Original Article
Genotoxicity effect, antioxidant and biomechanical correlation: Experimental study of agarose–chitosan bone graft substitute in New Zealand white rabbit model
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(8) 800–809 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914547247 pih.sagepub.com
Samira Jebahi*,1,2,3, Ghada Ben Saleh4, Mongi Saoudi3, Salma Besaleh2, Hassane Oudadesse1, Moufida Mhadbi2, Tarek Rebai2, Hassib Keskes2 and Abdelfattah El Feki3
Abstract Bone loss associated with skeletal trauma or metabolic diseases often requires bone grafting. In such situations, a biomaterial is necessary for migrated cells to produce new tissue. In this study, agarose–chitosan was implanted in the femoral condyle of New Zealand White rabbits that were divided into three groups: Group I was used as control; Groups II and III were used as implanted tissue with agarose–chitosan and presenting empty defects, respectively. This study evaluated the agarose–chitosan biocompatibility by determining the in vivo genotoxicity, oxidative stress balance that correlated with the hardness mechanical property. Moreover, the histopathological and quantitative elements analyzed by using inductively coupled plasma optical emission spectrometry were determined. After 30 days of implantation, the in vivo analysis of genotoxicity showed that agarose–chitosan did not induce chromosome aberration or micronucleus damage. A significant decrease in thiobarbituric and acid-reactive substance was observed after agarose–chitosan implantation in the bone tissue. Superoxide dismutase, catalase and glutathione peroxidase were significantly enhanced in agarose–chitosan–treated group compared with that of control group. A negative correlation coefficient of the mechanical property with malonyldialdehyde level was detected (R = 20.998). The histological study exhibited a significantly increased angiogenesis and newly formed tissue. No presence of inflammatory process, necrotic or fibrous tissue was detected. Major and trace elements such as Ca, P, Zn, Mg and Fe were increased significantly in the newly formed bone. These findings show that agarose–chitosan biomaterial implantation might be effective for treating trauma and bone regeneration.
Keywords Agarose–chitosan, graft biomaterial, antioxidative profile, bone regeneration, genotoxicity, mechanical hardness test
Date received: 7 November 2013; accepted: 14 July 2014
Introduction As a natural biomaterial, chitosan (CH) offers many biological activities.1,2 The importance of the chemical composition of CH has been explored in the fields of tissue engineering. Its adjustable amine content on the surface was explored for enhancing adhesion, chemotaxis and differentiation of bone cells. Further to cell attachment, one of the foremost challenges of tissue engineering is designing the scaffold that provides structure while the tissue regenerates. Polymer scaffolds applied as space filling agents and delivery vehicles for bioactive molecules help to maintain structures that organize cells and present stimuli to direct the
*
Samira Jebahi and Ghada Ben Saleh contributed equally to this work.
1
UMR CNRS 6226, University of Rennes 1, Rennes, France Histology, Orthopaedic and Traumatology Laboratory, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia 3 Animal Ecophysiology Laboratory, Department of Life Sciences, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia 4 Laboratory of Human Molecular Genetics, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia 2
Corresponding author: Samira Jebahi, UMR CNRS 6226, University of Rennes 1, Campus de Beaulieu, 263 av. du Ge´ne´ral Leclerc, 35042 Rennes, France. Email: [email protected]
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formation of a desired tissue. Agarose (Ag), a biodegradable polymer, has been selected as an organic matrix because it is a biocompatible material which acts as gelling agent. It exhibits macromolecular properties similar to those of the extracellular matrix and allows enough diffusion and transport of oxygen, essential nutrients and secretary products across its network and thus provides a friendly environment for cellular spreading and proliferation.3 During callus formation, fibroblast and collagen cells, new capillary vessels as well as other inflammatory cells increase the production of oxygen free radicals.4 Due to the attack of oxygen free radicals on the lipid component of membrane, the lipid peroxide content is elevated. To evaluate this, estimation of levels of many intermediate lipid peroxides and their end-products has been used to indirectly evaluate the oxidative stress. The most reliable indicators are malonyldialdehyde (MDA). After bone healing, woven bone is quickly formed and poorly organized with a more or less random arrangement of collagen fibers and mineral crystals. Thus, disruption of the balance between antioxidant defense and accumulation of reactive oxygen species (ROS) in the bone tissue results in disorders in the bone metabolism and mechanical strength.5 A relationship between the oxidative/antioxidative status, bone mineral density (BMD) and bone healing rate was noted in degenerative tissue.6 One study reported that a 54% and 70% decrease in type X collagen in the fracture callus in rats showed oxidative stress damage and suggested that this might have a role in defective rat bone healing.7 Therefore, we notice today a growing interest in the use of natural antioxidant agent to combat oxidative stress. Currently, there has been a great deal of research interest in the CH antioxidant activity.8 In fact, CH has obvious scavenging activities on superoxide radical. In addition, CH scavenging mechanism is reported to be related to the fact that the free radicals can react with the residual free amino groups NH2 to form stable macromolecule radicals, and the NH2 groups can form ammonium groups NH3+ by absorbing hydrogen.9 Although both CH and Ag have been the subject of intense studies and claimed to be a non-toxic biocompatible polymer in several reports,10 safety has not to date been comprehensively assessed in cytogenetic terms. An example of material used in modern dentistry and presenting a genotoxicity is methyl methacrylate (MMA), which is the mainstream material in denture bases. The sisterchromatid exchange frequencies were found to increase in the concentration of MMA. Genotoxicity tests are conducted via several methods, evaluating cell toxicity and identifying potential mutagens.11 Here, we chose to blend CH with Ag to investigate its potential application for bone regeneration. For that, this study aims to evaluate the Ag-CH biocompatibility through determining the profile of the genotoxicity in the bone morrow cells, oxidative stress and mechanical property after Ag-CH implantation in the femoral condyle of New Zealand white rabbit model.
Material and methods Preparation of Ag-CH biomaterial CH (degree of deacetylation (DDA): 75%–85%, molecular weight (MW): 50,000–190,000 g/mol; Sigma, France) was first dissolved in 2.5 wt% acetic acid solution. SeaPrep Ag (Sigma) was then added to CH solution and dissolved by heating the mixture in a 60 °C water bath.12 Ag and CH were adjusted to obtain biomaterial with Ag:CH ratio of 1:3.
Surgical protocol A total number of 170 New Zealand white rabbits weighing 1.6–1.9 kg were used for the experiments. The rabbits were anesthetized and positioned in a stereotactic apparatus. Surgery was performed under a combination of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight) applied intraperitoneally. An 8-mm-diameter defect was created on the lateral aspect of the femoral condyle using a refrigerated drill to avoid necrosis. The empty defect (ED) was filled with 20 mg of Ag-CH in the implanted group. The filling was done carefully in a retrograde fashion to ensure both minimal inclusion of air bubbles and direct implant–bone contact. The closure of the wounds was performed in layers (i.e. fascias and the subcutaneous tissue), using resorbable material (Vicryl 3/0; Ethicon, Germany) in a continuous fashion. After the surgical operation, all rabbits received subcutaneous analgesia (carprofen 10 mg/kg RimadylÒ) for three postoperative days. The animals were allowed to move freely in their cages without restriction. The animals were sacrificed at 7, 15 and 30 days postoperatively. The handling of the animals was approved by the Tunisian Ethical Committee for the care and use of laboratory animals. All rabbits were randomly divided into three groups: the first group (Group I) was used as control (CT), and Groups II and III were used as implanted tissue with Ag-CH and presenting EDs, respectively (Figure 1).
Morphological study of Ag-CH The scanning electron microscopy (SEM) (Jeol JSM 6301F) was used to identify the morphological changes and the interaction between bone and AgCH biomaterial. The collected samples were prefixed with 2.5% glutaraldehyde solution (phosphate buffer solution, pH 7.4) (Sigma) overnight and then washed with phosphate buffer solution (pH 7.4). Then, the samples were post fixed with 2% osmic acid solution (Sigma) (phosphate buffer solution, pH 7.4) for 90 min and dehydrated with an alcohol evaporating system. The samples were then freeze-dried with a freeze-dryer (JFD-300 Electron Optic Laboratory) and were carried out with a vapor deposition system (JFC-1200).
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Figure 1. Experimental protocols. CT: Group I used as control; Ag-CH: Group II used as implanted tissue with agarose–chitosan; ED: Group III presenting empty defects; MN: micronucleus; CA: chromosomal aberration.
Histopathological evaluation For histological analysis of the newly formed bone tissue, samples were included in a mixture of MMA and glycol methacrylate (GMA) without prior decalcification. In addition, the decalcified samples of Ag-CH– bone specimens were examined with hematoxylin and eosin staining. The histological observations in the rabbit condyle tissues after 15 and 30 days permitted us to study the repairing potency of the osseous substitute. The histological features, such as the intensity of inflammatory reaction, cellular activity, necrosis, hemorrhage, presence of connective tissue, apposition of tissue and presence of particles of biomaterial, were recorded in a descriptive way after observation with a light microscope (Olympus Optical, Japan).
Tissue preparation and stress oxidative Thiobarbituric acid–reactive substance measurements. The femoral condyles were removed and rapidly frozen by dry ice. The two tissues of all groups were minced and homogenized (100 mg/mL) at 4 °C in 0.1 mol/L TrisHCl buffer pH 7.4 and centrifuged at 3000 g for 10 min. The implanted femoral condyles of all groups were minced and homogenized (100 mg/mL) at 4 °C in 0.1 mol/L Tris-HCl buffer pH 7.4 and centrifuged at 3000 g for 10 min. Lipid peroxidation in the tissue homogenate was estimated by measuring thiobarbituric acid–reactive substances (TBARS) and was expressed in terms of malondialdehyde (MDA) content which is the end-product of lipid peroxidation.13 Antioxidant enzyme studies. Regarding the superoxide dismutase (SOD) activity, it was assayed by the spectrophotometric method of Marklund and Marklund.14 Glutathione peroxidase (GPx) activity was measured by the method described by Pagila and Valentine.15
Catalase (CAT) activity was assayed calorimetrically at 240 nm and expressed as moles of H2O2 consumed per minute per milligram of protein, as described by Aebi.16 The level of total protein was determined by the method of Lowry et al.17 using bovine serum albumin.
Genotoxic parameters Chromosomal aberration assay. The preparation of bone marrow cells and the assessment of chromosomal aberrations were carried out as described previously.18 Animals were yeast-stimulated overnight and injected with an antimitotic solution (vinblastine sulfate). After the sacrifice, both femurs were immediately dissected out and were cut at both ends with bone snips. Bone marrow was extracted and incubated for 18 min at 37 °C in 8 mL hypotonic KCl (0.075 M), then centrifuged and fixed twice with fresh methanol:glacial acetic acid (3:1). The cell suspensions were dropped on cold glass slides, and they were air-dried. They were allowed to air dry and then stained with 5% Giemsa solution (pH 6.8) for 15 min. All slides were coded before being scored. In total, 60 metaphases from each point were analyzed (a total of 100 metaphases per duplicate point). Only unstable aberrations were recorded, namely, gap (G), break (B), exchange (EXC), dicentric (DIC) and premature centromere division (PCD). In vivo micronucleus assay. The bone marrow was flushed out by gentle flushing and aspiration with fetal calf serum.19 The cell suspension was centrifuged and a small drop (3 mL) of the re-suspended cell pellet was spread on a microscope slide and stained in with MayGru¨nwald/Giemsa.20 Three slides per animal were stained with acridine orange (AO) and washed twice with phosphate buffer (pH 6.8) as described
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previously.21 Micronuclei (MNs) were scored with a Zeiss Axioskop microscope cat 1003 following previously proposed criteria.22 Approximately 2000 erythrocytes were scored per animal to estimate the frequency of micronucleated erythrocytes.
Hardness measurement The hardness of the bone–biomaterial junction was measured by using MEKTON Vickers Tester at a load of 0.5 kg. In this test, force–displacement data of a pyramidal diamond were pressed into the bone material and were recorded. Measurements are taken at three different locations in the center, biomaterial–bone interface and the edge of specimens. Each test is repeated at least five times to get a good average with minimum deviation. The Vickers hardness number (HV) is the ratio of the load applied to the indenter to the surface area of the indentation HV = 2Psin (h/2)/D2, where P is the applied load in kilograms, D is the mean length of the diagonals in millimeter and h is the angle (136°) between opposite faces of the diamond.23
Determination of bone mineral content using inductively coupled plasma optical emission spectrometry Femoral condyle was dried for 24 h at 65 °C. Dry bones were weighed accurately and placed in 25-mL tubes; 2 mL of nitric acid was added. One milliliter of 30% H2O2 (Sigma) was placed in the tube after 10 min. The volume of the mixture was made up to 500 mL with distilled water. Standard solutions of Ca, P, Mg, Zn and Fe were used to prepare the working standard solution and a blank solution. The element concentrations were detected using inductively coupled plasma optical emission spectrometry (ICP-OES; Ciros; Spectro Analytical Instrument, Germany).
Statistical analysis The statistical data analysis was made using Student’s t-test. All values were expressed as mean 6 standard error (SE). Simple linear regression analysis and Pearson’s correlation coefficient were used to assess the relationship between the mechanical property of the bone–Ag-CH and the oxidative stress marker.
Results Genotoxic parameters The DNA damage at the chromosomal level entails alterations in either chromosomal number or chromosome structure. Such alterations can be measured as chromosomal aberrations or MN frequency. MN assay in rabbit bone marrow receiving Ag-CH graft at day 30 did not show any significant difference as compared with those of CT rabbit group (p . 0.05) (Figure 2) and Ag-CH-treated rabbits. On the other hand, the chromosomal aberration assay showed that under this experimental condition, Ag-CH did not mark any DNA damage (Figure 3). In fact, the different types of chromosomal aberrations such as simple strand break (SSB), PCD, EXC, DIC and total chromosomal aberrations (TCA) did not show significant changes (p . 0.05) when compared with those of CT and ED groups (Table 1).
Oxidative damage in the bone tissue The detailed quantitative assessments of the oxidative stress bio-marker as well as the antioxidant enzyme profile after 15 days of the Ag-CH installation in the bone tissue are shown in Table 2. In fact, MDA levels in the femoral condyle tissue following 15 days of implantation were significantly different from those of the CT (p 4 0.05). SOD, CAT and GPx activities in the rabbit bone tissue showed a highly significant decrease when
Figure 2. Effect of Ag-CH exposition to the bone marrow (acridine orange staining; 1003) damaged erythrocyte contained many MN normal erythrocytes.
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Figure 3. Normal chromosomes in the control rabbit and dicentric chromosomes in the Ag-CH-treated bone marrow rabbits.
Table 1. The type and the frequency of chromosomal aberrations and the percentage of aberrant metaphases and micronucleus in bone morrow cells of CT and Ag-CH groups. Group
CT Ag-CH
Type of aberration SSB
PCD
EXC
DIC
GAP
0.000 0.000
0.050 0.000
0.025 0.029
0.000 0.030
0.000 0.023
TCA
Aberrant metaphase (%)
Micronucleus frequency (&)
0.075 0.200
7.50 9.00
0.028 0.034
SSB: simple strand break; PCD: premature centromere division; EXC: exchange; DIC: dicentric; TCA: total chromosomal aberrations.
Table 2. Effects of Ag-CH biomaterial on CAT, SOD and GPx activities in femoral condyle tissue and MDA level after 7, 15 and 30 days. Parameters TBARS (nmol MDA/mg prote´ine) SOD (Unite´/mg prote´ine) CAT (mmol/min/mg prote´ine) GPx (mmol/min GSH/mg prote´ine)
Control ED Ag-CH CT ED Ag-CH CT ED Ag-CH CT ED Ag-CH
7 days
15 days
30 days
6.8 6 01 6 0.05 15.20 6 0.09* 16.23 6 0.02* 14.25 6 1.3 8.36 6 0.2* 7.11 6 0.77* 12.18 6 1.01 7.18 6 0.81* 7.10 6 0.11* 31.9 6 05 22.20 6 0.01* 20.21 6 1.51*
6.24 6 0.07 14.51 6 0.04* 12.60 6 0.09* 13.38 6 2.03 8.33 6 0.5* 9.11 6 0.4* 13.02 6 1.91 9.31 6 0.91* 11.02 6 0.91 32.19 6 0.91 22.18 6 1.01* 23.13 6 1.21*
6.22 6 0.08 12.45 6 0.03* 7.63 6 0.04 13.00 6 0.9 9.27 6 0.33* 13.23 6 0.55 12.18 6 1.1 10.46 6 1.2 11.82 6 1.21 30.10 6 0.83 24.13 6 2.01* 28.99 6 2.01
SE: standard error; CT: control; MDA: malonyldialdehyde; TBARS: thiobarbituric acid–reactive substances; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; ED: empty defect; Ag-CH: agarose–chitosan. Values are given as mean 6 SE. *Significantly different enzymatic activity in group presenting empty defect and Ag-CH versus the CT group.
compared with those of CT rabbit tissues. After 30 days of implantation, there were significant changes in the bone tissue detected when compared with those of ED CT group.
Ag-CH–bone hardness measurements exhibited a similar transitional pattern to that of CT femoral condyle bone. It went up to even higher levels after 30 days and it reached 32.13 6 3.2 HV.
Bone hardness measurements
Correlations between the mechanical property and oxidative stress marker
The results of the hardness measurements of the CT femoral condyle and Ag-CH–bone after 7, 15 and 30 days of implantation are represented in Table 3. The
A negative correlation R = 20.998 was exhibited between the mechanical property of the bone–Ag-CH
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Table 3. Hardness values of the control femoral condyle, the implanted group with Ag-CH and group presenting ED. Hardness test (HV)
7 days
15 days
30 days
CT Ag-CH ED
34.89 6 3.3 18.17 6 3.7* 11.17 6 2.1*
35.17 6 6.1 23.23 6 1.1* 17.33 6 3.3*
35.90 6 5.3 32.13 6 3.2 21.43 6 4.7*
CT: control; ED: empty defect; Ag-CH: agarose–chitosan. *Significantly different values in the indicated group than CT.
graft and MDA content which is the end-product of lipid peroxidation.
Bone histopathological study Under SEM, Figure 4 allows to visualize the interaction between rabbit bone tissue and Ag-CH biomaterial. After 15 days of surgery, some defect regions were still occupied by implant (Figure 4(a)). Ag-CH has been shown to encourage cell spreading and improve the biocompatibility and interconnection with neighboring cells (Figure 4(b)–(d)). After 30 days of surgery, collagens were deposited in an orderly way (Figure 4(e)). These features demonstrate that Ag-CH was well tolerated by the host and the in-growth becomes more pronounced with increasing time periods of implantation. After 30 days of surgery, a fibrous tissue could be seen in the regenerated bone of ED group (Figure 5(a)). On the other hand, the staining section showed an interaction between Ag-CH and the bone tissue (Figure 5(b)). Moreover, the histological staining showed collagen fibers covered biomaterial (Figure 5(c)). No inflammatory cells such as macrophages were seen after 30 days. In this period of time, osteoid tissue with intense cell activity could be seen (Figure 5(d)). The results indicated that after 1 month of implantation, Ag-CH was associated with osteogenesis capability.
Determination of bone mineral content using ICP-OES The contents of Ca, P, Mg, Zn and Fe were significantly decreased in ED group when compared to that of Ag-CH-treated rabbits (p \ 0.01) as illustrated in Table 4. As a result of Ag-CH implantation, the content of Zn was significantly decreased when compared to that of the CT group. As regards Ca, P, Mg and Fe, no significant differences were observed in Ag-CH-treated rabbits when compared to those of the CT group.
Discussion This study investigated the effect of Ag-CH biomaterial on the bone healing. To our knowledge, there is no reported study about the Ag-CH potential of genotoxicity, antioxidant defense and the correlation with the mechanical property. For the standard biological evaluation of medical devices, the International Standard
Organization (ISO) has outlined the need for genotoxicity testing in ISO 10993-3.24 The chromosome abnormalities are a direct consequence of DNA damage such as double-strand breaks and misrepair of strand breaks. MNs were formed in dividing cells from chromosome fragments or whole chromosomes that were unable to engage with the mitotic spindle during mitosis.25 In this study, the data collected from bone marrow cells showed that Ag-CH did not significantly exert genotoxic effects. Similar results were observed by other authors using different ceramic compounds. Dental porcelain is considered as non-genotoxic when cultured with fibroblastic cells.26 However, some biomaterials such as MMA are clinically used in several orthopedic applications at present. In spite of this satisfactory clinical use, a quantitative analysis showed a dose-dependent increase in the number of chromosome aberrations. The aberration type induced by MMA was the chromatid-type aberrations.27 This indicated that MMA induced chromosome aberrations in late S or G2 phase. Genotoxic effects will eventually lead to abnormal and reduced cell growth, even if the cells initially appear cytocompatible. One study reported that the prepared CH with concentrations of 3.00 and 4.50 mg/mL induced mutation on Trichoderma.28 However, those techniques have some shortcomings (e.g. time investment, requirement for proliferating cell populations). In this study, after bone graft surgery, the increase in MDA level might influence bone through further intensification of oxidative processes and stimulation of osteoclastic bone resorption.29 The bone antioxidative barrier consists of enzymatic antioxidants such as GPx, SOD and CAT. GPx formed the main part in the enzymatic antioxidative defense system in the bone tissue.30 This enzyme is responsible for lipid peroxide detoxification and together with CAT acts for H2O2 detoxification, whereas SOD is responsible for dismutation of O22 to molecular oxygen and H2O2.31 Additionally, in this study, the major and trace element kinetic in the implanted bone was determined. It is noted that there is an important relationship between the antioxidative profile and the ossification kinetic. In fact, bone bioelements are necessary for proper function of the antioxidative enzymes such as Zn, Fe, Se and Cu. GPx is a Se-dependent enzyme, CAT contains Fe in its active center, whereas SOD activity is dependent on Zn and Cu32 In this study, the decrease in SOD activity in the bone tissue might be explained, at least
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Figure 4. SEM image in vivo shows (a) Ag-CH biomaterial and bone attachment at 15 days, (b)–(d) interconnection between AgCH biomaterial and neighboring cells and (e) collagen of the newly formed bone. Arrow indicates bone–Ag-CH interaction, *indicates osteoblasts, circle indicates the cells’ interaction and short head arrows indicate collagen.
partly, by this bioelement depletion. After 30 days of implantation, Fe trace content, performed in Ag-CH– bone area, revealed the presence of cavities previously filled with red blood cells. The Ca measurements in the newly formed bone indicated the deposition of significant amounts of mineralized matrix with a significant increase in Ca-P. Because oxidative stress negatively impacts bone healing, attempts to enhance the healing process have been investigated through the present mechanical bone correlation. In this study, an inverse
linear relationship between mechanical hardness and TBAR level (R = 20.998) was detected after 30 days of implantation. In a recent study, the increase in ROS release in bone exhibited consistent changes with impaired healing, including smaller calluses with decreased bone and reduced mechanical strength compared with those of CT bone.33 Moreover, an approximately 20% decrease in biomechanical strength in femurs related to oxidative stress in a rat model was reported.34 This study demonstrated that the disorders
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Figure 5. (a) Fibrous tissue with island of mineralized bone in the rabbit group presenting empty defect. (b) Bone–Ag-CH biomaterial interaction. (c) Collagen of the newly formed bone. (d) Osteoid tissue with intense cell activity showing effective osteogenesis. (a)–(c) Goldner’s trichrome staining (103 objective). Hematoxylin–eosin staining (403 and 1003 objective).
Table 4. Distribution of Ca, P, Zn, Mg and Fe in CT femoral condyle bone, after Ag-CH implantation and in group presenting ED during 15 and 30 days.
CT Ag-CH ED
15 days 30 days 15 days 30 days 15 days 30 days
Ca (mg/g)
P (mg/g)
Zn (mg/g)
Mg (mg/g)
Fe (mg/g)
255 6 50 250 6 80 245 6 45 255 6 78 $ 170 6 38*$ 200 6 17*
143 6 66 141 6 90 145 6 80 142 6 50 $ 91 6 17*$ 103 6 25*
0.26 6 0.07 0.25 6 0.09 0.16 6 0.08* 0.29 6 0.07 0.15 6 0.07*$ 0.17 6 0.05*
4.1 6 0.1 4.6 6 0.1 3.25 6 0.7 3.82 6 0.8 $ 1.95 6 0.08*$ 2.45 6 0.09*
760 6 40 720 6 40 790 6 20 720 6 30 $ 870 6 45*$ 810 6 45*
CT: control; ED: empty defect; Ag-CH: agarose–chitosan. *Significantly different level in the indicated group than CT. $ Significantly different level in the indicated group than Ag-CH group.
in the bone oxidative/antioxidative balance might affect the bone tissue by enhancing lipid peroxidation and oxidative protein damage. MDA has been reported to contribute to the destruction of collagen and proposed to serve as a measure of osteoclastic activity.35,36 As appropriate collagen network determines proper formation and bone biomechanical properties (hardness), its oxidative damage compromises the bone biomechanical properties making it susceptible to deformities and fractures.37 This study revealed that the bone graft weakened the antioxidative capacity of the bone tissue and led to oxidative stress during the first 15 days. The antioxidative stress profile was enhanced after 30 days of implantation. The deficits in bone repair were corrected by Ag-CH biomaterial treatment when compared with the non-implanted bone rabbits. In fact, CH has been
reported to stimulate the synthesis of collagen as well as inhibit its degradation.38 Moreover, many studies revealed that the scavenging mechanism might be related to the fact that hydroxyl group can react with active hydrogen atoms in CH to form a most stable macromolecule radical. The scavenging activities of CH derivatives against hydroxyl group might be derived from some or all of the following: (1) the hydroxyl groups in the polysaccharide unit can react with hydroxyl group by the typical H-abstraction reaction;39 (2) hydroxyl group can react with the residual free amino groups NH2 to form stable macromolecule radicals and40 (3) the NH2 groups can form NH3+ groups by absorbing hydrion from the biological fluid and then reacting with hydroxyl group through addition reaction.41 On the other hand, Ag has the property of
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self-gelation at low temperature and has good mechanical and elastic behavior. To assess whether the matrix proteoglycan (PG) deposited within the Ag material was mechanically and electromechanically functional, the time evolution of dynamic mechanical stiffness and oscillatory streaming potential were measured in uniaxial confined compression. The analysis showed PG-rich matrix and collagen fibrils developing around cells. The dynamic stiffness and oscillatory streaming potential increased many times.42 These results suggested the formation of a mechanically functional matrix. The present Ag-CH biomaterials are of great importance and practical usefulness because it may suggest that manipulation of Ag-CH in the bone tissue may be a useful approach in the prevention against tissue damage due to trauma and surgery.
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Conclusion Based on these results, the chromosomal aberration and the in vivo MN assays confirmed the absence of the genotoxicty. The in vivo results showed an increase in GPx, SOD and CAT activities in the bone tissue after 30 days of implantation. The Ag-CH biomaterial inhibited the disruption of the enzymatic antioxidative profile. The mechanical hardness test showed a significant improvement of bone strength. The histological study implies that Ag-CH could directly impact the morphological development of bone cells and provide a compatible environment for bone tissue. Therefore, all these results suggested that the developed Ag-CH as a promising bone healing might have potential applications for hard tissue repair. Declaration of conflicting interests No potential conflict of interest relevant to this article was reported.
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Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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megakaryocyte proliferation, differentiation, and maturation. Cell Death Dis 2013; 11: 722. Morikawa D, Nojiri H, Saita Y, et al. Cytoplasmic reactive oxygen species and SOD1 regulate bone mass during mechanical unloading. J Bone Miner Res 2013; 28: 2368–2380. Cervellati C, Bonaccorsi G, Cremonini E, et al. Oxidative stress and bone resorption interplay as a possible trigger for postmenopausal osteoporosis. Biomed Res Int 2014; 2014: 569563 (8 pp.). Topping R, Bolander M and Balian G. Type X collagen in fracture callus and the effects of experimental diabetes. Clin Orthop Relat Res 1994; 308: 220e8. Jebahi S, Oudadesse H, Ben Saleh G, et al. Chitosanbased bioglass composite for bone tissue healing: oxidative stress status and antiosteoporotic performance in a ovariectomized rat model. Korean J Chem Eng. Epub ahead of print 6 May 2014. DOI: 10.1007/s11814-0140072-9. Tian F, Liu Y, Hu K, et al. The depolymerization mechanism of chitosan by hydrogen peroxide. J Mater Sci 2003; 38: 4709–4712. Shuang F, Hou SX, Zhao YT, et al. Characterization of an injectable chitosan-demineralized bone matrix hybrid for healing critical-size long-bone defects in a rabbit model. Eur Rev Med Pharmacol Sci 2014; 18: 740–752. Bhamra MS and Case CP. Biological effects of metal-onmetal hip replacements. Proc IMechE, Part H: J Engineering in Medicine 2006; 220: 379–384. Cao Z, Gilbert RJ and He W. Simple agarose-chitosan gel composite system for enhanced neuronal growth in three dimensions. Biomacromolecules 2009; 10: 2954–2959. Buege JA and Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1984; 105: 302–310. Marklund S and Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and convenient assay for superoxide dismutase. Eur J Biochem 1975; 47: 469–474. Pagila DE and Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158–169. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121–126. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with Folin phenol reagent. J Biol Chem 1951; 193: 265–275. Boujbiha MA, Ben Salah G, Ben Feleh A, et al. Hematotoxicity and genotoxicity of mercuric chloride following subchronic exposure through drinking water in male rats. Biol Trace Elem Res 2012; 148: 76–82. Valette H, Dolle F, Bottlaender M, et al. Fluoro-A-85380 demonstrated no mutagenic properties in in vivo rat micronucleus and Ames tests. Nucl Med Biol 2002; 29: 849–853. D’Souza UJ and Narayana K. Mechanism of cytotoxicity of ribavirin in the rat bone marrow and testis. Indian J Physiol Pharmacol 2002; 46: 468–474. Hayashi M, Tice RR, MacGregor JT, et al. In vivo rodent erythrocyte micronucleus assay. Mutat Res 1994; 312: 293–304. Heddle JA, Fenech M, Hayashi M, et al. Reflections on the development of micronucleus assays. Mutagenesis 2011; 26: 3–10.
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23. Ling Q, Pikkwan F, Hongbin L, et al. Low intensity pulsed ultrasound increases the matrix hardness of the healing tissues at bone–tendon insertion—a partial patellectomy model in rabbits. Clin Biomech 2006; 21: 387–394. 24. Hassan A and Swaminathan D. Kajian In Vitro untuk Menilai Genotoksisiti Hidroksiapatit Tambah Nilai sebagai Bahan Tulang Gantian [An in vitro study to evaluate the genotoxicity of value added. Hydroxyapatite as a bone replacement material]. Sains Malays 2011; 40: 163–171. 25. AshaRani PV, Mun GLK, Hande MP, et al. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 2009; 3: 279–290. 26. Noushad M, Kannan TP, Husein A, et al. Genotoxicity evaluation of locally produced dental porcelain—an in vitro study using the Ames and Comet assays. Toxicol In Vitro 2009; 23: 1145–1150. 27. Yang H-W, Chou L-S, Chou M-Y, et al. Assessment of genetic damage by methyl methacrylate employing in vitro mammalian test system. Biomaterials 2003; 24: 2909–2914. 28. Keong LC and Halim AS. In vitro models in biocompatibility assessment for biomedical-grade chitosan derivatives in wound management. Int J Mol Sci 2009; 10: 1300–1313. 29. Jebahi S, Oudadesse H, El Feki H, et al. Antioxidative/ oxidative effects of strontium-doped bioactive glass as bone graft. In vivo assays in ovariectomised rats. J Appl Biomed 2012; 10: 195–209. 30. Sharma P, Bhushan Jha A, Dubey RS, et al. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012; 2012: 217037 (26pp.). 31. Wang GG, Li W, Lu XH, et al. Taurine attenuates oxidative stress and alleviates cardiac failure in type I diabetic rats. Croat Med J 2013; 54: 171–179. 32. Kumar BA, Reddy AG, Kumar PR, et al. Protective role of N-Acetyl L-Cysteine against reproductive toxicity due
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to interaction of lead and cadmium in male Wistar rats. J Nat Sci Biol Med 2013; 4: 414–419. Baek KH, Oh KW, Lee WY, et al. Association of oxidative stress with postmenopausal osteoporosis and the effect of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. Calcif Tissue Int 2010; 87: 226–235. Graves DT, Alblowi J, Paglia DN, et al. Impact of diabetes on fracture healing. J Exp Clin Med 2011; 3: 3–8. Sheweita SA and Khoshhal KI. Calcium metabolism and oxidative stress in bone fractures: role of antioxidants. Curr Drug Metab 2007; 8: 519–525. Cvijetic´ S, Grazio S, Gomzi M, et al. Muscle strength and bone density in patients with different rheumatic conditions: cross-sectional study. Croat Med J 2011; 52: 164–170. Estai MA, Suhaimi F, Shuid AN, et al. Biomechanical evaluation of fracture healing following administration of Piper sarmentosum in ovariectomised rats. Afr J Pharm Pharmaco 2012; 6: 148–156. Yan LP, Wang YJ, Ren L, et al. Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications. J Biomed Mater Res A 2010; 95: 465–475. Xie W, Xu P and Liu Q. Antioxidant activity of watersoluble chitosan derivatives. Bioorg Med Chem Lett 2001; 11: 1699–1701. Yan JK, Wang WQ, Ma HL, et al. Sulfation and enhanced antioxidant capacity of an exopolysaccharide produced by the medicinal fungus Cordyceps sinensis. Molecules 2013; 18: 167–177. Halliwell B, Murcia MA, Chirico S, et al. Free radicals and antioxidants in food and in vivo: what they do and how they work. Crit Rev Food Sci Nutr 1995; 35: 7–20. Buschmann MD, Gluzband YA, Grodzinsky AJ, et al. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res 1992; 10: 745–758.
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Genotoxicity effect, antioxidant and biomechanical correlation: Experimental study of agarose–chitosan bone graft substitute in New Zealand white rabbit model
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(8) 800–809 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914547247 pih.sagepub.com
Samira Jebahi*,1,2,3, Ghada Ben Saleh4, Mongi Saoudi3, Salma Besaleh2, Hassane Oudadesse1, Moufida Mhadbi2, Tarek Rebai2, Hassib Keskes2 and Abdelfattah El Feki3
Abstract Bone loss associated with skeletal trauma or metabolic diseases often requires bone grafting. In such situations, a biomaterial is necessary for migrated cells to produce new tissue. In this study, agarose–chitosan was implanted in the femoral condyle of New Zealand White rabbits that were divided into three groups: Group I was used as control; Groups II and III were used as implanted tissue with agarose–chitosan and presenting empty defects, respectively. This study evaluated the agarose–chitosan biocompatibility by determining the in vivo genotoxicity, oxidative stress balance that correlated with the hardness mechanical property. Moreover, the histopathological and quantitative elements analyzed by using inductively coupled plasma optical emission spectrometry were determined. After 30 days of implantation, the in vivo analysis of genotoxicity showed that agarose–chitosan did not induce chromosome aberration or micronucleus damage. A significant decrease in thiobarbituric and acid-reactive substance was observed after agarose–chitosan implantation in the bone tissue. Superoxide dismutase, catalase and glutathione peroxidase were significantly enhanced in agarose–chitosan–treated group compared with that of control group. A negative correlation coefficient of the mechanical property with malonyldialdehyde level was detected (R = 20.998). The histological study exhibited a significantly increased angiogenesis and newly formed tissue. No presence of inflammatory process, necrotic or fibrous tissue was detected. Major and trace elements such as Ca, P, Zn, Mg and Fe were increased significantly in the newly formed bone. These findings show that agarose–chitosan biomaterial implantation might be effective for treating trauma and bone regeneration.
Keywords Agarose–chitosan, graft biomaterial, antioxidative profile, bone regeneration, genotoxicity, mechanical hardness test
Date received: 7 November 2013; accepted: 14 July 2014
Introduction As a natural biomaterial, chitosan (CH) offers many biological activities.1,2 The importance of the chemical composition of CH has been explored in the fields of tissue engineering. Its adjustable amine content on the surface was explored for enhancing adhesion, chemotaxis and differentiation of bone cells. Further to cell attachment, one of the foremost challenges of tissue engineering is designing the scaffold that provides structure while the tissue regenerates. Polymer scaffolds applied as space filling agents and delivery vehicles for bioactive molecules help to maintain structures that organize cells and present stimuli to direct the
*
Samira Jebahi and Ghada Ben Saleh contributed equally to this work.
1
UMR CNRS 6226, University of Rennes 1, Rennes, France Histology, Orthopaedic and Traumatology Laboratory, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia 3 Animal Ecophysiology Laboratory, Department of Life Sciences, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia 4 Laboratory of Human Molecular Genetics, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia 2
Corresponding author: Samira Jebahi, UMR CNRS 6226, University of Rennes 1, Campus de Beaulieu, 263 av. du Ge´ne´ral Leclerc, 35042 Rennes, France. Email: [email protected]
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formation of a desired tissue. Agarose (Ag), a biodegradable polymer, has been selected as an organic matrix because it is a biocompatible material which acts as gelling agent. It exhibits macromolecular properties similar to those of the extracellular matrix and allows enough diffusion and transport of oxygen, essential nutrients and secretary products across its network and thus provides a friendly environment for cellular spreading and proliferation.3 During callus formation, fibroblast and collagen cells, new capillary vessels as well as other inflammatory cells increase the production of oxygen free radicals.4 Due to the attack of oxygen free radicals on the lipid component of membrane, the lipid peroxide content is elevated. To evaluate this, estimation of levels of many intermediate lipid peroxides and their end-products has been used to indirectly evaluate the oxidative stress. The most reliable indicators are malonyldialdehyde (MDA). After bone healing, woven bone is quickly formed and poorly organized with a more or less random arrangement of collagen fibers and mineral crystals. Thus, disruption of the balance between antioxidant defense and accumulation of reactive oxygen species (ROS) in the bone tissue results in disorders in the bone metabolism and mechanical strength.5 A relationship between the oxidative/antioxidative status, bone mineral density (BMD) and bone healing rate was noted in degenerative tissue.6 One study reported that a 54% and 70% decrease in type X collagen in the fracture callus in rats showed oxidative stress damage and suggested that this might have a role in defective rat bone healing.7 Therefore, we notice today a growing interest in the use of natural antioxidant agent to combat oxidative stress. Currently, there has been a great deal of research interest in the CH antioxidant activity.8 In fact, CH has obvious scavenging activities on superoxide radical. In addition, CH scavenging mechanism is reported to be related to the fact that the free radicals can react with the residual free amino groups NH2 to form stable macromolecule radicals, and the NH2 groups can form ammonium groups NH3+ by absorbing hydrogen.9 Although both CH and Ag have been the subject of intense studies and claimed to be a non-toxic biocompatible polymer in several reports,10 safety has not to date been comprehensively assessed in cytogenetic terms. An example of material used in modern dentistry and presenting a genotoxicity is methyl methacrylate (MMA), which is the mainstream material in denture bases. The sisterchromatid exchange frequencies were found to increase in the concentration of MMA. Genotoxicity tests are conducted via several methods, evaluating cell toxicity and identifying potential mutagens.11 Here, we chose to blend CH with Ag to investigate its potential application for bone regeneration. For that, this study aims to evaluate the Ag-CH biocompatibility through determining the profile of the genotoxicity in the bone morrow cells, oxidative stress and mechanical property after Ag-CH implantation in the femoral condyle of New Zealand white rabbit model.
Material and methods Preparation of Ag-CH biomaterial CH (degree of deacetylation (DDA): 75%–85%, molecular weight (MW): 50,000–190,000 g/mol; Sigma, France) was first dissolved in 2.5 wt% acetic acid solution. SeaPrep Ag (Sigma) was then added to CH solution and dissolved by heating the mixture in a 60 °C water bath.12 Ag and CH were adjusted to obtain biomaterial with Ag:CH ratio of 1:3.
Surgical protocol A total number of 170 New Zealand white rabbits weighing 1.6–1.9 kg were used for the experiments. The rabbits were anesthetized and positioned in a stereotactic apparatus. Surgery was performed under a combination of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight) applied intraperitoneally. An 8-mm-diameter defect was created on the lateral aspect of the femoral condyle using a refrigerated drill to avoid necrosis. The empty defect (ED) was filled with 20 mg of Ag-CH in the implanted group. The filling was done carefully in a retrograde fashion to ensure both minimal inclusion of air bubbles and direct implant–bone contact. The closure of the wounds was performed in layers (i.e. fascias and the subcutaneous tissue), using resorbable material (Vicryl 3/0; Ethicon, Germany) in a continuous fashion. After the surgical operation, all rabbits received subcutaneous analgesia (carprofen 10 mg/kg RimadylÒ) for three postoperative days. The animals were allowed to move freely in their cages without restriction. The animals were sacrificed at 7, 15 and 30 days postoperatively. The handling of the animals was approved by the Tunisian Ethical Committee for the care and use of laboratory animals. All rabbits were randomly divided into three groups: the first group (Group I) was used as control (CT), and Groups II and III were used as implanted tissue with Ag-CH and presenting EDs, respectively (Figure 1).
Morphological study of Ag-CH The scanning electron microscopy (SEM) (Jeol JSM 6301F) was used to identify the morphological changes and the interaction between bone and AgCH biomaterial. The collected samples were prefixed with 2.5% glutaraldehyde solution (phosphate buffer solution, pH 7.4) (Sigma) overnight and then washed with phosphate buffer solution (pH 7.4). Then, the samples were post fixed with 2% osmic acid solution (Sigma) (phosphate buffer solution, pH 7.4) for 90 min and dehydrated with an alcohol evaporating system. The samples were then freeze-dried with a freeze-dryer (JFD-300 Electron Optic Laboratory) and were carried out with a vapor deposition system (JFC-1200).
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Figure 1. Experimental protocols. CT: Group I used as control; Ag-CH: Group II used as implanted tissue with agarose–chitosan; ED: Group III presenting empty defects; MN: micronucleus; CA: chromosomal aberration.
Histopathological evaluation For histological analysis of the newly formed bone tissue, samples were included in a mixture of MMA and glycol methacrylate (GMA) without prior decalcification. In addition, the decalcified samples of Ag-CH– bone specimens were examined with hematoxylin and eosin staining. The histological observations in the rabbit condyle tissues after 15 and 30 days permitted us to study the repairing potency of the osseous substitute. The histological features, such as the intensity of inflammatory reaction, cellular activity, necrosis, hemorrhage, presence of connective tissue, apposition of tissue and presence of particles of biomaterial, were recorded in a descriptive way after observation with a light microscope (Olympus Optical, Japan).
Tissue preparation and stress oxidative Thiobarbituric acid–reactive substance measurements. The femoral condyles were removed and rapidly frozen by dry ice. The two tissues of all groups were minced and homogenized (100 mg/mL) at 4 °C in 0.1 mol/L TrisHCl buffer pH 7.4 and centrifuged at 3000 g for 10 min. The implanted femoral condyles of all groups were minced and homogenized (100 mg/mL) at 4 °C in 0.1 mol/L Tris-HCl buffer pH 7.4 and centrifuged at 3000 g for 10 min. Lipid peroxidation in the tissue homogenate was estimated by measuring thiobarbituric acid–reactive substances (TBARS) and was expressed in terms of malondialdehyde (MDA) content which is the end-product of lipid peroxidation.13 Antioxidant enzyme studies. Regarding the superoxide dismutase (SOD) activity, it was assayed by the spectrophotometric method of Marklund and Marklund.14 Glutathione peroxidase (GPx) activity was measured by the method described by Pagila and Valentine.15
Catalase (CAT) activity was assayed calorimetrically at 240 nm and expressed as moles of H2O2 consumed per minute per milligram of protein, as described by Aebi.16 The level of total protein was determined by the method of Lowry et al.17 using bovine serum albumin.
Genotoxic parameters Chromosomal aberration assay. The preparation of bone marrow cells and the assessment of chromosomal aberrations were carried out as described previously.18 Animals were yeast-stimulated overnight and injected with an antimitotic solution (vinblastine sulfate). After the sacrifice, both femurs were immediately dissected out and were cut at both ends with bone snips. Bone marrow was extracted and incubated for 18 min at 37 °C in 8 mL hypotonic KCl (0.075 M), then centrifuged and fixed twice with fresh methanol:glacial acetic acid (3:1). The cell suspensions were dropped on cold glass slides, and they were air-dried. They were allowed to air dry and then stained with 5% Giemsa solution (pH 6.8) for 15 min. All slides were coded before being scored. In total, 60 metaphases from each point were analyzed (a total of 100 metaphases per duplicate point). Only unstable aberrations were recorded, namely, gap (G), break (B), exchange (EXC), dicentric (DIC) and premature centromere division (PCD). In vivo micronucleus assay. The bone marrow was flushed out by gentle flushing and aspiration with fetal calf serum.19 The cell suspension was centrifuged and a small drop (3 mL) of the re-suspended cell pellet was spread on a microscope slide and stained in with MayGru¨nwald/Giemsa.20 Three slides per animal were stained with acridine orange (AO) and washed twice with phosphate buffer (pH 6.8) as described
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previously.21 Micronuclei (MNs) were scored with a Zeiss Axioskop microscope cat 1003 following previously proposed criteria.22 Approximately 2000 erythrocytes were scored per animal to estimate the frequency of micronucleated erythrocytes.
Hardness measurement The hardness of the bone–biomaterial junction was measured by using MEKTON Vickers Tester at a load of 0.5 kg. In this test, force–displacement data of a pyramidal diamond were pressed into the bone material and were recorded. Measurements are taken at three different locations in the center, biomaterial–bone interface and the edge of specimens. Each test is repeated at least five times to get a good average with minimum deviation. The Vickers hardness number (HV) is the ratio of the load applied to the indenter to the surface area of the indentation HV = 2Psin (h/2)/D2, where P is the applied load in kilograms, D is the mean length of the diagonals in millimeter and h is the angle (136°) between opposite faces of the diamond.23
Determination of bone mineral content using inductively coupled plasma optical emission spectrometry Femoral condyle was dried for 24 h at 65 °C. Dry bones were weighed accurately and placed in 25-mL tubes; 2 mL of nitric acid was added. One milliliter of 30% H2O2 (Sigma) was placed in the tube after 10 min. The volume of the mixture was made up to 500 mL with distilled water. Standard solutions of Ca, P, Mg, Zn and Fe were used to prepare the working standard solution and a blank solution. The element concentrations were detected using inductively coupled plasma optical emission spectrometry (ICP-OES; Ciros; Spectro Analytical Instrument, Germany).
Statistical analysis The statistical data analysis was made using Student’s t-test. All values were expressed as mean 6 standard error (SE). Simple linear regression analysis and Pearson’s correlation coefficient were used to assess the relationship between the mechanical property of the bone–Ag-CH and the oxidative stress marker.
Results Genotoxic parameters The DNA damage at the chromosomal level entails alterations in either chromosomal number or chromosome structure. Such alterations can be measured as chromosomal aberrations or MN frequency. MN assay in rabbit bone marrow receiving Ag-CH graft at day 30 did not show any significant difference as compared with those of CT rabbit group (p . 0.05) (Figure 2) and Ag-CH-treated rabbits. On the other hand, the chromosomal aberration assay showed that under this experimental condition, Ag-CH did not mark any DNA damage (Figure 3). In fact, the different types of chromosomal aberrations such as simple strand break (SSB), PCD, EXC, DIC and total chromosomal aberrations (TCA) did not show significant changes (p . 0.05) when compared with those of CT and ED groups (Table 1).
Oxidative damage in the bone tissue The detailed quantitative assessments of the oxidative stress bio-marker as well as the antioxidant enzyme profile after 15 days of the Ag-CH installation in the bone tissue are shown in Table 2. In fact, MDA levels in the femoral condyle tissue following 15 days of implantation were significantly different from those of the CT (p 4 0.05). SOD, CAT and GPx activities in the rabbit bone tissue showed a highly significant decrease when
Figure 2. Effect of Ag-CH exposition to the bone marrow (acridine orange staining; 1003) damaged erythrocyte contained many MN normal erythrocytes.
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Figure 3. Normal chromosomes in the control rabbit and dicentric chromosomes in the Ag-CH-treated bone marrow rabbits.
Table 1. The type and the frequency of chromosomal aberrations and the percentage of aberrant metaphases and micronucleus in bone morrow cells of CT and Ag-CH groups. Group
CT Ag-CH
Type of aberration SSB
PCD
EXC
DIC
GAP
0.000 0.000
0.050 0.000
0.025 0.029
0.000 0.030
0.000 0.023
TCA
Aberrant metaphase (%)
Micronucleus frequency (&)
0.075 0.200
7.50 9.00
0.028 0.034
SSB: simple strand break; PCD: premature centromere division; EXC: exchange; DIC: dicentric; TCA: total chromosomal aberrations.
Table 2. Effects of Ag-CH biomaterial on CAT, SOD and GPx activities in femoral condyle tissue and MDA level after 7, 15 and 30 days. Parameters TBARS (nmol MDA/mg prote´ine) SOD (Unite´/mg prote´ine) CAT (mmol/min/mg prote´ine) GPx (mmol/min GSH/mg prote´ine)
Control ED Ag-CH CT ED Ag-CH CT ED Ag-CH CT ED Ag-CH
7 days
15 days
30 days
6.8 6 01 6 0.05 15.20 6 0.09* 16.23 6 0.02* 14.25 6 1.3 8.36 6 0.2* 7.11 6 0.77* 12.18 6 1.01 7.18 6 0.81* 7.10 6 0.11* 31.9 6 05 22.20 6 0.01* 20.21 6 1.51*
6.24 6 0.07 14.51 6 0.04* 12.60 6 0.09* 13.38 6 2.03 8.33 6 0.5* 9.11 6 0.4* 13.02 6 1.91 9.31 6 0.91* 11.02 6 0.91 32.19 6 0.91 22.18 6 1.01* 23.13 6 1.21*
6.22 6 0.08 12.45 6 0.03* 7.63 6 0.04 13.00 6 0.9 9.27 6 0.33* 13.23 6 0.55 12.18 6 1.1 10.46 6 1.2 11.82 6 1.21 30.10 6 0.83 24.13 6 2.01* 28.99 6 2.01
SE: standard error; CT: control; MDA: malonyldialdehyde; TBARS: thiobarbituric acid–reactive substances; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; ED: empty defect; Ag-CH: agarose–chitosan. Values are given as mean 6 SE. *Significantly different enzymatic activity in group presenting empty defect and Ag-CH versus the CT group.
compared with those of CT rabbit tissues. After 30 days of implantation, there were significant changes in the bone tissue detected when compared with those of ED CT group.
Ag-CH–bone hardness measurements exhibited a similar transitional pattern to that of CT femoral condyle bone. It went up to even higher levels after 30 days and it reached 32.13 6 3.2 HV.
Bone hardness measurements
Correlations between the mechanical property and oxidative stress marker
The results of the hardness measurements of the CT femoral condyle and Ag-CH–bone after 7, 15 and 30 days of implantation are represented in Table 3. The
A negative correlation R = 20.998 was exhibited between the mechanical property of the bone–Ag-CH
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Table 3. Hardness values of the control femoral condyle, the implanted group with Ag-CH and group presenting ED. Hardness test (HV)
7 days
15 days
30 days
CT Ag-CH ED
34.89 6 3.3 18.17 6 3.7* 11.17 6 2.1*
35.17 6 6.1 23.23 6 1.1* 17.33 6 3.3*
35.90 6 5.3 32.13 6 3.2 21.43 6 4.7*
CT: control; ED: empty defect; Ag-CH: agarose–chitosan. *Significantly different values in the indicated group than CT.
graft and MDA content which is the end-product of lipid peroxidation.
Bone histopathological study Under SEM, Figure 4 allows to visualize the interaction between rabbit bone tissue and Ag-CH biomaterial. After 15 days of surgery, some defect regions were still occupied by implant (Figure 4(a)). Ag-CH has been shown to encourage cell spreading and improve the biocompatibility and interconnection with neighboring cells (Figure 4(b)–(d)). After 30 days of surgery, collagens were deposited in an orderly way (Figure 4(e)). These features demonstrate that Ag-CH was well tolerated by the host and the in-growth becomes more pronounced with increasing time periods of implantation. After 30 days of surgery, a fibrous tissue could be seen in the regenerated bone of ED group (Figure 5(a)). On the other hand, the staining section showed an interaction between Ag-CH and the bone tissue (Figure 5(b)). Moreover, the histological staining showed collagen fibers covered biomaterial (Figure 5(c)). No inflammatory cells such as macrophages were seen after 30 days. In this period of time, osteoid tissue with intense cell activity could be seen (Figure 5(d)). The results indicated that after 1 month of implantation, Ag-CH was associated with osteogenesis capability.
Determination of bone mineral content using ICP-OES The contents of Ca, P, Mg, Zn and Fe were significantly decreased in ED group when compared to that of Ag-CH-treated rabbits (p \ 0.01) as illustrated in Table 4. As a result of Ag-CH implantation, the content of Zn was significantly decreased when compared to that of the CT group. As regards Ca, P, Mg and Fe, no significant differences were observed in Ag-CH-treated rabbits when compared to those of the CT group.
Discussion This study investigated the effect of Ag-CH biomaterial on the bone healing. To our knowledge, there is no reported study about the Ag-CH potential of genotoxicity, antioxidant defense and the correlation with the mechanical property. For the standard biological evaluation of medical devices, the International Standard
Organization (ISO) has outlined the need for genotoxicity testing in ISO 10993-3.24 The chromosome abnormalities are a direct consequence of DNA damage such as double-strand breaks and misrepair of strand breaks. MNs were formed in dividing cells from chromosome fragments or whole chromosomes that were unable to engage with the mitotic spindle during mitosis.25 In this study, the data collected from bone marrow cells showed that Ag-CH did not significantly exert genotoxic effects. Similar results were observed by other authors using different ceramic compounds. Dental porcelain is considered as non-genotoxic when cultured with fibroblastic cells.26 However, some biomaterials such as MMA are clinically used in several orthopedic applications at present. In spite of this satisfactory clinical use, a quantitative analysis showed a dose-dependent increase in the number of chromosome aberrations. The aberration type induced by MMA was the chromatid-type aberrations.27 This indicated that MMA induced chromosome aberrations in late S or G2 phase. Genotoxic effects will eventually lead to abnormal and reduced cell growth, even if the cells initially appear cytocompatible. One study reported that the prepared CH with concentrations of 3.00 and 4.50 mg/mL induced mutation on Trichoderma.28 However, those techniques have some shortcomings (e.g. time investment, requirement for proliferating cell populations). In this study, after bone graft surgery, the increase in MDA level might influence bone through further intensification of oxidative processes and stimulation of osteoclastic bone resorption.29 The bone antioxidative barrier consists of enzymatic antioxidants such as GPx, SOD and CAT. GPx formed the main part in the enzymatic antioxidative defense system in the bone tissue.30 This enzyme is responsible for lipid peroxide detoxification and together with CAT acts for H2O2 detoxification, whereas SOD is responsible for dismutation of O22 to molecular oxygen and H2O2.31 Additionally, in this study, the major and trace element kinetic in the implanted bone was determined. It is noted that there is an important relationship between the antioxidative profile and the ossification kinetic. In fact, bone bioelements are necessary for proper function of the antioxidative enzymes such as Zn, Fe, Se and Cu. GPx is a Se-dependent enzyme, CAT contains Fe in its active center, whereas SOD activity is dependent on Zn and Cu32 In this study, the decrease in SOD activity in the bone tissue might be explained, at least
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Figure 4. SEM image in vivo shows (a) Ag-CH biomaterial and bone attachment at 15 days, (b)–(d) interconnection between AgCH biomaterial and neighboring cells and (e) collagen of the newly formed bone. Arrow indicates bone–Ag-CH interaction, *indicates osteoblasts, circle indicates the cells’ interaction and short head arrows indicate collagen.
partly, by this bioelement depletion. After 30 days of implantation, Fe trace content, performed in Ag-CH– bone area, revealed the presence of cavities previously filled with red blood cells. The Ca measurements in the newly formed bone indicated the deposition of significant amounts of mineralized matrix with a significant increase in Ca-P. Because oxidative stress negatively impacts bone healing, attempts to enhance the healing process have been investigated through the present mechanical bone correlation. In this study, an inverse
linear relationship between mechanical hardness and TBAR level (R = 20.998) was detected after 30 days of implantation. In a recent study, the increase in ROS release in bone exhibited consistent changes with impaired healing, including smaller calluses with decreased bone and reduced mechanical strength compared with those of CT bone.33 Moreover, an approximately 20% decrease in biomechanical strength in femurs related to oxidative stress in a rat model was reported.34 This study demonstrated that the disorders
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Figure 5. (a) Fibrous tissue with island of mineralized bone in the rabbit group presenting empty defect. (b) Bone–Ag-CH biomaterial interaction. (c) Collagen of the newly formed bone. (d) Osteoid tissue with intense cell activity showing effective osteogenesis. (a)–(c) Goldner’s trichrome staining (103 objective). Hematoxylin–eosin staining (403 and 1003 objective).
Table 4. Distribution of Ca, P, Zn, Mg and Fe in CT femoral condyle bone, after Ag-CH implantation and in group presenting ED during 15 and 30 days.
CT Ag-CH ED
15 days 30 days 15 days 30 days 15 days 30 days
Ca (mg/g)
P (mg/g)
Zn (mg/g)
Mg (mg/g)
Fe (mg/g)
255 6 50 250 6 80 245 6 45 255 6 78 $ 170 6 38*$ 200 6 17*
143 6 66 141 6 90 145 6 80 142 6 50 $ 91 6 17*$ 103 6 25*
0.26 6 0.07 0.25 6 0.09 0.16 6 0.08* 0.29 6 0.07 0.15 6 0.07*$ 0.17 6 0.05*
4.1 6 0.1 4.6 6 0.1 3.25 6 0.7 3.82 6 0.8 $ 1.95 6 0.08*$ 2.45 6 0.09*
760 6 40 720 6 40 790 6 20 720 6 30 $ 870 6 45*$ 810 6 45*
CT: control; ED: empty defect; Ag-CH: agarose–chitosan. *Significantly different level in the indicated group than CT. $ Significantly different level in the indicated group than Ag-CH group.
in the bone oxidative/antioxidative balance might affect the bone tissue by enhancing lipid peroxidation and oxidative protein damage. MDA has been reported to contribute to the destruction of collagen and proposed to serve as a measure of osteoclastic activity.35,36 As appropriate collagen network determines proper formation and bone biomechanical properties (hardness), its oxidative damage compromises the bone biomechanical properties making it susceptible to deformities and fractures.37 This study revealed that the bone graft weakened the antioxidative capacity of the bone tissue and led to oxidative stress during the first 15 days. The antioxidative stress profile was enhanced after 30 days of implantation. The deficits in bone repair were corrected by Ag-CH biomaterial treatment when compared with the non-implanted bone rabbits. In fact, CH has been
reported to stimulate the synthesis of collagen as well as inhibit its degradation.38 Moreover, many studies revealed that the scavenging mechanism might be related to the fact that hydroxyl group can react with active hydrogen atoms in CH to form a most stable macromolecule radical. The scavenging activities of CH derivatives against hydroxyl group might be derived from some or all of the following: (1) the hydroxyl groups in the polysaccharide unit can react with hydroxyl group by the typical H-abstraction reaction;39 (2) hydroxyl group can react with the residual free amino groups NH2 to form stable macromolecule radicals and40 (3) the NH2 groups can form NH3+ groups by absorbing hydrion from the biological fluid and then reacting with hydroxyl group through addition reaction.41 On the other hand, Ag has the property of
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Proc IMechE Part H: J Engineering in Medicine 228(8)
self-gelation at low temperature and has good mechanical and elastic behavior. To assess whether the matrix proteoglycan (PG) deposited within the Ag material was mechanically and electromechanically functional, the time evolution of dynamic mechanical stiffness and oscillatory streaming potential were measured in uniaxial confined compression. The analysis showed PG-rich matrix and collagen fibrils developing around cells. The dynamic stiffness and oscillatory streaming potential increased many times.42 These results suggested the formation of a mechanically functional matrix. The present Ag-CH biomaterials are of great importance and practical usefulness because it may suggest that manipulation of Ag-CH in the bone tissue may be a useful approach in the prevention against tissue damage due to trauma and surgery.
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Conclusion Based on these results, the chromosomal aberration and the in vivo MN assays confirmed the absence of the genotoxicty. The in vivo results showed an increase in GPx, SOD and CAT activities in the bone tissue after 30 days of implantation. The Ag-CH biomaterial inhibited the disruption of the enzymatic antioxidative profile. The mechanical hardness test showed a significant improvement of bone strength. The histological study implies that Ag-CH could directly impact the morphological development of bone cells and provide a compatible environment for bone tissue. Therefore, all these results suggested that the developed Ag-CH as a promising bone healing might have potential applications for hard tissue repair. Declaration of conflicting interests No potential conflict of interest relevant to this article was reported.
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Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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