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Materials Science and Engineering C xxx (2013) xxxxxx

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Materials Science and Engineering C

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In vitro evaluation of osteoconductivity and cellular response of zirconia and alumina based ceramics
Ajoy Kumar Pandey a,, Falguni Pati b, Debika Mandal a, Santanu Dhara b, Koushik Biswas a
a b

Department of Metallurgical and Materials Engineering, Indian Institute of Technology, Kharagpur 721 302, India School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721 302, India

a r t i c l e

i n f o

a b s t r a c t
Developed ceria/yttria stabilized zirconia and ceria/yttria stabilized zirconia toughened alumina supported formation of apatite layer when immersed in simulated body uid without any prior surface treatment. The formed mineral layer was conrmed as hydroxyapatite through X-ray diffraction patterns. The calcium/phosphate atomic ratio obtained from energy dispersive X-ray spectroscopy was found to be little less (Ca/P = 1.5) than that of pure hydroxyapatite (Ca/P = 1.7) which indicates the probability of mixed type calcium-phosphate compound formation. The achieved thickness of apatite layer was estimated through a surface prolometer and as high as ~17 m thickness was found after 28 days of soaking. The biocompatibility of the developed materials was ensured through in vitro human osteoblast like cell (MG63) culture on ceramic discs. The morphology of attached cells was characterized through scanning electron microscopy and uorescent microscopy which show multilayered interconnected cell growth within 8 days of culture period. Moreover, differentiation of MG63 cells was evaluated through MTT assay, total protein content and alkaline phosphatase activity. 2013 Elsevier B.V. All rights reserved.

Article history: Received 31 May 2011 Received in revised form 8 April 2013 Accepted 13 May 2013 Available online xxxx Keywords: Bio-ceramic Osteoconduction In vitro biocompatibility Cell culture Bioactivity

1. Introduction Alumina and zirconia based bioceramics have found their wide applications in load bearing orthopedics (total hip and knee replacement) and as dental implants [14]. Due to high corrosion resistance, excellent hardness, high Young's modulus, adequate mechanical strength and bio-inertness; alumina is a preferred choice for such biomedical applications [1,2]. Moreover, alumina is prone to form a surface hydroxide layer while implanted. This lm acts as lubricant which effectively reduces the friction and wear of the material [2]. However, intrinsic brittleness and higher fracture rate of alumina have limited the range of applications and it is only suitable where mechanical load bearing capabilities are less stringent [3]. Best way to overcome these problems of alumina is to add a second phase having higher toughness without deteriorating the other properties of alumina. Introduction of zirconia in the alumina matrix (called zirconia toughened alumina) improves its mechanical properties. In zirconia toughened alumina, alumina imparts high hardness and stabilized zirconia provides toughness. Thus, aluminazirconia particulate composite have improved mechanical properties with higher resistance to ageing. Owing to modulus mismatch between alumina matrix and zirconia dopant in the composite, crack path is always attracted towards less stiff zirconia grain during propagation of crack. This introduces transformation toughening of zirconia in the composite resulting enhanced fracture toughness [4]. This composite may be important for many load
Corresponding author. Tel.: +91 3222 226678. E-mail address: ajoy100@gmail.com (A.K. Pandey). 0928-4931/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.05.032

bearing biological applications. However, osteoconduction/bioactivity of these synthetic materials is important for their integration in vivo. A synthetic material essentially requires formation of bonelike apatite layer on its surface in vivo to ensure in vitro bond formation to living bone [5]. The bioactivity of bio-ceramics can be anticipated by in vitro appetite forming ability in a simulated body uid (SBF) with ion concentrations nearly equal to those of human blood plasma [57]. The degree of bioactivity depends upon the formation of bond to living bone through apatite layer formation on the surface [8]. It is already reported that apatite formation using SBF is induced by certain functional groups like TaOH [9], SiOH [10], TiOH [11], NbOH [12], COOH [13], PO4H2 [13], ZrOH [14] and AlOH [15]. However, researchers have controversy regarding the apatite formatting ability of AlOH [16,17]. Many researchers induce such hydroxide groups on the surface by chemical treatment before soaking in SBF using some chemical reagent called nucleating agent. Commonly used nucleating agents are ethanolic solutions HS(CH2)11X (X = CH3, COOH, CONH2, OH or NH2) [13], H3PO4, NaOH, H2SO4 or HCl [15,16]. On the other hand, some reports have showed that there are no effects of nucleating agent on the nucleation of apatite on ceramics. According to them, ZrOH or the AlOH (hydrate bonds) bond which is abundant on the surface helps nucleating apatite through calcium and subsequent phosphate ion deposition [15,18]. For tissue integration in vivo, biocompatibility of these materials is prerequisite which can be realized by their cellular responses through in vitro cell culture study and different cellular assay. The cellular responses largely depend upon the surface chemistry and topography

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Table 1 Sintering schedule adopted for different systems and their corresponding grain size and hardness value. System CSZ YSZ CSZ-TA YSZ-TA Sintering schedule 1500 C for 1 h and 1400 C for 2 h 1450 C for 30 min and 1250 C for 14 h 1550 C for 1 h and 1450 C for 2 h 1500 C for 1 h and 1400 C for 2 h Hardness (VHN) 950 20 1364 11 1730 16 1800 10 Average grain size (m) 4.3 0.78 Alumina grains Zirconia grains Alumina grains Zirconia grains 1.77 1.74 1.19 0.88

condition, following two step sintering process. The sintering schedule and the average grain size obtained are represented in Table 1. 2.2. SBF treatment SBF used in this study is the n-SBF solution which was prepared by liquid mixing process as described by Tadakama et al. [24]. In this process Ca and P solutions are prepared separately by dissolving different reagents in a proper sequence and maintaining the pH of the solution at 7.25. Cleaned and polished samples were placed inside a glass beaker, SBF was added into it and then the whole assembly was placed inside a water bath which maintains a constant temperature of 37.5 C. The beakers were covered with aluminum foil to prevent addition of evaporated and condensed normal water from the water bath (water may evaporate, condense on the top of chamber and get into the beaker). The soaking time of the specimens was varied and test was carried out for a total duration of 28 days. After every alternate day the SBF solution was replaced with fresh one and after every 7 days one sample was taken out for characterization. 2.3. Characterization of mineral deposited layers After removing the samples from the SBF, it was gently washed with distilled water and dried at 40 C and observed under scanning electron microscope (SEM) (SUPRA-40, Carl Zeiss, Germany) attached with dispersive X-ray spectrometer (EDX) (Oxford Instruments Ltd., UK). Before SEM observation, the dried sample was coated with very thin layer of gold. Apatite formation was conrmed from the Ca/P ratio of EDX result and also from the X-ray diffraction (XRD) patterns (CuK radiation, step size 0.05 (2) and time per step 2.5 (s)) of the surface obtained from high resolution X-ray diffractometer (PANalytical, XPert PRO, Phillips, The Netherlands). The thickness of the apatite layer after different time interval of soaking was estimated through the surface scan using a surface proler (Veeco Dektak 150 Surface Prolometer, USA). The surface scan was started from the apatite and carried out up to the bare surface. As the formed apatite surface thickness was varying from point to point, average roughness value on the apatite surface was taken while reporting the apatite thickness. Fig. 1 shows a typical example of how apatite thickness was estimated. 2.4. Cell culture study Human osteoblast like cell MG-63 (human osteosarcoma cell line) obtained from the National Centre for Cell Science (NCCS, Pune, India) was cultured in 25 cm2 tissue culture ask (Costar, Corning Inc.) using Dulbeccos modied Eagles medium (DMEM, Himedia, Mumbai, India) supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 2 mM Na-pyruvate and 1% penicillin-streptomycin (A002A, Himedia, Mumbai, India). Cells were incubated inside an incubator at 37 C with 5% CO2 atmosphere and 100% relative humidity. The cells were sub-cultured when they reached 90% conuence and experiments were carried out on cells from passage 4 through 20. Polished ceramic discs were washed and sterilized in an autoclave at 121 C for 30 min before placing them inside a 6-well cell culture plate. The cells, with cell density of 105 cells/well, were seeded into the well xed with ceramic discs. Plates were incubated in standard culture conditions (37 C, 5% CO2 atmosphere and 100% relative humidity) for 2 h to ensure cell adhesion and then the culture medium was added to the well. The culture medium was changed every alternate day. The culture was carried out for a total duration of 16 days. 2.5. Cell proliferation assay

of implants [19]. Prior to cell attachment, proteins adsorb to the surface of the implants through different ionic and van der Waals interactions. These proteins have polypeptide cues which promote cell adhesion through cell surface receptor. Cell attachment is the primary step for adherent cell line to take part in cell proliferation, differentiation and maturation which are important to tissue integration of the implants [19,20]. In the present study, several alumina and zirconia based composite samples were prepared for possible biological application. Osteoconduction study of the developed samples was carried out by immersing them under SBF at 37 C resulting deposition of apatite like minerals layer on the surface. The layer was further inspected by SEM and EDX. The phases of the deposited minerals were studied by XRD. Further, MG63 human osteoblasts like cells were cultured in vitro to study their biocompatibility. For biocompatibility, cellular proliferation and differentiation on the samples surface was assessed by MTT, ALP and total protein content. 2. Materials and methods 2.1. Material development Homogeneously distributed nano sized 14 mol% ceria stabilized zirconia (CSZ), 8 mol% yttria stabilized zirconia (YSZ), 15 wt% zirconia (stabilized with 14 mol% ceria) toughened alumina (CSZ-TA) and 15 wt% zirconia (stabilized with 8 mol% yttria) toughened alumina (YSZ-TA) powder were synthesized by co-precipitation techniques from their respective nitrate salts dissolving in proportionate quantities as described elsewhere [2123]. The synthesized powders were calcined at different temperatures and compressed uni-axially to pallets of = 10 mm and t = 3 mm at 600 MPa. The pallets were sintered in conventional electrical heating furnace in pressure less

Fig. 1. Typical plot of surface proler data in case of CSZ-TA specimen showing apatite thickness after 21 day of soaking.

The cells were allowed to attach to the discs for 3 and 16 days after seeding. The density of attached cells on the discs was assayed

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Fig. 2. SEM images of the specimens after sintering, polishing and thermal etching showing degree of densication and variation in grain size observed in (a) CSZ (b) YSZ-TA (c) CSZ-TA and (d) YSZ samples. All images are of different magnication as indicated in the images.

by following the standard method of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, or MTT assay. The medium of all wells were replaced with a mixture of 360 l fresh medium and 40 l MTT solutions (5 mg/ml) in PBS and then it was incubated in 5% (v/v) CO2 in air at 37 C for 4 h. The derivatives were dissolved with 400 l dimethyl sulfoxide for 15 min with shaking at room temperature. The wells were centrifuged for 5 min at 1600 rpm to eliminate the particles which can interfere with the optical density. Finally the absorbance was measured at 570 nm with a microplate reader (GENios, Germany). 2.6. Protein content estimation Bicinchoninic acid (BCA) protein assay was used to determine the total protein concentration [25]. To estimate the protein content, reactive solution of BCA and CuSO4 of green coloration were used. Cu2 + ions of CuSO4 are reduced to Cu+ by the proteins in the cell suspension. Reduced Cu+ ion forms a complex with BCA. The crimson coloration of this complex is directly proportional to the protein contents. A standard protein concentration curve was developed using bovine serum albumin as a standard. The protein concentration was determined from the absorbance at 562 nm, read by a spectrophotometer (Shimatzu, Japan). 2.7. Alkaline phosphatase assay The catalytic activity of alkaline phosphatase (ALP) of cells was assessed by measuring the release of p-nitrophenol from p-nitrophenolphosphate spectrophotometrically at 405 nm [26]. The seeded scaffolds were rinsed with PBS, transferred into eppendorf tubes and were lysed in 100 l of extraction buffer containing 2 mM MgCl2 and 1% Triton X-100 in a shaker for 30 min at 37 C after 3 and 7 days of culture. Aliquots of 50 l were incubated with 100 l of p-nitrophenyl phosphate (pNP) solution at 37 C for 30 min. 100 l of 0.5 N NaOH was used to stop the reaction and absorbance was read on a micro plate reader (Recorders and Medicare Systems, India). ALP activity was estimated from a developed standard curve using pNP values ranging from 0 to 600 mol and was expressed as mol of pNP produced/ml/h [27].

2.8. Cell morphology study Morphology of the cells attached to ceramic discs was studied using scanning electron microscope (SEM) (SUPRA-40, Carl Zeiss, Germany). Samples for microscopic observations were prepared by quickly washing the specimens two times with PBS and then soaking in 2.5% glutaraldehyde in PBS solution for 1 h at room temperature. After soaking, the specimens were dehydrated using an ascending series of ethanol aqueous solutions (50100%) at room temperature followed by drying in vacuum. Before SEM observation, the specimens were coated with very thin layer of gold. For uorescence microscopy, after soaking the samples in 4% formaldehyde solution in PBS, the cells were stained with rhodamine-phalloidin (red) for actin laments and Hoechst 33342 (blue) for nuclei and observed under uorescence microscope (Zeiss Axio Observer Z1, Carl Zeiss, Germany) with ApoTome attachment at 200X magnication. 3. Results and discussion 3.1. Surface topography of the substrates Microstructure for four kinds of specimens namely ceria stabilized zirconia (CSZ), yttria stabilizes zirconia (YSZ), ceria stabilized zirconia toughened alumina (CSZ-TA) and yttria stabilized zirconia toughened alumina (YSZ-TA) achieved after calcinations, compaction and sintering (~99% theoretical density was ensured) of co-precipitated powders are shown in Fig. 2. The details of sample preparation and material properties are described in our earlier communications [2123]. The sintered specimens were polished metallographically using ascending grades of emery papers and nal polishing was done using 0.25 m sized diamond paste on cloths to achieve the average roughness value (Ra) around 0.03 m. From the Fig. 2 and Table 1 as well, it is clear that CSZ has the largest grain size and YSZ have the smallest one. 3.2. Apatite formation on surface SEM micrographs of the sintered specimen surfaces after immersion in SBF at different time interval are shown in Figs. 3 and 4. One can observe from the gures that after 7 days nucleation of precipitates has started. After 14 days the nucleation rate has increased

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Fig. 3. SEM images of hydroxyapatite formed on the surface of CSZ (a, c, e, g) and YSZ (b, d, f, h) specimens at different time of soaking. The soaking time is marked on the gures. Inset images show higher magnication views.

many times and almost the whole surface was surrounded with newly nucleated minerals layer. During 3rd and 4th week, the mineral layer has further grown up and increased layer thickness. One can notice some crack on the thick layer of apatite which is supposed to appear due to the shrinkage of apatite layer while drying. The chemical nature of the formed minerals layer was examined through EDX analysis. Table 2 represents the variation of Ca/P ratio with soaking time for four types of specimens. From Table 2, it is clear that there was variation in Ca/P atomic ratio among four different specimen types after 1st week of immersion in SBF at 37 C at pH 7.4. Interestingly, the composition of deposited mineral was perhaps marginally different after 2nd weeks onwards as seen from Ca/P atomic ratio (Table 2). During the rst seven days of soaking, the Ca/P ratio was found far below than that of pure hydroxyapatite. Samples containing alumina (CSZ-TA and YSZ-TA) was having relatively less Ca/P ratio than that of without alumina (YSZ and CSZ). During 3rd

and 4th week of soaking, the Ca/P ratio increases to 1.4 irrespective of the composition but still did not reach to the Ca/P ratio of hydroxyapatite (1.6). But the XRD pattern taken after 4th week clearly shows some apatite peaks (Fig. 5). In Fig. 5 the two broad peaks 26 and 32 (2) are the main characteristic peaks of low crystalline apatite which is similar to biological apatite. From the existence of apatite peaks in XRD and less Ca/P ratio (compared to hydroxyapatite) in the EDX, it seems some other oxides of calciumphosphate (Tricalcium phosphate (Ca/P = 1.5), octacalcium phosphate (Ca/P = 1.0), dicalcium phosphate dehydrate (Ca/P = 1.0) etc.) having higher phosphate content (low Ca/P ratio) might have also formed along with hydroxyapatite. This differential growth of hydroxyapatite during the 1st and 2nd week in different samples is also reected in Figs. 3 and 4. If we compare the population of apatite at second week in Figs. 3 and 4 we observe that the population is signicantly high for CSZ and YSZ specimens compared to CSZ-TA and YSZ-TA specimens. However after 3rd

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Fig. 4. SEM images of hydroxyapatite formed on the surface of CSZ-TA (a, c, e, g) and YSZ-TA (b, d, f, h) specimens at different time of soaking. The soaking time is marked on the gures. Inset images show higher magnication views.

and 4th week the difference is not signicant. From the above analysis it seems alumina is prohibiting precipitate while immersed. According to Barrere et al., in physiological condition, only negatively charged HPO2 can be deposited on the surface of alumina and it does not 4 show any afnity to Ca2+ ions [17]. For this reason, one may observe poor Ca/P ratio for alumina containing specimens at the beginning. Actually, zirconia grains act as nucleation site and promote biomimetic growth of calcium phosphate minerals. At the beginning, island type cauliower like growth starts which cover the entire surface through bridging the gap. After three weeks of treatment, a thick continuous deposition of calcium phosphate minerals takes place. The thickness of apatite layer achieved after 21 and 28 days of soaking is shown in Table 3. It is encouraging to note that the coating thickness was found to be maximum for CSZ and minimum for YSZ-TA among the four kinds of specimens. The coating thickness was moderate for both YSZ and CSZ-TA specimens.

Calciumphosphate compound nucleates on the surface and its concentration increases with increasing in soaking time through more and more fresh deposition and growth of the earlier deposited apatite. The ZrOH group is supposed to act as a nucleation cite for apatite and once the nucleation is started; it grows spontaneously by consuming the calcium, phosphate and hydroxide ions of surrounding SBF solution

Table 2 Variation of Ca/P atomic ratio of deposited layer with soaking time for different composition. 7 Days CSZ YSZ CSZ-TA YSZ-TA 1.00 1.12 0.72 0.62 0.22 0.17 0.09 0.10 14 Days 1.33 1.37 1.29 1.31 0.12 0.08 0.01 0.05 21 Days 1.39 1.39 1.39 1.38 0.08 0.01 0.09 0.09 28 Days 1.46 1.45 1.43 1.44 0.10 0.02 0.05 0.02

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Fig. 5. XRD patterns of the samples after 28 days of soaking in SBF, presence of 26 and 32 (2) peaks ensure formation of hydroxyapatite.

[28]. As the SBF is highly saturated with phosphate and hydroxide ions it helps in precipitation [15]. It is reported that the degree of super-saturation increases with the increase in calcium or phosphate ion concentration, pH of the solution and alkali, calcium, or phosphate ion release from the zirconia surface resulting increased rate of apatite nucleation and growth [18]. 3.3. Cell attachment and morphology The morphology of the attached cells on the material surface was also evaluated under SEM to assess the cytocompatibility. Typical morphology of attached human osteoblast like cells observed under SEM after 3 day and 8 day of culture are shown in Fig. 6. All the four substrates supported intimate cellular attachment to the substrate by cellular extension and their continuous growth. After 3 day of culture, cells were connected to each other by lamellipodia and covered the surface of the substrates. After 8 day, colonized multilayered cells with numerous cellcell contacts were observed. No signicant morphological difference of the osteoblast like cell was evidenced between alumina and zirconia based ceramic. Similar cell morphology was also reported by other researchers [29] in case of alumina and zirconia based ceramics.
Table 3 Apatite thickness measured through surface proler after 21 and 28 days. Measured apatite thickness (m) Days 21 days 28 days CSZ 8.0 0.55 17.79 1.4 YSZ 7.8 0.73 17.12 1.2 CSZ-TA 6.10 1.04 14.8 0.63 YSZ-TA 5.84 0.54 14.03 0.41

Cell attachment on the materials was evaluated through uorescence microscopy. Fig. 7 shows the attachment of MG63 cells on the developed material surface. As it can be seen in Fig. 7, cells proliferated rapidly and became conuent at day 8. Cells were observed to attach rmly on the surface of the materials. Further, the cells were able to contact each other with the cellular protrusions and extensions. The uorescence microscopic study was in agreement with the MTT assay and SEM microscopic study.

3.4. Cellular proliferation, differentiation and total protein assay In vitro biocompatibility of the developed ceria/yttria stabilized zirconia and ceria/yttria stabilized zirconia toughened alumina was investigated using MG63 cells. The cell proliferation and viability were determined by MTT assay at scheduled intervals, which relies on the mitochondrial activity of vital cells and represents a parameter for their metabolic activity [30]. The results of a direct-contact cytotoxicity assay using cells cultured on the materials are shown in Fig. 8. Cell viability is expressed as the absorbance at 590 nm. In case of CSZ and CSZ-TA specimens, there were similar results with MTT assay compare to control (polystyrene tissue culture plate) but it was relatively higher in case of YSZ and YSZ-TA specimens. Typical trend of total protein content and ALP activity with the increase in culture time is represented in Fig. 8. Alkaline phosphate activity was lower in control with all specimen assessed at different time intervals. But, total protein content was lower with ceramics samples in comparison to the control. It is also interesting to note that amongst all the ceramics samples types, YSZ-TA exhibited better cellular response in terms of cell proliferation and differentiation.

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Fig. 6. SEM images of specimen surfaces revealing the morphology of human osteoblasts cell adhered to the surface after 3 day (a, c, e, g) and 8 day (b, d, f, h) of cell culture on (ab) CSZ, (cd) YSZ, (ef) CSZ-TA and (gh) YSZ-TA specimens. Inset images at the center of each image show the higher magnication view. A: SEM images of specimen surfaces revealing the morphology of human osteoblasts cell adhered to the surface after 3 day (a, b, c, d) and 8 day (e, f, g, h) of cell culture on (ab and ef) CSZ, and (cd and gh) YSZ specimens. Right side images are the higher magnication view of left side images. B: SEM images of specimen surfaces revealing the morphology of human osteoblasts cell adhered to the surface after 3 day (a, b, c, d) and 8 day (e, f, g, h) of cell culture on (ab and ef) CSZ-TA, and (cd and gh) YSZ-TA specimens. Right side images are the higher magnication view of left side images.

In these ceramics specimens, addition of ceria or alumina probably reduces the biological activity compared to yttria stabilized zirconia. 4. Conclusions Prepared CSZ, YSZ, CSZ-TA and YSZ-TA materials promotes growth of apatite like layer while immersed in SBF without addition of nucleating agents. The growth of layer thickness was a function of soaking period. Mineral layer thickness up to ~ 1417 m found after 28 days of soaking. The EDX and XRD analysis revealed, the mineral layer was of mixed type calcium phosphate compound along with hydroxyapatite. Rate of nucleation was relatively poor for alumina containing specimens at the beginning but at the later stages almost similar growth was evidenced. In Zirconia, ZrOH bonds were abundant on the surface of the composite which might have helped this

accelerated nucleation of hydroxyapatite in comparison to AlOH. The formation of apatite like mineral layer supported bioactivity of prepared materials in vivo. In vitro cellular response of the developed materials are quiet appreciable. Multi layered, interconnected human osteoblast like cell attached on the surface, proliferation and differentiation was satisfactory indicating biocompatibility of the fabricated materials. Acknowledgements We are pleases to acknowledge the nancial support from Department of Biotechnology Ministry of Science and Technology, New Delhi, India (Sanction Ref. No. BT/PR9385/MED/32/10/2007) and technical or infrastructural supports from Raunak Das, Medical Image Processing Lab of School of Medical Science and Technology,

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Fig. 7. Fluorescent microscopic image showing attachment of MG63 human osteoblast cell on ceramic disc after 15 days of culture. Samples were stained with rhodamine-phalloidin (red) for actin laments and Hoechst 33342 (blue) for nuclei. Original magnications 200X. [6] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, J. Biomed. Mater. Res. (A) 24 (1990) 721734. [7] S. Fujibayashi, M. Neo, H.-M. Kim, T. Kokubo, T. Nakamura, Biomaterials 24 (2003) 13491356. [8] M. Neo, S. Kotani, T. Nakamura, T. Yamamuro, C. Ohtsuki, T. Kokubo, Y. Bando, J. Biomed. Mater. Res. (A) 26 (1992) 14191432. [9] T. Miyazaki, H.-M. Kim, T. Kokubo, H. Kato, T. Nakamura, J. Sol-Gel Sci. Technol. 21 (2001) 8388. [10] P. Li, C. Ohtsuki, T. Kokubo, K. Nakanishi, N. Soga, T. Nakamura, T. Yamamuro, J. Am. Ceram. Soc. 75 (1992) 20942097. [11] T. Kokubo, Acta Mater. 46 (1998) 25192527. [12] T. Miyazaki, H.-M. Kim, T. Kokubo, C. Ohtsuki, H. Kato, T. Nakamura, J. Ceram. Soc. Jpn. 109 (2001) 929933. [13] M. Tanahashi, T. Matsuda, J. Biomed. Mater. Res. (A) 34 (1997) 305315. [14] M. Uchida, H.-M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, Biomaterials 23 (2002) 313317. [15] A.A. Aguiar, V. Ussui, C. Ribeiro, M.A. Scapin, D.R. Ricci, N.B. de Lima, Mater. Sci. Forum. 591593 (2008) 697702. [16] M. Uchida, H.-M. Kim, T. Kokubo, M. Nawa, T. Asano, K. Tanaka, T. Nakamura, J. Biomed. Mater. Res. (A) 60 (2002) 277282. [17] F. Barrre, A. Lebugle, C.A. van-Blitterswijk, K. de-Groot, P. Layrolle, C. Rey, J. Mater. Sci. Mater. Med. 14 (2003) 419425. [18] M. Uchida, H.-M. Kim, T. Kokubo, F. Miyaji, T. Nakamura, J. Am. Ceram. Soc. 84 (2001) 20412044. [19] K. Anselme, Biomaterials 21 (2000) 667681. [20] H.-C. Ko, J.-S. Han, M. Bchle, J.-H. Jang, S.-W. Shin, D.-J. Kim, Dent. Mater. 23 (2007) 13491355. [21] A.K. Pandey, K. Biswas, Ceram. Int. 37 (2011) 257264. [22] A.K. Pandey, U.R. Jena, K. Biswas, Mater. Chem. Phys. (2013), (under review). [23] A.K. Pandey, D. Mandal, K. Biswas, Mater. Des. (2013), (under review). [24] H. Takadama, M.T. Hashimoto, Y. Takigawa, M. Mineo, T. Kokubo, Ceram. Eng. Sci. Proc. 25 (2004) 571576. [25] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 7685. [26] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265275. [27] G.R. Beck, E.C. Sullivan, E. Moran, B. Zerler, J. Cell. Biochem. 68 (1998) 269280. [28] C. Ohtsuki, T. Kokubo, T. Yamamuro, J. Non-Cryst. Solids 143 (1992) 8492. [29] Y. Josset, Z. Oum'Hamed, A. Zarrinpour, M. Lorenzato, J.J. Adnet, D. Laurent-Maquin, J. Biomed. Mater. Res.(A) 47 (1999) 481493. [30] J.-L. Pariente, B.-S. Kim, A. Atala, J. Biomed. Mater. Res.(A) 55 (2001) 3339.

Fig. 8. Plot of MTT assay, total protein content and alkaline phosphate activity on different specimens after 8 and 16 day of osteoblast (MG63) cell culture.

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Please cite this article as: A.K. Pandey, et al., Mater. Sci. Eng., C (2013), http://dx.doi.org/10.1016/j.msec.2013.05.032