3rd International Conferences and Workshops on Basic and Applied Sciences 2010 ISBN: 978-979-19096-1-7

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The Effect Of Composition Co:Cr Variation to Mechanical Properties And Cytotoxicity Co-Cr-HA Composite

Aminatun, Siswanto, Fitri Wijayanti*

*Departement of Physics Airlangga University, Surabaya, Indonesia

e-mail: aminatun_unair@yahoo.co.id

Abstract

Sintesis of composite Co-Cr-HA (Cobalt Composite Implants / CIC) created by varying the composition of Co: Cr respectively (82%: 18%), (79%: 21%), (76%: 24%), (73%: 27%), (70%: 30%) of added 5% HA from the Co-Cr alloy. Manufacturing process involves mixing, pressing and sintering. The process of mixing ingredients produced using milling machines with a speed of 600 rpm for 30 minutes and then pressed at a pressure of 370 MPa and sintering carried out at a temperature of 1000C for 3 hours. XRD test results showed that the diffraction pattern of crystal form and the emergence of phase oxide layer CoO, Co3O4 and CrO2 on the surface of the CIC result of sintering processes that do not vacuum. Sample hardness value of 351.096 13.339 kgf/mm2 to 490.130 76.540 kgf/mm2. Compressive streght increases with the addition of the composition of Cr, the highest value indicated the composition of CIC in the ratio of Co: Cr by 70%: 30% of 102.979 0.153 N/mm2. Test on fibroblast cells by MTT assay method obtained results that the percentage of living cells with the highest composition of 95.837%. Overall characterization of CIC showed good results, it is shown by the percentage of living cells is still above the tolerance limit of 60%. KeyWords: Cobalt Implant Composite (CIC), Pressing, Sintering, Hardness, Compressive Streght

1 Introduction

Types of biomaterials used in medical applications and dentistry are a metal (alloy), ceramics, polymers, and composites. Biomaterials are used to replace or repair a tissue or organ function which damaged. Material from the type of metal's mostly used as bone implant biomaterials are stainless steels (316L-steel), Co-Cr alloy, pure Ti (cpTi), and Ti alloy (Ti-6Al-4V) (Shamsul, et al. 2007).

Titanium has been widely used in medical and dentistry because of its good like having a relatively

low level of density, corrosion resistance, and acceptable to the body or does not cause hypersensitivity. However, based on its mechanical strength, the Ti still cannot be used in several medical applications (Shamsul, et.al, 2007). Besides the presence of Al and 4% V ions in titanium alloys provide long-term health problems. Low resistance of titanium alloys which can accelerate ions presence is harmful to the body (Goenharto, et al, 2008).

The development of new biomaterials-based alloys or composites that can be applied in medical safety is required. Found Co-Cr alloy and has been used as base material for the first time in the medical dental implants due to the planting of Co is very good resistance in the oral environment (Samsul, et al., 2007). Furthermore, studies done about the addition of hydroxyapatite to the Co-Cr alloy.

Shamsul et al., 2007, utilized Cobalt Composite Implants (CIC) which is a Co-Cr alloy with a ceramic hydroxyapatite (HA). The study varying the composition of the HA between 0% to 20% added to the Co-Cr alloy. On the variation of 5% HA, otherwise have better properties when compared with the other composition variations. In that study said that the composite Co-Cr-HA with the addition of 5% HA can be applied in the biomedical as bone implants, but not exposed to the metal alloy composition of Co-Cr in the composite. It is therefore necessary to modify the composition of Co-Cr in order to know the potential of CIC with the mechanical properties in accordance with human bones.

2 Methodology

2.1 Process Samples Sample preparation in this process involves mixing powders Co, Cr and HA. Material is weighed according to the composition in Table (1) and then mixed using a milling machine equipped with speed control device that is dimmer. The addition of alcohol is very helpful in smoothing the mixture,

Aminatun, The Effect of Composition Co:Cr Variations to Mechanical Properties and Cytotoxicity Co-Cr-HA Composite

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due to the materials used (cobalt, chromium and HA) is insoluble in water. And then, the sample milled using 600 rpm of speed for 30 minutes (Shamsul, 2007). Afterwards, the samples are removed from the tube milling and then dried, because the sample in wet state after being mixed with alcohol. Samples in the form of powder is weighed according to the needs of characterization then pressed with a pressure of about 370 MPa. Samples sintered at 1000oC for 3 hours with the rate of increase in 20 C / min so as to achieve 1000oC, and held for 2 hours (Shamsul, 2007), then performed the characterization. Furthermore, on the whole sample were characterized by XRD test, Vickers hardness and compressive strength.

Table 1: CIC constituent raw material composition in % of weight

Sample Co (%) Cr (%) HA (%) I 82 18 5 II 79 21 5 III 76 24 5 IV 73 27 5 V 70 30 5

2.2 Cytotoxicity testing Cell BHK 21 (Baby Hamster Kidney 21) clone 13 is saved at temperature of 85C in Nunc vials with eagle medium, carried out thawing stored is placed in an incubator at 37C to melt . Then centrifuged at 3000 rpm for 10 minutes, the precipitate is a cell culture; storage media are located on the top and discarded. 20 ml of cell culture is inserted into a small roux bottle added by grower medium of eagle and also added by 10% of calf serum, and then inserted into a CO2 incubator at 37C for 2 x 24 hours, cells will be full.

Cell cultures were divided into micro plate 96 well each 1.5 ml plus eagle medium and 10% of bovine serum, then put in a CO2 incubator with a temperature of 37C for 2x24 hours. Once confluent the media was removed and washed 2 times with PBS solution, monolayer culture which is clean is then added back with the eagle media and the EMS 5% which was mixed with a sterile CIC sample. Another one that is used as a control is cell culture medium without CIC compound, and then all of a micro plate is stored again in incubator CO2 for 24 hours.

Monolayer cell cultures that have been removed from the CO2 incubator, then the media was removed and washed with PBS 5 times to clean waste products of metabolism of cells. Then, ditrypsinasi cell culture with versence trypsin 0.25% to deteriorate the cell attached to the wall of the bottle. Once added with MTT, cell cultures in

the incubation for 4 hours and then add in DMSO (dimethyl Sulfoxida). After 15 minutes, the cell cultures in a shaker and then read using Elisa Reader Spectrophotometer. The percentage number of live cells for MTT test can be calculated by the following equation:

%100% xolmediacontrODlcellcontroOD

olmediacontrODtreatmentODlivecell

+

+=

With, % Live cells = percentage of cells after treatment, treatment OD = optical density values of samples after treatment, media control OD = optical density value of media control, cell control OD = optical density value of control cells.

3 Result

3.1 The test of X-ray Diffractometer (XRD) XRD test was conducted to identify synthesized compounds through a search match using the program Crystal Impact analyst. Results of search match XRD data shown in Figure (1), (2), (3), (4) and (5)

Figure 1: Search match curve CIC-I

Figure 2: Search match curve CIC-II

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Figure 3: Search match curve CIC-III

Figure 4: Search match curve CIC-IV

Figure 5: Search match curve CIC-V

Based on Figure (1), (2), (3), (4) and (5), search match results show that in each sample appears compounds CoO (cobalt oxide), Co3O4 (cobalt oxide), CrO2 (chromium oxide) and HA (CA10 (PO4)6 (OH)2). The presence of oxides in each constituent compound of CIC as a result of the sintering process during sample preparation is not done in a vacuum furnace (air vacuum), so that the oxygen in the furnace will bind to the elements Co and Cr to form CoO, Co3O4, CrO2.

3.2 Mechanical properties

Table 2: The test results of mechanical properties of CIC

Sample Vickers Hardness/ VHN (kgf/mm2)

Compressive strength (N/mm)

I 371,463 12,404 79,589 0,122

II 351,096 23,105 94,627 0,140

III 426,077 85,500 99,152 0,152

IV 490,130 132,572 102,668 0,157

V 392,888 19,341 102,979 0,153

3.3 Microvickers Hardness Measurement of hardness value of CIC is using a hardness tester with a suppressor on the sample surface with a 136 angle of diamond pyramid-shaped. The amount of loads should be greater than the yield strength of metals to plastic deformation occurs. VHN values taken on 3 (three) points of the sample surface so that CIC hardness values obtained as in Table (2) and Figure 6.

Grafik Kekerasan CIC terhadapVariasi komposisi Co-Cr

I II

IIIIV

V

0

100

200

300

400

500

600

0 1 2 3 4 5 6

CIC

VHN (kgf/mm)

Figure 6: Vickers Hardness CIC

Increasing Cr composition should increase the value of CIC hardness because of the nature of individual Cr is harder than the Co. But apparently at the highest composition of Cr (30%), hardness value is decreased. As shown in Figure (6). The presence of oxide compounds CoO, Co3O4, CrO2, believed to be the cause of the hardness decreases. Besides, the samples do not appear homogeneous, as seen from the standard deviation of hardness value is still quite large.

3.4 Compressive strength Compressive strength is the maximum stress that can be accepted by a material in the form of compression or tension without failure. The tools used are autograph equipped with a Control / Measure Unit. This tool provides a force from above or from below the surface of the sample so that the magnitude of P (force) when the specimen

Vickers Hardness Vs Composition Co-Cr Variation

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is damaged will be read on the instrument so that the compressive strength of CIC can be calculated, as shown in Table (2).

Grafik Kekuatan Tekan CIC Terhadap

Variasi komposisi Co-Cr

I

II IIIIV V

0

20

40

60

80

100

120

0 1 2 3 4 5 6CIC

(N/mm)

Figure 7: Compressive Strength of CIC

Figure (7) show a good correlation between the compressive strength of the composition of Co-Cr variation. The images provide information that the compressive strength value of the CIC will continue to rise if the composition of Cr increase (decrease compositions Co.), this is in line with the density of the CIC. Value of compressive strength of human cortical bone by 30-160 N/mm2 then from the result above shows that the value of compressive strength CIC 79.589 N/mm2 - 102.979 N/mm2 have compatibility with these networks.

3.5 The results of cytotoxicity test Elisa Reader readings indicate that the CIC is not toxic to fibroblast cells (cell lines), this is indicated by the percentage of living cells is still above 60 percent ( 60%), the optical density of the treatment is still close to the optical density of control.

Table 3: The percentage of fibroblasts with MTT test

Sampel Optical Density (OD) % Living Cells (OD)

I 0,177 0 033 78,606 II 0,191 0 036 81,317 III 0,209 0 034 84,947 IV 0,221 0 040 87,294 V 0,266 0 043 95,837 Table (3) is the result of calculating the value of the optical density of each sample CIC invested on the eagle and the EMS media containing BHK-21 fibroblast cells. Optical density can be interpreted as the ability of a material to absorb the light. OD value equivalent to the number of live cells within the CIC. The higher the OD value then the more and more of the living cell, so that the table shows that most living cells is occur in cells with a CIC V environment .

Figure 8: Grafic % Live Cell of CIC

Figure (8) shows the cells can live in the CIC environment, so that CIC can be applied in biomaterials. According to in vitro studies have been conducted, stating that the cobalt is toxic to humans as in cell lines in culture medium.

However, chromium and alloys CoCr are well tolerated without significant toxicity by cell lines with concentrations below 50%. In this study, the composition of chromium is added at 18-30%, with the hope of Co-Cr composition variations on the CIC does not cause any toxic properties. If associated with XRD results, which make up the CIC of CrO2 that have properties not toxic because they do not contain Cr6+ ions that are dangerous to human tissue. Most living cells are shown by the CIC V with OD value of 0.266 0.043 where the value of its OD close to the OD in the control cells, namely the composition with the highest Cr (composition Co lowest).

4 Conclusions

1. Variation in the composition of Co-Cr affects the mechanical properties (hardness and compressive strength) as well as cytotoxicity CIC.

2. Value of hardness and compressive strength increases as an increasing of Cr composition (decrease in Co) and the value of both in accordance with the cortical bone.

3. Toxicity value decreases as an increasing of Cr composition (decrease in Co). The entire samples in this study are not toxic indicated by the percentage of cells that lived more than 60%.

References

[1] Black, Jonathan, Hastings, Garth, Handbook of Biomaterials Properties, First Edition, London: Chapman and Hall, 1998.

Compressive strength Vs Composition Co-Cr Variation

Citotoxicity Vs Composition Co-Cr Variation

%Live cell

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[2] Budiatin, Aniek Setiya, Autograph sample inspection procedure, SP4 increase in the ISS program, LDB Airlangga University, Surabaya, 2008.

[3] Goenharto, Siniawati and Achmad Syefei, Titanium Bracket, Part Ortodonsia Faculty of Dentistry Airlangga University Surabaya, Indonesia, 2008.

[4] Ismiawati, Ike Dyah, Analysis of Mechanical Properties and Structure of Dental Enamel Hydroxyapatite Crystals on the result of ND-YAG Laser Exposure, Physics Department- Faculty of Mathematics and Natural Sciences, Airlangga University, 2008.

[5] Nuriana, Wahidin, Synthesis of Titanium-hydroxyapatite: A Quality Improvement Efforts Synthesis of hydroxyapatite as a Bone, Dissertation Graduate Airlangga University, 2005.

[6] Rachadini, Novianita, Test Citotoxicity Siwak Sawdust Extract (Salvadora persica) in Cell Culture Using MTT Essay, Experimental laboratory, Faculty of Dentistry, Airlangga University, 2007.

[7] Buddy D. Ratner, Allan S. Hoffman, Biomaterials Science: An introduction to materials in medicine, Academic Press: USA, 1996.

[8] Park, Joon B. Bronzino, Joseph, Biomaterials Prinsiples and Applications, Vol 1.USA: CRC Press, 1996

[9] Shamsul A.Z, Nur Hidayah, and C. M. Ruzaldi. Fibrication and Properties of Cobalt-Chromium Composite HAP. School of Materials Engineering. Universiti Malaysia Perlis. International Journal of Science ISSN 09734589 Volume 3, Number 1 (2008), pp 35-31 Research India Publication.

[10] Shamsul, AZ, Nur Hidayah, and CM Ruzaldi, Characterization of Cobalt-Chromium-HAP Biomaterials for Biomedical Applications, School of Materials Engineering. Universiti Malaysia Perlis. Journal of Apllied Science Research, 3 (11): 1544-553.2007, INSInet Publication.

[11] Sidhartha, Winiati, Sitoksisitas three kemasa eugenol against human fibroblast cells against gilgiva. Research institutes, University of Indonesia. Faculty of Dentistry. University of Indonesia, Jakarta, 2002.