ASIAN J. EXP. BIOL. SCI.

VOL 3(2) 2012: 303-310

© Society of Applied Sciences

ORIGINAL ARTICLE

Preliminary Biocompatibility and Bioactivity Properties of Alumina-

Zirconia-Hydroxyapatite Composite
*1
Rodica Rogojan, 2Ecaterina Andronescu, 3Maria
4
Cristina Munteanu, 3Mihaela Radu, 3Florina
1
Gisela Gaina, Roxana Trusca
National Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Bucharest, Romania
2
Faculty of Applied Chemistry
3
and Materials Science, Polytechnic University of Bucharest, Romania
Faculty of Biology,
4
University of Bucharest, Romania
Metav, Bucharest, Romania

ABSTRACT
Composites combine the properties of two or more components. The purpose of this study was to evaluate the preclinical in
vitro and in vivo biocompatibility of alumina-zirconia-hydroxyapatite ceramic nanomaterial for use in the biomedical
field.The ceramic nanocomposite, prepared by sol-gel and pirosol methods and sinterized at different temperature values:
1200oC, 1400oC and 1600oC were tested in vitro using a culture medium containing human osteoblastic cell line hFOB and in
vivo by implanting the ceramic composite cylindrical plates, in the parietal bones of Wistar rats. These tests have shown that
implants sintered at temperatures of 1400oC and 1600oC compared with those sintered at 1200oC showed good
biocompatibility and bioactivity properties.
Keywords: ceramic nanocomposite, in vitro, in vivo, biocompatibility, implant

INTRODUCTION
Research in the field of ceramics used in orthopedics was focused on several important aspects concerning the
mechanical, chemical and physical behavior of these materials in the physiological environment. Biocompatibility
studies and compressive strength, tear or corrosion tests aimed at obtaining a long-term use of these implantable
materials, eliminating their subsequent rejection by the human body. The properties of the ceramic materials are
strongly influenced by the chemical and physical characteristics of raw powders, such as particle size, surface area,
chemical homogeneity and extent agglomeration.
It is well known that biomaterials like hydroxyapatite (HA), alumina (Al2O3) and zirconia (ZrO2) are frequently used in
the field of bone reconstruction. The preparation of a composite material using simple compounds like alumina,
zirconia and hydroxyapatite, each of them with its own advantages or disadvantages as biomaterials, offers the
possibility of improving the properties of each single component [1].
The purpose of this study is to compare the biological properties of alumina-zirconia-hydroxyapatite based composite
materials, prepared by nonconventional methods such as sol-gel and pirosol processes, and sinterized at different
temperature values (1200oC, 1400oC and 1600oC). Biomaterials obtained combines the advantages of zirconia and
alumina as reinforcement material for hydroxyapatite (possesses high biocompatibility and bioactivity properties but
has a weak mechanical strength) [2-10] with the excellent mechanical properties of ZrO2 [11-12] and the chemical
inertness of Al2O3 [13- 14].
The biocompatibility and bioactivity properties of the cylindrical plate composite material samples were examined in
vitro using the hFOB human osteoblastic line culture medium and tested in vivo as implants in the parietal bone of
Wistar rats.

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

MATERIALS AND MEHODS

The alumina, zirconia and hydroxyapatite powders were prepared by sol-gel [15–17] and pirosol methods [18-20].
The precursor solutions are given in Table 1.
Table 1. Composite precursor solution

Material Precursor solution

Sol-gel method Pirosol method
Alumina Aluminium chloride + Ethanol Aluminium chloride + Distilled
water
Zirconia Zirconium chloride + Calcium Zirconium chloride + Calcium
chloride + Ethanol chloride + Distilled water
Hydroxyapatite Calcium azotate + Phosphor Calcium azotate + Phosphor
pentoxide + Ethanol pentoxide + Distilled water

In order to prepare the composite material, the powders obtained by pirosol or sol-gel method were combined in
an alumina:zirconia:hydroxyapatite mass ratio of 25:25:50%.
Ceramic composite plates were prepared by pressing and cylindrical 2
shaping the alumina-zirconia-hydroxyapatite
powder mixture (Carver press; applied force value of 2000
o
kgf/cm
o
). The plates
o
were weighed and sinterized for a two
hour interval at the following temperature values: 1200 C, 1400 C and 1600 C (Table 2).
Duplicate ceramic samples obtained at each sinterization temperature value were used in order to evaluate the
composite material in vitro bioactivity and in vivo biocompatibility.
For the in vitro tests, the cylindrical alumina-zirconia-hydroxyapatite plate dimensions were of 10±0.5 mm in
diameter and 1 mm in thickness, while for the in vivo tests, the samples measured 5±0.5 mm in diameter and 1 mm in
thickness.
Table 2. Composite name, temperature and synthesis method

Name composite Synthesis of Synthesis of

sample method temperature
1 Sol-gel method 1200oC
2 1400oC
3 1600oC
4 Pirosol method 1200oC
5 1400oC
6 1600oC

The in vitro testing

The first step in the assessment of biocompatibility of new biomaterials is in vitro toxicity tests that include
cytotoxicity.
Placing the osteoblastic cell line into the culture medium:
The utilized hFOB line is formed of human fetal cells which are capable of osteoblastic differentiation and bone
structure formation. The frozen cells (-80oC, liquid nitrogen atmosphere) were rapidly defrosted (at 37°C), and the
resulted cell suspension was distributed into centrifuge tubes containing a 5 ml volume of growth medium containing
a mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 medium (DMEM:F12=1:1), supplementary being
added glutamine (2.5 mM), sodium piruvate (0.5 mM), sodium bicarbonate (1.2 g/l), G418 antibiotic (0.3 mg/l)
(aminoglycoside antibiotic similar in structure to gentamycin), and phosphate buffered saline solution (PBS, pH 7.4)
containing 10% fetal bovine serum (FBS). 2
The tubes were centrifuged at 1500 rpm for 10 minutes, and the resulted sediment was resuspended in 75 cm flasks.
The cell culture medium was incubated at 34ºC, in a 5% CO2 humid atmosphere. The final solution pH value was
7.2–7.4. The cell culture medium was refreshed every 2-3 days. The cells were multiplied by passing through the
tripsinization process: tripsine powder (0.25%), EDTA (0.03%), DMEM/F12 medium, o
and phosphate buffered saline
solution (PBS, pH 7.4) containing 10% fetal bovine serum (FBS, preheated at 34 C).
The cells insemination was made to each well, in the presence, in the presence of the ceramic samples and a blank.

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

Before testing them, the composite samples were washed with a 75% 4ethanol solution and sterilized using UV laminar
flow. Each plate pit containes 1 ml cellular suspension having a 5x10 cells/pit density. In order to accomplish the cell
adhering, the each well were incubated for 24 hours, at 34ºC, in a 5% CO2 atmosphere. Each plate of the pit was
microscopically analyzed and photographed.
The in vivo testing
For in vivo study we used a total of six rats Wistar breed. The in vivo study animals were administered an anesthetic
injection (1 ml of 2% lidocaine/adrenaline solution). A trephine was used to remove a parietal bone plug (with a 5 mm
diameter). The test ceramic materials were sterilized by autoclaving at 120°C for 20 minutes and surgically placed in
the central region of rat's parietal zone. The wound was sutured in two layers (PGA).
After the implanting procedure, the rats were kept in special cages for 40 days (Figure 1). During this time interval,
they received a normal food and water ratio. After this period the animals were slaughtered (Figure 2.) and harvested
implant and bone tissue around it.All the experimental procedures were performed according to the International and
European Laws on animal testing.

RESULTS AND DISCUSSIONS

The ceramic composite samples tested in vitro were characterized using an Olympus binocular microscope, while the
sample materials tested in vivo were analyzed by means of scanning electron microscopy - Quanta Inspect F
microscope (1.2 nm resolution) with field emission gun (FEG) and by means of X-ray dispersive energy
spectrophotometer (EDAX), having a MnK resolution of 133 eV (Figures 3-9).
In order to perform the SEM analysis, the samples were cut into thin slices of 20 μm with a manually cryotom
505 N and placed on the adhesive carbon band and covered with a thin gold layer, the microscope visualization being
made at different magnification values (Figure 10.).

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

Figure 3.Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 1, after 24 hours
of incubation in culture medium

Figure 4. Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 2, after 24 hours
of incubation in culture medium

Figure 5. Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 3, after 24 hours
of incubation in culture medium

Figure 6. Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 4, after 24 hours
of incubation in culture medium

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

Figure 7. Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 5, after 24 hours
of incubation in culture medium

Figure 8. Images for phase contrast microscopy for the hFOB osteoblasts (10X objective) in the presence of sample 6, after 24 hours
of incubation in culture medium

Figure 9. Images for phase contrast microscopy for the hFOB osteoblasts cells (10X objective), after 24 hours of incubation in
culture medium (blank)

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

a b

c d

e f
Figure 10. Scanning electron microscopy images and images intensity distribution in the surface relative to X
rays characteristic C, O Al, P, Ca, Zr on a micro area that includes the interaction between bone and composite
material

The optical microscopy images are very suggestive.

o
The samples sinterized at 1200 C are characterized by the absence of the fibroblasts, this negative phenomenon being
probably induced by the presence in the composite material of a toxic component, due to insufficient densification
(Figure 10 – a,b), for both methods of synthesis. The samples sinterized at 1400oC by sol-gel and pirosol methods
allowed line hFOB cell proliferation by the presence of a high density of fibroblasts as compared to the blank, being
o
thus the most biocompatible materials included in the test (Figure 10 – c,d). For the samples sinterized at 1600 C by

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Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

sol-gel and pirosol methods have found high levels of colonization with a blank look similar (Figure 10 – e,f) [21-22].
The rats were continuously monitorized in order to observe any physiologic or comportamental alteration.During the
40 days study interval, no animal death was registered, and there were no important variations of the rats weight. SEM
images of the bone implanted samples, which were cut into thin slices for microscopic visualization, show that the
composite material had a good compatibility with the physiologic medium [23]. Moreover, the 1400oC sinterized
ceramics (Figure 10 – c,d) presented a very good penetration at the bone-composite interface. At this level, a newly
grown bone tissue can be observed [24]. These bioimplants morphologic analysis revealed the presence of an
intensified periostal proliferation process, thus pointing out their role as growth factor for bone tissue. At the bone-
implant interface, branches of prolifered bone tissue penetrate the implant and generate a lacunar structure, which is a
characteristic of osteogenesis. This phenomenon offers the bone progenitor support which will penetrate the implant,
the processesso
being evidently similar to those observed during bone fracture repairing [25].
For the 1600 C sinterized materials, the fibroblasts pseudopodic penetration into the implant bodies is relatively slow
as compared to the other tested samples. This phenomenon ca be explained by the high material density and,
consequently its reduced porosity (Figure 10 – e,f).
Taking into account the chemical composition of the tested samples, the images obtained by X-ray dispersive energy
spectrophotometry were evaluated and the micro areas containing the composite material constituents were analyzed.
These areas were observed from the qualitative compositional point of view in order to identify the components.
Figure 10 illustrate the EDAX spectra for the composite materials and atest the presence at the bone-implant interface
for C, O, Al, P, Ca, Zr and in low quantities, for Na, Mg, Si and K.
The tests presented in this paper are preliminary and orientative. In order to obtain more conclusions, the study should
be made using a larger sample lot.

CONCLUSIONS
In this article, the biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite nanocomposites
(mass ratio of 25:25:50%) obtained at 1200oC, 1400oC and 1600oC by sol-gel and pirosol process were examined in
vitro and in vivo tests. o o
In vitro and in vivo test results indicated that the materials sintered at 1400 C and 1600 C were biocompatible, as
o
compared with those sintered at temperatures of 1200 C, which showed cell death, which could affect cell
metabolism. o o
Nanocomposite ceramics based on alumina-zirconia and hydroxyapatite, sintered at 1400 C and 1600 C, showed
excellent bioactive qualities and possess promising properties for their application as biomedical materials for bone
repair.

REFERENCES
[1] Ducheyne, P., Marcolongo, M. and Schepers, E., Hench, L.L. and Wilson, J. (1993) An Introduction to Bioceramic, Singapore (1993), pp.
281–297;
[2]. Chris Arts J.J., Verdonschot N., Schreurs B., et al (2006) The use of a bioresorbable nano-crystalline hydroxyapatite paste in acetabular
bone impaction grafting, Biomaterials, 27, pp. 1110 – 1118;
[3]. Johnson G. S., Mucalo M. R., Lorier M. A., et al.(2000) The processing and characterization of animal derived bone to yield materials with
biomedical applications. Part. II: Milled bone powders, reprecipitated hydroxyapatite and the potential uses of these materials, J. Mater.
Med. 11(4), pp. 425 – 741;
[4]. Simon, V.,Muresan, D., Popa, C. and Simion, S. (2005) Microscopic analysis of sintered Titanium-Hydroxyapatite implant materials,
Journal of Optoelectronics and Advance Materials, 7(6): 2823 – 2826;
[5]. Jian-Ming Ruan and Grant, M. H. Biocompatibility evaluation in vitro. Part III: Cytotoxicity expression of human and animal osteoblasts
on the biomaterials, Journal of Central South University of Technology 8(3): 147-155;
[6]. Maugars, Y., Péru, L., Berthelot, J.M., Delecrin, J., Prost, A. and Daculsi G. (1994) Air pouch model in the rat for evaluating articular
phlogogenic capacity of ceramics, 120(2):88-93;
[7]. Racquel, Z., LeGeros, Guy Daculsi and John P. LeGeros (2008) Bioactive Bioceramics, Orthopedic Biology and Medicine,
Musculoskeletal Tissue Regeneration, 2, Pages 153-181;
[8]. Hench, L.L. (1991) Bioceramics: from concept to clinic. J. Am. Ceram. Soc., 74 p. 1487;
[9]. Suchanek, W. and Yoshimura, M. (1998) Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue
replacement implants. J. Mater. Res., 13 1;
[10] Mobasherpour, I., Soulati Heshajin, M., Kazemzadeh, A. and Zakeri, M. (2007) Synthesis of nanocrystalline hydroxyapatite by using
precipitation method, J. Alloys Compd., 430 1–2, pp. 330–333;
[11]. Tsukuma, K., Ueda, K. and Shimada, M. (1985) High-temperature strength and fracture toughness of Y2O3-partially-stabilized ZrO2/Al2O3
composites, J. Am. Ceram. Soc., 681;
[12]. Grigoryev, O.N., Firstov, S.A. Babiy, O.A. and Homenko, G.E. (1994) Effect of zirconia (3 mol% yttria) additive on mechanical properties
and structure of alumina ceramics. J. Mater. Sci., 29, p. 4633;
[13].Champion, E., Gautier, S. and Bernache-Assollant, D. (1996) Characterization of hot pressed Al2O3-platelet reinforced hydroxyapatite
composites (biomaterial for orthopedics), J. Mater. Sci. Mater. Med., 7, p. 125;
[14]. Birsan M., Andronescu E., Birsan C., Ghitulica C., Pall G. (2007) Alumina based Composites with Possible Medical Applications, Journa
of Optoelectronics and Advanced Materials 9 (8) :2634 – 2637;
[15]. Rodica Rogojan, Ecaterina Andronescu, Cristina Ghiţulică, Bogdan Ştefan Vasile (2011) Synthesis and characterization of alumina nano-
powder obtained by sol-gel method, U.P.B. Sci. Bull., Series B, 73 (2), ISSN 1454-2331;

ASIAN J. EXP. BIOL. SCI. VOL 3 (2) 2012 309

Preliminary biocompatibility and bioactivity properties of alumina-zirconia-hydroxyapatite composite.......................................Rodica Rogojan et al.

[16].Rodica Rogojan, Ecaterina Andronescu, Irina Iliescu, Roxana Truşcă, Bogdan Ştefan Vasile (2011) Synthesis and characterization of calcia
stabilized zirconia nano-powder, obtained by sol-gel method, Romanian Journal of Materials, 41 (3), 240-247;
[17].Rajabi, A. H., Behnamghader, A., Kazemzadeh, A. and Moztaarzadeh, F. (2006) Synthesis and characterization of nanocrystalline
hydroxyapatite powder via sol-gel method, 3RD Kuala Lampur International Conference on Biomedical Engineering, IFMBE Procedings,
Vol. 15, Part 5, p. 149-151;
[18].Marco Antonio Alvarez-Perez et al. (2009) In vitro studies of osteoblasts response onto zinc aluminate ceramic films. Mat. Res., 12(4) :
509-515;
[19].Vallet-Regi, M., Nicolopoulos, S., Roman, J., Martinez, J.L., Gonzalez-Calbert, J.M. (1997) Structural Characterization of ZrO2
nanoparticles obtained by aerosol pyrolysis, J. Mater. Chem., 7(6), 1017-1022;
[20].Elena Dinu, Elisabeth Siebert, Ecaterina Andronescu, Elisabeth Djurado, Laurent Dessemond, Cristina Ghitulica (2008)
Nanocristallites Obtained Through the Pyrosol Method, Physica Status Solidi (a), 205/6, pp. 1488 – 1493.
[21]. Thomas J. Webster, and Richard W. Siegel (1999) Osteoblast adhesion on nanophase ceramics, Biomaterials, 20, 1221-1227;
[22].Yousefpour, M., Askari, N., Abdollah-Pour, H., Amanzadeh, A. and.Riahi, N (2011) Investigation on biological properties of dental
implant by Ce-TZP/Al2O3/HA bio-nano-composites, Digest Journal of Nanomaterials and Biostructures, 6(2): 675 – 681;
[23]. Chang, Y.S., Oka,M., Nakamura, T. and Gu, H.O.(1996) Bone remodeling around implanted ceramics, J Biomed Mater Res.,30(1):117-
24;
[24]. Kong, Y.M., Bae, C.J., Lee, S.H., Kim, H.W. and Kim, H.E.(2005) Improvement in biocompatibility of ZrO2-Al2O3 nano-composite by
addition of HA, Biomaterials, 26(5):509-17;
[25]. Takemoto, M., Fujibayashi, S., Neo, M., Suzuki, J., Kokubo, T. and Nakamura, T.(2006) Bone-bonding ability of a hydroxyapatite coated
zirconia-alumina nanocomposite with a microporous surface, J Biomed Mater Res A. 15;78(4):693-701.

Corresponding Author: Rodica Rogojan, National Institute of Rehabilitation, Physical Medicine

and Balneoclimatology, Buchar est, Romania , E -mail: rorogojan@yahoo.com

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