RSC Advances

PAPER

Fabrication of di

aDepartment of Chemistry, Periyar Univ

dhanaraj_gopi@yahoo.com; Fax: +91 427 2bDepartment of Physics, School of Basic an

Tamilnadu, Thiruvarur 610101, IndiacDepartment of Zoology, School of Life Scie

IndiadCentre for Nanoscience and Nanotechnology

Cite this: RSC Adv., 2015, 5, 47341

Received 30th March 2015Accepted 14th May 2015

DOI: 10.1039/c5ra05624a

www.rsc.org/advances

This journal is © The Royal Society of C

valent ion substitutedhydroxyapatite/gelatin nanocomposite coating onelectron beam treated titanium: mechanical,anticorrosive, antibacterial and bioactiveevaluations

A. Karthika,a L. Kavitha,b M. Surendiran,a S. Kannanc and D. Gopi*ad

The key property in the fabrication of a biomaterial is to facilitate the replacement and/or regeneration of

damaged tissues and organs. To obtain such a biomaterial, we fabricated a triple mineral (strontium,

magnesium and zinc) substituted hydroxyapatite/gelatin (M-HAP/Gel) nanocomposite coating on

electron beam treated titanium (Ti) metal. The influence of gelatin concentration in M-HAP was studied

to investigate its effect on morphological changes, crystallinity, mechanical and anticorrosion properties.

The M-HAP/Gel nanocomposite coating (with 3 wt% gelatin) on treated Ti resulted in better mechanical

and anticorrosion properties as a consequence of the electron beam treatment of Ti. A reduced number

of bacterial colonies were observed for the M-HAP/Gel composite against Staphylococcus aureus (S.

aureus) and Escherichia coli (E. coli) microbes which evidences a lower chance of implant failure after

implantation. Moreover, the cell proliferation assay, live/dead staining of MT3C3-E1 cells and cell viability

of fibroblast stem cells on the resultant nanocomposite revealed that the M-HAP/Gel composite will

definitely be an effective implant material for better cell growth in orthopedic applications.

1. Introduction

Over the last few decades, bioceramics have attracted growingattention among researchers because of their increasing appli-cation in treating damaged and infected bone. The mainrequirement for a bioceramic material to be suitable to replacetraumatized, damaged, or lost bone is that it should mimic theproperties of natural bone.1 Recently, hydroxyapatite (HAP,Ca10(PO4)6(OH)2) has been broadly used as a biomaterial due toits similar characteristics to natural bone.2 The HAP structurecan accommodate a great variety of other substituents. Thus itcan readily accept minerals to mimic bone like material. Amongmanymineral ions, strontium (Sr) is an important trace elementin the human body and enhances preosteoblast proliferationand decreases bone resorption.3 In addition, Sr-HAP can exhibitbetter mechanical properties than pure HAP.4–7 Zinc (Zn) isrequired for humans as an essential element and its require-ment is estimated to be 15 mg per day.8 Also, Zn has

ersity, Salem 636011, India. E-mail:

345124; Tel: +91 427 2345766

d Applied Sciences, Central University of

nces, Periyar University, Salem-636 011,

, Periyar University, Salem 636011, India

hemistry 2015

antibacterial activity, which minimizes the bacterial load on theimplant surface aer orthopedic implantation and improvesthe mechanical strength.9,10 Similarly, magnesium (Mg) directlystimulates osteoblast proliferation and helps with the miner-alization of calcied tissues which indirectly controls mineralmetabolism.11–13 Previously, Gopi et al., have developed amineral substituted HAP coating over titanium alloy by a pulsedelectrodeposition method.14

In order to fasten/improve biological interactions with thesurrounding tissues, nowadays, biopolymers such as chitosan,gelatin, etc., have been used. In particular, gelatin, a naturalpolymer, is composed of a unique sequence of amino acids suchas glycine, proline and hydroxyproline that stimulate woundhealing and intensify osteointegration as well as three dimen-sional tissue regeneration.15,16Moreover, gelatin is obtained by acontrolled hydrolysis of brous insoluble protein, collagen,which is the major constituent of skin, bones and tissues.17

Gelatin is currently used in pharmaceuticals, wound dressingsand adhesives in clinics due to its good cytocompatibility, non-antigenicity, plasticity and adhesiveness.18–21 Furthermore, it iscompletely resorbable in vivo, and its presence with bioceramicmaterial can resemble bone due to the organic and inorganicphases. The addition of gelatin to the bioceramic not only canimprove the biological interactions, but also can be expected tobe enhance the mechanical properties of the composite.22

Taking into account the importance of these mineral ions, Sr,

RSC Adv., 2015, 5, 47341–47352 | 47341

RSC Advances Paper

Mg and Zn are substituted into HAP and fabricated as acomposite with gelatin for better bioactivity for osteoblast cellsin vitro and to improve the biocompatibility along with havinggood mechanical properties.

The growth of bioceramic coating on implant materials withgood adhesion strength has become a major eld in biomate-rials research. Also, the achievement of osseointegration issignicantly affected by the surface nature of implants. So, thesurface of implants such as titanium (Ti) and its alloys, 316Lstainless steel, etc., was modied using some modicationtechniques,23 which has resulted in better adhesion strengthalong with good corrosion resistance. Among the varioussurface modication techniques, electron beam treatment hasbeen accepted as the best technique for the surface modica-tion of implant materials. Moreover, the electron beam treat-ment improves the physical, chemical and mechanicalproperties of metals as was reported by Dong et al.,23 andProskurovsky et al.24 Here, we employed high energy low currentelectron beam treatment on Ti metal to obtain an increasedroughness of the metal surface, which could lead to highadhesion strength for the coating.25,26 Also, Lee et al.,27 havefabricated Zr-based bulk metallic glass (BMG)/Ti surfacecomposites by high-energy electron beam irradiation andreported that the BMG composite has excellent bondingstrength as well as very high hardness and wear resistance.

A composite biomaterial of HAP and gelatin is expected toshow improved osteoconductivity and biodegradation alongwith mechanical strength for orthopedic usage.3 A nano-structured composite could show better adhesion of osteoblastcell at the implant tissue interface. Also, the nanostructuredcomposites have mechanical properties similar to bone, sincebone itself is a nanostructured composite.28 Zandi et al.,29 haveevaluated the biocompatibility of a gelatin coated nano-HAPscaffold using mesenchymal stem cells. They reported that theprepared scaffolds possessed desirable biocompatibility, highbioactivity with better cell attachment and proliferation andsufficient mechanical strength in comparison with a noncoatedHAP sample. Recently, Liu et al.,30 fabricated gelatin function-alized graphene oxide for mineralization of HAP, and reported indetail about MC3T3-E1 cells osteogenic activity in terms of thecell spreading, growth, and alkaline phosphatase activity.

Chang et al.,31 have developed a HAP–gelatin system and theyhave proved the chemical binding between hydroxyapatite andgelatin molecules, which mimics natural bone microstructure.Moreover, the bioactivity and corrosion performances of thestrontium substituted calcium phosphate and gelatincomposite were studied by Huang et al.,32 and it has beenreported that the resultant coating exhibited better cyto-compatibility than Ca–P coating. Though they developed thecoating, they reported only 5 MPa as bond strength which isinsufficient for load bearing applications. Though the devel-oped composite coating can exhibit good biological activities,the interaction between the bioceramic and implant materialhas to reach appreciable adhesion strength. For that reason, theimplant material could be subjected to electron beam irradia-tion to obtain a roughened surface, which leads to an adhesivecoating.

47342 | RSC Adv., 2015, 5, 47341–47352

The pulsed electrodeposition (PED) technique has beenemployed for the development of composite coatings since it hasgood advantages for obtaining better coating than other depo-sition methods.33 The authors employed the PED technique forthe development of a M-HAP/Gel composite on the Ti implantmaterial. The objective of the present work is to develop a moreadhesive M-HAP/Gel nanocomposite coating on a HELCDEBtreated Ti surface and to study the effect of gelatin concentrationin the M-HAP/Gel nanocomposite on various properties and toobtain a better bioimplant material. The in vitro corrosionbehavior of theM-HAP/Gel nanocomposite coating onHELCDEBtreated Ti has been evaluated in simulated body uid and themechanical property of the nanocomposite was studied in rela-tion to adhesion strength. The antibacterial ability of the nano-composite was tested against S. aureus (Gram-positivebacterium) and E. coli (Gram-negative bacterium). The MT3C3-E1 preosteoblast cells were cultured on the M-HAP/Gel nano-composite coating to evaluate the viability and proliferation byMTT and live/dead cell assay. The apatite growth ability that isthe bioactivity over the composite surface was investigatedthrough SBF immersion study for various days.

2. Materials and methods2.1 Preparation of Ti substrate

The substrates were pure Ti metal (99.99%) with a size of 10 �10 � 3 mm3. These substrates were polished thoroughly with400–1200 silicon carbide sheets, then cleaned ultrasonicallywith acetone to remove residual particles from grit and dried inair at room temperature. Aer this process, the substrates weresurface treated using a 700 keV DC accelerator with an electronbeam of current 1.5 mA at an energy of 700 keV to enhance themechanical properties and corrosion resistance of the Ti metal.The detailed procedure for the electron beam treatment wasprovided in our previous report.26

2.2 Preparation of M-HAP/Gel composite coatings

Analytical grade Ca(NO)3$6H2O (0.294 M), Sr(NO3)2$6H2O(0.042 M), Mg(NO3)2$6H2O (0.042 M) and Zn(NO3)2 (0.042 M)were dissolved separately in deionized (DI) water. (NH4)2HPO4

(0.25 M) was dissolved in DI water and the solutions were mixedin the molar ratio of 1.67. Then, gelatin solutions of 1, 2, 3 and 4wt% were added to the electrolytes in the presence of a nitrogenatmosphere and the pH of the solutions maintained at 4.5. Theelectrolytes were stirred uniformly keeping the temperature at65 �C and using a magnetic stirrer and a thermostat. Then, theresultant electrolytes were used for further processes.

A regular three electrode system was used for the pulsedelectrodeposition of M-HAP/Gel composite using CHI 760C (CHInstruments, USA), where HELCDEB treated Ti substrate servedas working electrode, platinum electrode as counter electrodeand saturated calomel electrode (SCE) act as reference elec-trode. The deposition was performed for 1 h on a HELCDEBtreated Ti sample in galvanostatic mode with a current densityof 1.0 mA cm�2. As per our previous study,26 the optimumconditions of 1 s pulse on time and 4 s pulse off time with

This journal is © The Royal Society of Chemistry 2015

Paper RSC Advances

respect to the current density of 1.0 mA cm�2 and 0 mA cm�2

were used. When starting the deposition process, 2000 ppm ofH2O2 was added into the electrolyte solutions to reduce the H2

gas bubbles. Aer the coating process, the specimens wereremoved from the electrolyte and washed with deionized water,and then dried. The in situ formation of the M-HAP/gelatinnanocomposite is illustrated in Scheme 1.

2.3 Characterization of the composite coatings

The back bone of the coatingmaterial was analyzed using Fouriertransform infrared spectroscopy (FT-IR) using a Bruker Tensor27. The FT-IR spectrum was recorded in the 4000–400 cm�1

region with 4 cm�1 resolution by using the KBr pellet technique.The phases were characterized by powder X-ray diffraction(PANalytical) with CuKa radiation in the 2q range 20–60�. Thesurface topography and actual composition of the as-developedcomposite samples were observed by F E I Quanta FEG 200-high resolution scanning electron microscope (HRSEM) equip-ped with energy dispersive X-ray (EDX) spectrometry.

2.4 Adhesion strength

The M-HAP/Gel composite coatings on HELCDEB treated Tispecimens were tested for adhesion strength by the pull-out testaccording to the American Society for Testing Materials (ASTM)international standard F1044-05 (ref. 34) and the experimentswere repeated for ve times for every coated material. Thespecimens were exposed to tests at a constant cross-head speedusing a universal testing machine (Model 5569, Instron).

2.5 Electrochemical studies

In this study, potentiodynamic polarization measurement wascarried out for the HELCDEB treated Ti at 700 keV and M-HAP/

Scheme 1 In situ pulsed electrodeposition of M-HAP/Gel nanocompos

This journal is © The Royal Society of Chemistry 2015

Gel composite coatings on HELCDEB treated Ti specimens insimulated body uid. The ion concentrations and pH of thesimulated body uid were maintained nearly equal to that ofhuman blood plasma. The SBF solution was prepared by usingKokubo’s procedure.35 The polarization study was performed ina CHI 760C (CH Instruments, USA) in which HELCDEB treatedTi at 700 keV with an exposed surface area of 1 cm2 was used asthe working electrode. Potentiodynamic polarization was per-formed in a potential range of�1.2 to 1.2 V vs. SCE at a scan rateof 0.001 V s�1. The electrochemical impedance spectroscopic(EIS) measurements were carried out with a frequency rangingfrom 10�2 to 105 Hz. Nyquist and Bode plots were obtained aerthe specimens were immersed in the SBF solution for 1 h. Thepotentiodynamic polarization and EIS were repeated at leastthree times.

2.6 Antibacterial activity

The antibacterial activity of the M-HAP/Gel composite coatingwas tested against S. aureus (ATCC 25923) and E. coli (ATCC25922) by the colony count quantitative method. Inoculums ofthe microorganism were prepared from fresh overnight broth(peptone broth) incubated at 37 �C. The nanocomposite wasmixed with molten MacConkey agar at varying nal concen-trations (25, 50 and 100 mg mL�1). Serial dilution (1/104) of latelog phase bacteria (OD600¼ 2.0) were then plated onto solidiednanocomposite agar plates and incubated at 37 �C for 24 h.36

Error bars were calculated for colony count based on the stan-dard errors by t-test. To determine the antibacterial property ofthe material, the antibacterial reduction percentage was calcu-lated using the formula37

R ¼ [(C0 � C)/C0] � 100 (1)

ite on HELCDEB treated Ti.

RSC Adv., 2015, 5, 47341–47352 | 47343

Fig. 1 Schematic representation of the formation of M-HAP/Gel composite.

Fig. 2 FT-IR spectra of (a) pure gelatin and (b) M-HAP/Gel nano-composite coating. Fig. 3 X-ray diffraction patterns of (a) pure gelatin and (b) M-HAP/Gel

nanocomposite coating.

RSC Advances Paper

where, “R” is the microbial reduction percentage, “C0” is thenumber of microbial colonies on the control plate (withoutnanocomposite) and “C” is the number of microbial colonies onthe M-HAP/Gel nanocomposite samples.

2.7 Cell proliferation assay and live/dead staining

MC3T3-E1 subclone 4 mouse osteoblast cell lines (ATCC) werecultured in Eagles minimum essential medium (a-MEM, Invi-trogen), supplemented with 10% Fetal Bovine Serum (FBS,Invitrogen), 10 mg mL�1 Streptomycin (Invitrogen), 10 mg mL�1

Penicillin and the culture was incubated at 37 �C in a humidi-ed atmosphere with 5% CO2. The medium was changed everytwo days until 80% to 90% conuency and then the cells weresub-cultured. The cell monolayer was washed twice with PBSsolution and detached from the culture ask by incubating with

47344 | RSC Adv., 2015, 5, 47341–47352

0.25% trypsin–EDTA (Gibco) solution for 3 min. DetachedMC3T3-E1 cells of 1 � 104 (Passage 16) were seeded in each wellof 48 well tissue culture plates.

Cell proliferation was studied on the M-HAP/Gel nano-composite sample and control sample (without nano-composite), using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay aer 1, 4 and 7 days. Eachtime, 400 mL of MTT reagent (1 mg mL�1) was added to eachwell and incubated for 4 h under the same conditions. Finally,the MTT reagent was removed and 400 mL of dimethyl sulfoxide(DMSO) (Sigma-Aldrich) was added to dissolve the formasancrystals and the absorbance was measured at 570 nm (ref. 38)on a spectrophotometric microplate reader. The proliferationrate of cells was quantied by measuring the optical density(OD).39

This journal is © The Royal Society of Chemistry 2015

Fig. 4 Morphological features of (a) 700 keV treated Ti, M-HAP/Gelcomposite coating on 700 keV treated Ti at different gelatinconcentrations of (b) 1 wt% (c) 2 wt% (d) 3 wt% (e) 4 wt% and EDSspectrum of M-HAP/Gel composite coating with 3 wt% of gelatin.

Fig. 5 Potentiodynamic polarization curves of untreated, 700 keVtreated Ti andM-HAP/Gel composite coatings on 700 keV treated Ti atvarious gelatin concentrations.

Fig. 6 Impedance spectra of untreated, 700 keV treated Ti and M-HAP/Gel composite coatings on 700 keV treated Ti at various gelatinconcentrations in simulated body fluid: (a) Nyquist, (b) Bode imped-ance and (c) Bode phase plots.

Paper RSC Advances

Aer each culture, the cells on the M-HAP/Gel nano-composite sample were stained using a live/dead assay kit(Molecular Probes) and the results are compared with controlsamples, containing calcein AM and ethidium homodimer.

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 47341–47352 | 47345

Fig. 7 Petri dishes with MacConkey agar inoculated with (a) S. aureus and (b) E. coli, showing variable numbers of colonies when supplementedwith different amounts of M-HAP/Gel nanocomposite and (c) quantitative bacterial colony count of M-HAP/Gel nanocomposite against S.aureus and E. coli.

RSC Advances Paper

Every time, the media was discarded and the samples werewashed with PBS, live/dead solution containing 2 mM calceinAM and 4mM ethidium homodimer was added to each well andincubated at 37 �C for about 20 min. The stained cultures wereviewed using uorescent microscope Nickon Eclipse 80i.38

2.8. Fibroblast-stem cells

An MTT assay test was used to determine the viability andproliferation of broblast-like stem cells on M-HAP/Gel nano-composite coating on HELCDEB treated Ti. The cells werecultured in Dulbecco’s modied Eagle medium (DMEM, Gibco),which consisted of a minimal essential medium (Hi MediaLaboratories), supplemented with 10% fetal bovine serum (FBS,Biowest, France) in 96-well tissue culture plates. The cell culturewas incubated at 37 �C under a humidied atmosphere of 5%CO2 and 95% air for one day. The dual layer coated sampleswere sterilized in an autoclave at 120 �C for 2 h and then placedin 96-well tissue culture plates. The cells were seeded onto theM-HAP/Gel nanocomposite coatings and were maintained at 37�C under 5% CO2. The culture media in each well was replacedevery day with immersion extracts supplemented with 10% FBS,

47346 | RSC Adv., 2015, 5, 47341–47352

and incubated at 37 �C in an atmosphere of 5% CO2 and 95%air. The MTT assay was carried out as a function of incubationtime for 1, 4 and 7 days. An MTT (10 mL) solution containing 5mg of thiazolyl blue tetrazolium bromide powder was thenadded into each well on day 1, 4 and 7. Aer an incubationperiod, 100 mL of 10% sodium dodecyl sulphate (Sigma-Aldrich, USA) in 0.01 M HCl (Sigma-Aldrich, UK) was addedinto each well and incubated at 37 �C in an atmosphere of 5%CO2 and 95% air for 24 h. To determine the cell viability of thesamples, the absorbance was measured using an ELISA micro-plate reader at 570 nm wavelength. Cell viability (%) wascalculated with respect to control wells (without nano-composite) using the following formula:40

% Cell viability ¼ [A]test/[A]control � 100. (2)

2.9 Statistical analysis

In this study all the experimental groups were carried out intriplicate and the results are presented as average � standard

This journal is © The Royal Society of Chemistry 2015

Fig. 8 (a) Optical microscopic images showing proliferation ofMC3T3-E1 cells on control and M-HAP/Gel nanocomposite coating at(a) day 1, (b) day 4, (c) day 7 and (b) optical density measurementillustrating MC3T3-E1 cell proliferation on the M-HAP/Gel nano-composite coating and control. Asterisk denotes a significant differ-ence compared with control (P < 0.05).

Fig. 9 (a) Fluorescence micrographs of osteoblasts cultured on M-HAP/Gel nanocomposite coating for 1, 4 and 7 days and (b) MTT assayof osteoblasts incubated on M-HAP/Gel nanocomposite for 1, 4 and 7days (asterisk denotes a significant difference compared with control,P < 0.05).

Paper RSC Advances

deviation and were analyzed using a one factor ANOVA statis-tical study. The differences were considered statistically signif-icant if p < 0.05.

2.10 Immersion study in SBF solution

The apatite forming ability of the M-HAP/Gel composite coating(at an optimum concentration of gelatin) on HELCDEB treatedTi sample can be evaluated by immersing in simulated body

This journal is © The Royal Society of Chemistry 2015

uid at 37 �C for 1, 7 and 14 days. As mentioned earlier, thecomposition and preparation of SBF solution was according toKokubo’s protocol.35 In the fresh SBF solution (40 mL) con-taining beaker, the resultant composite coated samples weresoaked with an airtight lid for 1, 7 and 14 days and thetemperature was sustained at 37 �C in an incubator. The SBFsolutions were changed every two days to maintain the cationicconcentration in the test solution. Aer appropriate days ofimmersion, the samples were removed from SBF solution, then

RSC Adv., 2015, 5, 47341–47352 | 47347

Fig. 10 (a) Optical microscopic images showing the viability offibroblast stem cells on control and M-HAP/Gel nanocomposite at 4and 7 days of incubation and (b) % viability of fibroblast stem cells onM-HAP/Gel nanocomposite at 4 and 7 days of incubation (asteriskdenotes a significant difference compared with control, P < 0.05).

Fig. 11 SEM images of apatite growth on M-HAP/Gel nanocompositeafter (a) 1 day (b) 7 days and (c) 14 days of immersion in SBF solution.

RSC Advances Paper

gently washed with DI water and the formation of apatitegrowth on the composite coated HELCDEB treated Ti sampleswas evaluated by microscopic study (HRSEM).

47348 | RSC Adv., 2015, 5, 47341–47352

3. Results and discussion3.1 Mechanism of M-HAP and gelatin composite formation

A possible mechanism for M-HAP/Gel composite formation isshown in Fig. 1. From the gure, it is evident that thecomplexation of M2+ ions (Ca2+, Sr2+, Mg2+ and Zn2+) with M-HAP and gelatin takes place thereby forming a partial bondbetween the M2+ cation and gelatin moiety. Then, the as-formedcomplex (M2+ and gelatin) molecules interact with PO4

3� ions toform the M-HAP/Gel composite. During this formation process,the carboxylate (COO�) and amino (–NH–) groups in gelatin canform bonds with P–O and OH groups of M-HAP.41 Thus, it isevident that the above-mentioned process might take place inthe electrolyte solution while depositing on the HELCDEBtreated Ti surface.

3.2 FT-IR analysis of M-HAP/Gel composite coatings

The FT-IR spectra of pure gelatin and M-HAP/Gel compositecoating are shown in Fig. 2. The data of the M-HAP/Gelcomposite coating (Fig. 2b) is compared with that of puregelatin (Fig. 2a). The absorption peaks of phosphate ions areobserved at 563, 602, 1030 and 1081 cm�1. The small peak at496 cm�1 is attributed to the PO4

3� bending vibration. Thebroad bands that appear at 3403 and 667 cm�1 are mainly dueto the stretching and bending modes of the hydrogen-bondedH2O molecules. The characteristic –OH peaks of HAP ataround 3540 cm�1 correspond to the stretching mode. Thepeaks at 2923 and 2852 cm�1 are attributed to the presence ofthe C–H bond in the gelatin present in the M-HAP/Gelcomposite, which indicates that gelatin interacts with M-HAP.In addition, two bands are identied at 985 and 937 cm�1.These may be observed due to P–O–C aliphatic stretching andP–N–C stretching, which is evidence for the binding of phos-phate ions with gelatin.41 At the same time, the peak at 1642cm�1 corresponds to the carboxyl group of gelatin andcomposite, which is similar to the presence of type-I collagen inbiological tissue.42 The appearance of sharp peaks at 1531 and1727 cm�1 represent the N–H peak in the composite. Thus theobtained FT-IR spectra conrmed the formation of M-HAP/Gelcomposite coating and no other impurities were identied.

3.3 X-ray diffraction studies

Fig. 3 shows the X-ray diffraction patterns of pure gelatin andM-HAP/Gel composite coating. For pure gelatin, the characteristicbroad peak around 20� was observed, which is similar to theobservation by Kim et al.,43 and is also in good agreement withICDD card no. 09-432. The 2q values for the HAP peaks werefound to be 25.83, 31.54, 32.28, 32.80, 34.06, 39.89, 46.69, 48.27,49.26, 50.42, 52.11 and 53.11, respectively and the planes cor-responding to the 2q values were (002), (211), (112), (300), (202),(310), (222), (312), (213), (321), (402) and (004).26 These peaksexperienced a shi towards the lower diffraction angle whichmay be due to the inuence of themineral ion substitution suchas Sr2+ (radius, 0.112 nm), Mg2+ (0.072 nm) and Zn2+ (0.074 nm)in HAP (Ca2+ 0.100 nm), that leads to the distortion in the HAPlattice.44,45 In particular, the atomic radius of the Sr ion is larger

This journal is © The Royal Society of Chemistry 2015

Paper RSC Advances

than Ca, which induces a shi in the XRD pattern whilesubstituting it in the place of Ca. Along with other peaks, asmall peak was observed at 20�, which is evidence for thebinding of gelatin with the HAP moiety. Moreover, the highintensity peaks of M-HAP/Gel composite exhibit the crystallinenature of the coating material. This proves the fact that thegelatin concentration does not inuence the crystallinity of theHAP when compared with the previous report.46 No otherimpurity peaks were observed, indicating that the purity of theM-HAP/Gel composite is appreciable.

3.4 Morphological results of composite coatings

The surface topographic images of the 700 keV HELCDEB treatedTi andM-HAP/Gel composite coating on HELCDEB treated Ti aredisplayed is Fig. 4a–e. Fig. 4a shows the morphology of electronbeam irradiated Ti. The eruption seen on the surface of irradiatedTi might be obtained due to the surface changes that are occur-ring by superfast heating, melting and solidication of the Timetal. This modied surface will provide improved physico-chemical properties and bonding strength to the Ti substrate.The composite coatings on irradiated Ti result in the typicalmorphologies of M-HAP/Gel composite (with different concen-trations of gelatin). The M-HAP/Gel composite (with 1 wt%gelatin) exhibited a novel dumbbell shaped structure on theHELCDEB treated Ti surface (Fig. 4b) with some heterogeneoussurface coverage, which clearly indicates that the nucleation ofthe composite was not complete at the minimum concentrationof gelatin (i.e. 1 wt%) in M-HAP. On increasing the gelatinconcentration to 2 wt%, the dumbbell shaped composite waschanged into irregularly arranged petals, which indicates that therods/petals of the dumbbell shaped composite began to separateand were unevenly distributed (Fig. 4c). Interestingly, at 3 wt%gelatin concentration in the composite (Fig. 4d), a morphology ofuniformly organized nanosized (�20 nm) ower petals wasobtained, which were compactly packed on the irradiated surfacewithout any voids. The texture and uniform coverage of thecomposite coating may also be due to the deposition condition,i.e. the prolonged pulse off time. This coating on HELCDEBtreated Timay result in a better anticorrosion performance due tothe complete coverage of thematerial without any cracks or poreson the surface. As the concentration of gelatin was increased(Fig. 4e) to 4 wt% in M-HAP/Gel composite, an irregular petalstructure of composite with agglomeration was obtained. More-over, the surface of the coating was observed with pores on theHELCDEB treated Ti surface. The morphology of the compositediffers due to the higher concentration of the gelatin whichgreatly inuenced the texture and coverage of the coating. Fig. 4fshows the elemental analysis results (EDX) of M-HAP/Gel nano-composite coating (at 3 wt% of gelatin concentration) whichexhibited the presence of Sr, Mg, Zn, Ca, P, N and C (which is thebackbone of the gelatin), thereby supporting the formation of theM-HAP/Gel composite coating.

3.5 Adhesion strength of the composite coatings

The adhesion between the coating and substrate is the mostimportant factor for better mechanical interlocking and

This journal is © The Royal Society of Chemistry 2015

chemical bonding of the implant in the physiological solution.Thus, the pulsed electrodeposited M-HAP/Gel composite coat-ings were tested for their adhesion strength. The M-HAP/Gelcomposite-I (1 wt%) coating on HELCDEB treated Ti exhibiteda good adhesion strength of 22.8 � 0.9 MPa. The adhesionstrengths of the M-HAP/Gel composite-II (2 wt%) and M-HAP/Gel composite-III (nanocomposite) (3 wt%) coatings exhibited23.0 � 0.8 MPa and 23.8 � 0.6 MPa, respectively. Whereas, theadhesion strength for the M-HAP/Gel composite-IV (4 wt%)coating was found to be 23.1 � 0.7 MPa, which possessed lessadhesion value compared with the M-HAP/Gel composite-IIIcoating. The M-HAP/Gel composite-III coating has good adhe-sion strength which will be helpful for long term orthopedicapplications.

3.6 Potentiodynamic polarization measurements

The corrosion potential (Ecorr) and the corrosion current density(Icorr) of the HELCDEB treated Ti and M-HAP/Gel compositecoatings (at different concentrations of gelatin) were deter-mined from the polarization curves and are shown in Fig. 5. Asevidenced from the gure, the Ecorr and Icorr values of theHELCDEB treated Ti were found to be �0.09 � 0.003 V and 0.08� 0.003 mA cm�2, respectively. The M-HAP/Gel composite-Icoating on HELCDEB treated Ti showed a positive Ecorr value(0.235 V � 0.002) with the Icorr of 0.032 � 0.005 mA cm�2,whereas the M-HAP/Gel composite-II coating exhibited Ecorr of0.237 � 0.001 V and Icorr of 0.03 � 0.004 mA cm�2 values (thecorresponding curve is not provided). The values of Ecorr andIcorr of the M-HAP/Gel composite-III and M-HAP/Gel composite-IV coatings were 0.324 � 0.002 V, 0.01 � 0.007 mA cm�2, and0.269� 0.003 V, 0.027� 0.002 mA cm�2, respectively. The Ecorr ofthe composite coated materials were higher than that of theHELCDEB treated Ti, which showed the protective nature of thecoatings. In particular, the M-HAP/Gel composite-III coating onHELCDEB treated Ti exhibited a maximum positive shitowards the noble direction. The potentiodynamic polarizationstudies showed that the M-HAP/Gel composite-III coating onHELCDEB treated Ti had more corrosion potential, whichimplies the signicant anticorrosion behavior of the compositecoating.

The Nyquist plots of the HELCDEB treated Ti andM-HAP/Gelcomposite coated on HELCDEB treated Ti specimens atdifferent concentrations of gelatin are shown in Fig. 6a. Incomparison, the acquired polarization resistance (Rp) value forthe M-HAP/Gel composite-I shows a higher corrosion resistanceof 180.2 � 0.02 kU cm2 than that obtained for the HELCDEBtreated Ti (114.3 � 0.07 kU cm2) and untreated Ti (52.4 � 0.03kU cm2). The Rp value for the M-HAP/Gel composite-II wasfound at 182.8 � 0.01 kU cm2 (curve not provided) and M-HAP/Gel composite-III was obtained at 216.7 � 0.02 k U cm2. For M-HAP/Gel composite-IV, the Rp value was observed to be 195 �0.05 kU cm2. The highest Rp value was obtained for the M-HAP/Gel composite-III on HELCDEB treated Ti sample, indicatingthe better and signicant corrosion resistance of the coating.

Similarly the Bode plot for the M-HAP/Gel composite coatingon HELCDEB treated Ti at different concentrations of gelatin

RSC Adv., 2015, 5, 47341–47352 | 47349

RSC Advances Paper

exhibits greater total impedance (|Z|) than the HELCDEBtreated (174 � 0.03 kU cm2) and untreated Ti (65 � 0.03 kUcm2). The |Z| values for the M-HAP/Gel composite-I andcomposite-II were found to be 230 � 0.05 kU cm2 and 234.6 �0.04 kU cm2, respectively. The M-HAP/Gel composite-IIIexhibited around 425 � 0.07 kU cm2 for |Z| which was higherthan the |Z| of M-HAP/Gel composite-IV (270 � 0.03 kU cm2) asshown in Fig. 6b. When comparing all the samples, the M-HAP/Gel composite-III shows a shi towards the higher frequencyrange than the other coated samples and metal specimens.

The Bode phase angle also shows that the M-HAP/Gelcomposite-III has a high resistivity in simulated body uid.The phase angle for the HELCDEB treated Ti was obtained atthe low frequency region and also the uncoated Ti was obtainedat a lower frequency region than the other coated samples asshown in Fig. 6c. Hence, based on the above results, it is clar-ied that the M-HAP/Gel nanocomposite coating with 3 wt% ofgelatin resulted in a better anticorrosion performance in theSBF solution.

3.7 Antibacterial activity

The antibacterial efficacy of the M-HAP/Gel nanocompositecoating at various concentrations was tested against microbeslike S. aureus (Gram-positive bacterium) and E. coli (Gram-negative bacterium) by supplementing MacConkey agar(Fig. 7). The presence of M-HAP/Gel composite in the Mac-Conkey agar plates was able to inhibit the formation of bacterialcolonies for both the stains S. aureus and E. coli, compared withthe control (without composite material) samples. As shown inFig. 7a and b, the number of bacterial colonies was reduced onincreasing the nanocomposite concentration from 25 mg mL�1

to 100 mg mL�1. Noticeable antibacterial activity was obtainedfor S. aureus (Fig. 7a) compared to E. coli (Fig. 7b), as thenanocomposite was able to substantially reduce the number ofbacterial colonies at a nal concentration of 100 mg mL�1. Thisstudy clearly evidenced that the M-HAP/Gel nanocomposite canaffect the bacterial growth much more effectively. In addition,the Fig. 7c clearly indicates the quantitative reduction in colonycount with different concentrations of nanocomposite, whichdisplays a maximum bacterial reduction at the 100 mg mL�1 ofnanocomposite. Thus, the results strongly show higher anti-bacterial colony reduction by the multifaceted compositecoating which makes it possible to prevent implant failures.

3.8 Cytocompatibility

The cell proliferation on the M-HAP/Gel nanocomposite coatingwas evaluated against the MT3C3-E1 cells by MTT assay for 1, 4and 7 days and the results are shown in Fig. 8. On day 1, the cellshad started to spread on the control and composite surface(Fig. 8a). The MT3C3-E1 cells on the composite coating werespread well through cytoskeletal processes on day 4, whencompared with the control.47 Aer 7 days of culture, the multi-plicity of cells and amount of cells on the nanocompositesample (Fig. 8a) was higher than that on the control sample.The optical images suggest that the composite material hascompatibility with osteoblast cells and improves the cell growth

47350 | RSC Adv., 2015, 5, 47341–47352

behavior. Moreover, the MT3C3-E1 cells on the nanocompositecoating spread well through the cytoskeletal processes at alldays.44 The cellular viability (cell proliferation numbers) wasrepresented based on the absorbance value from theMTT assay,and the results are shown in Fig. 8b. The M-HAP/Gel nano-composite showed a statistically signicant increase in thecellular viability on day 7 compared with the control (p < 0.05),which suggests a strong positive effect on the cell proliferationof MC3T3-EI cells due to the presence of Zn ions in the nano-composite coating. This result is signicant evidence for thebenecial effect of biocompatibility of the composite coatingmaterial.

3.9 Live/dead cell assay

Fig. 9 shows the live/dead cells on the M-HAP/Gel nano-composite coating aer different numbers of days of incubationby uorescent microscopy. The cells started to spread on thenanocomposite surface which is evidenced from Fig. 9a. Theviable cells were present in greater numbers on the compositeaer day 1 of incubation and no dead cells were detected whencompared with the control. When prolonging the incubationperiod to 4 and 7 days, pronounced cells have been observed.From the image, it can be observed that the MC3T3-E1 cellsproliferated well and were spread at 7 days of incubation. Fromthemorphology of the cells it is well evident that cells seemed toprefer expanding on the nanocomposite surface, which actuallyenhances the metabolic condition of the cells (Fig. 9a).48–51

Fig. 9b shows proliferation data of the cells seeded on thenanocomposite aer different numbers of days such as 1, 4 and7 days of incubation, which showed more favorable adhesionand higher proliferation rate of MC3T3-E1 cells. The cellproliferation on the nanocomposite was signicantly (p < 0.05)higher compared with that on control (without nanocomposite)at 7 days of cell culture, which is mainly due to the presence ofmineral ions and gelatin in the nanocomposite.

3.10 Fibroblast stem cells

The viability of broblast stem cells on the nanocompositecoating was determined by MTT assay. The absorbance at awavelength of 570 nm is directly proportional to the number oflive cells in the broblast stem cells culture medium. The % ofviable cells of 125 mg mL�1 nanocomposite coating was calcu-lated with respect to the control for 4 and 7 days (Fig. 10b). Fromthe optical images, it is evident that the % of viable broblaststem cells are more for 4 and 7 days of incubation. At 7 days ofincubation, the nanocomposite coating exhibited higher/greater cell viability (99.7%). This result is further conrmedwith the optical microscopic images (Fig. 10a) for control andnanocomposite coating at 1, 4 and 7 days of incubation. Thegures evidenced that a greater number of cells were found tobe viable in the nanocomposite coating. The nanocompositecoating exhibited cell morphology similar to that of the controlgroup on day 4 and 7 of incubation and the cells werecompletely spread out, which revealed biocompatibility of thenanocomposite coating. Thus, it is proved that the nano-composite coating greatly improves the bioactivity and

This journal is © The Royal Society of Chemistry 2015

Paper RSC Advances

promotes cell growth and hence the nanocomposite coatedsample is believed to be able to be used as an orthopedicimplant with better osteoinductive properties.

3.11 Apatite forming ability of the nanocomposite coating

The bioactivity of the M-HAP/Gel nanocomposite coating wasconrmed by an SBF immersion study. The resulting apatitegrowth was evaluated aer immersing the coated sample in SBFfor various numbers of days such as 1, 7 and 14 days ofimmersion. The growth of apatite on the nanocompositecoating differs at each day. At day 1, the apatite started to growon the surface as ower petals as shown in Fig. 11a. The growthof spongy apatite was formed on the nanocomposite at 7 days ofimmersion (Fig. 11b). On increasing the immersion period to 14days, some spongy ower structures were observed as a result ofapatite growth (Fig. 11c). Usually, the apatite formation of thesample takes place through a sequence of chemical reactions.When the coated samples are soaked in the SBF solution, thenegative ions (PO4

3� and OH�) can easily attract the positiveions (Ca2+) and form Ca-rich amorphous calcium phosphate.Then, the surface gains the positively charged ions from the SBFsolution, which began to attract ions such as PO4

3� and OH�

forming Ca-poor amorphous calcium phosphate. This processleads to the formation of bone-like apatite growth on thenanocomposite surface.52 Thus, the micrographs display theapatite mineralization ability at 1, 7 and 14 days of immersionand revealed that the resultant nanocomposite coatingimproves the bioactivity in SBF solution.

4. Conclusions

The development of an M-HAP/Gel composite coating (withdifferent concentrations of gelatin) on HELCDEB treated Ti wassuccessfully achieved by the pulsed electrodeposition method.The presence of functional groups and the phase purity of theM-HAP/Gel composite were conrmed by FT-IR and XRDobservations, respectively. The typical nanostructured coatingmorphology with complete and uniform surface coverage wasachieved at 3 wt% gelatin. The M-HAP/Gel nanocompositecoating on HELCDEB treated Ti exhibited better adhesionstrength (23.8 � 0.6 MPa), when compared to that on theuntreated Ti, which evidenced that the HELCDEB treatment onTi surface helps to improve the adhesion strength of the coatingmaterial. Moreover, the electrochemical studies revealed thatthe resultant composite coating exhibited excellent anticorro-sion properties in simulated body uid. The bacteriocidalability of the nanocomposite resulted in more bacterial colonyreduction at 100 mg mL�1 for both bacteria (S. aureus and E.coli). The cell proliferation assay, live/dead staining of MT3C3-E1 cells and cell viability of broblast stem cells on the resul-tant nanocomposite coating showed better biocompatibilityand bioactivity aer 7 days of incubation. Furthermore, themineralization study of the M-HAP/Gel nanocomposite per-formed at 14 days, demonstrated the excellent apatite growthwith bone mimicking character. Thus, the M-HAP/Gel nano-composite coating on HELCDEB treated Ti can be an alternative

This journal is © The Royal Society of Chemistry 2015

implant material with improved properties for orthopedicapplication.

Acknowledgements

One of the authors D. Gopi acknowledges major nancialsupport from the Department of Science and Technology, NewDelhi, India (DST-TSD, Ref. no.: DST/TSG/NTS/2011/73), DST-EMEQ, Ref. no.: SB/EMEQ-185/2013) and Defence Researchand Development Organisation, New Delhi, India, (DRDO, no.ERIP/ER/1103949/M/01/1513). D. Gopi also acknowledges UGC,New Delhi, India for the Research Award (Ref. no. F. 30-1/2013(SA-II)/RA-2012-14-NEW-SC-TAM-3240. The authorsacknowledge Industrial and Medical Accelerator Section, RajaRamanna Centre for Advanced Technology (RRCAT), Indore(MP), India for the support to carry out high energy electronbeam surface treatment.

References

1 C. T. Laurencin, A. M. A. Ambrosio, M. D. Borden andJ. A. Cooper, Annu. Rev. Biomed. Eng., 1999, 1, 19.

2 Y. Huang, H. Zeng, X. Wang and D. Wang, Appl. Surf. Sci.,2014, 290, 353.

3 E. Bonnelye, A. Chabadel, F. Saltel and P. Jurdic, Bone, 2008,42, 129.

4 D. Rajeswari, D. Gopi, S. Ramya and L. Kavitha, RSC Adv.,2014, 4, 61525.

5 D. Gopi, D. Rajeswari, S. Ramya, M. Sekar, R. Pramod,J. Dwivedi, L. Kavitha and R. Ramaseshan, Appl. Surf. Sci.,2013, 286, 83–90.

6 W. Xia, C. Lindahl, J. Lausmaa, P. Borchardt, A. Ballo,P. Thomsen and H. Engqvist, Acta Biomater., 2010, 6, 1591.

7 D. Gopi, N. Murugan, S. Ramya, E. Shinyjoy and L. Kavitha,RSC Adv., 2015, 5, 27402.

8 H. Tapiero and K. D. Tew, Biomed. Pharmacother., 2003, 57,399.

9 V. Stanic, S. Dimitrijevi, J. A. Stankovic, M. Mitri, B. Joki,I. B. Plecas and S. Raicevi, Appl. Surf. Sci., 2010, 256, 6083.

10 G. Sun, J. Ma and S. Zhang, Mater. Sci. Eng., C, 2014, 39, 67–72.

11 L. Xu, G. Yu, E. Zhang, F. Pan and K. Yang, J. Biomed. Mater.Res., Part A, 2007, 83, 703.

12 W. L. Suchanek, K. Byrappa, P. Shuk, R. E. Riman, V. F. Janasand K. S. TenHuisen, Biomaterials, 2004, 25, 4647.

13 R. Z. LeGeros, Calcium phosphates in oral biology andmedicine, Karger AG, Basel, Switzerland, 1991.

14 D. Gopi, A. Karthika, S. Nithiya and L. Kavitha, Mater. Chem.Phys., 2014, 144, 75.

15 M. Sivakumar and K. Panduranga Rao, Biomaterials, 2002,23, 3175.

16 G. A. Digenis, T. B. Gold and V. P. Shah, J. Pharm. Sci., 1994,83, 915.

17 S. Teng, L. Chen, Y. Guo and J. Shi, J. Inorg. Biochem., 2007,101, 686.

18 V. Chiono, E. Pulieri, G. Vozzi, G. Ciardelli, A. Ahluwalia andP. Giusti, J. Mater. Sci.: Mater. Med., 2008, 19, 89.

RSC Adv., 2015, 5, 47341–47352 | 47351

RSC Advances Paper

19 M. Cheng, J. Deng, F. Yang, Y. Gong, N. Zhao and X. Zhang,Biomaterials, 2003, 24, 2871.

20 A. A. Haroun and V. Migonney, Int. J. Biol. Macromol., 2010,46, 310.

21 P. I. Rose, in Encyclopedia of Polymers Science andEngineering, ed. H. F. Mark, Wiley, New York, 1987, p. 488.

22 A. Bigi, B. Bracci and S. Panzavolta, Biomaterials, 2004, 25,2893.

23 C. Dong, A. Wu, S. Hao, J. Zou, Z. Liu, P. Zhong, A. Zhang,T. Xu, J. Chen, J. Xu, Q. Liu and Z. Zhou, Surf. Coat.Technol., 2003, 163–164, 620.

24 D. I. Proskurovsky, V. P. Rotshtein, G. E. Ozur, A. B. Markov,D. S. Nazarov, V. A. Shulov, F. Yu, R. Ivanov and G. Buchheit,J. Vac. Sci. Technol., A, 1998, 16, 2480.

25 D. Gopi, D. Rajeswari, S. Ramya, M. Sekar, R. Pramod,J. Dwivedi, L. Kavitha and R. Ramaseshan, Appl. Surf. Sci.,2013, 286, 83.

26 D. Gopi, A. Karthika, M. Sekar, L. Kavitha, R. Pramod andJ. Dwivedi, Mater. Lett., 2013, 105, 216.

27 K. Lee, D.-H. Nam, S. Lee and N. J. Kim, Surf. Coat. Technol.,2006, 201, 1620.

28 T. J. Webster, C. Ergun, R. H. Doremus, R. W. Siegel andR. Bizios, Biomaterials, 2000, 21, 1803.

29 M. Zandi, H. Mirzadeh, C. Mayer, H. Urch,M. B. Eslaminejad, F. Bagheri and H. Mivehchi, J. Biomed.Mater. Res., Part A, 2010, 92, 1244.

30 H. Liu, J. Cheng, F. Chen, D. Bai, C. Shao, J. Wang, P. Xi andZ. Zeng, Nanoscale, 2014, 6, 5315.

31 M. C. Chang, C. C. Ko andW. H. Douglas, Biomaterials, 2003,24, 2853.

32 Y. Huang, Y. Yan, X. Pang, Q. Ding and S. Han, Appl. Surf.Sci., 2013, 282, 583.

33 D. Gopi, J. Indira and L. Kavitha, Surf. Coat. Technol., 2012,206, 2859.

34 ASTM standard F 1044-05, ASTM International, WestConshohocken, PA.

35 T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi andT. Yamamuto, J. Biomed. Mater. Res., 1990, 24, 721.

47352 | RSC Adv., 2015, 5, 47341–47352

36 M. Liong, B. France, K. A. Bradley and J. I. Zink, Adv. Mater.,2009, 21, 1684.

37 N. D. Ravi, R. Balu and T. S. Sampath Kumar, J. Am. Ceram.Soc., 2012, 95, 2700.

38 E. Kolanthai, V. S. D. Colon, P. A. Sindu, V. S. Chandra,K. R. Karthikeyan, M. S. Babu, S. M. Sundaram,M. Palanichamy and S. N. Kalkura, RSC Adv., 2015, 5, 18301.

39 Y. Huang, Q. Ding, X. Pang, S. Hana and Y. Yan, Appl. Surf.Sci., 2013, 282, 456.

40 D. Gopi, N. Murugan, S. Ramya, E. Shinyjoy and L. Kavitha,RSC Adv., 2015, 5, 27402.

41 M. Chen, J. Tan, Y. Lian and D. Liu, Appl. Surf. Sci., 2008, 254,2730.

42 J. Hong, X. Qiu, J. Sun, M. Deng, X. Chen and X. Jing,Polymer, 2004, 45, 6699.

43 H.-W. Kim, J. C. Knowles and H.-E. Kim, J. Biomed. Mater.Res., Part A, 2005, 72, 136.

44 D. Gopi, E. Shinyjoy and L. Kavitha, Ceram. Interfaces, 2015,41, 5454.

45 D. Gopi, S. Ramya, D. Rajeswari, P. Karthikeyan andL. Kavitha, Colloids Surf., A, 2014, 451, 172.

46 D. Gopi, A. Karthika, D. Rajeswari, L. Kavitha, R. Pramod andJ. Dwivedi, RSC Adv., 2014, 4, 34751.

47 Y. Lu, A. Zhu, W. Wang and H. Shi, Appl. Surf. Sci., 2010, 256,7228.

48 D. Milovac, T. C. Gamboa-Martinez, M. Ivankovic,G. G. Ferrer and H. Ivankovic, Mater. Sci. Eng., C, 2014, 42,262.

49 B. Li, Y. Han and K. Qi, ACS Appl. Mater. Interfaces, 2014, 4,18258.

50 P. M. Favi, R. S. Benson, N. R. Neilsen, R. L. Hammonds,C. C. Bates, C. P. Stephens and M. S. Dhar, Mater. Sci. Eng.,C, 2013, 33, 1935.

51 X. Li, J. Xie, X. Yuan and Y. Xia, Langmuir, 2008, 24, 14145.52 H. M. Kim, F. Miyaji, T. Kokubo, C. Ohtsuki and

T. Nakamura, J. Am. Ceram. Soc., 1995, 78, 2405.

This journal is © The Royal Society of Chemistry 2015