcagccgcagctcccaaccacReverse primercgccagcctccagcactgttt

Alkaline phosphatase Forward primer 156 bp 25 bp, 21 bptgcgcagagaaagagaaagaccccaReverse primerggcagccgtcactgtggagac

GAPDH Forward primer 153 bp 20 bp, 20 bpaattgagcccgcagcctcccReverse primerccaggcgcccaatacgacca

2.17. Analysis of in vitro immuno-response of modified Ti surfaces

TNF-a, IL-1b and nitric oxide (NO) production in vitroThe immunogenicity of the on pristine Ti, Ti-SS and Ti-SS-RGD were evaluated

by in vitro quantitative determination of TNF-a, IL-1b and NO concentrations in cellculture supernatant. Two sets of experiment were designed to analyse in vitroimmuno-response of different Ti substrate. Briefly macrophage (105 cells/cm2) andmacrophage-osteoblast co-culture in 2:1 ratio cells were seeded on different coatedsamples. After 24 h of cell seeding, the complete media was replaced with incom-plete one. Cell supernatant was collected to analyse TNF-a and IL-1b level using TNF-a and IL-1b quantification ELISA kit (Invitrogen). Data accumulation was done at450 nm and 600 nm using a microplate reader. To quantify the NO production cellsupernatant were collected and incubated with Griess Reagent (Sigma) for 10 min.Data accumulation was done at 548 nm and 600 nm using microplate reader.

3. Results

3.1. Analysis of topography of modified surfaces

Silane molecule, 3-Aminopropyltriethoxysilane (APTES) wasused to modify surface properties of Ti substrate that creates freeprimary amine group at end (eNH2). This amine group is thenattached to glutaraldehyde as linker molecule as its free aldehydegroup react with the terminal amine group of APTES. Free aldehydegroup remained of glutaraldehyde further reacts with aminegroups of the desired protein molecules. The schematic illustrationof the surface immobilization of bioactive sericin and RGD peptideto Ti substrate is shown in Fig. 1. The surface morphologies ofpristine Ti, Ti-SS and Ti-SS-RGDwere investigated by SEM and AFM.The typical SEMmicrographs of pristine Ti and Ti immobilized withsericin protein are shown in Fig. 2. The pristine Ti revealed thepresence of regular parallel grooves on the surface Fig. 2A (a)however, morphologies of chemically treated titanium followed byimmobilization is totally different from pristine Ti surface. Theimmobilized protein on Ti surface exhibits ordered porous top-ography structure as shown in the Fig. 2A (b). In AFM images(Fig. 2B(a-c)) immobilized Ti surface with sericin and sericin/RGD,show the nanoscale topography. Several aggregates with hill-valleystructure were observed on immobilized surfaces Fig. 2B(b,c)compared to pristine Ti (Fig. 2B(a)). Quantitative AFM analysiswhichwas calculated using AFMWSxM 4.0, software demonstratedRRMS (root-mean square roughness) value of 42.92 nm, 3.55 and6.28 nm for Ti, Ti-SS and Ti-SS-RGD surfaces respectively. Ra(average roughness) value of Ti, TI-SS and TI-SS-RGD are 31.48, 2.78and 4.86 respectively. Both RRMS and Ra values for Ti-SS and Ti-SS-RGD were reduced after protein immobilization.

The influence of the modification on the chemical compositionof the Ti surface was determined using XPS measurements shownin Fig. 3. Table 2 shows the chemical composition present on thesurface of the samples. After immobilization Ti signal reduces onthe surface of Ti-SS and Ti-SS-RGD. There is also an increase ofnitrogen and carbon content on the samples that have beenimmobilized with sericin and RGD, this data indicates the presenceof the protein on the surface. All binding energies were referencedto the C 1s hydrocarbon peak at 284.6 eV. In pristine Ti, Ti2p peak(w460 eV) was observed but after sericin/RGD immobilization Ti2ppeaks further decreased. Similarly new N1s peaks (w400 eV)appeared corresponding to Ti-SS and TI-SS-RGD samples indicatingthe successful immobilization of sericin. C2p peaks (284.6 eV)increased in Ti substrates after immobilization. Presence of C2ppeak in Ti sample may be due to from unavoidable hydrocarboncontamination. There was no significant difference in the peak ofsericin and sericin/RGD immobilized Ti substrate. This result wasconsistent with that of the FTIR spectra. FTIR analysis was used tocharacterize the presence of sericin and sericin/RGD on immobi-lized Ti surfaces (Fig. 4). FTIR indicates the presence of specificfunctional groups on the grafted surface. Presence of amides I (C]O

Fig. 1. Schematic diagram for the immobilization of silk sericin and coupling of RGD peptide to Ti substrate.

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stretching vibration) at 1650 cm�1, amides II at 1560 cm�1 and CH2(CH bending vibration) at 1450 cm�1 was observed.

3.2. Cell proliferation

MG-63 osteoblast-like cells were cultured on different Ti sub-strate to assess the cellesurface interactions such as adhesion,proliferation, cell viability and spreading. The results of adhesion

Fig. 2. (A) SEM micrographs of the surface morphology of (a) pristine titanium (b) after imimmobilized titanium and (c) sericin/RGD immobilized titanium.

and proliferation are shown in Figs. 5 and 6, respectively. Cellsstarted to adhere on the coatings after 1 day of culture and spreadwell which revealed that the coatings were able to support adhe-sion and spreading of osteoblasts. Fig. 5 shows the SEM images ofthe morphology of attached osteoblast cells on glass and differentTi substrates. Fig. 6 show confocal images of the attached cells andshows uniform distribution of cells in Ti-SS, Ti-SS-RGD and glass.While in Ti surface the cells seems to be stretched. The morphology

mobilization with sericin; (B) 2D and 3D images of (a) uncoated titanium, (b) sericin

Fig. 3. XPS core-level spectra of a) Ti2p, b) C1s, c) O1s, d) N1s, e) S2p and f) Si2p of different modified titanium substrates.

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of individual osteoblast cell exhibited spreaded structure with actinfilaments in sericin and sericin-RGD immobilised Ti substratescomparable to glass.

3.3. Metabolic activity and live dead assay

Cells, cultured on Ti-SS and Ti-SS-RGD substrate demonstratedhigh numbers of live (green) cells compared to pristine Ti andhardly any dead (red) cells were observed (Fig. 7A). No apparentdifference in the number of live (green) cells was observed betweenTi-SS and Ti-SS-RGD sample at day 5 of culture. The results of live/dead assay show that most MG-63 cells on all Ti substrates werealive indicating all Ti substrates are safe and suitable materials forthe growth of osteoblasts. The Alamar Blue assay indicated thatcells on Ti-SS-RGD substrate had the highest proliferation rate,followed by Ti-SS and Ti after 3 days of culture (Fig. 7B). Thenumber of cells in different Ti substrate increases over time in theevaluated period as indicated by the Alamar Blue reduction %. Itwas seen that at early time point the cell density on the Ti waslower as compared to Ti-SS and Ti-SS-RGD samples. Coupling ofRGD sequence to sericin immobilized Ti substrate has enhanced cell

Table 2The chemical composition of the developed surfaces as determined by XPS after ionbombardment.

% at Ti Ti-SS Ti-SS-RGD

Ti2p 26.9 13.8 16.8O1s 52.7 46.3 50.3C1s 16.4 29.5 24.6N1s 1.4 8.2 6.8Si2p 2.6 2.1 1.4S 0.1 0.1 0.1

attachment thus increasing proliferation rate. At day 14 the pro-liferation of Ti-SS-RGD (w72%) was significantly higher than pris-tine Ti (w49%) followed by Ti-SS (w62%).

3.4. Calcium deposition

Mineralization of the cells after 7 and 14 days culture on thedifferent substrates was assessed by staining with Alizarin Red, andthe results are presented in Fig. 8. The stains are attributed to the

Fig. 4. FTIR spectra of different Ti substrates and sericin protein powder.

Fig. 5. SEM pictographs of adhered osteoblast cells on TCP and on different Ti substrates (a, e), (b, f), (c, g) and (d, h) represents growth and morphology on TCP, Ti, Ti-SS and Ti-SS-RGD surfaces respectively.

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calcium nodules deposited by the cells. On the immobilized sericinand sericin/RGD Ti, mineralization is higher as shown by the densecoverage of calcium deposits on the Ti-SS (Fig. 8b, e) and Ti-SS-RGD(Fig. 8c, f) substrates compared to pristine Ti (Fig. 8a, d).

Fig. 6. Confocal micrograph of day 1 and day 3 growth and morphology of osteoblast cells onand morphology on TCP, Ti, Ti-SS and Ti-SS-RGD surfaces respectively.

3.5. RT-PCR analysis

To test the effect of sericin coating on Ti on osteoblast differ-entiation, osteoblast-specific transcriptional gene markers in

TCP and on different Ti substrate. (a, e, i), (b, f, j), (c, g, k) and (d, h, l) represents growth

Fig. 7. (A) The viability of osteoblast on different Ti substrates using live/dead assay with confocal microscope of day 1 and 5 culture; (a, d) represents viability on Ti, (b, e) Ti-SS, and(c, f) Ti-SS-RGD respectively. Live cells produced green fluorescence and dead cells red fluorescence. Scale bars 100 mm. (B) Alamar blue assay of osteoblast cells cultured for 1, 3, 7and 14 days on different Ti substrates (n ¼ 3, Mean � standard deviation). (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

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MG-63 cells exposed to different Ti substrate for 7 days wereexamined by RT-PCR. Genes coding for proteins involved in boneturnover: BSP, OC and ALP were up-regulated in MG-63 cells grownin Ti-SS and Ti-SS-RGD substrates. Level of mRNA for GAPDH had noeffect which was amplified as an internal control (Fig. 9A). Thedensitometric value of different RT-PCR product normalised byGAPDH using Image J software value is shown in Fig. 9B. TheGAPDH normalised value clearly shows high expression of BSP, OCand ALP genes in Ti-SS-RGD and Ti-SS followed by Ti.

3.6. TNF a, IL-1b and NO production

Murine macrophage-like cells RAW 264.7 and osteoblast-macrophage co culture for 24 h were used to evaluate the effectof different surface coating on Ti substrate on macrophage activa-tion and secretion of pro-inflammatory cytokines IL-1b, TNF-a andNO. Distinctly different cytokine profiles were observed arounddifferent Ti substrates in macrophage and osteoblast-macrophageco-culture as shown in Fig. 10. We found that the release of TNF-

Fig. 8. Optical microscopy images of Alizarin Red stained osteoblasts after culturing for 7 and 14 days on (a, d) Ti, (b, e) Ti-SS and (c, f) TieSSeRGD respectively. Scale bar ¼ 100 mm.

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a and IL-1b was enhanced by pristine titanium by macrophageswhen compared toTCP.While in co culture the release of TNF-a andIL-1b in all samples were comparable to TCP. The data obtained forimmobilized surface were not significantly different from TCP. Thisshowed that the immobilization did not induce any toxic effect.Very low nitric oxide production was observed in Ti-SS and Ti-SS-RGD substrate. Nitric oxide production was compared to nonstimulated control (treated with PBS, pH 7.4) where there wasminimal nitric oxide production. Nitric oxide was more in all Tisubstrate by macrophage alone than macrophage osteoblast coculture. In both type of culture, pristine Ti produces higher NO incomparison to Ti-SS and Ti-SS-RGD.

4. Discussion

Our aim was the surface functionalization of titanium withbioactive sericin protein which can interact with target cells topromote growth and integration of titanium implant materials.Recent studies have shown that nanoscale dimension influences

Fig. 9. (A) Expression of bone-related genes in osteoblastic cultures on different Ti surfaces.reaction (RT-PCR) showed for bone sialoprotein (BSP), osteocalcin (OC), alkaline phosphatasusing Image j software.

cell adhesion and osteogenic differentiation as well as nano-structured titanium implant promote early osteoblastic differenti-ation of hMSC and, consequently, rapid osseointegration oftitanium implants [40]. The success of surface functionalizationwasconfirmed by combined techniques of SEM, AFM, XPS and FTIR. Theadsorbed protein seems to form globular aggregates. The Ti surface(as received) appeared with large smoother areas along withmicrocavities. However the coated surface with the proteinappeared to form aggregate within the deeper surface of the par-allel grooves of pristine Ti giving more homogeneous roughnesspattern. The data shows uniform and efficient protein immobili-zation was constructed on titanium surface. One of the importantfactors in osseointegration is the finer detail of surface texture andits effect on bone response ie, the formation of a direct contactbetween implant and surrounding bone can possibly direct theimplant development [41]. Recently the importance of nanometresized surface implants are exemplified in biomaterial science.However since decades, implant surface topography at the micro-metre level of resolution is considered the parameter for implant

Representative electrophoretic images from the reverse transcriptase polymerase chaine (ALP) and GAPDH. B) GAPDH normalized densitometric quantification of PCR product

Fig. 10. (A) TNF a production, (B) IL-1b production and C) nitric oxide production by RAW 264.7 murine macrophages and macrophage-osteoblast co culture on different Tisubstrates with tissue culture plates (TCP as negative control) for day 1. The TNF-a, IL-1 b and Nitric oxide production by the cells estimated from a standard graph. Data representsmean standard deviation (n ¼ 3, *P < 0.05).

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success. Some of the studies indicated that surface roughness in-fluence cell differentiation [42]. However, direct comparisons cannot be made between in vivo outcomes or clinical results within vitro studies. It has been recommended that only a very specificsurface topography with a Ra value between 1 and 1.5 mm providesan optimal surface for bone integration [43].

The surface chemical compositions of substrates strongly in-fluence protein adsorption process and cell attachment [44].Chemical nature of surface functionalized titanium substrate wascharacterized by XPS and FTIR. All immobilized substrates exhibi-ted peaks corresponding to carbon and nitrogen resulting fromimmobilization of sericin protein. Additionally, FTIR showed peaksfor amide I and amide II significant peak for proteins.

In vitro cell proliferation and metabolic activity investigationperformed on surface functionalized titanium substrates displayedgood cell proliferation and cell viability tendency. The number ofcells adhered to sericin and sericin/RGD immobilized titaniumwasstatistically significantly higher (*p < 0.05) than pristine titanium.The result suggests that the presence of sericin has promotedgrowth and proliferation at the long termwhile RGD had enhancedthe initial attachment of the cells to the surface. Moreover, theimmobilized RGD in addition to sericin have shown to have a pro-found combinatorial effect on osteoblast adhesion and prolifera-tion. Hence the osteoblast growth and proliferation enhancingproperties of sericin and sericin/RGD immobilized Ti substrate areexpected to have good potential in orthopaedic applications.

In vitro and in vivo osteoblast differentiation basically consists ofthree stages comprising of cell proliferation, matrix maturation andmatrix mineralization [45]. Cell proliferation directs the productionof several detectable amounts of extracellular matrix proteins(procollagen I, TGF-b, and fibronectin) [46]. While maximalexpression of alkaline phosphatase (AP) is characteristic feature ofmatrix maturation. During initial matrix mineralization, genes forosteocalcin (OC), bone sialoprotein (BSP), and osteopontin (OPN)are expressed and vast extracellular calcium deposits are produced.Successful in vitro bone formation can be detected by stainingcalcium deposits using Alizarin Red S giving bright orange-red. Theexpression of alkaline phosphatase and extracellular matrix proteinosteocalcin and bone sialoprotein was increased in Ti-SS and Ti-SS-RGD immobilized surface compared to Ti. Moreover at day 14,mineralized nodule formation as determined by alizarin redstaining, was increased in Ti-SS and Ti-SS-RGD.

When implant material come in contact with in vivo situationit comes in contact with inflammatory cells, such as macrophages.

The macrophage has a major role in normal wound healing andthe reparative process around implants. Macrophages may impairosteoblast function due to osteolysis caused by release of bone-associated cytokines thereby causes aseptic loosening of ortho-paedic implant surgery (e.g. joint prostheses). Cytokines are,soluble proteins produced in response to an antigen and functionas chemical messengers for regulating the innate and adaptiveimmune systems. TNF-a, IL-1b and NO production by RAW 264.7and osteoblast-macrophage co culture for 24 h were not sig-nificantly different from TCP. This shows that the immobilizationdid not induce any toxic effect. TNF-a and IL-1b are pro-inflammatory cytokines and during the initiation of the inflam-mation they act synergistically. TNF-a is the main mediator inresponse to Gram-negative bacteria and its concentration reflectsthe amount of bacteria and the stage of inflammation. Increasedlevel of TNF-a has been reported to result in programmed celldeath (apoptosis) as well as enhance phagocytosis of neutrophilsundergoing apoptosis [47]. Whereas IL-1b is related to tissuedestruction, stimulate bone resorption and induction of tissue-degrading proteinases. Sericin is well-characterized as non in-flammatory protein [34,35]. These results suggest that sericin hasno significant inflammatory response after immobilization topristine Ti.

We have demonstrated that the immobilization of titaniumsurface with sericin protein and sericin/RGD generates a bioactivenano-topography. This study clearly shows a marked difference ininitial cell attachment and spreading of osteoblast between pristineand modified surfaces. This has effects on early events of osteo-genesis, and augments bone formation. It is emphasized here thatsericin is available freely in textile industries as a waste productabundantly. It will be cost effective biomaterial.

5. Conclusion

The surface functionalization approaches can be used for boneprostheses applications in order to accelerate bone healing. Theresults support the concept of surface modification with bioactivesilk protein sericin and RGD peptides for enhancing the osseoin-tegration and osteoconduction of orthopaedic titanium implants.Immobilization of two ormore domains that enhance adhesion andactivity may have a synergetic effect of the osseointegration ofimplants. Many of the basic mechanisms of cellematrix interactionare unclear and demands further investigation. The results indicatethat the immobilized sericin and sericin/RGD titanium substrate are

S. Nayak et al. / Biomaterials 34 (2013) 2855e28642864

having the potential to enhance the osseointegration in orthopae-dic applications.

Acknowledgement

This work is supported by Department of Biotechnology and itsBioinformatics facilities (also Senior Research Fellowship to SN) andDepartment of Science and Technology, Government of India, NewDelhi.

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