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Effect of hydroxyapatite coated bioactive glass and ethyl vinyl acetate on antioxidant defense mechanism, oxidative DNA damage and chromosomal anomalies.

Introduction

Oxidative stress is an abnormal phenomenon occurring inside our cells or tissues when production of free radicals exceeds their antioxidant capacity and may results in a series of events which deregulates the cellular functions leading to various pathological conditions [1]. Excess of free radicals damage essential macromolecules like proteins, lipids and DNA present in the cell, leading to abnormalities. A major defense mechanism in the body is the antioxidant enzymes, which catalyze decomposition of free radicals. The major antioxidant enzymes include glutathione reductase and glutathione peroxidase which differs from each other in structure, tissue distribution and cofactor requirement. Glutathione reductase, also known as GSR or GR, is responsible for reducing glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant [2]. "Glutathione peroxidase (GPx) is a selenocysteine-dependent enzyme that protects against oxidative injury and is the most important hydrogen peroxide ([H.sub.2][O.sub.2])-removing enzyme in the mammalian cells [3]. Glutathione itself plays a major role as a reductant in oxidant-reduction processes, and also serves in detoxification [4].

All ROS have the potential to interact with cellular components including DNA bases or the deoxyribosyl backbone of DNA to produce damaged bases or strand breaks. The hydroxylation of the deoxyguanosine residue in DNA by an oxygen radical, such as the hydroxyl radical [5], results in the formation of 8-hydroxy-2_deoxyguanosine (8-OHdG). 8-OHdG is a conventional model to demonstrate ROS-induced DNA damage, because this modified base is important in mutagenesis and carcinogenesis [6].

The use of artificial implants may be a permanent solution for individuals having dysfunctions. This dysfunction can be compensated by medical devices produced with special biomaterials. The potential noxious effects of leachable materials from the implants as well as effects of chemical elements present in the composition of medical implants are a constant concern in patients fitted with medical devices [7, 8].

Hydroxyapatite is one of the most widely used calcium phosphate-based bioceramics. Bioactive Glass (BGA) and Ethyl Vinyl Acetate (EVA) are two biomaterials used as an ideal bone substitute [9]. The use of Hydroxyapatite coated BGA and EVA particles promotes a much faster proliferation of new bone tissues comparable to that occurring after the use of autogenous bone graft. These biomaterials have shown the ability to help in bone regeneration and clinical insertion gain, with better results than other materials available [10].

In a particular tissue, the relationship between the level of oxidative stress and the protective role that the antioxidant enzymes is vital for normal functioning although the mechanisms of operation is not as simple as it looks. Oxidative stress was assayed by examining lipid peroxidation, antioxidant enzymes and oxidative DNA damage. The present investigation was designed to evaluate the oxidative stress of BGA and EVA by using the above mentioned parameters.

Materials and Methods

Chemicals and Reagents

Thiobarbituric acid (TBA), reduced glutathione (GSH), oxidized glutathione (GSSG), dithio-bis-2-nitrobenzoic acid (DTNB), hydrogen peroxide ([H.sub.2][O.sub.2]), disodium hydrogen phosphate ([Na.sub.2]HP[O.sub.4]), sodium dihydrogen phosphate ([Na.sub.2]HP[O.sub.4]), ethylene triamine tetra acetic acid (EDTA), agarose, RNase, ethanol, bromophenol blue, ethidium bromide, physiological saline, DNeasy blood and tissue Mini Kit (Qiagen, Hilden, Germany) and High sensitive 8-OHdG check ELISA kit (Japan Institute for the Control of Aging, Fukuroi, Japan or Genox Corp., Baltimore, MD). All the chemicals and reagents used were of analytical grade.

Equipments

Rotor stator homogenizer at 900 rpm (Polytron, PT 3100), freezer mill, 96-well plate (Nunc), micro-plate reader (Asys Expert plus, Austria), Refrigerated centrifuge (Eppendorf, 5810R, USA), UV visible spectrophotometer (UV-1601, Shimadzu, Japan), and ABX Microsoft automated biochemical analyzer.

Experimental animals

Wistar rats (weighing 170-230g) and Albino Rabbits (above 2000g) were used in this study. The animals were procured from the Division of Laboratory Animal Sciences of Biomedical Technology Wing, Sree Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India.

Individual animals were identified with tattoo marks on its ears (rabbit) and picric acid marks on rats. In addition to this, each animal cage was identified by labels having details such as study number, study name, animal number(s), date of experiment initiation and experiment completion. All the animals were acclimatized for a period of 5 days before initiation of experiment

Animal husbandry and welfare

All animals were handled humanely, without making pain or distress and with due care for their welfare. The care and management of the animals will comply with the regulations of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Govt. of India. All the animal experiments were carried out after prior approval from Institutional Animal Ethics Committee and in accordance with approved institutional protocol.

Animals were maintained in a controlled environment with a temperature 22 [+ or -]3oC, humidity of 30-70% and a light/dark cycle of 12 hrs. The animals were provided with commercially available feed and aqua guard filtered fresh drinking water, ad libitum.

Biomaterials for experiment

The biomaterials used in all aspects were Bioactive Glass and Ethyl Vinyl acetate from Bioceramics and Polymer Processing Laboratories, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India.

Biomaterials extract preparations

Two grams of the two biomaterials were extracted in 10ml of physiological saline. The extraction was carried out at a temperature of 70oC in an incubator shaker for 24hr. After the incubation period extracts were collected in a sterile beaker and were used for different assays.

Implantation of HA-BGA in rabbit femur bone

The animals were grouped into two; one set as control and other as the test. All the animals were anaesthetized with ketamine (2ml/kg) and xylazine (0.25ml/kg) following atropine sulphate (0.25ml/kg) subcutaneously and diazepam (0.4ml/kg) pre- medication, by intramuscular administration and place the animal on the surgical table. Swab the clipped skin lightly but thoroughly with 70% alcohol followed by betadine solution. Expose the cortex region of each femur and 2.0 mm size holes were made by drilling using low speed drill with profuse irrigation with saline. Both the test and control materials were made into cylindrical shape of 2.0 mm in diameter and 6.0 mm in length. The implants were inserted into the holes and the wounds were closed using sutures.

Implantation of HAP-EVA in rat brain

The animals were randomly divided into different groups with eight numbers of animals in each group. All animals were anesthetized by means of intramuscular injection of ketamine hydrochloride 100 mg/kg (Ketalar, 5% solution, Eczacibasi with Parke-Davis, Levent, Istanbul, Turkey) and xylazine 5 mg/kg IM (Rompun, 2% solution, Bayer, Istanbul, Turkey). They were then fixed on the table and their scalps were shaved and cleaned with 10% Povidone iodine. A calvarial defect was performed with a high-speed drill on the right parietal bone of all rats. The dura mater was kept intact during this procedure. The implants were inserted into the holes and the wounds were closed using sutures.

All the implanted animals were observed for an observation period of 1, 4, 12, 26 and 52 weeks. After the experimental periods the animals were sacrificed bone, brain and liver were rapidly excised, washed in normal ice-cold physiological saline, and immediately placed in an ice bath.

Measurement of enzymatic activities

For antioxidant assays 10% liver homogenates were made in Tris-HCl (0.1M, pH 7.4) using an ice-chilled glass homogenizing vessel in a rotor stator homogenizer at 900 rpm (Polytron, PT 3100). The suspended mixtures were centrifuged at 3500 rpm for 10 min at 4 [degrees]C in a refrigerated centrifuge (Eppendorf, 5810R). The resultant supernatants were maintained in an ice bath until used for the estimation of total protein and different antioxidant parameters using standard protocols with slight modifications. Enzymic antioxidant status alters due to oxidative stress mediated material leachants. Alterations in the levels reflect the severity of the damage. The various biochemical parameters were measured in UV visible spectrophotometer (UV-1601, Shimadzu, Japan).

Estimation of protein in liver homogenate

Determination of protein concentration was performed in liver homogenate by the method of Lowry et al, [11] with bovine serum albumin as standard and expressed as mg/ml.

Assay for Lipid Peroxidation (LPO)

The extent of LPO was determined as the concentration of malondialdehyde (MDA) generated by the thiobarbituric acid reactive substances (TBARS), as described by Ohkawa et al., 1979 [12]. The amount of malondialdehyde (MDA) formed was measured spectrophotometrically at 532 nm.

Assay for Reduced glutathione (GSH)

GSH level in the liver homogenate was determined by the method of Moron et al., 1979 [13] with slight modifications in which Ellman's reagent or DTNB (5, 5'- dithiobis-(2-nitrobenzoic acid), reacts with GSH to form a spectrophotometrically detectable product at 412 nm. The change in absorbance at 412 nm is a linear function of the GSH concentration in the reaction mixture and is based on the reaction of GSH with DTNB to give a compound that absorbs at 412 nm. The amount of GSH was expressed as n mole / mg protein.

Assay for glutathione reductase (GR)

Determination of glutathione reductase activity was performed as previously described by Mize and Langdon, 1962 [14]. Briefly, this assay measures the rate of NADPH oxidation to NADP+, which is accompanied by a decrease in absorbance at 340nm, so GR activity can be monitored spectrophotometrically. Thus, one GR unit is defined as the reduction of one [micro]M of GSSG per minute at 25[degrees]C and pH 7.6.

Assay for glutathione peroxidase ([GP.sub.x])

Activity of GPX was assayed by the method described by Rotruck et al., 1973 [15]. The remaining GSH after the enzyme catalyzed reaction was complexes with 5, 5'dithiobis 2-nitrobenzoic acid (DTNB) that absorbs at maximum wavelength of 412 nm. Enzyme activity was expressed as [micro]g of GSH consumed /min/mg protein.

Genomic DNA extraction

For genomic DNA isolation, the bone samples were cleaned and powered in freezer mill. The powered samples were stirred using magnetic stirrer in 0.5 M EDTA for decalcification. Decalcified samples were used for genomic DNA isolation.

Genomic DNA was extracted from rat brain and decalcified bone tissue using the DNeasy blood and tissue Mini Kit (Qiagen, Hilden, Germany) with minor modifications in manufacturer's protocol. Each tissue was incubated overnight at 56[degrees]C with 180il Buffer ATL and 50il proteinase K (20mg/ml). 10il RNase (1mg/ml) was added and incubated at room temperature for 5min. Incubated samples were treated with 230il of AL buffer at 70 [degrees]C for 10min. Ethanol (230il of 99%) was added to the suspension, mixed thoroughly by vortexing. The mixture was applied to the DNeasy spin column sitting in a 2ml centrifuge tube and centrifuged at 8000rpm for 2min. DNA binds to the silica spin filter while contaminants pass through. Remaining contaminants and enzyme inhibitors are removed by a wash step. Pure DNA is then eluted in a low salt Tris buffer (pH-8) to allow for pH stabilization of the DNAin storage. To maximize DNA yield, two successive elution steps were performed each with 50il elution buffer.

Determination of DNA concentration

Purity check: After ensuring complete solubility of DNA, the purity factor (260/280nm) was determined spectrophotometrically (UV-1601, Shimadzu, Japan). The concentration of 50[micro]g/ml was taken as one unit of optical absorption for double stranded--helical DNA at 260nm with optical path of 1cm.

Agarose gel electrophoresis : The integrity was checked on 0.7% agarose gel stained with ethidium bromide (10mg/mL) by loading 12 [micro]l of DNA preparation (2[micro]l extracted DNA, 2[micro]l gel loading buffer containing 25% bromophenol blue and 30% glycerol, 8[micro]l sterilized deionized water). The electrophoresis was carried out at 100 V using 1x TAE buffer for 30min. After electrophoresis, the gel was observed under an UV lamp in a gel documentation system (Alpha Innotech).

Analysis of Brain and bone 8-OHdG

8-OHdG in brain and bone DNA digests was determined by a competitive Immunosorbent assay using High sensitive 8-OHdG check (Japan Institute for the Control of Aging, Fukuroi, Japan or Genox Corp., Baltimore, MD). Samples pretreatment includes DNA were dissolved in water at a concentration of 50 [micro]g and made into single stranded by incubating at 95[degrees]C for 5min and rapidly chilling on ice. Samples were then digested into nucleosides, by incubating the denatured DNA with 10 [micro]l 0.5M sodium acetate, 1.25[micro]l 1M magnesium chloride and 1 [micro]l DNease I for 10min at room temperature. The reaction mixtures were then centrifuged for 1min at 2000rpm and the supernatants were collected and stored for further study.

50 [micro]l of pretreated samples along with standards were placed in the 96-well plate (Nunc). Assays were carried out as per the manufacture's instruction. At the end of experiment the color developed is proportional to the amount of antibody in the plate, which in turn is inversely related to the amount of 8-OHdG in the serum sample. Lower color means higher amounts of 8-OHdG. Results are expressed in nanograms per milliliter. The absorbance of the reaction mixture was read at 450 nm with a micro-plate reader (Asys Expert plus, Austria). Curve fitting was done with Digi Swift software. The unknown concentration of 8-OHdG in each sample was determined by generating standard curves for each lot of assay reagents from standardized samples contained in each ELISA kit. The mean of each subject's three samples was computed and then interpret by statistical analyses.

Peripheral blood lymphocyte culture and GTG Banding

Heparinized human whole blood (0.4 mL) was cultured at 37[degrees]C for 72 h in 10 mL Karyotyping medium. After 48 h cells were treated with physiological saline extract of the biomaterials EVA and BGA and CP at a dose of 100 ig/culture [16] was used as positive controls. The treatment lasted for 24 h in the absence of S9 mix and 3 h in the presence of S9. The S9 fraction was used at a final concentration of 5% in RPMI medium. After 3 h of treatment with material extracts and CP in the presence of S9, the cells were replaced with fresh karyotype medium. Cells were arrested with colchicine (1 ig/culture) for 2 h. Chromosome preparations were made by incubating the cell suspension with 0.075 M KCl at 37[degrees]C for 13 min, followed by a fixation step using freshly prepared mixture of 3:1 methanol: acetic acid solution. GTG banding was performed by incubating the glass slides on 0.025% trypsin for 10-20s, followed by rinsing the slides with phosphate-buffered saline and staining using 2% Giemsa stain for 10 min. The slides were rinsed with water and were air dried. The dried slides were then mounted using DPX and were then screened for chromosomal aberrations using automated metaphase finder (Carl Zeiss, Germany). Respective lymphocyte culture slides with metaphase chromosomes were analyzed.

Statistical analysis

All the samples were run in duplicate; differences were statistically assessed using student t-test. The results obtained were expressed as Mean [+ or -] Standard deviation (SD). For all comparisons, a pd" 0.05 was considered for a statistically significant analysis.

Results

For the present investigation control material used is ultra high molecular weight polyethylene (UHM) for both the biomaterials and hydroxyapatite coated EVA and BGA were implanted as the test biomaterials.

Lipid peroxidation

The amount of malondialdehyde in control and four different observation period of rats implanted with the biomaterial EVA is demonstrated in figure 1. The level of LPO present in the 4th week (44.7 nmol/mg protein) is observed to be higher than the control value (35.12 nmol/ mg protein). The amount found to be decreasing in the subsequent period. Except 26th week all other periods were found to be slightly significant (pd"0.01).

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The LPO level in liver homogenates of control and four different observation period of rabbits implanted with BGA are demonstrated in figure 2. Similar to that of EVA the amount of malondialdehyde in 4th week (22.76 nmol/ mg protein) was seemed to be increased when compared with the respective control. The concentration of LPO in the later periods was decreasing and observed to reach the control values (15.99 nmol/mg protein). All the periods were found to be slightly significant pd"0.01.

Reduced glutathione

Figure 3 depicts the concentration of GSH in control and four different observation period of rats implanted with the biomaterial EVA. The amount of GSH in the 4th week was 4.04860 nmol/mg protein and seemed to be increasing in the succeeding periods. The concentration of GSH in the 52nd week (4.183 nmol/mg protein) is found to be similar to that of the control value. All the period were found to be nonsignificant pe"0.05.

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The GSH concentration in control and four different observation period of rabbits implanted with BGA is represented in figure 4. GSH concentration in liver homogenate was observed to be increasing in all the observation period when compared to that of the control value 5.971 nmol/mg protein. All the periods were found to be slightly significant p<0.01.

Glutathione reductase

The concentration of GR in rats liver homogenate implanted with EVA and control are shown in figure 5. The amount of GR in the control (0.61514 units/mg protein) and 26th week were observed to be similar. The level of GR in 4th week was decreased to 0.5567 units/ mg protein and increased to 0.625 units/mg protein when compared with the control. All the period were found to be nonsignificant pd"0.05.

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Figure 6 shows the level of GR in control and four different observation period of rabbit implanted with BGA. GR concentration found to be increasing in all the observation period with that of the respective control value (0.699 units/mg protein). The concentration of GR in 12th week was found to be slightly significant pd"0.01 and all other periods were nonsignificant with the control value pd"0.05.

Glutathione peroxidase

GPx expression in the liver homogenate of control and rats implanted with EVA are represented in figure 7. The level of GPx in the 4th week (0.443 units/mg protein) is found to be increased when compared with that of the respective control (0.437 units/mg protein). But the level observed to be decreased in the 12th week (0.415 units/ mg protein) and increasing in the following period. All the observation period were found to be nonsignificant pd"0.05.

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Figure 8 demonstrates the level of GPx concentration in the liver homogenate of control and the four different observation period of rabbits implanted with BGA. The expression of GPx in the 4th week (0.638 units/mg protein) was increased when compared with that of the control value (0.218 units/mg protein) and seemed to be decreasing in the subsequent weeks. The 12th and 26th weeks were observed to be slightly significant with the control pd"0.01 and the 4th and 52nd week were found to be nonsignificant pd"0.05.

Influence of the biomaterials on 8-OHdG

Figure 9 depicts the amount of 8-OHdG in the genomic DNA isolated from the control and four different observation period of rats brain implanted with EVA. The concentration of 8-OHdG was found to be increasing in the 4th week (2.226 8-OHdG/ 105 dG) and decreasing in the 12th week (2.209 8-OHdG/ 105 dG) when compared to that of the control (2.2185 8-OHdG/ 105 dG). The level found to be increasing in the succeeding periods. Except 12th week all other periods were found to be nonsignificant pd"0.05.

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The concentration of 8-OHdG in the genomic DNA isolated from the control and four different observation period of rabbits femur bone implanted with BGA is presented in figure 10. The expression of 8-OHdG was found to be increasing in all the observation period. The level observed to be decreased in the 4th week (1.7205 8-OHdG/ 105 dG) and increasing in the following weeks when compared with the control value (1.8358-OHdG/ 105 dG). All the weeks observed to be nonsignificant (pe"0.05).

G banding of human chromosomes

The cyclophosphamide and physiological saline treated human lymphocyte cultures are shown in figure 11 and 12. The former shows a break in the 11q 13.1 position of chromosome 11 and 16q 22 of chromosome 16 and structural deformalities in the 12q 11.1 of chromosome 12 and 7q 31.1.1 of chromosome 7. The physiological saline treated lymphocyte culture represents the normal chromosomal G banding. Exposure of physiological saline extracts of EVA for 72 hours with and without the metabolic activator S9 is presented in figure 13 and 14. Similarly, lymphocytes culture treated with physiological saline extract of BGA for 72 hours with and without metabolic activator S9 is presented in figure 15 and 16. Both biomaterials did not show up any significant chromosomal aberrations in the GTG banded chromosomes.

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Discussion

A cellular oxidative stress condition is determined by an imbalance between the generation of free radicals and the antioxidant defense capacity of the cell and can affect major cellular components including lipids, proteins and DNA. The free radicals can also influence the gene expression profile by affecting intracellular signal transduction pathway [17]. Hepatic damage is associated with the increases in tissue lipid peroxidation and also the depletion in the tissue glutathione levels [18]. Hydroxyl radicals is highly reactive and one of the most oxidizing species in living systems and have the ability to react with cellular bio molecules [19].

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Lipid peroxidation is a major harmful consequence of ROS formation [20] as it reflects irreversible oxidative changes of cell membranes. Malondialdehyde is the principal and the most studied product of polyunsaturated fatty acid peroxidation. In the present investigation, rats implanted with EVA shows slightly significance except for 26th week (pd"0.01). But all the observation period of rabbits implanted with BGA were found to be slightly significant ((pd"0.01). In the first week of observation, their found an in the increase the level of peroxidation of lipids in both the biomaterials may be due to the oxidative stress created by the implantation. But the results explain that even though there occurs a minute peroxidation in the cell membrane, it found to overcome in the due course of time. Hence the leachants from the biomaterials shows less damage to the hepatocytes.

[FIGURE 16 OMITTED]

According to Jiangxue 2006 [21], GSH plays a vital role in the protection of cells against oxidative stress and acts as an important water-phase non-enzymatic antioxidant and an essential cofactor for antioxidant enzymes taking part in cellular redox reactions. The level of GSH in the case of EVA shows no significant differences. Since there is no significant difference in the GSH level, it protects cellular proteins against oxidation through glutathione redox cycle and also directly detoxifies reactive oxygen species and/or neutralizes reactive intermediate species. Although BGA implantation results in a slight significance, it also did not show much damage. GSH depletion is likely to have led to the accumulation of peroxides, which could be transformed to ROS through the Fenton reaction [22]. Hence their was no adverse damage on GSH, both the materials had no effect on fenton pathway.

Glutathione reductase [23] is an important enzyme for the maintenance of glutathione in the reduced form, and possibly for controlling the redox state of NADP in tissues if GSSG is available. The GSH/GSSG concentration ratio in hepatocytes depends on the change in GPx/GR ratio. Since there is a positive correlation between the rate of oxidation of GSH and the glutathioine peroxidase activity, this enzyme is responsible for maintaining cell integrity by protecting it against the deleterious effects of peroxides [24]. However, the in vivo results of the present study showed that the activity of GPx and GR of rats implanted with the biomaterial EVA shows no significance (Pd"0.05) with the control values which explains the capability of these enzymes to provide a continuous flow of reduced form of glutathione as a substrate for endogenous antioxidant enzyme. In case of BGA both the enzymes shows slight significance with the control and the results found to be to be adaptive changes in the cellular glutathione system as well as GPx and GR. These two enzymes are the two most important in the GSH-GSSG cycle and may be activated by increased hydrogen and/ or lipid peroxide production [25]. The expression of GR explains the capability of this enzyme to provide a continuous flow of reduced form of glutathione as a substrate for endogenous antioxidant enzyme like glutathione peroxidase which plays a critical role in combating oxidative stress.

The result of the present study demonstrates that the concentration of 8-OHdG in the brain tissues of rat implanted with EVA and rabbit femur bone implanted with BGA respectively does not have any significant difference from their respective controls. In most of the Oxidative stress studies 8-OHdG has been used as a biomarker for indicating the level of oxidative DNA damage. Methylation of cytosine in DNA results in the conversion of guanine to 8-OHdG which may alters the gene expression and can cause carcinogenesis [26]. Fenton pathway results in hydroxyl radicals, a highly destructive ROS which are capable of inducing a wide array of mutagenic single-base substitutions and DNA adducts [27], From the present study it was revealed that since their was no leachable material from both the biomaterials that were able of altering the C-8 position of guanine, the biomaterials does not have any influence on the Fenton pathway and also carcinogenesis.

GTG-banding of chromosomes is the most commonly used banding technique for the detection of numerical and structural chromosomal abnormalities. The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light. Most importantly, G banding produces reproducible patterns for each chromosome, and these patterns were shared between individuals in the species [13]. In the present study, lymphocytes treated with the mutagen cyclophosphamide shows breaks and structural anomalies in the chromosomes but the physiological saline extract treated culture do not show any aberrations. Lymphocytes culture treated with physiological saline extract of EVA and BGA with and without S9 did not show any aberration indicates that the material is non-toxic.

In summary, the concerted experiment revealed that the implantation of EVA and BGA in rat and rabbit femur bone has not induce excessive production of free radicals, demonstrated by the level of enzymatic and non enzymatic antioxidants in liver homogenate and 8-OHdG in brain and bone tissues, compared to the controls in our study. The results of the G- banding also shows no significant alteration in the chromosome structure. Hence, these studies support that the biomaterial Hydroxyapatite coated EVA and BGA, can be safely used as an effective bone substitute in the medical field.

Funding

This work was supported by Indian Council of Medical Research (grant numbers No.5/20/1 (Bio)/07-NCD-I), New Delhi

Acknowledgments

Authors are thankful to the Director and the Head BMT Wing, SCTIMST for providing the facilities to carry out the work. We are gratified to Dr. P. Ramesh, Dr. H. K. Varma, Scientists of SCTIMST for providing the materials. The authors gratefully acknowledge S. Shaji and G. Harikumar for their technical assistance.

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Anjana Vaman VS, Tinu SK, Geetha CS

Toxicology Division, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695 012, Kerala, India

Mohanan PV Corresponding Author, mohanpv@sctimst.ac.in

Received 31 March 2012; Accepted 20 April 2012; Available online 8 May 2012
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Title Annotation:Original Article
Author:Anjana, Vaman V.S.; Tinu, S.K.; Geetha, C.S.; Mohanan, P.V.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:9INDI
Date:Apr 1, 2012
Words:5320
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