Biotechnology & Biomaterials Investigation on Biological Properties of Dental Implant by Ce-TZP/Al 2 O 3 / HA Bio-nano-composites

Early years, bio-nano-composites materials are interesting in advanced engineering applications fields. In order to, biological properties of the 10Ce-TZP/Al 2 O 3 nano-composite was evaluated by the addition of HA nanopowder for medical applications. In this research, a bio-nano-composite consisting of CeO2- stabilized zirconia (Ce-TZP), Al 2 O 3 and Hydroxyapatite (HA) powders proposed characterized by X-ray diffraction(XRD),the and and cellular response test performed with osteoblastic cell-lines cell-lines the morphology of the with

Alumina and zirconia have excellent biocompatibility. The main advantages of Al 2 O 3 is its high hardness and wear resistance, while ZrO 2 exhibits higher strength and fracture toughness and the composite consisting of the two has higher ductility and fracture toughness than each constituent [12]. Some interest has been paid to composites comprising of hydroxyapatite and another phase of high mechanical properties and bio-inertness, such as alumina and zirconia [13]. Cerium oxide or Ceria is used to stabiles the tetragonal polycrystalline structure of zirconia at room temperature, the product called Ceria-partially stabilized zirconia (Ce-TZP) [15]. Ce-TZP/Al 2 O 3 is a composite in which Ce-TZP particles are added to Al2O3 matrix as the second phase to modify the fracture behavior of composite because, it has high fracture toughness [14]. In additional, Ce-TZP/Al 2 O 3 nano-composites could be superior to Y-TZP in clinical use [16,17]. The biocompatibility of zirconia-alumina (ZA) nano-composites in load-bearing applications such as dental/orthopedic implants was significantly enhanced by the addition of bioactive HA [4].
In this work, the effect of HA is studied on biological properties of Ce-TZP/Al 2 O 3 . In order to, bio-nano-composites were fabricated by mechanical milling of pre-synthesized nano-scaled constituents, and cold pressing and sintering Phase analysis; strength and biological properties of produced composites were evaluated by different characterization methods.

Experimental
For this study, we used of Al 2 O 3 , HA and ZrO 2 nanopowders syn-thesized by sol-gel, chemical precipitation and hydrothermal methods (with particle sizes 112,9,12 nm respectively). The process Schematic is given in Figure 1.
As is observed ZA nano -composite powders were prepared by fast milling machine (sharifi co. iran) of 70 vol% 10Ce-TZP and 30 vol% Al 2 O 3 powder in ethanol for 8 hr. Obtained powder was mixed with HA (up to 40vol%) in ethanol by fast milling machine again for 8 hr. Mixed slurry was dehydrated, crushed and sieved, and uniaxial cold pressed at a pressure of 200 MPa by universal test machine (Zwick roll-Z100). Cold pressed samples were sintered at 1600°C for 2 hr by electrical furnace (Vecstar). The density of the specimens was measured by the Archimedes method [ASTM B311-08]. Crystalline phase analysis was performed by XRD (Bruker model D8). The bending strength was measured by means of three-point bend test with universal test machine (Zwick roll-Z100) [ASTM C1674-08].
In order to investigation the proliferation behavior on the ZA-HA composites, the samples used for cell tests were prepared from disc specimens with 10mm diameter that after polishing both sides with diamond slurries down to 1 mm, the specimens were washed and sterilized at 121 o C for 20 minutes. The samples were placed in a 24 well tissue culture plate and seeded with 25μl of the 3x10 4 cells/cm 2 MG63 (National Cell Bank of Iran-no NCBI C…) cell density suspension.

Abstract
Early years, bio-nano-composites materials are interesting in advanced engineering applications fields. In order to, biological properties of the 10Ce-TZP/Al 2 O 3 nano-composite was evaluated by the addition of HA nanopowder for medical applications. In this research, a bio-nano-composite consisting of CeO2-stabilized zirconia (Ce-TZP), Al 2 O 3 and Hydroxyapatite (HA) powders proposed to use for medical application .The first step, nano-powders of Ce-TZP and Al 2 O 3 , were blended by fast milling machine. Then, nano-powder of HA was added to the alumina -zirconia (ZA) mixture from 10 to 40 vol%, and homogenized again. In final step, mixed powder was cold pressed and sintered at1600°C for 120 min. Crystal phase of the sintered samples were characterized by X-ray diffraction(XRD),the density and bending strength were measured and cellular response test was performed with osteoblastic cell-lines by MG63 cell-lines and the morphology of the proliferated cells was observed with FESEM too.
From the strength and cellular response evaluations of samples, specimens with 30 vol% HA showed the best result, also XRD patterns confirmed this results.
Cells were allowed to adhere for 2 hours then flooded with 1 ml of Dulbecco's modified Eagle's medium (DMEM, Gibco-USA), 2mM L-glutamine (Gibco), 100 IU/ml of penicillin and 100µg/ml of streptomycin (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco-USA) and placed back into the incubator. The fabricated specimens, including pure HA as a positive control, were placed into a 24-well plate, which was followed by plating on all the discs.
The cells were cultured for 7 days in a humidified incubator under an atmosphere of 5% CO 2 at 37 o C. After detaching the cells from the specimens with a trypsin-EDTA solution (0.25% trypsin, 0.05% EDTA-Gibco) and staining with 0.4% trypan blue, the living cells were counted using a hemocytometer (Paul Marienfeld GmbH & Co-Germany). Each set of tests was performed in triplicate.
The results were expressed as means with standard errors. The data were then analyzed by one-way ANOVA, followed by the post hoc paired Student's two-tailed t-test. The significance levels were notified significant (*, if p< 0.05) and highly significant (**, if p<0.01) in the tables. Figure 2 shows the densities of the ZA nano-composites containing various amounts of HA. Compared to the theoretical density, which was calculated by the rule of mixture, the density of the specimens with a low HA content almost matched with the theoretical value. However, as the amounts of HA were increased, the discrepancy between the measured and theoretical densities increased.  Figure (3-a). With addition of 10, 20, 30 and 40 vol% HA ( Figure (3-b-c-d-e), respectively), as well as the peaks corresponding to HA, TCP peaks, were also detected, that TCP component was originated from the reaction between HA and ZrO 2 . The bending strengths of the nanocomposites are shown in Figure 4, as the HA content was increased up to 40vol% the strength decreased of 850 to 137 MPa.

Results
Biocompatibility tests are in progress. Figure 5 illustrates Cell proliferation results on the specimens after 7 days culture. The number of cells was significantly increased by the addition of 10% HA to the ZA (ANOVA, p<0.05). The number of cells increased steadily with further addition of HA (ANOVA, p±0.001). When 40% HA was added to the ZA, the proliferation rate on that specimen became comparable to that on the pure HA means of wells±S.D.  Figuer 6 show morphology of the proliferated cells on the specimens. As was observed specimens with30vol%HA show most of the cell proliferation.

Discussion
As a bio-inert implant material, ZA nano-composite is considered to have sufficient strength. However, its biocompatibility such as satisfactory osseointegration and faster bone regeneration has to be improved by the addition of calcium phosphates such as HA, and TCP, [1,4]. TCP component was originated from the reaction between HA and ZrO 2 . Both HA and TCP are formed of calcium and phosphate component, which are essential to satisfactory osseointegration and faster bone regeneration to be achieved [4,7]. Just as shown in the XRD patterns ,intensities of the HA and TCP peaks increased steadily with increasing HA content.

Conclusion
Hydroxyapatite (HA)-zirconia-alumina (ZA) bio-nano-composites were produced by mechanical blending of separately synthesized nano-scaled powder. Composite materials were characterized by phase analysis and cellular response test. From mechanical and biological properties evaluation of the 10Ce-TZP/Al 2 O 3 /HA bio nano composites, specimens with 30 vol% of HA, was found to be the optimal composition for biological applications.