In Vitro Evaluation of Osteoconductivity and Cellular Response of Zirconia and Alumina Based Ceramics

8/14/2019 In Vitro Evaluation of Osteoconductivity and Cellular Response of Zirconia and Alumina Based Ceramics

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In vitroevaluation of osteoconductivity and cellular response of zirconia and aluminabased ceramics

Ajoy Kumar Pandey a,, Falguni Pati b, Debika Mandal a, Santanu Dhara b, Koushik Biswas a

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

a b s t r a c ta r t i c l e i n f o

Article history:

Received 31 May 2011Received in revised form 8 April 2013

Accepted 13 May 2013

Available online xxxx

Keywords:

Bio-ceramic

Osteoconduction

In vitrobiocompatibility

Cell culture

Bioactivity

Developed ceria/yttriastabilized zirconia and ceria/yttriastabilized zirconia toughened aluminasupported forma-

tion of apatite layer when immersed in simulated body uid without any prior surface treatment. The formed

minerallayerwas conrmed as hydroxyapatite throughX-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 ~17m thickness was found after 28 days of soaking. The biocompatibility of the developed materials was

ensured through in vitro human osteoblast likecell (MG63) culture on ceramic discs. The morphology of attached

cells wascharacterized through scanning electronmicroscopyand uorescent microscopy which show multilay-

ered 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.

1. Introduction

Alumina and zirconia based bioceramics have found their wide ap-

plications 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 appli-

cations[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 im-

parts 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 com-

posite, 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

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 format-

ting 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 integrationin 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

Materials Science and Engineering C xxx (2013) xxxxxx

Corresponding author. Tel.: +91 3222 226678.

E-mail address:[email protected](A.K. Pandey).

MSC-04053; No of Pages 8

0928-4931/$ see front matter 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.msec.2013.05.032

Contents lists available at SciVerse ScienceDirect

Materials Science and Engineering C

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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
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of implants[19]. Prior to cell attachment, proteins adsorb to the sur-

face of the implants through different ionic and van der Waals inter-

actions. 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, differenti-

ation and maturation which are important to tissue integration of the

implants[19,20].

In the present study, several alumina and zirconia based com-

posite 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 vitroto 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% zirco-

nia (stabilized with 14 mol% ceria) toughened alumina (CSZ-TA)and 15 wt% zirconia (stabilized with 8 mol% yttria) toughened alumi-

na (YSZ-TA) powder were synthesized by co-precipitation techniques

from their respective nitrate salts dissolving in proportionate quanti-

ties 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

condition, following two step sintering process. The sintering sched-

ule 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 dif-

ferent reagents in a proper sequence and maintaining the pH of thesolution 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 tempera-

ture of 37.5 C. The beakers were covered with aluminum foil to pre-

vent 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 var-

ied 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 fromthe 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 dis-

persive 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 fromthe 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. 1shows a typicalexample 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

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

Table 1

Sintering schedule adopted for different systems and their corresponding grain size

and hardness value.

System Sintering schedule Hardness (VHN) Average grain size (m)

CSZ 1500 C for 1 h and

1400 C for 2 h

950 20 4.3

YSZ 1450 C for 30 min

and 1250 C for 14 h

1364 11 0.78

CSZ-TA 1550 C for 1 h and

1450 C for 2 h

1730 16 Alumina grains 1.77

Zirconia grains 1.74YSZ-TA 1500 C for 1 h and

1400 C for 2 h

1800 10 Alumina grains 1.19

Zirconia grains 0.88

Fig. 1.Typical plot of surface proler data in case of CSZ-TA specimen showing apatite

thickness after 21 day of soaking.

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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

40l 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 tem-

perature. The wells were centrifuged for 5 min at 1600 rpm to elimi-

nate 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 CuSO4are reduced to Cu+ by the proteins in the cell sus-

pension. Reduced Cu+ ion forms a complex with BCA. The crimson

coloration of this complex is directly proportional to the protein con-

tents. 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 spectropho-

tometer (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

MgCl2and 1% Triton X-100 in a shaker for 30 min at 37 C after 3 and

7 days of culture. Aliquots of 50l were incubated with 100l of

p-nitrophenyl phosphate (pNP) solution at 37 C for 30 min. 100l 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 asmol 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 speci-

mens were coated with very thin layer of gold. For uorescence

microscopy, after soaking the samples in 4% formaldehyde solutionin 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 areshown inFig. 2. The details of sample preparation and material proper-

ties are described in our earlier communications[2123].

The sintered specimens were polished metallographically using as-

cending grades of emery papers and nal polishing was done using

0.25m sized diamond paste on cloths to achieve the average rough-

ness value (Ra) around 0.03 m. From theFig. 2andTable 1as well, it

is clearthatCSZ has the largest grain size and YSZ havethe smallest one.

3.2. Apatite formation on surface

SEM micrographs of the sintered specimen surfaces after immer-

sion 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 precipi-

tates has started. After 14 days the nucleation rate has increased

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 magni cation as indicated in the images.

3A.K. Pandey et al. / Materials Science and Engineering C xxx (2013) xxxxxx



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many times and almost the whole surface was surrounded with newly

nucleated minerals layer. During 3rd and 4th week, the mineral layerhas 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.

FromTable 2, it is clear that there was variation in Ca/P atomic ratio

among fourdifferent specimentypes 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 sevendays of soaking,

the Ca/P ratio was found far below than that of pure hydroxyapatite.

Samples containingalumina (CSZ-TA and YSZ-TA) was havingrelatively

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 hydroxyapa-tite (1.6). But the XRD pattern taken after 4th week clearly shows

some apatite peaks (Fig. 5). InFig. 5the 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 phos-

phate 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 inFigs. 3 and 4.If we com-

pare the population of apatite at second week in Figs. 3 and 4we

observe that the population is signicantly high for CSZ and YSZ spec-

imens compared to CSZ-TA and YSZ-TA specimens. However after 3rd

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.




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and 4th week the differenceis not signicant. From the above analysis it

seems alumina is prohibiting precipitate while immersed. Accordingto Barrere et al., in physiological condition, only negatively charged

HPO42 can be deposited on the surface of alumina and it does not

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 inTable 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 con-

centration increases with increasing in soaking time through more andmore 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

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.

Table 2

Variation of Ca/P atomic ratio of deposited layer with soaking time for different

composition.

7 Days 14 Days 21 Days 28 Days

CSZ 1.00 0.22 1.33 0.12 1.39 0.08 1.46 0.10

YSZ 1.12 0.17 1.37 0.08 1.39 0.01 1.45 0.02

CSZ-TA 0.72 0.09 1.29 0.01 1.39 0.09 1.43 0.05

YSZ-TA 0.62 0.10 1.31 0.05 1.38 0.09 1.44 0.02




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[28]. As the SBF is highly saturated with phosphate and hydroxide

ions it helps in precipitation [15]. It is reported that the degree ofsuper-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 lamellipodiaand 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.

Cell attachment on the materials was evaluated through uores-

cence microscopy.Fig. 7shows the attachment of MG63 cells on thedeveloped material surface. As it can be seen in Fig. 7, cells prolifer-

ated 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 exten-

sions. 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 relieson the mitochondrial activity of vital cells and represents a parameter

for their metabolic activity[30]. The results of a direct-contact cyto-

toxicity 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.

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.

Table 3

Apatite thickness measured through surface proler after 21 and 28 days.

Measured apatite thickness (m)

Days CSZ YSZ CSZ-TA YSZ-TA

21 days 8.0 0.55 7.8 0.73 6.10 1.04 5.84 0.54

28 days 17.79 1.4 17.12 1.2 14.8 0.63 14.03 0.41




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In these ceramics specimens, addition of ceria or alumina probablyreduces 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 nucle-

ating agents. The growth of layer thickness was a function of soaking

period. Mineral layer thickness up to ~1417m found after 28 days

of soaking. The EDX and XRD analysis revealed, the mineral layer was

of mixed type calcium phosphate compound along with hydroxyapa-

tite. 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 materialsin 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,

Fig. 6. SEMimagesof specimensurfacesrevealingthe morphologyof human osteoblastscell adheredto the surface after3 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 (a b 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 cellcultureon (ab a nd ef)CSZ-TA,and(cdandgh) YSZ-TA specimens. Right sideimagesare thehigher magnication viewof leftside images.




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IIT Kharagpur and Mr. Dilip Chakraborty of Metallurgical and Materials

Engineering, IIT Kharagpur.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://

dx.doi.org/10.1016/j.msec.2013.05.032.

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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 actinlaments and Hoechst 33342 (blue) for n uclei. Original magnications 200X.

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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