Modulation of Protein Adsorption and Cell Proliferation on

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Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites Rahul Kumar Upadhyay, Sharmistha Naskar, Nitu Bhaskar, Suryasarathi Bose, and Bikramjit Basu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00946 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on May 2, 2016

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ACS Applied Materials & Interfaces

Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites a±

c±

a

b*

Rahul Upadhyay , Sharmistha Naskar , Nitu Bhaskar , Suryasarathi Bose , Bikramjit a,c,* Basu

Author Address a

Laboratory for Biomaterials, Materials Research Center, Indian Institute of Science,Bangalore560012, India

b

Department of Materials Engineering, Indian Institute of Science, Bangalore-560012, India

c

Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India

KEYWORDS: cytocompatibility; graphene oxide; HDPE; nanocomposites; grafting; toxicity; osteoblast; hMSC

± These authors made equal contributions (RU and SN) *Joint corresponding authors E-mail addresses: [email protected] (B. Basu), [email protected] (S. Bose)

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Abstract The uniform dispersion of nanoparticles in a polymer matrix, together with an enhancement of interfacial adhesion is indispensable towards achieving better mechanical properties in the nanocomposites. In the context to biomedical applications, the type and amount of nanoparticles can potentially influence the biocompatibility. In order to address these issues, High Density Polyethylene (HDPE) based composites reinforced with graphene oxide (GO) were prepared by melt mixing followed by compression moulding. In an attempt to tailor the dispersion and to improve the interfacial adhesion, polyethylene (PE) was immobilized onto GO sheets by nucleophilic addition-elimination reaction. A good combination of yield strength (ca. 20 MPa), elastic modulus (ca. 600 MPa) and an outstanding elongation at failure (ca. 70 %) were recorded with 3 wt % polyethylene grafted graphene oxide (PE-g-GO) reinforced HDPE composites. Considering the relevance of protein adsorption as a biophysical precursor to cell adhesion, the protein adsorption isotherms of bovine serum albumin (BSA) were determined to realise three times higher equilibrium constant (Keq) for PE-g-GO reinforced HDPE composites as compared to GO reinforced composites. In order to assess the cytocompatibility, osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) were grown on HDPE/GO and HDPE/PE-g-GO composites, in vitro. The statistically significant increase in metabolically active cell over different time periods in culture for upto 6 days in MC3T3 and 7 days for hMSCs was observed irrespective of the substrate composition. Such observation indicated that HDPE with GO or PE-g-GO addition upto 3 wt % can be used as cell growth substrate. The extensive proliferation of cells with oriented growth pattern also supported the fact that tailored GO addition can support cellular functionality, in vitro. Taken together, the experimental results suggest that the PE-g-GO in HDPE can effectively be utilised to enhance both mechanical and cytocompatibility properties and can further be explored for potential biomedical applications.


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1. Introduction Natural bone is a composite of collagen, a polymer matrix and hydroxyapatite, an inorganic reinforcement.1-2 The load bearing capabilities of the bone gain needs to be critically considered in developing an artificial bone implant.3-4 The mechanical properties of the implant materials must match with natural bone and this aspect is more significant for hip and knee joint.5 If the implant has higher mechanical properties than natural bone then it can cause stress shielding in patients.6-7 This has been reported as one of the major problems that can lead to bone loss or in revision surgery.8-9 The wear resistance of the implant material has always been important concern as the wear debris generated from the implant can cause osteolysis, which damages the integrity of the implant due to aseptic loosening.10 The biocompatibility of the implant material is therefore very important in order to avoid toxic effects.11-12 In the last two decades, the biomaterials research has witnessed impressive progress in reference to developing joint/bone replacements, based on polymeric materials.13-15 Various polymers like High Density Polyethylene (HDPE), Ultra High Molecular Weight Polyethylene (UHMWPE) etc. are being investigated for biomedical applications.16-20 However, the strength, elastic modulus need to be enhanced without compromising biocompatibility properties. In order to overcome these issues, the hybrid composites of HDPE with graphene oxide (GO) and polyethylene grafted graphene oxide (PE-g-GO) have been developed in the present study. GO is a two-dimensional nanosheet composed of hydrophobic π domains in the core region and ionized groups around the edges.21-22 These are the features that enhance its interactions with proteins through hydrophobic and electrostatic interaction.23-25 In biomedical field, GO has been used for applications such as tissue engineering, drug delivery, gene delivery and bioimaging.26-29 It is envisaged that the dispersion of nanoparticles in a polymer matrix decides the mechanical and cytocompatibility properties of the developed implant.30-32 Therefore, polyethylene is grafted onto GO for enhancing the interfacial adhesion with the matrix and henceforth, the dispersion.



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As far as the processing aspect is concerned, melt mixing has been reported to be superior than other methods like solution blending33-34, because it facilitates a better dispersion of nanoparticles in the polymer matrix. During melt mixing, in-situ grafting between the reactive functional group present either on the polymer or on the nanoparticle facilitates enhanced interfacial adhesion between the matrix and the nanoparticles. Although, earlier studies have reported the improvement in modulus and strength of UHMWPE and HDPE based composites, but retaining the above mentioned properties at higher filler content still remains a challenge.35-36 Extensive efforts have been made to improve the dispersion and interfacial adhesion in the biocomposites for their applicability as acetabular cup liner used in total hip replacement.27, 37 Significant research has been pursued on joint/bone replacements using HDPE/HA/Al2O3 and HA/UHMPE composites but the dispersion of ceramic fillers and the interfacial adhesion still remains a key challenge even with the use of coupling agents.38-39 In the present study, PE chain is immobilized onto GO surface to improve the structural properties and enhance the biocompatibility. The stress transfer from the matrix to the filler is expected to improve which eventually will result in enhancing the structural properties to a considerable extent. Efforts were therefore made to improve the mechanical properties of HDPE based composites in which surface modified GO nanoparticles were melt mixed with HDPE. The mechanical and cytocompatibility properties were assessed and compared with the composites filled with unmodified GO sheets. Protein adsorption studies have been performed as it is precursor to cell growth on any substrate. Cell viability and cell adhesion analysis were evaluated for HDPE/GO and HDPE/PE-g-GO composites. Mouse osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) were selected for all the in vitro experiments. It was observed that osteoblast and hMSC growth/adhesion systematically increases with increasing GO content and substantial cell growth was observed for PE-g-GO based composites


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as compared to unmodified GO based composites. Taken together this study opens up new avenues in designing unique polymeric substrates with enhanced cell growth.

2. Experimental section 2.1. Materials and methods High density polyethylene (HDPE) of density 0.95 g/cc was obtained from Swasan Chemicals (India). Graphite flakes, maleated polyethylene (PE-g-MAH), 4,4-methylene dianiline (MDA), Bovine Serum Albumin (BSA) and WST-1 Assay (Roche) were procured from Sigma Aldrich (USA).The solvents tetrahydrofuran (THF), xylene, toluene, ethanol, and methanol were obtained from Merck (India). 2.1.1 Biocomposite Processing 2.1.1.1 Synthesis of Graphene Oxide (GO) GO was synthesized from graphitic flakes by modified Hummers method.21,40 Briefly, the graphite flakes were mixed with concentrated H2SO4 and H3PO4 (9:1 ratio) in a water bath. KMnO4 was added very slowly to oxidize the mixture. This reaction mixture was stirred for 24 h at room temperature. After completion of the reaction, the mixture was poured into deionized water and stirred for 2 h. 5-10 ml of H2O2 (35 %) was added dropwise into the solution until the colour turns bright yellow. The solution was filtered and the residue obtained was washed in succession with deionized water, HCl and ethanol. The final residue obtained was vacuum dried at room temperature. 2.1.1.2 Synthesis of amine functionalized graphene oxide (GO-NH2) Amine functionalized graphene oxide was synthesized through nucleophilic substitution reaction by direct coupling of 4,4′- methylenedianiline (MDA) onto GO by a procedure described earlier.41 Briefly, 500 mg of GO was dispersed in 200 ml of THF using a probe sonication 5 ACS Paragon Plus Environment


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method. Then, 500 mg of MDA was later added to it and the resultant mixture was refluxed at 80⁰C (oil bath) in N₂ atmosphere for 6 h. The mixture obtained was centrifuged and washed thrice with ethanol. After drying overnight under vacuum at 55⁰C, the sample of GO-NH2 was obtained. Table 1 shows the elemental analysis of the as synthesized GO-NH2. 2.1.1.3 Synthesis of polyethylene grafted graphene oxide (PE-g-GO) PE-g-MAH and GO-NH2 were blended in HAAKE mini lab melt extruder at a speed of 60 rpm at 100°C in a weight ratio 3:1 to ensure a complete conversion of amine groups of GO-NH2 to imide groups. Scheme 1 shows different steps involved in the synthesis of PE-g-GO. From FTIR spectra (figure 1), it is confirmed that there is no free amine group in PE-g-GO suggesting that all amine groups of GO-NH2 are converted to imide groups. To further validate the complete conversion of all free amine to imide group, we performed CHNS (vario MICRO CUBE CHNS analyzer) analysis of GO-NH2, the mass percentage of C, H, N and O are summarized in table 1. The source of Nitrogen in GO-NH2 was amine group only. Mass percentage of Nitrogen is found to be 2.93 % i.e. 1 gram of GO-NH2 contains 29.3 mg of reactive amine Nitrogen in GO-NH2. Saponification value of PE-g-MAH (sigma Aldrich) is 32 mg KOH/g i.e. 1 gram of PE-g-MAH contains 16 mg reactive anhydride oxygen because two mole of KOH react with one mole of di carboxylic acid to form anhydride group. Hence, a ratio of 1:1 (PE-g-MAH and GO-NH2,) will not completely convert all free amine to imide group due to excess presence of amine group in GO-NH2 compared to the reactive anhydride group in PE-g-MAH. As a result, we used a ratio of 3:1 (PE-g-MAH and GO-NH2, respectively) to ensure a complete conversion of all free amine to imide group.


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Table 1: Compositional analysis of the amine functionalized graphene oxide (GO-NH2)

Sample Amine functionalized graphene oxide ( GO-NH2 )

Element

Mass Percentage

C

51.01

H

3.84

N

2.93

S

2.08a

O

40.14b

a The small percentage of sulphur could be due to contamination. b The oxygen percentage was calculated by subtracting the sum of percentage of C, H, N and S from 100.

The bubble formation in the extruded samples indicated the release of water vapour during the imidization reaction. The blending process was continued for another two cycles, until the bubble formation was ceased. The product (PE-g-GO) was further confirmed by FTIR.



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Scheme 1. Synthetic approach followed in grafting of polyethylene on graphene oxide (GO). PE-g-MAH: polyethylene grafted maleic anhydride; MDA: 4,4-methylene dianiline ; PE-g-GO: polyethylene grafted graphene oxide 2.1.1.4. Preparation of nanocomposites All the nanocomposites (HDPE/GO and HDPE/PE-g-GO) were prepared by using HAAKE minilab melt extruder at rotation speed of 60 rpm maintained at 220°C for the duration of 20 min. The extruded composites were then subsequently compression moulded at 150 °C for 2 min at pressure of 10 Psi. 2.1.2 Mechanical Characterization Uniaxial tensile testing was carried out using INSTRON 5967 in an effort to measure the yield strength, elastic modulus and % elongation until failure. All the samples were prepared according to ASTM standard, D-638 type-V for tensile testing. In particular the dumbbell shape specimens were tested under a cross head speed of 5 mm/min and the loading was continued till specimens were fractured. 2.1.3. Structural and morphological analysis of GO and PE-g-GO The attenuated total internal reflection fourier transform infrared spectra (ATR-FTIR, PerkinElmer) of GO and PE-g-GO were recorded from 4500 to 500 cm−1. Raman spectroscopy (Horiba LabRAM HR) on GO and PE-g-GO were carried out with a 532 nm monochromatic laser. The percentage grafting was evaluated using Netzsch STA 409 PC Thermogravimetry Analyzer (TGA). Dispersed GO and PE-g-GO nanoparticles were drop casted on a silicon wafer and dried in vacuum for atomic force microscopy analysis (AFM, NanoWizardR 3, JPK Instruments).The SEM micrographs of tensile fractured composites were acquired using FEI, QUANTA (USA) at 10 kV (see figure S2, supporting information).The electron transparent thin sections of ca. 60 nm of selected composites were obtained using Ultramicrotome (Leica) in liquid nitrogen 8 ACS Paragon Plus Environment

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atmosphere using antistatic device. Transmission electron microscopy (TEM) was carried out on a Tecnai G2 Spirit Bio-TWIN TEM instrument (120 kV) to observe the dispersion of the GO and PE-g-GO in the HDPE matrix. 2.1.4. Protein adsorption studies The adsorption of proteins from cell culture medium onto any solid surface is critical for cellular adhesion as the cell membrane receptors bind to the adsorbed proteins which further in downstream interacts with cytoskeleton complex.42 Hence, the adsorption of proteins together with other factors dictates the biological response of a material. In the present study, the adsorbed amount of protein was determined using solution depletion method. 43-44 The change in the concentration of the protein in bulk solution, prior to and after adsorption, gives an account of amount of protein adsorbed onto the material surface. Bovine serum albumin (BSA, A2058, Sigma-Aldrich) was chosen as the model protein for the adsorption studies as albumin is the most abundant protein in the circulatory system. 2. 1.4.1. Determination of the time required to attain equilibrium It is necessary to determine the time required for each sample type to attain equilibrium. A known concentration of BSA solution (50 µg/ml) was prepared in which disc shaped substrates, with a surface area of 1cm2, were soaked for a long period of time. During this period, readings were taken at 280 nm wavelength using a multi-mode plate reader (Eppendorf AF2200) at every 1 h interval. 200µl of the solution was taken in each well of a 96 well plate to determine the optical density (OD). The substrates were kept in this manner until the OD was constant as a function of time. 2.1.4.2. Quantitative estimation of the adsorbed protein BSA solution of 20 graded concentrations (maximum concentration was 1000 µg/ml, minimum concentration was 50 µg/ml, with an increment of 50 µg/ml) was prepared in deionized water. 9 ACS Paragon Plus Environment


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Very thin discs of ca.1 cm2 of surface area, were taken as substrates for the protein adsorption studies. Prior to protein adsorption, the surface of the substrates were cleaned by ultrasonication. These substrates were immersed and kept undisturbed in 1 ml of the prepared protein solution, in order to allow protein adsorption. After 24 h, absorbance of the spent bulk solution was measured at 280 nm of wavelength using the multi-mode plate reader by taking 200µl in each well of a 96 well plate. Standard curve was plotted to estimate the amount of protein left in the solution. Subsequently equilibrium constant (Keq) and surface molecular concentration (Cm) were determined from the obtained data. The amount of adsorbed protein onto the surface of the substrates was quantitatively determined as the difference ∆Cb, of the protein prior to and after adsorption,

ΔC = C = C − C

(1)

where, Cs is the surface concentration (µg/cm2), Cbi is the initial bulk concentration (µg/ml) and Cbf is the final bulk concentration (µg/ml) after the allowed duration of adsorption. A standard curve with optical density (OD) versus protein concentration (C) (see figure S4 of supporting information) was plotted from which the following equation was obtained after fitting with Origin Pro 9.1 software.

OD = 0.254 ln( C ) + 0.001

(2)

where, coefficient of determination (R2) is 0.9912. This equation was used to calculate the protein concentration from the measured OD as well as the amount of protein adsorbed onto the substrate. 2.1.4.3 Determination of Keq The Keq values for different sample composites were evaluated. It is envisaged that the phenomenon of adsorption of albumin onto hydrophobic surfaces follow Langmuir’s isotherm45, which is described as 10 ACS Paragon Plus Environment

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C =

!" % #$ !"

(3)

where Cs is the weight of protein adsorbed per unit area of surface, M is the molecular weight of the adsorbing protein molecule, Keq is an equilibrium adsorption constant, Cb is the bulk protein concentration in solution, A is the surface area per site, and NA is Avogadro's number.45A linear form of the equation can be derived as,

!" !&

=

#

!'

!

+ !"

'

(4)

A linear relation between Cb/Cs vs Cb can be clearly expected. The equilibrium constant (Keq) and monolayer concentration (Cm) are obtained from the slope divided by the intercept and inverse of slope, respectively. 2.1.5. Cell Culture Two cell lines, mouse osteoblast precursor cells (MC3T3) and human mesenchymal stem cell (hMSC) were selected to observe their responses towards all the substrate (HDPE, HDPE/GO and HDPE/PE-g-GO composites). Both the studies were planned, in respect to their culture duration in such a way that they would be considered as two independent studies. Three observations were planned at every alternate day. Each study was repeated thrice with triplicates. 2.1.5.1 Mouse osteoblast precursor cell (MC3T3) culture MC3T3 is an osteoblast precursor cell line derived from Mus musculus (mouse) calvaria.46 The reasons behind choosing MC3T3 cell line include (a) they have behaviour similar to primary calvarial osteoblasts; (b) osteoblast cell line is ideal in context of the use of a biomaterial as a



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bone implant; (c) they have good adherence on both lower and higher stiffness surfaces without compromising the reproducibility of the experiments. All the cell culture experiments were conducted with cells at passage of 2-7. Prior to seeding, cells were revived from cryo-preserved stock and expanded in tissue culture graded T25 flask (Eppendorf, Germany) containing complete culture media, which was αMEM (Alpha Minimum Essential Medium; Invitrogen), supplemented with 10 % fetal bovine serum (FBS; Invitrogen) and 1 % antibiotic antimycotic solution (Sigma Aldrich). Cells were maintained in an environment of 5 % CO2 incubator (Sanyo, MCO-18AC, USA) with 37°C temperature and 95 % humidification. Revival took 3 days during which after 2 days the media had been changed once. As the cells were 70-80 % confluent, they were enzymatically lifted from the T-25 flask by using 2 ml of 0.05 % TrypsinEDTA (Invitrogen) and subcultured for further use. The cells were concentrated by centrifugation at 1100 rpm for 3 minutes, re-suspended in known amount of media, counted with a Neubauer chamber, and then diluted in complete culture media to a cell density of ~104 cells per ml of media. The single cell suspension thus prepared has been used for seeding onto the samples. Disc shaped, thin substrates of all the samples (HDPE, HDPE/GO and HDPE/PE-g-GO composites) were prepared with a diameter of ca. 11mm for the cell culture experiments. The substrate size was taken in the manner that they would fit at the bottom of 48-well plate leaving no or negligible uncovered area. The substrates were sterilized exposing them in UV for 1 h while keeping them dipped in absolute ethanol (Merck, Germany). Gelatin (0.2 %) coated glass cover slips had been used as control. Before cell seeding, all the substrates were rinsed properly with phosphate buffer saline (1X PBS; pH 7.2-7.4) so that no trace of ethanol was left. Each well containing the substrate was seeded with 200 µl of the prepared cell suspension and maintained in the cultured for 2 days, 4 days and 6 days. 2.1.5.2 Human mesenchymal stem cell (hMSC) culture


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Human mesenchymal stem cells (hMSCs) were cultured as a part of this investigation with respect to the cytocompatibility of the substrates (HDPE, HDPE/GO and HDPE/PE-g-GO composites) and were considered to be justified in reference to the application of the biomaterials.47 hMSC cell line were procured from Institute for Regenerative medicine, Texas A&M HSC COM, USA and the necessary approval from the Institutional Committee for Stem Cell Research and Therapy (IC-SCRT), IISc, Bangalore was obtained before carrying out hMSC related experiments. Important to mention, the rationale of considering hMSC as an experimental cell line was that, these cells have been shown to be multipotent, capable of differentiating down to osteogenic lineages along with adipogenic and chondrogenic lineages. The cells were cultured using complete culture medium containing αMEM (Alpha Modified Eagle's Medium; Invitrogen) supplemented with 20 % Mesenchymal Stem Cell - Fetal bovine serum (MSC-FBS; Invitrogen), 1 % antibiotic antimycotic solution (Sigma) and 2 mM l-glutamine (Invitrogen). Cells were maintained at a temperature of 37 °C in a saturated humidified atmosphere containing 5 % CO2. Prior to seeding they were revived in a tissue culture graded T25 flask (Eppendorf, Germany). Upon reaching 70–80 % confluency, the cells were harvested using 0.05 % TrypsinEDTA (Invitrogen) and sub-cultured for further use. Cells were concentrated by centrifuging at 900 rpm for 3 minutes and then re-suspended in the complete growth medium, in order to 4

achieve single cell suspension of cell concentration of ca. 10 cells per ml of media. Seeding condition was kept same as of MC3T3. The culture duration for the hMSC was planned for 3 days, 5 days and 7 days. 2.1.6. Cell Viability using WST-1Assay Cell viability was evaluated biochemically using water-soluble tetrazolium salts (WST-1, 4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene-disulfonate,Roche).48 As WST-1 assay has no interaction with graphene oxide, hence this assay has been chosen over MTT



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(3(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. The WST-1 assay was performed after the prescribed culture durations of 2 days, 4 days and 6 days for MC3T3 and 3 days, 5 days and 7 days for hMSC. Both the cell types were cultured in 48 well plates on the substrates fitted and covering the bottom of each well. Approximately 2×103 cells/well were seeded on the sterilized samples in 48 well plates and subsequently placed in 5 % CO2 incubator at 37°C and 95 % humidified atmosphere. The substrates were transferred to a new plate after being washed thrice with 1X PBS. To perform the colorimetric WST-1 (Roche) assay, 200 µl of 10 % v/v solution of WST-1 and complete culture medium was prepared and added to each of the wells. After incubating for 4 hr at 37°C in 5 % of CO2 atmosphere along with saturated humidity, the optical density (OD) of the developed color was measured by a microplate reader (iMark, Bio-rad laboratories, India) at a wavelength of 450 nm. The absorbance value provides a direct correlation to the number of viable cells in each well. The measurements were obtained by averaging the data from three wells repeated thrice. Cell viability was calculated by using the following equation:

% Cell Viability =

012 1341250 3 670 189:0;341250 3 670 :12< 012 1341250 3 670 532643:;341250 3 670 :12

Modulation of Protein Adsorption and Cell Proliferation on

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