Accepted Manuscript Title: Deposition of superparamagnetic nanohydroxyapatite on iron-fibrin substrates: preparation, characterization, cytocompatibility and bioactivity studies Author: Weslen S. Vedakumari Vishnu M. Priya Thotapalli P. Sastry PII: DOI: Reference:
S0927-7765(14)00219-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.021 COLSUB 6398
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-1-2014 2-4-2014 23-4-2014
Please cite this article as: W.S. Vedakumari, V.M. Priya, T.P. Sastry, Deposition of superparamagnetic nanohydroxyapatite on iron-fibrin substrates: preparation, characterization, cytocompatibility, and bioactivity studies, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Deposition of superparamagnetic nanohydroxyapatite on iron-fibrin substrates: preparation, characterization, cytocompatibility, and bioactivity studies Weslen S Vedakumaria, Vishnu M Priyab, Thotapalli P Sastrya,* Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India. PES Institute of Technology, Banashankari Stage III, Bangalore 560 085, India.
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*Corresponding Author
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Email address: [email protected]
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Tel: +91-9444382361; Fax: +91-44-24912150
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AUTHOR INFORMATION
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ABSTRACT In the present study, nanosized hydroxyapatite (nHAp) was formed on iron-fibrin substrates and its physico-chemical properties were characterized. The prepared iron-fibrin-
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nanohydroxyapatite (IF-nHAp) composite was needle shaped with an average width of about 30 nm and length of 80 nm. The vibrating sample magnetometer (VSM) was used to evaluate
linked
immunosorbent
bio/immunocompatibility
and
assay)
MTT
were
performed
to
evaluate
the
its
(3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium
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(Enzyme
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the superparamagnetic behaviour of the nanocomposite, IF-nHAp. Hemolysis and ELISA
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bromide) assay using osteoblast cells was performed to scrutinize its proliferative potential. Alkaline phosphatase activity (ALP) and calcium deposition were studied to investigate the
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osteogenic property of the nanocomposite. RT-PCR (real time-polymerase chain reaction) was used to quantify the mRNA levels of ALP, OC (osteocalcin), and OPN (osteopontin)
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genes involved in the osteogenic differentiation and matrix mineralization. Further, the bone
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bonding ability of IF-nHAp was observed by the deposition of apatite layers on the composite incubated in simulated body fluid (SBF).
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Keywords: iron-fibrin; nanohydroxyapatite; immunocompatibility; RT-PCR; osteogenesis.
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1. INTRODUCTION During the last few years, various important developments have taken place in the field of bone tissue engineering. However, present therapies such as bone grafts or surrogates still
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have numerous restrictions and no adequate bone substitute has been developed, making orthopaedic and bone reconstructive surgeries difficult. On the other hand, tissue engineering
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has proved to be a promising alternative to the existing therapies of bone regeneration [1], [2] and [3]. The main aim of tissue engineering, unlike the traditional biomaterial approach, is to
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promote the formation of new functional tissues; and this has shown great promise in
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providing biological alternatives [4].
Hydroxyapatite (HAp) forms the main inorganic component of natural bones and binds to
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them directly. Previous studies have established the osteoconductive and osteoinductive properties of HAp, apart from the fact that it possesses fine biocompatibility and absorption
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capabilities [5]. To be better used as an implant material in bone graft substitution, HAp
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needs to be further enhanced. We utilised fibrin (a unique bone forming protein) and iron (a metallic element) to produce osteogenetically efficient HAp. Fibrin is an insoluble protein, a
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fundamental component of hemostasis and serves as a biological scaffold for cell attachment and proliferation [6]. It has been widely used for developing different nanocomposite materials for tissue engineering applications [7]. The present study utilizes bovine fibrin, a major solid waste in Indian slaughter houses, for the preparation of HAp. Iron/iron oxide has been extensively used for a wide range of applications, which include magnetic resonance imaging, drug delivery, biosensors, and electronics [8]. It has been proved recently that Fe3+ ions influence the solubility of HAp molecules [9]. Further nanostructured hydroxyapatite shows improved adhesion and propagation of osteoblasts;[10] hence, fibrin (F) bioconjugated with iron (I) was used for the synthesis of nanohydroxyapatite (nHAp). The prepared ironfibrin-nanohydroxyapatite composite (IF-nHAp) was characterized using transmission 3
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electron microscope (TEM), scanning electron microscope (SEM), fourier transfrom infra red spectroscopy (FTIR) and X-ray diffraction (XRD); its osteogenic potency was studied in MG-63 cells using alkaline and alizarin red assays.
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2. Materials and methods 2.1. Materials
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Bovine blood was collected from a municipal slaughter house and stirred using a glass rod to obtain fibrin. The isolated fibrin was washed completely with distilled water and treated with
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using a blender, and stored at −20 °C until used [11].
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sodium acetate and hydrogen peroxide. It was once again washed with distilled water, ground
For the preparation of iron oxide nanoparticles, iron(II) chloride and iron(III) chloride (in the
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ratio of 1:2), dissolved in HCl, were added dropwise into ammonium hydroxide solution under nitrogen atmosphere. The solution was heated to 100 °C to remove excess NH4OH and
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2.2. Synthesis of IF-nHAp
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the product obtained was centrifuged and washed with distilled water [12].
Five hundred milligram of fibrin (F) was dissolved in 5 ml of 1N NaOH. The pH of this
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solution was reduced to 8 using 1N NaOH. When pH of the solution reached 8, iron oxide nanoparticles (I), dispersed in water, were added drop-wise under constant stirring. The pH of this solution was further reduced to 7, to obtain a black coloured precipitate that was centrifuged and washed twice with distilled water. The resultant iron-fibrin nanocomposite (IF) was lyophilized and used for the synthesis of hydroxyapatite. Five hundred milligram of IF was dispersed in 5ml of distilled water. Equal volumes of calcium chloride and orthophosphoric acid were added drop-wise to this and stirred for 1 h at room temperature [13]. The pH of the resulting solution was raised to 10 by drop-wise addition of ammonia, which led to the formation of a pale brown coloured precipitate (IF-
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nHAp). The precipitate obtained was washed repeatedly with distilled water, vacuum dried at 100 °C for 5 h, and used for characterization. 2.3. Characterization
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To examine the size and shape of the nanoparticles, a drop of IF-nHAp was placed on the surface of a carbon-coated copper grid, air dried, and observed under Philips TECHNAI-10
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TEM. The morphology of IF-nHAp was observed under a JEOL-JSM 6390 SEM attached to a Phonix Energy dispersive X-ray spectroscopy (EDX). For this, the sample was coated with
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gold at 2.5 kV, 20–25 mA for 120 s. Functional group analysis was performed using an ABB
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MB3000 FTIR, with wave number in the range of 4000–500 cm−1. XRD was used to determine the phase analysis of IF-nHAp using a Rigaku Miniflex II desktop X-ray
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Diffractometer with Cu Kα radiation (40 kV, 100 mA). A Universal TGA-V4 thermal analyser (TA Instruments) was used to define the thermal decomposition temperature of the
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sample with a heating rate of 10 °C/min. Differential scanning calorimetric (DSC) analysis
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was carried out using a Universal V4.4A differential scanning calorimeter (TA Instruments). The vibrating sample magnetometer (VSM) was used to measure the magnetic properties of
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IF-nHAp using a Lakeshore VSM 7410 at room temperature. 2.4. Hemolytic assay
Hemolytic assay was carried out to study the toxicity of IF-nHAp on red blood cells (RBCs). For this study, human blood samples were collected with consent from healthy individuals and centrifuged at 3000 rpm for 10 min. The pellet containing the RBCs was diluted with PBS to obtain a 5% hematocrit. A hundred microliters of the RBC suspension was treated with 25, 50, 100, 250 μg/ml of IF-nHAp and incubated at 37 °C for 60 min. RBC treated with PBS was used as control. After incubation, the samples were centrifuged and their optical density was read at 540 nm, the absorption maxima of hemoglobin [14].
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2.5. Enzyme linked immunosorbent assay (ELISA) Enzyme linked immunosorbent assay (ELISA) was carried out to determine the complement activation in human serum after IF-nHAp treatment. The serum complement split products,
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C3a and C4d, were examined using ELISA kits obtained from Quidel Corporation, San Diego, California, USA. Human serum was treated with 250 μg/ml of IF-nHAp and
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placed in a shaking incubator at 37 °C for 1 h. PBS solution containing 0.05 % Tween-20 protein stabilizers and 0.035 % ProClin 300 was added to stop the reaction. Human serum,
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incubated with zymosan, served as positive control and, without any treatment, served as
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negative control. 2.6. Cell viability
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MG-63, the human osteosarcoma cell line, was obtained from National Centre for Cell Sciences, India. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
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supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin with
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5% CO2. MTT (3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was used to evaluate the viability of MG-63 cells at predetermined time intervals (1, 3, and 5 days).
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1× 104 cells were seeded in a 96-well plate and incubated overnight. The cells were then treated with 25, 50, 100, 250 μg/ml of nHAp; cells without any treatment served as control. After each exposure, the cell culture medium was discarded and incubated with 100 μl of 1mg/ml MTT [15]. After 4h of incubation, the unreduced MTT was removed and 100 μl of Dimethylsulfoxide (DMSO) was added. Metabolically active cells reduce the yellow coloured tetrazolium, MTT, to purple coloured formazan, which can be read spectrophotometrically at 570 nm (Biorad). It should be noted the assay was carried out in culture medium containing 10% FBS, therefore the surface of nanoparticles would have been coated by corona and the viability of MG-63 cells took place in the presence of corona. 2.7. Alkaline phosphatase enzyme activity (ALP) 6
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For measuring the ALP activity, MG-63 cells were treated with 25, 50, 100, 250 μg/ml of IFnHAp for 1, 7, and 14 days. After each treatment, cells were lysed and centrifuged at 15,000 rpm for 15 min. To the cell culture supernatant, 200 μl of 5 mM p-nitrophenol phosphate was
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added and incubated at 37 °C. After 15 min, 0.02 mM NaOH was added to stop the reaction and the absorbance was measured at 405 nm [16].
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2.8. Real-time PCR (RT-PCR)
RT-PCR was used to quantify the mRNA levels of ALP, osteopontin (OPN), and osteocalcin
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(OC) after IF-nHAp treatment. In short, MG-63 cells were incubated with 250 μg/ml of
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IF-nHAp for 14 days followed by total RNA isolation using TRIzol reagent (Invitrogen Life Technologies, USA). RT-PCR was performed using ABI 7300 Real-Time PCR System
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(Applied Biosystems, CA) using SYBR PrimeScript™ RT-PCR Kit (Takara, Japan). Glyceraldehyde phosphatedehydrogenase (GAPDH) was used as the housekeeping gene for
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calculating the relative expression of ALP, OPN, and OC.
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The following primers were used for the PCR reactions: ALP [forward: 5'-AGGCAGGATTGACCACGG-3']
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[reverse: 5'-TGTAGTTCTGCTCATGGA-3']
OPN [forward: 5'-CGACGGCCGAGGTGATAGCTT-3'] [reverse: 5'-CATGGCTGGTCTTCCCGTTGCC-3']
OC [forward: 5'-AAAGCCCAGCGACTCTC-3'] [reverse: 5-CTAAACGGTGGTGCCATAGAT-3']
GAPDH [forward: 5'-AACCCATCACCATCTTCCAGG-3'] [reverse: 5-GCCTTCTCCATGGTGGTGAA-3'] 2.9. Alizarin red S (ARS) staining ARS staining was carried out to study the ability of MG-63 cells to form mineralized nodules and calcium deposits after incubation with nHAp. The cells were treated with 25, 50, 100, 7
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250 μg/ml of IF-nHAp for 21 days. After treatment the cells were washed with PBS and fixed in ice cold ethanol for 1h. They were then stained with 40 mM Alizarin red-S solution for 30 min, washed thoroughly with distilled water, and observed under a microscope. The cells
nm [17].
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2.10. In vitro bioactivity through stimulated body fluid (SBF)
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were also treated with 10% cetylpyridinium chloride and their optical density was read at 450
In vitro bioactivity test was performed to study the growth of apatite on IF-nHAp
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nanocomposite. 100 mg of the powdered sample was made into 1mm thick pellets of 10 mm
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diameter. They were weighed (W1) and soaked in 10 ml of stimulated body fluid (SBF) kept in airtight plastic containers to avoid change in pH and microbial contamination [18]. The
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SBF solution was changed once in every two days for a period of three weeks. After three weeks of incubation, the pellets were removed from SBF, air dried, and weighed (W2). The
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percentage of weight gain was calculated by ((W2 − W1)/(W1)) x 100 and the surface
2.11. Statistical analysis
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morphology of the samples before and after incubation were viewed under SEM.
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SPSS software version 13.0 was used to carry out the statistical analysis. Data were expressed as mean ± standard deviation (n = 3). One way analysis of variance (ANOVA) with Duncan’s test for multiple comparisons was used to evaluate the parameters. P values < 0.05 were considered significant.
3. Results and discussion 3.1. Characterization
In the present study, fibrin was dissolved in an alkaline solution followed by pH reduction in the presence of iron oxide nanoparticles. When pH reached 7, fibrin precipitated encapsulating the iron oxide nanoparticles. Further, the Ca2+ ions of calcium chloride complexed with the carboxylic groups of fibrin formed nHAp in the presence of 8
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orthophosphoric acid and ammonium hydroxide [19]. TEM is a semiquantitative method that helps to determine the size and morphology of the nanoparticles. Fig. 1A shows well-defined needle shaped IF-nHAp nanoparticles with an average width of 30 nm and length of 80 nm.
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TEM results also prove the embedding of iron oxide nanoparticles within nHAp. Thus the prepared nanocomposite can be used in the form of injectable particles, which could be
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directed to different sites of the body to treat bone or related defects.
SEM findings (Fig. 1B) are in good agreement with the TEM results. The particles tend to
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aggregate because of their high surface energy and the increased ripening time required for
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the growth of nHAp [20]. Figure 1C shows the EDX of IF-nHAp with Ca/P ratio as 1.68, a value that is similar to that of bone HAp [21]. Moreover, the presence of iron oxide is
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confirmed in the spectrum.
FTIR was used to determine the functional groups present in the IF-nHAp nanocomposite.
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The presence of a broad band (Fig. 2A) at 3457 cm-1 may be assigned to the absorbed water
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molecules. The absorption bands at 962 cm-1, 1033 cm-1, and 1098 cm-1 can be attributed to the υ1, υ3, and υ4 stretching modes of the phosphate groups of hydroxyapatite with their
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values coinciding with those reported in the literature [19]. The characteristic amide bands of fibrin are observed at 1644 cm-1 and 1420 cm-1 whereas the bands at 567 cm-1 and 470 cm-1 are allocated to the iron oxide [22]. The XRD pattern (Fig. 2B) is in good accord with the standard JCPDS value of HAp (09−0432). The peaks are broad showing the nanocrystalline nature of nHAp. The peaks at 25.9°, 32.19°, 32.2°, 34.09°, and 46.7° are characteristic of nHAP and those at 35.7° and 64° are characteristic of iron oxide. Reduction in peak intensity of iron oxide may be a result of its loading within the nHAp [23]. The TGA curve of IF-nHAp shows the loss of water molecules between 100 and 400 °C (Fig. 2C), with no apparent weight loss after 400°C. About 85% of the sample was stable at 800°C, 9
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which proves that the prepared nanocomposite is comparable to that of bone HAp. The differential scanning calorimetric thermogram (Fig. 2D) shows the evaporation of water molecules at 115 °C with no specific change thereafter.
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VSM was used to study the diamagnetic property of the prepared IF-nHAp nanocomposite. A saturation magnetization (Ms) of 5.29 emu/g (Fig. 3) was observed proving their
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superparamagnetic behavior. Drugs such as antibiotics or anticancer agents can be coupled to these nanocomposites and delivered at the site of infection. Additionally, magnetic resonance
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imaging (MRI) can be used to track these nanoparticles; and their distribution can also be
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controlled using an appropriate magnetic field. 3.2. Hemolytic assay
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Hemolysis occurs when a foreign antigen comes in contact with a RBC leading to the rupture of the cell membrane and resulting in the discharge of hemoglobin. Therefore it is quite
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essential to perform hemolytic assay to determine the interaction of a material with RBC.
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According to the ASTM standard F756, a material exhibiting less than 5% of hemolysis is non-hemolytic, 5−10% is slightly hemolytic, and higher than 10% is highly hemolytic. The
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different concentrations of the nanocomposite (Fig. 4A) used in our study showed no hemolytic activity (less than 2%), which may be because of their rigid nature. It has been suggested that a rigid molecule is less vulnerable to bind to the membrane of a RBC, which could be a probable explanation for such low hemolytic activity [24]. 3.3. ELISA
The human complement system is a collection of 30 different plasma proteins, which plays an important role in the innate branch of the immune system. These proteins circulate in blood in an inactive form and get activated when they come in contact with foreign materials or antigens [25]. So, it is highly recommended to investigate the relationship between the IFnHAp nanocomposite and the complement system. We examined the instigation of 10
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complement split products – C3a and C4d – under normal in vitro conditions. It has been proved that the complement system is triggered through any of the following pathways – the classical pathway, the alternative pathway, and the lectin pathway. The alternative pathway is
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triggered when complement factor C3 cleaves into C3a and C3b*; hence C3a is used as a biomarker for alternative pathway. C4d, a split product of complement factor C4 is
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discharged when the classical/lectin pathway gets initiated and hence, acts as the biomarker for classical/lectin pathways [26]. When ELISA (Fig. 4B) was performed, no change was
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seen in the level of C3a and C4d, which confirms the applicability of the IF-nHAp for bio-
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nanotechnological applications. 3.4. Cell viability
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MTT assay was performed to resolve the cytocompatibility of IF-nHAp with MG-63 cells. MTT is an easy and rapid method used to establish mitochondrial impairment and cell
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proliferation. Figure 5A represents the viability of MG-63 cells in the presence of different
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concentrations of IF-nHAp at various time intervals. On days 1 and 3, almost 95–98% of the cells were viable with osteoblast densities analogous with that of the control. However,
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significant increase in the number of cells was observed on day 5, which might be because of the presence of fibrin within the nanocomposite. It has been proved that fibrin provides signals and binding sites for various growth factors, integrins, and extracellular matrix components leading to successive cellular division and tissue formation, which might be the potential cause for such increased cell numbers in IFnHAp treated cells [27, 28]. Moreover treated cells displayed normal morphology as the control indicating the nontoxic and biocompatible property of the nanocomposite (Fig. 5B). With respect to the concept of “cell vision”, the nanocomposite which is non-toxic to MG-63 cells may show different toxicity profile when any other cell type was used for the study. Thus it is proposed that toxicity profile of any material is strongly dependent on the cell type 11
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and in the current study the prepared nanocomposite showed good safety profile for MG-63 cells. 3.5. ALP activity
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Among the most important osteogenic features, the upregulation of ALP activity is a crucial event that happens during the initial stages of osteogenesis. ALP supports osteogenic
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differentiation and mineralization by generating inorganic phosphates (Pi), the inducer of hydroxyapatite formation [29]. Therefore it is imperative to study the level of ALP expressed
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during the course of bone formation. As shown in Figure 6A, the level of ALP was expressed
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at similar levels as those of the control on day 1. However, the level of ALP increased significantly on days 7 and 14 in IF-nHAp treated cells, thus proving their osteogenic
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potency. Endogenous expression of growth factors due to the interaction of cell surface with the chemical groups present on the nanocomposite may be a possible reason for such
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3.6. RT-PCR
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increased ALP acitivity in treated cells [30].
Several genes such as ALP, OPN and OC are expressed during new bone formation and
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hence, are used as indicators of osteogenic differentiation. ALP acts as an early marker of osteoblast lineages and induces hydroxyapatite formation by generating inorganic phosphates [31]. OC forms the most profuse, bone specific, non-collagenous protein and serves as an indicator of osteogenic maturation and bone formation [32]. OPN is generated by preosteoblasts, osteoblasts, and osteocytes and is deemed to play a key feature in bone remodelling [33]. The RT-PCR (Fig. 6B) result of IF-nHAp treated cells shows about 12.2, 8.3, and 2.5 times increase in the mRNA levels of ALP, OC and OPN genes, respectively. This significant increase serves as the foundation for the impending mineralization as they continue to form bone like nodules, which can further develop into complete 3D mineralized structures. 12
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3.7. ARS Staining Deposition of calcium ions is one of the most essential markers of bone formation and osteoblast differentiation. ARS staining was performed to determine the mineral deposition of
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MG-63 cells in the presence of the IF-nHAp nanocomposite. ARS chelates Ca2+ ions and helps in concurrent assessment of mineral distribution through microscopy and spectroscopy
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[17]. Osteoblasts cultured with IF-nHAp showed 2–5 times (Fig. 7A) increase in calcium deposition than that of the control. The microscopic images (Fig. 7B) also show the presence
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of red coloured calcium ions in all the concentrations with the maximum observed at 250 μg.
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The above results are consistent with those obtained for ALP activity and thus substantiate improved differentiation of osteoblasts in the presence of IF-nHAp composite.
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3.8. In vitro bioactivity through SBF
When considering osseo-integration, the main feature of an implant is to bind to the living
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bone by forming a bone-like apatite on its surface. This apatite is believed to trigger the cell
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signalling proteins and pathways involved in bone formation [34]. Therefore an easy method to assess the bone-bonding ability of a material is to immerse it in SBF, a solution with ion
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concentrations similar to that of human blood plasma [18]. Figure 8 A, B, C and D show the SEM images of apatite coatings deposited on the surface of the nanocomposite after 21 days of incubation in SBF. These mineralized nodules are irregular shaped calcospherites with a mean diameter of 8–9 µm. EDX analysis (Fig. 8 E) shows the Ca/P ratio as 1.43, thus proving the formation of a Ca-rich apatite on the surface of the SBF soaked nanocomposite. The formation of apatite layers was further verified using the weight gained by the samples before and after soaking in SBF, which revealed 50–60% increase in the nett sample weight at the end of 21 days. In general HAp, which is negatively charged (because of the presence of the phosphate and hydroxyl groups) interacts with the calcium ions present in SBF and forms a positively charged calcium-rich intermediate (CRI). This calcium-rich intermediate further 13
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interacts with the negatively charged phosphate ions of SBF and cyrstallizes to form a bone like apatite [35]. 4. Conclusion
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From the present study, it is clear that iron-fibrin nanocomposites can act as substrates for the growth of nHAp crystals. The prepared IF-nHAp composite was needle shaped with an
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average width of about 30 nm and length of 80 nm. FTIR, XRD and VSM results proved the loading of iron within the nHAp, thus confirming their promising use in the area of magnetic
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field assisted orthopaedic tissue engineering. Hemolytic assay and ELISA proved the Further IF-nHAp offered a
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hemo/immunocompatible properties of the nanocomposite.
suitable environment to support osteoblast viability, spreading, and proliferation. Osteoblasts
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cultured in the presence of IF-nHAp showed enhanced ALP activity and calcium deposition, which are the chief factors of mineralization and bone formation. In addition, the
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upregulation of genes involved in osteogenic differentiation and matrix mineralization prove
Acknowledgment
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the potential use of IF-nHAp nanocomposite in the field of bone tissue engineering.
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Weslen S Vedakumari acknowledges the funding support granted by the Department of Science and Technology, India. 5. References [1] [2]
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[27] A. C. Brown, T. H. Barker Acta Biomater., 2013, doi.org/10.1016/j.actbio.2013.09.008 [28] T. Kitajima, M. Sakuragi, H. Hasuda, T. Ozu, Y. Ito Acta Biomater. 5(2009), p. 2623
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[29] T. Osathanon, C. M. Giachelli, M. J. Somerman Biomaterials, 30(2009), p. 4513
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[30] S. Kocabey, H. Ceylan, A. B. Tekinay, M. O. Guler Acta Biomater., 9(2013), p. 9075 [31] M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M.
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A. Moorman, D. W. Simonetti, S. Craig, D. R. Marshak Science, 284(1999), p. 143
[32] P. Ducy, C. Desbois, B. Boyce, G. Pinero, B. Story, C. Dunstan, E. Smith, J. Bonadio, S. Goldstein, C. Gundberg, A. Bradley, G. Karsenty Nature, 382(1996), p. 448
[33] W. T. Butler Connect. Tissue Res., 23(1989), p. 123 [34] P. S. Vanzillotta, M. S. Sader, I. N. Bastos, A. Soares Gde Dent. Mater., 22(2006), p. 275
[35] R. V. Suganthi, S. Prakash Parthiban, K. Elayaraja, E. K.; Girija, P. Kulariya, Y. S. Katharria, F. Singh, K. Asokan, D. Kanjilal, S. Narayana Kalkura J. Mater. Sci. Mater. Med., 20 (2009), p. 271
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FIGURE LEGENDS Fig. 1. (A) TEM image showing needle shaped IF-nHAp nanoparticles with average width of 30 nm and length of 80 nm. The arrow mark depicts iron oxide nanoparticles embedded
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within nHAp. (B) SEM micrograph of IF-nHAp showing well defined nanoparticles. (C) EDX spectrum of IF-nHAp with Ca/P ratio as 1.68, the value similar to that of bone HAp.
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Fig. 2. (A) FTIR spectrum of IF-nHAp showing the characteristic peaks of nHAp, fibrin and iron oxide respectively. (B) X-ray diffraction pattern of IF-nHAp with typical peaks of nHAp
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and iron oxide respectively. (C) TGA curve of IF- nHAp. (D) DSC thermogram of IF-nHAP.
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Fig. 3. Magnetization curve of IF-nHAp obtained using VSM.
Fig. 4. (A) Hemolytic assay for 25, 50, 100 and 250 μg of IF-nHAp respectively. (B) ELISA
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result showing the in vitro production of C3a and C4d complement split products after incubation with IF-nHAp. The asterisk (*) denotes a statistically significant difference from
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control (P< 0.05).
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Fig. 5. (A) MTT assay for 25, 50, 100 and 250 μg of IF-nHAp at different time intervals respectively. The asterisk (*) denotes a statistically significant difference from control
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(P< 0.05). (B) Microscopic images of MG-63 cells after 5 days of incubation with 25, 50, 100 and 250 μg of IF-nHAp respectively. Fig. 6. (A) ALP activity for 25, 50, 100 and 250 μg of IF-nHAp at different time intervals respectively. The asterisk (*) denotes a statistically significant difference from control (P< 0.05). (B) Expression levels of ALP, OC and OPN genes after IF-nHAp treatment. Fig. 7. (A) ARS assay for 25, 50, 100 and 250 μg of IF-nHAp respectively. The asterisk (*) denotes a statistically significant difference from control (P< 0.05). (B) ARS staining illustrating calcium deposition in the presence of various concentrations of IF-nHAp respectively.
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Fig. 8. (A, B, C, D) SEM images with different magnifications of the SBF soaked
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IF-nHAp. (E) EDX spectrum of SBF soaked IF-nHAp with Ca/P ratio as 1.43.
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GRAPHICAL ABSTRACT
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HIGHLIGHTS Iron-fibrin- nanohydroxyapatite (IF-nHAp) composite was prepared using a novel method. VSM result proved the superparamagnetic behaviour of the nanocomposite.
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MTT, Haemolysis and ELISA proved the bio/immunocompatibility of the nanocomposite.
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RT-PCR was used to quantify the mRNA levels of ALP, OC, OPN genes.
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S0927-7765(14)00219-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.021 COLSUB 6398
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-1-2014 2-4-2014 23-4-2014
Please cite this article as: W.S. Vedakumari, V.M. Priya, T.P. Sastry, Deposition of superparamagnetic nanohydroxyapatite on iron-fibrin substrates: preparation, characterization, cytocompatibility, and bioactivity studies, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Deposition of superparamagnetic nanohydroxyapatite on iron-fibrin substrates: preparation, characterization, cytocompatibility, and bioactivity studies Weslen S Vedakumaria, Vishnu M Priyab, Thotapalli P Sastrya,* Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India. PES Institute of Technology, Banashankari Stage III, Bangalore 560 085, India.
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*Corresponding Author
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Email address: [email protected]
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Tel: +91-9444382361; Fax: +91-44-24912150
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AUTHOR INFORMATION
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ABSTRACT In the present study, nanosized hydroxyapatite (nHAp) was formed on iron-fibrin substrates and its physico-chemical properties were characterized. The prepared iron-fibrin-
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nanohydroxyapatite (IF-nHAp) composite was needle shaped with an average width of about 30 nm and length of 80 nm. The vibrating sample magnetometer (VSM) was used to evaluate
linked
immunosorbent
bio/immunocompatibility
and
assay)
MTT
were
performed
to
evaluate
the
its
(3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium
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(Enzyme
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the superparamagnetic behaviour of the nanocomposite, IF-nHAp. Hemolysis and ELISA
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bromide) assay using osteoblast cells was performed to scrutinize its proliferative potential. Alkaline phosphatase activity (ALP) and calcium deposition were studied to investigate the
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osteogenic property of the nanocomposite. RT-PCR (real time-polymerase chain reaction) was used to quantify the mRNA levels of ALP, OC (osteocalcin), and OPN (osteopontin)
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genes involved in the osteogenic differentiation and matrix mineralization. Further, the bone
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bonding ability of IF-nHAp was observed by the deposition of apatite layers on the composite incubated in simulated body fluid (SBF).
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Keywords: iron-fibrin; nanohydroxyapatite; immunocompatibility; RT-PCR; osteogenesis.
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1. INTRODUCTION During the last few years, various important developments have taken place in the field of bone tissue engineering. However, present therapies such as bone grafts or surrogates still
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have numerous restrictions and no adequate bone substitute has been developed, making orthopaedic and bone reconstructive surgeries difficult. On the other hand, tissue engineering
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has proved to be a promising alternative to the existing therapies of bone regeneration [1], [2] and [3]. The main aim of tissue engineering, unlike the traditional biomaterial approach, is to
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promote the formation of new functional tissues; and this has shown great promise in
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providing biological alternatives [4].
Hydroxyapatite (HAp) forms the main inorganic component of natural bones and binds to
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them directly. Previous studies have established the osteoconductive and osteoinductive properties of HAp, apart from the fact that it possesses fine biocompatibility and absorption
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capabilities [5]. To be better used as an implant material in bone graft substitution, HAp
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needs to be further enhanced. We utilised fibrin (a unique bone forming protein) and iron (a metallic element) to produce osteogenetically efficient HAp. Fibrin is an insoluble protein, a
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fundamental component of hemostasis and serves as a biological scaffold for cell attachment and proliferation [6]. It has been widely used for developing different nanocomposite materials for tissue engineering applications [7]. The present study utilizes bovine fibrin, a major solid waste in Indian slaughter houses, for the preparation of HAp. Iron/iron oxide has been extensively used for a wide range of applications, which include magnetic resonance imaging, drug delivery, biosensors, and electronics [8]. It has been proved recently that Fe3+ ions influence the solubility of HAp molecules [9]. Further nanostructured hydroxyapatite shows improved adhesion and propagation of osteoblasts;[10] hence, fibrin (F) bioconjugated with iron (I) was used for the synthesis of nanohydroxyapatite (nHAp). The prepared ironfibrin-nanohydroxyapatite composite (IF-nHAp) was characterized using transmission 3
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electron microscope (TEM), scanning electron microscope (SEM), fourier transfrom infra red spectroscopy (FTIR) and X-ray diffraction (XRD); its osteogenic potency was studied in MG-63 cells using alkaline and alizarin red assays.
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2. Materials and methods 2.1. Materials
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Bovine blood was collected from a municipal slaughter house and stirred using a glass rod to obtain fibrin. The isolated fibrin was washed completely with distilled water and treated with
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using a blender, and stored at −20 °C until used [11].
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sodium acetate and hydrogen peroxide. It was once again washed with distilled water, ground
For the preparation of iron oxide nanoparticles, iron(II) chloride and iron(III) chloride (in the
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ratio of 1:2), dissolved in HCl, were added dropwise into ammonium hydroxide solution under nitrogen atmosphere. The solution was heated to 100 °C to remove excess NH4OH and
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2.2. Synthesis of IF-nHAp
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the product obtained was centrifuged and washed with distilled water [12].
Five hundred milligram of fibrin (F) was dissolved in 5 ml of 1N NaOH. The pH of this
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solution was reduced to 8 using 1N NaOH. When pH of the solution reached 8, iron oxide nanoparticles (I), dispersed in water, were added drop-wise under constant stirring. The pH of this solution was further reduced to 7, to obtain a black coloured precipitate that was centrifuged and washed twice with distilled water. The resultant iron-fibrin nanocomposite (IF) was lyophilized and used for the synthesis of hydroxyapatite. Five hundred milligram of IF was dispersed in 5ml of distilled water. Equal volumes of calcium chloride and orthophosphoric acid were added drop-wise to this and stirred for 1 h at room temperature [13]. The pH of the resulting solution was raised to 10 by drop-wise addition of ammonia, which led to the formation of a pale brown coloured precipitate (IF-
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nHAp). The precipitate obtained was washed repeatedly with distilled water, vacuum dried at 100 °C for 5 h, and used for characterization. 2.3. Characterization
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To examine the size and shape of the nanoparticles, a drop of IF-nHAp was placed on the surface of a carbon-coated copper grid, air dried, and observed under Philips TECHNAI-10
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TEM. The morphology of IF-nHAp was observed under a JEOL-JSM 6390 SEM attached to a Phonix Energy dispersive X-ray spectroscopy (EDX). For this, the sample was coated with
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gold at 2.5 kV, 20–25 mA for 120 s. Functional group analysis was performed using an ABB
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MB3000 FTIR, with wave number in the range of 4000–500 cm−1. XRD was used to determine the phase analysis of IF-nHAp using a Rigaku Miniflex II desktop X-ray
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Diffractometer with Cu Kα radiation (40 kV, 100 mA). A Universal TGA-V4 thermal analyser (TA Instruments) was used to define the thermal decomposition temperature of the
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sample with a heating rate of 10 °C/min. Differential scanning calorimetric (DSC) analysis
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was carried out using a Universal V4.4A differential scanning calorimeter (TA Instruments). The vibrating sample magnetometer (VSM) was used to measure the magnetic properties of
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IF-nHAp using a Lakeshore VSM 7410 at room temperature. 2.4. Hemolytic assay
Hemolytic assay was carried out to study the toxicity of IF-nHAp on red blood cells (RBCs). For this study, human blood samples were collected with consent from healthy individuals and centrifuged at 3000 rpm for 10 min. The pellet containing the RBCs was diluted with PBS to obtain a 5% hematocrit. A hundred microliters of the RBC suspension was treated with 25, 50, 100, 250 μg/ml of IF-nHAp and incubated at 37 °C for 60 min. RBC treated with PBS was used as control. After incubation, the samples were centrifuged and their optical density was read at 540 nm, the absorption maxima of hemoglobin [14].
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2.5. Enzyme linked immunosorbent assay (ELISA) Enzyme linked immunosorbent assay (ELISA) was carried out to determine the complement activation in human serum after IF-nHAp treatment. The serum complement split products,
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C3a and C4d, were examined using ELISA kits obtained from Quidel Corporation, San Diego, California, USA. Human serum was treated with 250 μg/ml of IF-nHAp and
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placed in a shaking incubator at 37 °C for 1 h. PBS solution containing 0.05 % Tween-20 protein stabilizers and 0.035 % ProClin 300 was added to stop the reaction. Human serum,
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incubated with zymosan, served as positive control and, without any treatment, served as
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negative control. 2.6. Cell viability
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MG-63, the human osteosarcoma cell line, was obtained from National Centre for Cell Sciences, India. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
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supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin with
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5% CO2. MTT (3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was used to evaluate the viability of MG-63 cells at predetermined time intervals (1, 3, and 5 days).
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1× 104 cells were seeded in a 96-well plate and incubated overnight. The cells were then treated with 25, 50, 100, 250 μg/ml of nHAp; cells without any treatment served as control. After each exposure, the cell culture medium was discarded and incubated with 100 μl of 1mg/ml MTT [15]. After 4h of incubation, the unreduced MTT was removed and 100 μl of Dimethylsulfoxide (DMSO) was added. Metabolically active cells reduce the yellow coloured tetrazolium, MTT, to purple coloured formazan, which can be read spectrophotometrically at 570 nm (Biorad). It should be noted the assay was carried out in culture medium containing 10% FBS, therefore the surface of nanoparticles would have been coated by corona and the viability of MG-63 cells took place in the presence of corona. 2.7. Alkaline phosphatase enzyme activity (ALP) 6
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For measuring the ALP activity, MG-63 cells were treated with 25, 50, 100, 250 μg/ml of IFnHAp for 1, 7, and 14 days. After each treatment, cells were lysed and centrifuged at 15,000 rpm for 15 min. To the cell culture supernatant, 200 μl of 5 mM p-nitrophenol phosphate was
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added and incubated at 37 °C. After 15 min, 0.02 mM NaOH was added to stop the reaction and the absorbance was measured at 405 nm [16].
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2.8. Real-time PCR (RT-PCR)
RT-PCR was used to quantify the mRNA levels of ALP, osteopontin (OPN), and osteocalcin
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(OC) after IF-nHAp treatment. In short, MG-63 cells were incubated with 250 μg/ml of
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IF-nHAp for 14 days followed by total RNA isolation using TRIzol reagent (Invitrogen Life Technologies, USA). RT-PCR was performed using ABI 7300 Real-Time PCR System
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(Applied Biosystems, CA) using SYBR PrimeScript™ RT-PCR Kit (Takara, Japan). Glyceraldehyde phosphatedehydrogenase (GAPDH) was used as the housekeeping gene for
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calculating the relative expression of ALP, OPN, and OC.
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The following primers were used for the PCR reactions: ALP [forward: 5'-AGGCAGGATTGACCACGG-3']
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[reverse: 5'-TGTAGTTCTGCTCATGGA-3']
OPN [forward: 5'-CGACGGCCGAGGTGATAGCTT-3'] [reverse: 5'-CATGGCTGGTCTTCCCGTTGCC-3']
OC [forward: 5'-AAAGCCCAGCGACTCTC-3'] [reverse: 5-CTAAACGGTGGTGCCATAGAT-3']
GAPDH [forward: 5'-AACCCATCACCATCTTCCAGG-3'] [reverse: 5-GCCTTCTCCATGGTGGTGAA-3'] 2.9. Alizarin red S (ARS) staining ARS staining was carried out to study the ability of MG-63 cells to form mineralized nodules and calcium deposits after incubation with nHAp. The cells were treated with 25, 50, 100, 7
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250 μg/ml of IF-nHAp for 21 days. After treatment the cells were washed with PBS and fixed in ice cold ethanol for 1h. They were then stained with 40 mM Alizarin red-S solution for 30 min, washed thoroughly with distilled water, and observed under a microscope. The cells
nm [17].
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2.10. In vitro bioactivity through stimulated body fluid (SBF)
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were also treated with 10% cetylpyridinium chloride and their optical density was read at 450
In vitro bioactivity test was performed to study the growth of apatite on IF-nHAp
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nanocomposite. 100 mg of the powdered sample was made into 1mm thick pellets of 10 mm
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diameter. They were weighed (W1) and soaked in 10 ml of stimulated body fluid (SBF) kept in airtight plastic containers to avoid change in pH and microbial contamination [18]. The
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SBF solution was changed once in every two days for a period of three weeks. After three weeks of incubation, the pellets were removed from SBF, air dried, and weighed (W2). The
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percentage of weight gain was calculated by ((W2 − W1)/(W1)) x 100 and the surface
2.11. Statistical analysis
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morphology of the samples before and after incubation were viewed under SEM.
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SPSS software version 13.0 was used to carry out the statistical analysis. Data were expressed as mean ± standard deviation (n = 3). One way analysis of variance (ANOVA) with Duncan’s test for multiple comparisons was used to evaluate the parameters. P values < 0.05 were considered significant.
3. Results and discussion 3.1. Characterization
In the present study, fibrin was dissolved in an alkaline solution followed by pH reduction in the presence of iron oxide nanoparticles. When pH reached 7, fibrin precipitated encapsulating the iron oxide nanoparticles. Further, the Ca2+ ions of calcium chloride complexed with the carboxylic groups of fibrin formed nHAp in the presence of 8
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orthophosphoric acid and ammonium hydroxide [19]. TEM is a semiquantitative method that helps to determine the size and morphology of the nanoparticles. Fig. 1A shows well-defined needle shaped IF-nHAp nanoparticles with an average width of 30 nm and length of 80 nm.
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TEM results also prove the embedding of iron oxide nanoparticles within nHAp. Thus the prepared nanocomposite can be used in the form of injectable particles, which could be
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directed to different sites of the body to treat bone or related defects.
SEM findings (Fig. 1B) are in good agreement with the TEM results. The particles tend to
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aggregate because of their high surface energy and the increased ripening time required for
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the growth of nHAp [20]. Figure 1C shows the EDX of IF-nHAp with Ca/P ratio as 1.68, a value that is similar to that of bone HAp [21]. Moreover, the presence of iron oxide is
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confirmed in the spectrum.
FTIR was used to determine the functional groups present in the IF-nHAp nanocomposite.
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The presence of a broad band (Fig. 2A) at 3457 cm-1 may be assigned to the absorbed water
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molecules. The absorption bands at 962 cm-1, 1033 cm-1, and 1098 cm-1 can be attributed to the υ1, υ3, and υ4 stretching modes of the phosphate groups of hydroxyapatite with their
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values coinciding with those reported in the literature [19]. The characteristic amide bands of fibrin are observed at 1644 cm-1 and 1420 cm-1 whereas the bands at 567 cm-1 and 470 cm-1 are allocated to the iron oxide [22]. The XRD pattern (Fig. 2B) is in good accord with the standard JCPDS value of HAp (09−0432). The peaks are broad showing the nanocrystalline nature of nHAp. The peaks at 25.9°, 32.19°, 32.2°, 34.09°, and 46.7° are characteristic of nHAP and those at 35.7° and 64° are characteristic of iron oxide. Reduction in peak intensity of iron oxide may be a result of its loading within the nHAp [23]. The TGA curve of IF-nHAp shows the loss of water molecules between 100 and 400 °C (Fig. 2C), with no apparent weight loss after 400°C. About 85% of the sample was stable at 800°C, 9
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which proves that the prepared nanocomposite is comparable to that of bone HAp. The differential scanning calorimetric thermogram (Fig. 2D) shows the evaporation of water molecules at 115 °C with no specific change thereafter.
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VSM was used to study the diamagnetic property of the prepared IF-nHAp nanocomposite. A saturation magnetization (Ms) of 5.29 emu/g (Fig. 3) was observed proving their
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superparamagnetic behavior. Drugs such as antibiotics or anticancer agents can be coupled to these nanocomposites and delivered at the site of infection. Additionally, magnetic resonance
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imaging (MRI) can be used to track these nanoparticles; and their distribution can also be
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controlled using an appropriate magnetic field. 3.2. Hemolytic assay
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Hemolysis occurs when a foreign antigen comes in contact with a RBC leading to the rupture of the cell membrane and resulting in the discharge of hemoglobin. Therefore it is quite
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essential to perform hemolytic assay to determine the interaction of a material with RBC.
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According to the ASTM standard F756, a material exhibiting less than 5% of hemolysis is non-hemolytic, 5−10% is slightly hemolytic, and higher than 10% is highly hemolytic. The
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different concentrations of the nanocomposite (Fig. 4A) used in our study showed no hemolytic activity (less than 2%), which may be because of their rigid nature. It has been suggested that a rigid molecule is less vulnerable to bind to the membrane of a RBC, which could be a probable explanation for such low hemolytic activity [24]. 3.3. ELISA
The human complement system is a collection of 30 different plasma proteins, which plays an important role in the innate branch of the immune system. These proteins circulate in blood in an inactive form and get activated when they come in contact with foreign materials or antigens [25]. So, it is highly recommended to investigate the relationship between the IFnHAp nanocomposite and the complement system. We examined the instigation of 10
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complement split products – C3a and C4d – under normal in vitro conditions. It has been proved that the complement system is triggered through any of the following pathways – the classical pathway, the alternative pathway, and the lectin pathway. The alternative pathway is
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triggered when complement factor C3 cleaves into C3a and C3b*; hence C3a is used as a biomarker for alternative pathway. C4d, a split product of complement factor C4 is
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discharged when the classical/lectin pathway gets initiated and hence, acts as the biomarker for classical/lectin pathways [26]. When ELISA (Fig. 4B) was performed, no change was
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seen in the level of C3a and C4d, which confirms the applicability of the IF-nHAp for bio-
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nanotechnological applications. 3.4. Cell viability
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MTT assay was performed to resolve the cytocompatibility of IF-nHAp with MG-63 cells. MTT is an easy and rapid method used to establish mitochondrial impairment and cell
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proliferation. Figure 5A represents the viability of MG-63 cells in the presence of different
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concentrations of IF-nHAp at various time intervals. On days 1 and 3, almost 95–98% of the cells were viable with osteoblast densities analogous with that of the control. However,
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significant increase in the number of cells was observed on day 5, which might be because of the presence of fibrin within the nanocomposite. It has been proved that fibrin provides signals and binding sites for various growth factors, integrins, and extracellular matrix components leading to successive cellular division and tissue formation, which might be the potential cause for such increased cell numbers in IFnHAp treated cells [27, 28]. Moreover treated cells displayed normal morphology as the control indicating the nontoxic and biocompatible property of the nanocomposite (Fig. 5B). With respect to the concept of “cell vision”, the nanocomposite which is non-toxic to MG-63 cells may show different toxicity profile when any other cell type was used for the study. Thus it is proposed that toxicity profile of any material is strongly dependent on the cell type 11
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and in the current study the prepared nanocomposite showed good safety profile for MG-63 cells. 3.5. ALP activity
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Among the most important osteogenic features, the upregulation of ALP activity is a crucial event that happens during the initial stages of osteogenesis. ALP supports osteogenic
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differentiation and mineralization by generating inorganic phosphates (Pi), the inducer of hydroxyapatite formation [29]. Therefore it is imperative to study the level of ALP expressed
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during the course of bone formation. As shown in Figure 6A, the level of ALP was expressed
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at similar levels as those of the control on day 1. However, the level of ALP increased significantly on days 7 and 14 in IF-nHAp treated cells, thus proving their osteogenic
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potency. Endogenous expression of growth factors due to the interaction of cell surface with the chemical groups present on the nanocomposite may be a possible reason for such
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3.6. RT-PCR
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increased ALP acitivity in treated cells [30].
Several genes such as ALP, OPN and OC are expressed during new bone formation and
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hence, are used as indicators of osteogenic differentiation. ALP acts as an early marker of osteoblast lineages and induces hydroxyapatite formation by generating inorganic phosphates [31]. OC forms the most profuse, bone specific, non-collagenous protein and serves as an indicator of osteogenic maturation and bone formation [32]. OPN is generated by preosteoblasts, osteoblasts, and osteocytes and is deemed to play a key feature in bone remodelling [33]. The RT-PCR (Fig. 6B) result of IF-nHAp treated cells shows about 12.2, 8.3, and 2.5 times increase in the mRNA levels of ALP, OC and OPN genes, respectively. This significant increase serves as the foundation for the impending mineralization as they continue to form bone like nodules, which can further develop into complete 3D mineralized structures. 12
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3.7. ARS Staining Deposition of calcium ions is one of the most essential markers of bone formation and osteoblast differentiation. ARS staining was performed to determine the mineral deposition of
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MG-63 cells in the presence of the IF-nHAp nanocomposite. ARS chelates Ca2+ ions and helps in concurrent assessment of mineral distribution through microscopy and spectroscopy
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[17]. Osteoblasts cultured with IF-nHAp showed 2–5 times (Fig. 7A) increase in calcium deposition than that of the control. The microscopic images (Fig. 7B) also show the presence
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of red coloured calcium ions in all the concentrations with the maximum observed at 250 μg.
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The above results are consistent with those obtained for ALP activity and thus substantiate improved differentiation of osteoblasts in the presence of IF-nHAp composite.
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3.8. In vitro bioactivity through SBF
When considering osseo-integration, the main feature of an implant is to bind to the living
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bone by forming a bone-like apatite on its surface. This apatite is believed to trigger the cell
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signalling proteins and pathways involved in bone formation [34]. Therefore an easy method to assess the bone-bonding ability of a material is to immerse it in SBF, a solution with ion
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concentrations similar to that of human blood plasma [18]. Figure 8 A, B, C and D show the SEM images of apatite coatings deposited on the surface of the nanocomposite after 21 days of incubation in SBF. These mineralized nodules are irregular shaped calcospherites with a mean diameter of 8–9 µm. EDX analysis (Fig. 8 E) shows the Ca/P ratio as 1.43, thus proving the formation of a Ca-rich apatite on the surface of the SBF soaked nanocomposite. The formation of apatite layers was further verified using the weight gained by the samples before and after soaking in SBF, which revealed 50–60% increase in the nett sample weight at the end of 21 days. In general HAp, which is negatively charged (because of the presence of the phosphate and hydroxyl groups) interacts with the calcium ions present in SBF and forms a positively charged calcium-rich intermediate (CRI). This calcium-rich intermediate further 13
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interacts with the negatively charged phosphate ions of SBF and cyrstallizes to form a bone like apatite [35]. 4. Conclusion
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From the present study, it is clear that iron-fibrin nanocomposites can act as substrates for the growth of nHAp crystals. The prepared IF-nHAp composite was needle shaped with an
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average width of about 30 nm and length of 80 nm. FTIR, XRD and VSM results proved the loading of iron within the nHAp, thus confirming their promising use in the area of magnetic
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field assisted orthopaedic tissue engineering. Hemolytic assay and ELISA proved the Further IF-nHAp offered a
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hemo/immunocompatible properties of the nanocomposite.
suitable environment to support osteoblast viability, spreading, and proliferation. Osteoblasts
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cultured in the presence of IF-nHAp showed enhanced ALP activity and calcium deposition, which are the chief factors of mineralization and bone formation. In addition, the
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upregulation of genes involved in osteogenic differentiation and matrix mineralization prove
Acknowledgment
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the potential use of IF-nHAp nanocomposite in the field of bone tissue engineering.
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Weslen S Vedakumari acknowledges the funding support granted by the Department of Science and Technology, India. 5. References [1] [2]
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FIGURE LEGENDS Fig. 1. (A) TEM image showing needle shaped IF-nHAp nanoparticles with average width of 30 nm and length of 80 nm. The arrow mark depicts iron oxide nanoparticles embedded
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within nHAp. (B) SEM micrograph of IF-nHAp showing well defined nanoparticles. (C) EDX spectrum of IF-nHAp with Ca/P ratio as 1.68, the value similar to that of bone HAp.
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Fig. 2. (A) FTIR spectrum of IF-nHAp showing the characteristic peaks of nHAp, fibrin and iron oxide respectively. (B) X-ray diffraction pattern of IF-nHAp with typical peaks of nHAp
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and iron oxide respectively. (C) TGA curve of IF- nHAp. (D) DSC thermogram of IF-nHAP.
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Fig. 3. Magnetization curve of IF-nHAp obtained using VSM.
Fig. 4. (A) Hemolytic assay for 25, 50, 100 and 250 μg of IF-nHAp respectively. (B) ELISA
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result showing the in vitro production of C3a and C4d complement split products after incubation with IF-nHAp. The asterisk (*) denotes a statistically significant difference from
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control (P< 0.05).
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Fig. 5. (A) MTT assay for 25, 50, 100 and 250 μg of IF-nHAp at different time intervals respectively. The asterisk (*) denotes a statistically significant difference from control
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(P< 0.05). (B) Microscopic images of MG-63 cells after 5 days of incubation with 25, 50, 100 and 250 μg of IF-nHAp respectively. Fig. 6. (A) ALP activity for 25, 50, 100 and 250 μg of IF-nHAp at different time intervals respectively. The asterisk (*) denotes a statistically significant difference from control (P< 0.05). (B) Expression levels of ALP, OC and OPN genes after IF-nHAp treatment. Fig. 7. (A) ARS assay for 25, 50, 100 and 250 μg of IF-nHAp respectively. The asterisk (*) denotes a statistically significant difference from control (P< 0.05). (B) ARS staining illustrating calcium deposition in the presence of various concentrations of IF-nHAp respectively.
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Fig. 8. (A, B, C, D) SEM images with different magnifications of the SBF soaked
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IF-nHAp. (E) EDX spectrum of SBF soaked IF-nHAp with Ca/P ratio as 1.43.
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GRAPHICAL ABSTRACT
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HIGHLIGHTS Iron-fibrin- nanohydroxyapatite (IF-nHAp) composite was prepared using a novel method. VSM result proved the superparamagnetic behaviour of the nanocomposite.
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MTT, Haemolysis and ELISA proved the bio/immunocompatibility of the nanocomposite.
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RT-PCR was used to quantify the mRNA levels of ALP, OC, OPN genes.
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