γ-Fe2O3 nanoparticles filled polyvinyl alcohol as potential biomaterial for tissue engineering scaffold

journal of the mechanical behavior of biomedical materials 49 (2015) 90–104

Available online at www.sciencedirect.com

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Research Paper

γ-Fe2O3 nanoparticles filled polyvinyl alcohol as potential biomaterial for tissue engineering scaffold Nor Hasrul Akhmal Ngadimana, Ani Idrisb,n, Muhammad Irfanb, Denni Kurniawana, Noordin Mohd Yusofa, Rozita Nasirib a

Faculty of Mechanical Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia

b

ar t ic l e in f o

abs tra ct

Article history:

Maghemite (γ-Fe2O3) nanoparticle with its unique magnetic properties is recently known to

Received 27 February 2015

enhance the cell growth rate. In this study, γ-Fe2O3 is mixed into polyvinyl alcohol (PVA)

Received in revised form

matrix and then electrospun to form nanofibers. Design of experiments was used to

28 April 2015

determine the optimum parameter settings for the electrospinning process so as to produce

Accepted 30 April 2015

elctrospun mats with the preferred characteristics such as good morphology, Young’s

Available online 9 May 2015

modulus and porosity. The input factors of the electrospinnning process were nanoparticles

Keywords:

content (1–5%), voltage (25–35 kV), and flow rate (1–3 ml/h) while the responses considered

Electrospinning

were Young’s modulus and porosity. Empirical models for both responses as a function of

Tissue engineering

the input factors were developed and the optimum input factors setting were determined,

Biomaterials

and found to be at 5% nanoparticle content, 35 kV voltage, and 1 ml/h volume flow rate. The

Nanofiber

characteristics and performance of the optimum PVA/γ-Fe2O3 nanofiber mats were com-

Magnetic nanoparticle

pared with those of neat PVA nanofiber mats in terms of morphology, thermal properties, and hydrophilicity. The PVA/γ-Fe2O3 nanofiber mats exhibited higher fiber diameter and surface roughness yet similar thermal properties and hydrophilicity compared to neat PVA PVA/γ-Fe2O3 nanofiber mats. Biocompatibility test by exposing the nanofiber mats with human blood cells was performed. In terms of clotting time, the PVA/γ-Fe2O3 nanofibers exhibited similar behavior with neat PVA. The PVA/γ-Fe2O3 nanofibers also showed higher cells proliferation rate when MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was done using human skin fibroblast cells. Thus, the PVA/γ-Fe2O3 electrospun nanofibers can be a promising biomaterial for tissue engineering scaffolds. & 2015 Elsevier Ltd. All rights reserved.

n

Corresponding author. Tel.: þ607 5535603; fax: þ607 5588166. E-mail address: [email protected] (A. Idris).

http://dx.doi.org/10.1016/j.jmbbm.2015.04.029 1751-6161/& 2015 Elsevier Ltd. All rights reserved.

journal of the mechanical behavior of biomedical materials 49 (2015) 90–104

1.

Introduction

Tissue engineering (TE) combines living cells with synthetic or natural support or scaffolds that is biodegradable so as to develop a three dimensional living construct that is architecturally functional and mechanically equivalent to or better than the tissue that needs to be replaced (Stock and Vacanti, 2001). The regenerated cells are seeded into a scaffold which provides structural support and 3D geometry template according to the damaged tissue to be healed. The scaffold can also act as a reservoir for bioactive molecules to facilitate cell growth and proliferation within it. The scaffold gradually degrades with time, replaced by the newly formed tissue from the seeded cells which now become fully functioning and is ready to be transplanted to the patient (Venugopal et al., 2008). Considering its function, scaffold must meet biological and nutritional needs for a specific cell population. There are a number of factors such as biocompatibility (Mikos and Temenoff, 2000; Lanza et al., 2011), biodegradability (Ma, 2004), large surface area to volume ratio (Thomson et al., 1995), good mechanical integrity (Lanza et al., 2011), high porosity and proper pore size (Lanza et al., 2011; Ma, 2004). Suitable surface properties that must be considered when selecting materials and fabrication process for tissue engineering scaffolds includes promoting cell adhesion, growth, and proliferation (Ma, 2004; Ramakrishna, 2005). Polyvinyl alcohol (PVA) is a semi-crystalline polymer that possesses good chemical and thermal stability (Qin and Wang, 2006). The synthetic polymer is soluble in water, nontoxic, biocompatible, and biodegradable. Therefore, it has the potential among the pool of biomaterials to be used for tissue engineering scaffolds. And indeed PVA has been extensively used as the main material to construct bone scaffold in tissue engineering applications (Asran et al., 2010; Gao et al., 2012; Linh et al., 2010; Shafiee et al., 2011; Qi et al., 2013; Wei et al., 2011). Furthermore, PVA based scaffold is known to provide mechanical stability and flexibility compared to conventional scaffolds made from natural polymers (Schmedlen et al., 2002; Sailaja et al., 2009). Despite its potential, PVA, like any other biomaterials, still needs improvements to be the ideal scaffold material. Blending with other biomaterials is one way towards this and is of interest in this study. Magnetic nanoparticles which have been used in biomedical applications (Arbab et al., 2003; Wilhelm et al., 2003; Shimizu et al., 2007; Attaluri et al., 2011; Akiyama et al., 2010) such as cell sheet construction, cell expansion, magnetic cell seeding, cancer hyperthermia treatment, and drug delivery vehicle can be a possible option to enhance the properties of PVA when blended with it. Availability of magnetic nanoparticles within PVA scaffold will also increase its rigidity favourably (Shao et al., 2013; Idris et al., 2012). Of particular interest, this study introduces the use of γ-Fe2O3 magnetic nanoparticles as the filler in PVA matrices blend. Electrospinning is a potential process used to fabricate the scaffold due to its simplicity and ability to produce fibres made from many types of polymers at nanometer scale. The fibers fabricated have similar dimension with the extracellular matrix of human tissue with critical structural and instructive component almost replicating the structure of natural human tissue

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(Hong Thien et al., 2012; Nelson et al., 2012; Rainer et al., 2011; Sill and von Recum, 2008; Li et al., 2012; Vaquette and CooperWhite, 2013). The fabricated nanofibers tend to favor cell adhesion, proliferation, migration, and differentiation due to the high surface area to volume ratio of the nanofibers combined with the microporous structure; all of them are highly desired properties for tissue engineering applications (Bhattarai et al., 2004; Liao et al., 2008). Electrospinning was successfully used to fabricate various magnetic nanofibrous bone scaffolds such as iron(III) oxide (Fe3O4)/polyurethane (PU) (Amarjargal et al., 2013), superparamagnetic iron(III) oxide nanoprticles (SPIONs)/polylactide (PLA) (Li et al., 2014), iron(III) oxide (Fe3O4)/ polycaprolactone(PCL) (Gloria et al., 2013), iron(III) oxide (Fe3O4)/ poly(lactide-co-glycolide) (PLGA) (Lai et al., 2012), and magnetic nanoparticles (MNP)/polycaprolactone(PCL) (Singh et al., 2014). Most of the researchers have blended the iron(III) oxide (Fe3O4) with their polymers to enhance their properties. Results revealed that the presence of magnetic nanoparticles has an effect on osteoinduction even without external magnetic force. Its singularity characteristic orientation movement in magnetic field has become of great interest for many applications. Wu et al. (2010) reported that the introduction of magnetic nanoparticles to CaP bioceramics could promote the bone formation and growth in vitro and in vivo. Meng et al. (2010) also demonstrated that the addition of magnetic nanoparticles in hydroxyapatite nanoparticles (nHA)/poly (D, L-lactide) (PLA) composite nanofibrous films could induce a significantly higher proliferation rate and faster differentiation of osteoblast cells (Meng et al., 2010). In this study maghemite, γ-Fe2O3 nanoparticles which are much finer in size with diameter (less than 13 nm) have stronger magnetic field properties compared with Fe3O4. Thus γ-Fe2O3 will have better performance as a bone tissue engineering scaffold compared with Fe3O4 due to larger surface area provided by the very fine nanoparticles and also its superparamagnetic properties because those characteristics will enhance the cell growth rate. Currently, most of the electrospun bone scaffolds are still in the in vitro testing stage. Some modification are required to transform the nanofibrous films into a 3D form before performing the vivo testing as well as clinical use. In order to fabricate PVA/γ-Fe2O3 nanofibers with favorable characteristics, the parameters of the electrospinning process need to be optimized. Previous studies have reported the range of parameter settings of the electrospinning process on various polymeric blends using fiber diameter as their sole response for consideration (Abdal-hay et al., 2013; Brun et al., 2011; Ha et al., 2013; Haslauer et al., 2012; Salifu et al., 2011; Wang and Wang, 2012; Zhang, 2011). In fact, we have also done some work on electrospinning process for PVA blends (Fallahiarezoudar et al., 2015). However, to the best of our knowledge there has not been any research that considers porosity and mechanical strength of the electrospun mat as responses. Therefore, the main objectives of this study are to introduce a new biomaterial consisting of PVA/γ-Fe2O3 blend and to determine the optimum parameter setting of the electrospinning process in order to fabricate nanofibers with high mechanical properties in terms of Young’s modulus and porosity. The input factors investigated are nanoparticle content, flow rate, and voltage applied. The interaction between scaffold and cells is also part of the investigation.

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journal of the mechanical behavior of biomedical materials 49 (2015) 90 –104

2.

Materials and methods

2.1.

Materials

Chemicals used in this study were of analytical and they are as follows: sulfuric acid (H2SO4) (QRëC), iron(II) chloride (FeCl2) (98% purity, Sigma Aldrich), iron(III) chloride (FeCl3) (45% purity, Riedel-de Haen), nitric acid (HNO3) (65% purity, QRëC), acetone (QRëC), ammonia solution (NH3) (25% purity, Merck), hydrochloric acid (HCl) (37% purity, QRëC), polyvinyl alcohol (PVA) (99þ% purity, Sigma-Aldrich), phosphate buffered saline (PBS, Gibco, USA), human red blood cells (HRBCs), human skin fibroblast cell line (HSF1184) penicillin-streptomycin, Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Gibco, USA), penicillin (Gibco, USA), streptomycin (Gibco, USA), trypsinase (Sigma, USA).

2.2.

Synthesizing of γ-Fe2O3

The co-precipitation method (Massart, 1981) was chosen as a method for synthesizing γ-Fe2O3. The synthesis of these magnetic nanoparticles was by alkaline co-precipitation of a stoichiometric ferrous chloride (FeCl3) and ferric chloride (FeCl2) in ammonium hydroxide solution (NH4OH). The black precipitate obtained called magnetite (Fe3O4) was obtained initially and was further acidified using nitric acid (HNO3) and oxidized at 100 1C in a solution of ferric nitrate (Fe (NO3)3  9H2O) to form γ-Fe2O3. The purpose of the last step is to make sure that the nanoparticles obtained were stable and can be used for further study. The particles will be cautiously cleaned for a few times in order to reduce the ionic strength of the acidic anionic ferrofluid (pH¼2). The nanoparticles were coated with citric acid to prevent agglomeration. The ferrofluid obtained is stable due to the existence of surface charges which induced screened electro-static

repulsions between the particles (Idris et al., 2010). They were spherical in shape and their size was analyzed using TEM and was in the range of 8 nm–13 nm as described in our previous studies (Idris et al., 2010; Ngadiman et al., 2014).

2.3. Fabrication of PVA/γ-Fe2O3 electrospun nanofiber mats by electrospinning process Fig. 1 shows the schematic diagram of the electrospinning process. The syringe pump pushed the polymer solution (γ-Fe2O3 filled PVA) through the tube into the needle of the electrospinning machine. The polymer was stored in the 30 ml syringe before being pushed by the syringe pump to the stainless steel gauge needle with an inner diameter of 0.60 mm. The function of the syringe pump is to make sure the flow of polymer is constant and controllable. The needle was connected to a high dc voltage power supply. The distance from the tip to the collector was controlled and kept at 8.0 cm in length and the drum rotating speed was kept constant at 1500 rpm as the optimum setting so as to achieve finer fiber diameter as reported previously (Fallahiarezoudar et al., 2015). The grounded counter electrode was attached on the rotating collector. The electrospun nanofiber was collected using rotating collector which was covered with aluminum foil so as to ease its removal from the drum which was in the form of a thin nanofiber mat. The entire fibrous electrospun nanofiber mat obtained was dried in vacuum to remove residual solvent. The PVA concentration was fixed at 10 wt/v%.

2.4.

Design of experiments

In this study, factorial design was used to determine the significant factors and the optimize parameters setting for the electrospinning process in order to produce high strength and high porosity electrospun mat for bone scaffolds. Table 1 shows the low and high levels for the three factors investigated. In this

Fig. 1 – The schematic diagram of the electrospinning process.

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journal of the mechanical behavior of biomedical materials 49 (2015) 90–104

Table 1 – Factors and levels for the factors investigated. No.

Factor

1 2 3

Level

Nanoparticle content (%) Voltage (kV) Flow rate (ml/h)

case, the 2-level full factorial design was used to evaluate the effect of process factors on the Young’s Modulus and porosity of the electrospun nanofiber mats. Factors involved in this model are nanoparticle content, voltage supply, and volume flow rate. The range of each parameters are 1 v/v%–5 v/v% for nanoparticle content, 25–35 kV for voltage and 1–3 ml/h for flow rate values as shown in Table 1. The range of each parameter was selected based on the previous report (Fallahiarezoudar et al., 2015). Three center points were added to test the linearity assumption of estimated model. Commercially available statistical software (Version 7 of Design Experts (StatEase, Inc.)) was used to do experimental design and to analyze the results.

2.4.1.

Young modulus

The nanofiber mat was cut into a dumb bell shape according to ASTM D638-10 Type V and the tensile strength test was performed using a LRX Tensile Machine at a 10 mm/min crosshead speed at room temperature. At least five sample measurements were performed so as to ensure the reproducibility of the data. The young modulus was calculated by using Eq. (1), E¼

F=A0 L=L0

ð1Þ

where E is the young modulus, F the force exerted on nanofiber mat under tension, A0 the thickness of the nanofiber mat, L amount by which the length of the nanofiber mat changes and L0 the original length of the nanofiber mat.

2.4.2.

Porosity

Nanofiber mat were maintained in distilled water for 3 min, then it was weighed after wiping superficial water with filter paper. The wet nanofiber mat was placed in a freezer dryer for 72 h before measuring the dry weight. The porosity of membrane was calculated using Eq. (2) pð%Þ ¼

ðQ1 Q2Þ  100 Ahðapparent densityÞ

ð2Þ

The apparent density was calculated using equation as ρ¼ Q1/Ah, where p is the porosity of membrane, ρ the apparent density, Q1 the wet sample weight (g), Q2 the dry sample weight (g), A the square of membrane (cm2) and h is the thickness of membrane (μm). In order to minimize experimental error, each membrane was measured for three times and calculated average (Zheng et al., 2006).

2.5.

Characterization of electrospun nanofibrous mats

The morphology of the electrospun nanofibrous mats were observed under field-emission scanning electron microscopy

Low ( 1)

Center (0)

High (þ1)

1 25 1

3 30 2

5 35 3

(FE-SEM, JEOL JSM-7500F). The FE-SEM equipment was also equipped with an energy-dispersive X-ray (EDX) system so that EDX could be used to determine the elemental composition at selected spots of the sample surface. X-ray diffraction (XRD, Bruker, D8 Advance X-ray) patterns of the PVA/γ-Fe2O3 electrospun nanofiber were examined to study its crystal structure and they were compared with those of neat PVA electrospun nanofiber. Fourier transform infrared spectroscopy (FTIR, Nicolet 8700) was carried out to detect various chemical bonds in the blend. Calorimetric measurement was taken to examine the changes in crystalline caused by the iron oxide nanoparticle addition using a differential scanning calorimeter (DSC, TA Instrument Q1000). Atomic Force Microscopy (AFM, SII Nanotechnology SPMSPI3800N) was used to analyze the surface roughness of the nanofibers with 3D micrographs. For AFM, the contact mode was used and the scanning area of the nanofiber mat was approximately 5 μm  5 μm. F roughness, ‘Ra’ and ‘RMS’ values represent the mean roughness and root mean square roughness of the fiber’s surface, respectively, whereas P–V is the mean difference in heights between the five highest peaks and the five lowest valleys. Optical contact angle measurement system (KSV Instruments CAM 101 optical Contact Angle Meter) was used to determine the surface hydrophilicity of the electrospun mats.

2.6.

Biocompatibility

Blood under normal conditions contacts an endothelium (interior surface of blood vessels) with antithrombotic and anticoagulant properties. Neither PVA nor PVA/γ-Fe2O3 electrospun nanofibers has any endothelium function so they become foreign substances which trigger a multifaceted series of events of blood proteins, platelets, leukocytes, which complements thrombus formation resulting in fibrin matrix. Delayed fibrin formation with its less fibers accumulation favors biocompatibility and shorter clotting time indicates faster conversion of fibrinogen into insoluble fibrin protein which then leads to thrombus (blood clotting). The contact of blood with poor biocompatible tissue scaffold can disturb the chemistry of blood and can decrease or increase the clotting factor. The decrement of some clotting factors can cause bleeding disorders that lead to high risk of abnormal bleeding even with a small brush. Conditions such as lupus anticoagulant syndrome or antiphospholipid antibody syndrome develop when the immune system mistakenly attack blood clotting factors due to its structural changes. This can cause the blood to clot easily in veins and arteries and patient can die. The presence of tissue scaffolds is considered as an intruder for blood and body immune system which may disturb the blood chemistry thus

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the study of coagulation factor is an important parameter to judge the effect of tissue scaffold upon contact with blood. The following test were performed as an indicator to the biocompatibility of scaffolds prepared.

2.6.1. Blood clotting times for coagulation factors assay (PT, TT, APTT) The anti-thrombogenicity, pro-thrombin time (PT) for an intrinsic pathway, thrombin time (TT) for extrinsic pathway and the activated partial thromboplastin time (APTT) for both intrinsic and extrinsic pathway of the electrospun nanofiber mats were determined by a semi-automated blood coagulation analyzer Clot SP (All Eight (Malaysia) Sdn Bhd). Stago kits of neoplastin 2, thrombin 2, CK Prest 2 and fibrinogen 2 were used for the determination of PT, TT, and APTT, respectively. The electrospun nanofiber mats (0.5 cm  0.5 cm, three pieces) were immersed in 0.2 ml of phosphate buffered saline (PBS) solution (pH¼ 7.4) for 1 h. The PBS solution was then removed and the electrospun nanofiber mats were incubated in 100–200 ml of fresh healthy human blood plasma for 30 min at 37 1C in a transparent plastic tube. According to their test methods, PT, TT and APTT were measured, and each of the 3 mats were measured thrice (Morti et al., 2003). APTT and PT are usually used to inspect the intrinsic pathway (a clot form in response to an poor biocompatible vessel wall) and reveal the bioactivity of intrinsic blood coagulation factors whereas TT is used to measure the extent of an extrinsic (fibrin clot formation in response to tissue injury) pathway of blood clotting (Su et al., 2008).

2.6.2.

Effect of CaCl2 ion on fibrin formation

Fibrin is a protein that is involved in the clotting of blood and calcium ions are known to increase the rate of formation of fibrin. Its suggested that neutralizing the Caþþ ions can maintain the coagulation chemistry of blood and some of anticoagulants (e.g. acid-citrate-dextrose) can bind with calcium ion which lead to higher coagulation time. This test is important in order to measure the anticoagulant properties of the scaffolds. In this study, the both PVA and PVA/γ-Fe2O3 membranes were dipped in CaCl2 solutions and the performance of tissue scaffold neutralizing effect of calcium chloride solution was studied. The plasma of human blood was diluted with Stago Owner Koller buffer solutions at the ratio of 1:9. Electrospun mats of size 1 cm  1 cm which were immersed in PBS were incubated at 37 1C and dipped in 200 ml diluted plasma and 100 ml of 25 mM CaCl2 aqueous solution for half an hour. Then effect of Caþþ in terms of seconds on different electrospun mats was determined using Clot SP instrument.

2.6.3.

Plasma recalcification time (PRT)

Electrospun nanofiber mats (2 cm  2 cm) were immersed in the PBS and equilibrated at 37 1C for 1 h. Then, platelet poor plasma (PPP) was placed on the electrospun nanofiber mats attached to a watch glass and incubated statically at 37 1C. 100 ml of 25 mM CaCl2 aqueous solution was then added to the PPP and the plasma solution was monitored for clotting by manually dipping in a stainless-steel hook coated with silicone into the solution to detect fibrin threads. Clotting time was recorded at the first sign of fibrin formation on the

hook. The test was repeated three times for each sample to get a reliable value (Li et al., 2012).

2.6.4.

Hemocompatibility test

Human red blood cells (HRBCs) were collected by removing the serum and centrifuging at 3000 rpm for 3 min. In PBS, HRBCs were washed and diluted with PBS ten times at the ratio of 2:8. Water was used as the negative control and PBS buffer as the positive control. Then, 2 mg of electrospun nanofiber sample was dipped into the above mixture and incubated for 60 min at 37 1C, followed by centrifugation (10,000 rpm, 2 min). The absorbance was determined at 540 nm by UV–Vis spectrometer. The hemolytic percentage (HP) was calculated using Eq. (3), HP ð%Þ ¼

Dt  Dnc  100 Dpc Dnc

ð3Þ

where Dt is the absorbance of the test samples, Dpc and Dnc are the absorbances of the positive and negative control, respectively. The data was expressed as mean7SD, nZ3.

2.6.5.

MTT assay

The number of viable cells in the PVA/γ-Fe2O3 electrospun nanofiber mats was measured by a MTT assay. The mechanism of the MTT assay is that metabolically active cells will react with a tetrazolium salt in the MTT agent to produce a soluble formazan dye that can be absorbed in a wavelength of 490 nm (Wataha et al., 1992). The assay was performed by using human skin fibroblast cell line (HSF1184). The optical density (OD) of the solution was measured with a microplate reader (Multiskan MK3, Thermo Electron Corporation) after 72 h of incubation. All the experiments were performed in triplicate. TCPs (tissue culture plates) were used as control.

3.

Results and discussion

3.1.

ANOVA analysis

In this study, PVA/γ-Fe2O3 nanofibers were fabricated by using electrospinning process for tissue engineering scaffolds application. Mechanical integrity and porosity are the main elements in the development of the scaffolds. Mechanical integrity will determine the strength of scaffolds and porosity will affect the cell growth rate on the scaffolds. Thus, optimizing electrospinning process parameters in order to develop nanofibers which have good mechanical properties and porosity becomes an objective in this study. Table 2 lists the complete experimental layout of the designed experiments, consisting of a total of 11 experimental trials and the responses obtained from the electrospinning process based on these experimental trials. The tests for significance of each model, individual model terms and lack-of-fit were performed to evaluate the model obtained. p-value was set at 0.05 for a factor to be considered significant. Table 3 shows the details of the analysis of variance (ANOVA) test results for Young’s Modulus. The result shows that the p-value for the model iso0.01 which indicates that the model was significant and consequently desirable (Montgomery, 2004). The main effects, nanoparticle content (A), voltage supply (B), and flow

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Table 2 – Complete design layout of and the responses obtained from the electrospinning process. Std. run no.

1 2 3 4 5 6 7 8 9 10 11

Run

9 1 11 8 7 2 10 6 5 4 3

Block

1 1 1 1 1 1 1 1 1 1 1

Input factors

Responses

A—Nanoparticle content (%)

B–Voltage supply (kV)

C–Flow rate (ml/h)

Young’s modulus (MPa)

Porosity (%)

1 5 1 5 1 5 1 5 3 3 3

25 25 35 35 25 25 35 35 30 30 30

1 1 1 1 3 3 3 3 2 2 2

117.14 153.93 94.35 135.80 141.31 173.73 126.08 163.26 136.61 137.27 133.30

90.34 84.94 91.99 88.80 87.48 82.49 88.94 83.99 85.64 87.35 88.93

Table 3 – ANOVA results (partial sum of squares) for Young’s Modulus (MPa). Source

Sum of squares

Degree of freedom

Mean square

F value

p-Value

Model A—Nanoparticle content B—Voltage C—Flow rate Curvature Residual Lack of fit Pure error

4597 2659 555 1383 11 55 46 9

3 1 1 1 1 6 4 2

1532.2 2658.8 554.9 1382. 9 10.8 9.1 11.4 4.5

168.4 292.2 61.0 152.0 1.2

o0.01 o0.01 o0.01 o0.01 0.32

2.5

0.30

Significant

Not significant Not significant

Fig. 2 – Pareto chart for (a) Young’s modulus and (b) porosity.

rate (C) are found to be significant. The nanoparticle content (A) is the most significant factor influencing the Young’s Modulus of the electrospun nanofiber mats, followed by flow rate (C) and voltage (B) as illustrated in the pareto chart (Fig. 2a). Table 3 shows the ANOVA results for Young’s Modulus response which reveals that the model’s lack-of-fit was not significant which was desirable. Its R-squared value was 0.99

which was close to 1 which was also desirable. These results suggest that the model was able to explain about 99% of the variation in the response. The adequate precision which compared the range of predicted point to design point was 39.0, which was greater than the minimum desired value of 4. The curvature was not significant which indicates that there was no need for a 2nd order model to be fitted (Long-Wee et al., 2013).

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The same procedure was applied on the porosity results and the ANOVA is presented in Table 4. The model and all main input factors are significant. Ranking of influence of

input factors is the similar to Young’s modulus, based on the Pareto chart (Fig. 2b). The model’s lack-of-fit was not significant, its R-square value was 0.9132 which was close to 1,

Table 4 – ANOVA results (partial sum of squares) for porosity (%). Source

Sum of squares

Degree of freedom

Mean square

F value

p-Value

Model A—Nanoparticle content B—Voltage C—Flow rate Curvature Residual Lack of fit Pure error Cor total

73.57 42.92 8.97 21.68 9.1  10  3 7.68 2.26 5.41 81.25

3 1 1 1 1 6 4 2 10

73.57 42.92 8.97 21.68 9.1  10  3 1.28 0.57 2.71

19.17 33.54 7.01 16.95 7.113  10  3

0.0018 0.0012 0.0382 0.0062 0.9355

Not significant

0.21

0.9132

Not significant

Significant

Fig. 3 – Normal probability plot of residual for (a) Young’s modulus and (b) porosity as well as plots of residuals vs. predicted response for (c) Young’s modulus and (d) porosity.

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journal of the mechanical behavior of biomedical materials 49 (2015) 90–104

and its adequate precision was 13.2, all indicating that the model can adequately predict the response on hand. The curvature for this model was also not significant. Eqs. (4) and (5) are the final empirical models in terms of actual factors for Young’s modulus and porosity: Young’s modulus ¼ 134:28 þ 9:12nA 1:67nB þ 13:15nC

ð4Þ

Porosity ¼ 87:791:16nA þ 0:21nB1:65nC

ð5Þ

where A is the nanoparticle content, B is the voltage, and C is the flow rate. The normal probability plots of the residuals and the plots of the residuals versus the predicted response for Young’s modulus and porosity are shown in Fig. 3. A check on the former plot revealed that the residuals generally fall on a straight line implying that the errors are distributed normally. Latter plots revealed that there were no obvious pattern and unusual structure which implied that the models proposed were adequate and there was no reason to suspect any violation of the independence or constant variance assumption.

3.2.

Confirmation test

In order to verify the adequacy of the estimated model, six confirmation runs were performed. The test conditions for the first three confirmation runs were among the parameter settings that were performed previously whilst the remaining three confirmation runs were conditions that have not been used previously but are within the range investigated. The Young’s modulus and porosity of the selected experiments were

predicted together with the 95% prediction interval. The predicted values and the associated prediction interval are based on the models developed. The predicted and the actual experimental values were compared and the percentage error calculated. All these values are presented in Table 5. The percentage error range between the actual and predicted value for Young’s modulus and porosity are less than 10% which is acceptable. The empirical models can also be used to determine the optimum setting, within the range investigated for the input factors, to obtain the most desired responses. In this case, as maximum Young’s modulus and porosity are desired, the parameters setting should be at 5% nanoparticle content, 35 kV voltage, and 1 ml/h volume flow rate. The Young Modulus obtained at the optimum process parameter settings was 139 MPa with a porosity of 88.8%. As a comparison, Wei et al. (2011) have developed bone TE scaffolds using PVA blend with Fe3O4/chitosan by electrospinning process. Highest young modulus reported for their 5% amount of Fe3O4 in their experiments was 58.4 MPa. In this experiment, by using 5% of γ-Fe2O3, the young modulus obtained was 139 MPa. This proved that γ-Fe2O3 improved the mechanical properties of PVA.

3.3.

Characterization of electrospun nanofibrous mats

The PVA/γ-Fe2O3 electrospun nanofiber mats fabricated at the optimum parameters setting was further characterized, with neat PVA electrospun mats as control. From Fig. 4, the corresponding XRD pattern shows that the PVA/γ-Fe2O3 electrospun nanofibers possess one broad diffraction peak at 20.41 and six

Table 5 – Confirmation run results. No.

1 2 3 4 5 6

Nanoparticle content (%)

5 1 3 5 5 3

Voltage supply (kV)

35 35 30 25 35 35

Flow rate (ml/h)

1 3 2 2 2 3

Young’s modulus

Porosity

Actual

Predicted

Error (%)

Actual

Predicted

Error (%)

139 130 134 163 150 141

135 125 138 165 148 143

2.9 3.9 3.1 1.3 1.1 1.3

88.8 89.8 88.7 80.2 83.2 88.9

87.8 88.5 87.1 83.8 86.0 86.4

1.1 1.4 1.8  4.5  3.3 2.9

Fig. 4 – XRD patterns of electrospun fibrous scaffolds of PVA/γ-Fe2O3.

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sharp diffraction peaks at 30.01, 35.51, 391, 57.0 and 63.01. The broad peak indicates the semi-crystalline nature of PVA and the lateral order structure of its molecular chain. The XRD result revealed six sharp peaks which correspond to the crystal planes of inverse cubic spinel structural of γFe2O3 (Wei et al., 2011). This result is supported by EDX analysis that reveals the existence of iron in the composition as shown in Fig. 5. Fig. 5 exhibits the elemental compositions of neat PVA and 5% nanoparticle content PVA/γ-Fe2O3. The base elements in neat PVA are carbon and oxygen while iron was also observed in the blend’s composition. The FTIR spectra of neat PVA and PVA/γ-Fe2O3 electrospun nanofibers are shown in Fig. 6. For neat PVA, the characteristic bands present in the 2800–3200 cm  1 and 1300 1500 cm  1 are due to the stretching and deformation vibrations of methyl/methylene/methine (CH3/CH2/CH). The intense band at 3100 cm  1 3600 cm  1 is attributed to hydroxyl groups in each polymeric unit whereas peaks at 1088 and 847 cm  1 belong to –(C–O) and –(C–C) resonance of PVA (Qi et al., 2013; Zheng et al., 2001). For PVA/γ-Fe2O3 blend, no prominent difference was found compared with pure PVA except for

the presence of characteristic absorption of –(Fe–O) bond at 600 cm  1 indicating the presence of γ-Fe2O3 in the electrospun nanofiber mats (Wei et al., 2011) and the peaks became more sharper at 2800–3200 cm  1. From the FTIR spectra, the presence of characteristic absorption –(Fe–O) bond at 600 cm  1 indicates the presence of γ-Fe2O3 in the electrospun nanofiber mats (Wei et al., 2011) and the peaks became sharper at 2800–3200 cm  1. Regarding thermal properties, Fig. 7 represents the DSC results of PVA and PVA/γ-Fe2O3 electrospun nanofibers. In both spectra, three peaks are more prominent, with A and D represent the PVA’s melting state. From the DSC result, addition of γ-Fe2O3 in the PVA reduces the heat enthalpy (ΔHf) and increases the glass transition temperature (Tg) of the electrospun nanofibers, as expected from a blend with semi crystalline polymer matrix (Nor et al., 2012). Surface roughness which is directly proportional with the cell adhesion; the fundamental step in bone regeneration and which influences the morphology and capacity of cell proliferation and differentiation (Ma et al., 2005), was measured using AFM. Fig. 8 represents the AFM results of both electrospun nanofibers where

Fig. 5 – EDX graphs of elements present in the (a) neat PVA and (b) 5% nanoparticle content electrospun nanofiber mats.

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Fig. 6 – FTIR spectra of neat PVA and PVA/γ-Fe2O3 electrospun nanofibers.

Fig. 7 – DSC heating curves of (a) PVA and (b) PVA/γ-Fe2O3. A, B, C, D, E and F represent peaks and ΔHf, ΔCp, and Tg.

different colors show dissimilar surface roughness. The surface roughness of neat PVA electrospun nanofibers is lower than that of PVA/γ-Fe2O3 one from 2.8 nm to 3.9 nm, suggested due to the presence of nanoparticles on the latter’s surface. The PVA/γ-Fe2O3 nanofiber mat has higher surface roughness than PVA is likely to provide better cell adhesion. Another indication of biocompatibility is the hydrophilicity of the electrospun nanofibers (Wei et al., 2011). The water

contact angle was used to test the hydrophilicity of nanofibrous mats. The water contact angle was used to test the hydrophilicity of the nanofibrous mats and its value must be less than 901 (Su et al., 2012; Zhang et al., 2009; Ma et al., 2008). Results revealed that the neat PVA is highly hydrophilic with its contact angle of 44.5170.11 while PVA/γ-Fe2O3 blend’s contact angle is 48.2171.21. The blend is slightly less hydrophilic due to the presence of magnetic nanoparticles.

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3.4.

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Micrograph images

Fig. 9(a–d) shows the FE-SEM images of the electrospun nanofiber mats at the optimum conditions which exhibits homogenous, smooth and continuous surface morphology. Images of PVA/γ-Fe2O3 show well distributed spots of nanoparticles in the electrospun nanofiber. The fiber diameter of PVA/γ-Fe2O3 with 5% nanoparticle content is larger (average of 286761 nm) compared to neat PVA (average of 219723 nm). For FE-SEM analysis, PVA/γ-Fe2O3 electrospun nanofiber mats are having thicker fiber diameter compared to PVA electrospun nanofiber mats. The presence of nanoparticle influenced the size of fiber diameter. Zooming on the PVA/γFe2O3 electrospun nanofibers, the nanoparticles are uniformly distributed indicating that the nanoparticles are well coated and they do not agglomerate. Such uniform distribution ensures the fiber has a uniform superparamagnetic properties which can enhance cell proliferation rate. There

are no beads formation which reflects appropriate setting of electrospinning parameters.

3.5. mats

Blood biocompatibility of electrospun nanofibrous

3.5.1.

Blood clotting time (APTT, PT and TT)

The in vitro analysis findings on the blood compatibility of the electrospun nanofibers revealed that the addition of γ-Fe2O3 slightly reduced the clotting time, likely due to its low hydrophilicity. Fig. 10 illustrates the APTT, PT and TT results of neat PVA and PVA/γ-Fe2O3 electrospun nanofibers. In terms of concentration of fibrin and time, it can be said that both PVA and PVA/γ-Fe2O3 electrospun nanofibers exhibit similar results.

3.5.2.

Effect of CaCl2 ion on fibrin formations

Fig. 11 represents the test results of first fibrin formation within the electospun nanofiber mats. From Fig. 11, it is observed that the concentration of fibrin formation (Fig. 11A) on PVA/γ-Fe2O3

Fig. 8 – AFM results of surface roughness, line profile, front and 3D micrograph of (a) neat PVA and (b) PVA/γ-Fe2O3 electrospun nanofibers.

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Fig. 9 – FE-SEM images of the electrospun nanofibrous mats (a) PVA/γ-Fe2O3 with 5% nanoparticle loading at 15 K magnification, (b) PVA/γ-Fe2O3 with 5% nanoparticle loading at 50 K magnification, (c) neat PVA at 15 K magnification and (d) neat PVA at 50 K magnification.

nanofibers in contact with human blood plasma. Neat PVA showed higher plasma recalcification time but lower hemolytic percentage compared with PVA/γ-Fe2O3 nanofibers. Plasma recalcification time (PRT) is an indicator of intrinsic coagulation cascade activation (Kim et al., 2000) and hemo-lytic test can predict the destruction ratio of red blood cells. Addition of γ-Fe2O3 showed slightly reduced PRT time but similar HP ratio compared with neat PVA.

3.6.

Fig. 10 – Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time of the electrospun nanofiber mats.

nanofiber mat is higher compared with neat PVA. However the result is vice versa for fibrin formation time (Fig. 11B) and the effect of CaCl2 on fibrin formation in terms of time (Fig. 11C). In Fig. 11C, the PVA/γ-Fe2O3 electrospun nanofiber mat shows lower fibrin formation time compared to the PVA based nanofiber mat. The findings suggested that PVA/γ-Fe2O3 electrospun nanofiber mat has better tendency to neutralize Caþþ ions thus exhibiting better anticoagulant properties.

3.5.3. Plasma recalcification time (PRT) and hemo-lytic percentage (HP) Fig. 12 represents the plasma recalcification time (PRT) and hemo-lytic percentage (HP) of PVA and PVA/γ-Fe2O3

MTT assays

Fig. 13 shows the results of relative cell viability towards type of electrospun nanofiber mats used. The proliferation rate of cells on PVA/γ-Fe2O3 nanofibers (92%) was higher than that of neat PVA (80%). During the first day after cell seeding, there was no significant difference among the HSF1184 cell viability. As culture time increases, cell proliferation on the electrospun nanofibers also becomes higher. From the results of MTT assay, PVA itself is a biocompatible material. By adding magnetic nanoparticle in the PVA, the proliferation rate of HSF1184 cells accelerates. Similar result was reported by previous researches about the osteoinductive effect towards magnetic nanoparticles. Wei et al. (2011) stated that the presence of magnetic nanoparticles develop a great number of tiny magnetic fields, which would subsequently express osteoinductive effect of static magnetic fields. It can be said that each magnetic nanoparticle acts as a single magnetic nanofield. Therefore, when it was integrated with the matrix, it created the micro-environment in the pores or on the surface of the blend which in turn produced the great number of tiny magnetic fields which promote the

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Fig. 11 – Record of the first fibrin detection point by using Clot Sp instrument. (A) and (B) represent the concentration and time when the first fibrin was first detected, respectively, whereas (C) shows the effect of CaCl2 on fibrin formation.

Fig. 12 – Plasma recalfication time (PRT) and hemo-lytic percentage (HP) recorded from PVA and PVA/γ-Fe2O3 electrospun nanofibers contact with human blood plasma.

cell proliferation rate. Besides that, magnetic nanoparticles have a large surface area to volume ratio. Its presence in PVA increases cell area attachment thus allowing more cells to anchor; accommodating a large number of cells (Liao et al., 2008). The presence of (γ-Fe2O3) also enhanced the mechanical properties and also biocompatibility of the materials. This positive result is important in order to perform further investigation on the use of magnetic nanoparticles, in particular γ-Fe2O3 for tissue engineering scaffolds.

4.

Conclusions

A new biomaterial PVA/γ-Fe2O3 was introduced and its nanofibers were fabricated using electrospinning process. In order to obtain electrospun nanofiber mats which have

maximum Young’s modulus and porosity the electospinning parameters need to be set at the optimum settings of 5% nanoparticle content, 1 ml/h volume flow rate, and 35 kV voltage. The PVA/γ-Fe2O3 nanofibers obtained have average fiber diameter of 286761 nm which was higher than that of neat PVA nanofibers (219723 nm). The addition of γ-Fe2O3 nanoparticles in the PVA electrospun nanofibers also increased the surface roughness which is good for cells attachment. The hydrophilicity of the electrospun nanofiber mat was proven by its water contact angle value which was well below 901. The PVA/γ-Fe2O3 electrospun nanofibers also exhibited good biocompatibility properties depicted by the various blood clotting tests and MTT assays. The skin fibroblast cells proliferation rate was higher in PVA/γ-Fe2O3 compared to neat PVA nanofiber mats. The findings showed PVA/ γ-Fe2O3 nanafibrous mat is a suitable material for tissue engineering scaffolds.

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Fig. 13 – Result of relative cell viability towards type of electrospun nanofibers used.

Acknowledgements The authors wish to thank the Ministry of Higher Education (MOHE), Universiti Teknologi Malaysia (UTM) and Research Management Center, UTM for the financial support to this work through the Geran Universiti Penyelidikan (GUP) funding number Q.J130000.2524.06H09.

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