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ORIGINAL ARTICLE

Effect Of Surface Characteristics Of Anodized Ti-6Al-4V Implant Material On Osteoblast Attachment And Proliferation

K.K. Saju, Sasidharan Vidyanand, N.H. Jayadas, Jackson James, M.K. Jayaraj

Cochin University of Science & Technology

Address for Correspondence:

K.K. Saju
Cochin University of Science & Technology, Kochi, India.

Phone:  
Fax    :
E-mail:
kksaju@cusat.ac.in
 

Abstract:

Anodized implant surfaces which exhibit surface parameters like roughness, micro porosities etc are found to result in faster bone formation around a metallic implant. Cell adhesion on to an implant surface is found to be based on protein adhesion molecules and these molecules are learnt to favor a more hydrophobic surface. In this study Ti6Al4V implant material was anodized to varying levels and cell adhesion to these surfaces were studied by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The degree of influence of surface wet ability on cell adhesion was compared with other surface parameters like roughness, micro porosities etc. The anodized Ti6Al4V sample which showed maximum hydrophobic nature also exhibited higher surface roughness and porosities which are favorable for osteoblast adhesion and showed highest cell viability. However it was noted that the anodized samples with lesser hydrophobic nature than even the control surface of plain polished Ti6Al4V also exhibited good cell adhesion characteristics in the cell viability studies. It was concluded that even though protein adhesion molecules favor a more hydrophobic anodized surface ,the other factors like surface roughness and micro porosities of surfaces compensates for a lesser hydrophobic anodized surface to give appreciable cell adhesion.

J.Orthopaedics 2009;6(1)e5

Keywords:

Ti6Al4V; Anodization; MTT assay; cell viability

Introduction:

Titanium and its alloys are the materials of choice for most dental and orthopedic implants due to its biocompatibility and excellent mechanical properties [1-3]. Among the Titanium alloys TiAl6V4 (Ti) is the most commonly used implant material [1-3]. Bone response and tissue integration with the implant material depends on the physical and chemical properties of the surface. Different surface modification techniques have been developed for increasing the surface properties of  (Ti), anodization being one of them[4].It has been well established that the adhesion of osteoblasts on to an implant surface is by the interaction of cell adhesion protein molecules and these molecules favor surfaces with comparatively lesser wetability, higher  roughness and porosities [5-8]. Anodized surfaces exhibit these characteristics but these parameters vary with varying degree of anodization. In this study Ti samples have been acid anodized at different voltages to varying degree of anodization and the influence of their surface properties and contact angle measurements were studied to assess their protein adhesion characteristics as compared to a control surface as well as an acid etched surface (deoxidized). The results have been counterchecked using a cell viability study of osteoblast cells on the surface by an MTT assay and confocal imaging.

Materials and Methods:

Medical grade Ti disks of 15 mm diameter and 2mm thickness were cleaned ultrasonically in acetone for 20 minutes and later cleaned in 70% ethanol solution and washed with distilled water. The samples were etched in knolls reagent (2ml HF (40%) and 4 ml HNO3 (66%) in 1000 ml of water) and rinsed in distilled water and dried in air. One set of etched sample was used for comparative studies.

2.1 Procedure for anodization [9-10]

Set up for anodization of the samples is shown in fig1.Ti was anodized in 200 g/L sulfuric acid, 5% trisodium phosphate, and 5% sodium bicarbonate (baking soda). The electrolyte was contained in a chemical resistant tank with Fume extraction. A D.C. electrical supply with voltage regulation from 2 to 100 volts and sheet lead cathodes were provided.

Fig 1: Set up for anodization of the Ti samples

The parts to be treated was immersed in the processing solution and connected as the anode to the electrical D.C. source. The temperature of the bath was maintained in the range of 20- 26C throughout the duration of treatment.

The cell voltage was varied between 50-75 volts and three anodized samples were obtained at 55 volts with yellow surface appearance (sample labelled as yellow),60 volts with pink surface appearance (sample labelled as pink) and 75 volts with blue surface appearance (sample labelled as blue) respectively . The time of treatment was 15 minutes for each sample. Immediately after removal from the anodizing bath, parts were washed thoroughly in clean running water, rinsed in clean hot water and allowed to dry.

2.2 Characterization of Ti samples [11]

2.2.1 Film Thickness

The thickness of the anodized surfaces and average roughness was measured using a Veeco Dektak 6M stylus profilometer.

 2.2.2 Surface Morphology

Surface morphology of the samples was examined using Scanning Electron Microscope (SEM) (Hitachi S-2500, Tokyo, Japan).

2.2.3 Contact angle measurement

The contact angles of the samples with three different fluids were measured using a NRL contact angle goniometer (USA) using the sessile drop method [12] in three well characterized liquids, water, formamide and di-iodomethane as per previous studies [13].

 2.3 Cell Viability studies

2.3.1 Cell Culture

Osteosarcoma cell line KHOS-NP (R-970-5) [NCCS] were grown in culture medium (DMEM+10%FBS+1mM NEAA) in a T-25 flask and incubated at 37oC for 2 days in a 5% C02 incubator (Thermo).    ~ 5x105cells were plated on to three samples each of anodized, etched and plain polished control samples of Ti in 12 well plates. The culture was incubated for 72 hours at 37oC in 5% CO2 incubator. The samples with attached cells were used for MTT assay and confocal microscopy.

2.3.2 MTT assay [14]

For MTT assay, all the samples were transferred to a fresh plate and 800 mL of MTT reagent was added to each well and incubated for 2 hours at 37OC. MTT transformed to dark blue formazan by mitochondrial dehydrogenises enabling cell viability to be assessed. 800 mL of lysis buffer (20%Sodium Dodecyl Sulphate 50% Dimethyl Formamide 30% Distilled water) was added to each well, mixed and incubated at 37oC for 4 hours. 200 mL of each sample was transferred to a fresh 96 well plate and the optical density of the solution was measured at 570nm in an ELISA microplate reader (Biorad USA). Analysis of optical variance was used to evaluate difference in cell viability between the groups [15]. 

2.3.3 Confocal Imaging

Osteosarcoma cells were grown on another set of anodized etched and control Ti samples in culture medium for 48 hours.  After which the cells were fixed with 4% Paraformaldehyde ( PFA), washed twice in 1X Phosphate Buffered Saline (pH 7.4) (PBS) and incubated in DAPI (4’, 6-Diamidino-2-phenyindole) (1:1000) for 10 minutes at room temperature (280C). Cells were imaged for nuclear visualization thereafter using a confocal microscope (Leica TCS SPE Germany).

Results and Discussion:

The plots of contact angle for the various implant surfaces are shown in fig 2. As can be seen the contact angle for the anodized sample (blue) at 75 volts is the highest in all fluids, whereas the samples anodized at lower voltages (pink and yellow) have lesser or comparable contact angles to that of the control sample as

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Fig 2: Plot of contact angles of the various samples in different mediums.

as well as the etched (deoxidized) sample. The plot of surface roughness and surface thickness of the samples are shown in fig 3 and fig 4. It is seen that the surface roughness and thickness varies almost linearly with the degree of anodization.

Fig 3: Plot of average surface roughness of the various samples.

Fig 4: Plot of surface thickness of various samples

The anodization thickness also increases linearly with the anodization voltages. The scanning electron micrographs of the various surfaces are shown in fig5-8.The sample anodized at the highest voltage of 75 volts exhibit highest micro-porosities and surface asperities followed by the ones anodized at lower voltages. Etched sample (deoxidized) shows distinct etch line topography.

5

SampleII-04(yellow) with mark

6

Sample IV-05(Pink) with mark

7

Etched Sample I-01

8

Fig 5-8: SEM micrographs of anodized and etched samples 5. Ti anodized at 75 volts, 6.Ti anodized at 60 volts, 7.Ti anodized at 55 volts, 8.HF etched Ti.

Variance analysis plot for the different samples subjected to MTT assay is shown in fig 9.It is seen that the sample anodized at highest voltage of 75 volts (blue) exhibits maximum cell viability followed by the sample anodized at lower voltages (pink and yellow) and the etched (deoxidized) and control sample. 

Fig 9: Optical variance analysis plot to assess the cell viability of the samples

The confocal images of the adhered nucleus to the various samples are shown in figs 10-14. 

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10

C:\Users\sony\Desktop\de-oxy.tif

11

C:\Users\sony\Desktop\yellow.tif

12

C:\Users\sony\Desktop\Blue.tif

13

C:\Users\sony\Desktop\t pink.tif

14

Fig 10-14: Confocal Visualization of nuclear density on various samples.Fig 10- Control TiAl6V4,Fig 11- Etched Sample,Fig 12- Anodized at 55V( Yellow),Fig 13-Anodized at 60 V(Pink),Fig 14-Anodized at 75 V(Blue).

The nuclear density as shown by the white patches is maximum for the blue sample and decreases for the pink and yellow samples. The etched sample showed nuclear density just lower than the pink sample. The nucleus density confirms the maximum cell adhesion characteristics of the sample anodized at 75 V followed by the sample anodized at lower voltages and comparable nuclear density for the etched sample to that of the pink sample. The cell viability of the control sample is found to be the lowest.

As made clear in previous studies, adhesion of cells on to substrates occur through adhesion molecules which are proteins [5-6] .These proteins favour adhesion to hydrophobic, rough and porous surfaces. Since the Ti surface anodized at 75 V (blue) exhibits highest hydrophobic nature, surface roughness and maximum porosities, it presents the ideal condition for cell adhesion and is established in the cell viability study. However even though the wetability of the other anodized (pink and yellow) and etched samples are only comparable or even less than that of the plain polished control sample they exhibited higher cell adhesion than the control sample. This can be attributed to the higher roughness and porous natures of these samples in relation with the control sample. Hence even though the adhesion protein molecules which determine the adhesion characteristics of  cells on to an implant surface is known to favour a hydrophobic surface ,the other surface conditions like roughness, micro-porosities ,thickness are seen to override the dependence of cell adhesion on hydrophobicity of an anodized implant surface.

Conclusions:

Ti substrates were anodized to varying degrees and their surface parameters for cell protein adhesion like wetability, roughness, micro-porosity and thickness were studied and the influence of wetability on cell adhesion was compared with the other surface parameters thro a MTT assay and confocal imaging of adhered nuclear density. An anodized surface which has the highest hydrophobic nature also exhibited maximum roughness and porosity and was seen to exhibit the highest cell adhesion characteristics. However other anodized surfaces which exhibited lesser hydrophobic nature than the control Ti surface also exhibited good cell adhesion. This shows the dependence of cell adhesion more on the surface parameters like roughness, micro porosities and thickness within this range than on the wetability of the surface. This factor can be considered for selection of anodized implant material for orthopaedic use. Further study is required to optimize for roughness and hydrophobicity of anodized implant material.

Acknowledgements:

The authors wish to place on record their gratitude to the various research scholars of Indian Institute of Science, Bangalore, India for having assisted in the contact angle measurements. Special thanks to the research scholars of the Optoelectronics laboratory of the Department of Physics Cochin University of Science and Technology, Kerala, India and Rajiv Gandhi Centre for Biotechnology Thiruvananthapuram, Kerala, India for facilitating the various surface analyses. Timely advises and suggestions given by Dr K.V.Menon, Amrita Institute of Medical Science , Kerala, India and Dr H.K.Varma, Sree Chitra Thirunal Institute of Medical Sciences Kerala, India are appreciated.

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This is a peer reviewed paper 

Please cite as: K.K. Saju: Effect Of Surface Characteristics Of Anodized Ti-6Al-4V Implant Material On Osteoblast Attachment And Proliferation

J.Orthopaedics 2009;6(1)e5

URL: http://www.jortho.org/2009/6/1/e5

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