Starch-Derived Nanographene Oxide Paves the Way for

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Starch derived nano-graphene oxide paves the way for electrospinnable and bioactive starch scaffolds for bone tissue engineering Duo Wu, Archana Samanta, Rajiv K. Srivastava, and Minna Hakkarainen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00195 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 31, 2017

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Starch Derived Nano-graphene Oxide Paves the Way for Electrospinnable and Bioactive Starch Scaffolds for Bone Tissue Engineering

Duo Wu,† Archana Samanta,‡ Rajiv K. Srivastava,*‡ Minna Hakkarainen*†

†

Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, SE-100

44 Stockholm, Sweden. ‡

Department of Textile Technology, Indian Institute of Technology Delhi, Hauz Khas, New

Delhi 110016, India. Email: [email protected], [email protected]

ACS Paragon Plus Environment

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ABSTRACT A straightforward process that enabled electrospinning of bioactive starch-based nanofiber scaffolds was developed by utilizing starch derived nano graphene oxide (nGO) as a property enhancer and formic acid as a solvent and esterification reagent. The reaction mechanism and process were followed by detailed spectroscopic investigation. Furthermore, the incorporation of nGO as a “green bioactive additive” endorsed starch nanofibrous scaffolds several advantageous functionalities including improved electrospinnability and thermal stability, good cytocompatibility, osteo-bioactivity, and retained biodegradability. The biodegradable starch/nGO nanofibers underwent simultaneous degradation and mineralization process during 1 week of cell culture and mineralization test, thus, mimicking the structure and function of extracellular matrices (ECMs) and indicating promise for bone tissue engineering applications.

KEYWORDS:

Biobased-material,

Starch,

Graphene

Oxide,

Biomineralization.


Nanofiber,

Scaffold,

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INTRODUCTION Tissue engineering is a promising approach to repair bone defects which are commonly caused by trauma and infection. Typically, a 3D biocompatible/biodegradable scaffold with osteo-cells is implanted in situ. The scaffold virtually functions as an artificial extracellular matrix (ECM) which is of interwoven fibrous structure. Necessarily, ECM consists of protein fibers. It offers network and template to support cell growth and guide cell behaviors. One major challenge in tissue engineering is to develop scaffolds that possess identical structure to natural ECMs. Of all the natural and synthetic polymers, starch has favorable properties to mimic natural ECMs. Firstly, starch is a naturally abundant and cost-effective polysaccharide and may act as an analog to polysaccharides present in vivo.1-2 Secondly, starch is indispensable energy storage for body; it will metabolize into glucose, and not elicit an immunological foreign body reaction. More importantly, starch possesses plenty of hydroxyl groups that can be easily tailored/modified to desired structure through physical or chemical bonding.3-5 However, pure starch materials are rather brittle and difficult to process and do not provide proper features required for tissue engineering. One of the delicate strategies to improve the properties of starch materials is compositing with nano-enhancers such as nanoclay, fibers and functional nanofillers.6-9 Thus-designed starch nanocomposites normally achieve improved mechanical properties and specific functionalities. Besides of proper material selection and modification, fibrous and porous structure is required for scaffold to further mimic ECMs. A number of manufacturing processes have been explored to fabricate fibrous matrices, e.g. self-assembly,10 phase separation11 and electrospinning12-15. Among these techniques, electrospinning has been widely considered as simplest and more easily upscalable means to fabricate ultrafine fibers similar to the fibrous structures in natural ECMs. Electrospinning has been employed to fabricate nanofibers from a variety of synthetic and natural polymers.16 However, it is a challenge to prepare electrospun


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fibers of starch due to its naturally poor electrospinnability. Several attempts to prepare starch-based fibers have been reported. In most cases, starch served as a filler rather than the principle fiber-forming polymer.17-20 Kong and Ziegler studied the electrospinning process of pure starch fibers with micro size from dimethyl sulfoxide (DMSO) via a modified electrowet-spinning.21-23 and further developed different post-processing treatments (annealing, cross-linking) to improve the fiber stability.24-26 Moreover, starch-esters emerged as an alternative to pure starch due to their enhanced mechanical properties. Lancuski reported a straightforward method for electrospinning starch-formate nanofiber from aqueous formic acid (FA) solvent.27 The prepared starch-formate fibermat was found more ductile than pure starch film. This provided a strategy for preparing starch nanofibrous scaffolds with sufficient mechanical properties for tissue engineering. Graphene oxide (GO), on the other hand, has become widely known as a multi-functional additive in polymer composites. In tissue engineering applications, it can promote hydroxyapatite (HAP) mineralization,28-30 imparting polymer/GO scaffolds inductivity and osteo-conductivity. Novel biobased graphene oxide nanodots were derived in our previous works.31-33 The production route includes microwave-assisted hydrothermal carbonization of starch

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or cellulose

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, and then further oxidation of the carbon residues to at lateral

dimension nanosized graphene oxide (nGO) dots. We have fabricated nGO in solution-casted PCL films

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and freeze-dried porous starch scaffolds.38 In both cases high inductivity for

HAP mineralization was demonstrated. This indicates that biobased nGO has immense potential as a nano-enhancer in tissue engineering scaffolds. Here, we aimed to develop a process for electrospinning fully starch-derived starch/nGO bioactive nanofiber scaffolds. Our hypothesis was that formic acid would act both as a solvent and esterification reagent to stabilize and strengthen the starch fibers. In addition nGO, as a promising nano-enhancer, could further improve the electrospinnability and fiber morphology of the obtained starch-


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formate fibers as well as introduce bioactivity by inducing HA mineralization. Further, the properties and bioactivity of the obtained starch/nGO nanofibrous scaffolds were evaluated.

EXPERIMENTAL Materials. High-amylose Hylon VII maize starch (Hylon ST) was obtained from Ingredion, U.K. The amylose content was 70% as determined by the provider. Nitric acid (70%) was purchased from Sigma Aldrich Chemie Gmbh (Steinheim, Germany, and St. Louis, USA). Formic acid (99% purity) was purchased from Sigma. The chemicals used for mineralization test were NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2-6H2O, HCl (1 mol/L), CaCl2, Na2SO4, (HOCH2)3CNH2 and NaN3.

Electrospinning. Starch based nGO was prepared according to our previous works,33,

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Briefly, starch was hydrothermally carbonized in dilute sulfuric acid (0.01 g mL-1 under microwave irradiation for 2 hours, then the obtained carbon residues were further oxidized in nitric acid (1:100 w/w) for 30 min at 90 °C. Cold water was poured in solution to stop the reaction and solvent was then removed by evaporation under reduced pressure. nGO was obtained after freeze-drying the remaining solvent. Hylon ST was dissolved in formic acid (99%) at 25% w/w concentration. The solution was magnetically stirred overnight at room temperature for complete dissolution and reaction between formic acid and starch. The viscous solution will from now be referred to as ST-FA solution. Afterwards, different amounts of nGO were added to the ST-FA solution to prepare 0%, 1%, 2.5% and 5% (nGO/Hylon ST, w/w) solutions. Electrospinning was performed on these four solutions to get ST fibers with four different nGO concentrations, ST-nGO0 fiber, ST-nGO1 fiber, STnGO2.5 fiber and ST-nGO5 fiber, respectively. Each solution was filled in 2 mL plastic syringe and electrospun at 20 cm distance from the collector, under a flow rate of 0.2 mL/h


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and a high voltage of 23 kV. Fibers were collected on both aluminum foil paper and on glass cover. Characterization Nuclear magnetic resonance (NMR). Hylon ST, dried ST-FA and ST fibers were analyzed by Bruker Advance 400 Fourier Transform nuclear magnetic resonance spectrometer (FT NMR) operating at 400 MHz at 25°C. 10-20 mg of each sample was dissolved in 1 mL DMSO. Fourier Transform Infrared Spectroscopy (FTIR). FT-IR spectra of Hylon ST, dried STFA, ST fiber and ST-nGO5 fiber were recorded by PerkinElmer Spectrum 2000 FTIR spectrometer (Norwalk, CT) equipped with attenuated total reflectance (ATR) accessory (golden gate) Grasbey Specac (Kent, United Kingdom) Scanning electron microscopy (SEM). To examine the morphology of the original ST-nGO fibers as well as the fibers after 7 days’ of cell culture and mineralization test, an Ultra-High Resolution FE-SEM Hitachi S-4800 was utilized. The samples were sputter coated with 3 nm gold layers before analysis. Transmission electron microscopy (TEM). Transmission electron microscopy images were obtained by a HITACHI HT7700 (High contrast mode). ST-nGO fibers were collected on holey 400 mesh copper grids (TED PELLA, INC.). Water contact angle measurements. The water contact angles of the ST and ST-nGO fibers (on glass cover) were measured using a contact angle and surface tension meter (KSV instruments Ltd.). A drop of Milli-Q water (5 mL) was placed on the surface of the sample and images of the water menisci were recorded by a digital camera. The contact angle of each sample was taken as the average of three measurements at different points. FTIR imaging. FTIR single-peak absorbance images of the ST-nGO fibers were recorded by using a PerkinElmer Spotlight 400 system equipped with an optical microscope (Bucks,


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U.K.). The absorbance images of hydroxyl group at 3407 cm-1 were used to evaluate the distribution of starch -OH in the fiber matrix. Thermogravimetric analysis (TGA). For TG/DTG curves of the four ST-nGO fibers, a Mettler-Toledo TGA/ SDTA 851e was utilized. 5-40 mg of each sample was placed in 70 µl alumina cup. The samples were heated at 10°C/min from 25 to 700°C in N2 environment. Cell viability test (MTT assays). MTT assays were performed to measure the cytotoxicity of the prepared ST-nGO fibers. Osteoblastic cells MG63 were cultured in Dulbecco's modified eagle medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin and 100 µg/mL of streptomycin at 37°C, with 5% CO2 and 95% relative humidity. The glass covers with Starch-NGO fibers were placed on the bottom of a 24 wells microtiter plate (NUNC A/S, Roskilde, Denmark). Clean glass cover was placed in the control group. The cells were seeded on top of the glass covers in the microtiter plate at a density of 104 cells/well and incubated in the DMEM/well (400 µL) for 24 h. Then, 400 µL of 1× resazurin solution in PBS was added to each assay well. The absorbance was measured using a microplate reader (FLUOstar OPTIMA, BMG LABTECH) at a wavelength of 560 nm. The cell viability (%) was calculated from 100 × ([A]test − [A]PEI)/[A]control, where [A]test, [A]PEI and [A]control represent the absorbance values of the wells with ST-nGO fibers, with PEI (positive control) and without fibers (negative control), respectively. The absorbance given was the average value measured from six wells in parallel for each sample. Morphology of the cells after 7 days of incubation with ST-nGO fibers was examined under optical microscopy at ×400 and with SEM. Mineralization test. To evaluate if the ST-nGO can facilitate mineralization on the surface of starch scaffolds, a Simulated Body Fluid (SBF) was prepared.39 A conventional SBF (pH=7.4) was used containing NaCl (7.996 g/L), NaHCO3 (0.350 g/L), KCl (0.224 g/L), K2HPO4·3H2O (0.228 g/L), MgCl2·6H2O (0.305 g/L), HCl (1 mol/L, 40 mL), CaCl2 (0.278 g/L), Na2SO4


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(0.071 g/L), and tris(Hydroxymethyl)aminomethane (Tris, 6.057 g/L). The concentrations of SBF were Na+ 142, K+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl− 147.8, HCO3− 4.2, HPO42− 1.0, and SO42− 0.5 (mmol/L). These concentrations simulate the concentrations in human blood plasma. STnGO fibers, both on aluminum paper (1 cm in diameter) and on glass cover, were immersed in SBF and kept at 37°C. The SBF was changed every day. After 1 week incubation all the samples were taken out and rinsed with distilled water. After being dried at room temperature, the surfaces of the aluminum paper and glass cover were characterized by SEM/EDS.

RESULTS & DISCUSSION A process was developed for electrospinning bioactive starch nanofibers by utilizing a closedloop approach where starch was first carbonized and further oxidized to bioactive nGO. The starch derived nGO was then blended in starch during the electrospinning process to enhance the properties of starch nanofibers and to introduce bioactivity (Scheme 1). Hylon ST was dissolved in formic acid (99%, FA) at 25 wt% concentration, and stirred overnight to get a viscous solution, ST-FA. Different amounts of nGO were blended into the ST-FA solutions to obtain ST, ST-nGO1, ST-nGO2.5 and ST-nGO5 solutions, which were immediately spun into nanofibers by electrospinning. NMR and FTIR were applied to investigate the reaction between Hylon ST and formic acid. The morphology, surface property and thermal stability of ST-nGO fibers were evaluated. Biological study and mineralization testing were performed to evaluate the potential of ST-nGO fibers as bone scaffold.


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Scheme 1. Schematic description of the process for electrospinning ST-nGO nanofibers

Electrospinning of starch. It is well documented that starch undergoes rapid esterification, so called o-formylation in FA.40-43 The reaction of FA with starch at room temperature is regioselective, generating monoformate esters at the C6 position of the glucose units of starch,41 and reaches equilibrium after around 8 h in 90% formic acid solution.43 NMR was applied to follow the reaction during the preparation of the ST-FA electrospinning solution. Hylon ST in Figure 1 shows typical starch 1H NMR spectrum. Starch is a mixture of amylose, a linear polymer of α(1-4) linked glucosyl units, and amylopectine, linear amylose segments connected by 5-6% α(1-6) branch points. Standard starch typically constitutes of 20-30 wt% amylose and 70-80 wt% amylopectin.44 However, Hylon ST contains 70% of linear amylose.


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Compared to standard starch, Hylon ST spares more -OH at position C6. The chemical shifts at 3.34-3.37 ppm, 3.57-3.65 ppm, 5.13 ppm, can be assigned to the CH protons from C4/C2, C5/C3/C6 and C1 anhydroglucose carbons, respectively. Peaks at 4.60, 5.41, and 5.50 are due to protons from the -OH of position C6, C3 and C2 carbons, respectively. The largest peak at 3.2 ppm is attributed to the inevitably bonded water in the Hylon ST sample. ST-FA, represents Hylon ST dissolved in formic acid (25 wt%) overnight. The single peak at 8.13 ppm shows that some formic acid remained after solvent drying. Next to formic acid, broad large peaks at higher chemical shift than the formic acid single peak were detected indicating formation of starch formate ester bonds. To confirm this, more FA was added in Hylon ST and ST-FA (DMSO-d6 solvent) NMR samples (Figure S1): As a result the FA single peak sharply increased, but no effect on the wide peaks beside formic acid was observed. In addition to appearance of new peaks, -OH at position C6 was sharply decreased by around 56%, strongly suggesting that esterification had taken place. Meanwhile, water peak was expanded divergently and decreased indicating that bonded water was partly released during the esterification reaction. Different starch concentrations and temperatures were tested to examine the influences on the esterification reactivity. It can be deduced from Figure S2 that lower concentration (15 wt%) of starch in FA could lead to smaller amount C6 -OH replacement (Figure S2, left). Higher temperature (60°C) on the other hand enhanced the esterification replacement (Figure S2, right). Higher starch concentration and elevated temperature, thus, seem to facilitate the reactivity between Hylon starch and FA. Furthermore, after electrospinning, ST fiber was derived from the STFA solution. Figure 1 shows that the solvent, FA, was mostly removed by electrospinning. This solvent removal is expected to be facilitated by the largely increased surface area of the formed fiber. No other significant changes were detected after electrospinning, which indicates that


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electrospinning has no significant influence on the starch-FA reaction. Lastly, addition of nGO decreased the reactivity of ST-FA esterification (chemical shift at 8.2 decreases, Figure 1), while it promoted the solubility of starch formate in DMSO-d6 solvent improving the spectral resolution.45 The abundant -COOH groups in nGO might compete with the -COOH groups of formic acid, thus reducing the reactivity. It was further confirmed by 1H NMR (see Figure S3), that higher concentration of nGO resulted in lower formate substitution. Increased amount of position C6 -OH was also detected, indicating that α-1, 6 linkages in amylopectin were broken down. In summary, starch formate was formed after dissolving Hylon starch in formic acid (25 wt%) overnight at room temperature. Electrospinning process effectively removed the unreacted formic acid, but addition of nGO reduced the esterification reactivity as well as broke down the amylopectin chains at the branching points.

Figure 1. 1H NMR spectra of Hylon ST, ST-FA, ST fiber and ST-nGO5 fiber. FTIR spectra in Figure 2 as parallel evidence demonstrate the reaction and changes described above. In the spectrum of Hylon ST, the hydroxyl groups of starch are present at 3460 cm-1. The band at 2926 cm-1 is indicative of the -CH2- stretching vibration. There are several discernible absorbencies between 1080 and 930 cm-1 that were attributed to C-O bond stretching. An intense band at 1637 cm-1 was assigned to the deformation vibration of the hydroxyl group of water,

34, 46

showing the inevitable water absorption of starch materials. In


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FTIR curve of ST-FA, there is a sharp -C=O- peak at 1710 cm-1, indicating unreacted formic acid that remained in ST-FA after drying. Electrospinning of starch nanofiber could remove this extra formic acid and expose the formate ester stretching vibration at 1740 cm-1.

Figure 2. FTIR spectra of Hylon ST, ST-FA, ST fiber and ST-nGO5 fiber. 13

C NMR was employed to further confirm the reaction between starch and formic acid. In the

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C NMR spectra of Hylon ST (Figure 3), all the expected carbon atoms in the structures of

the starch molecule can be detected and assigned. The signal at 100.14 ppm is attributed to the C1 of the anhydroglucose units. Signal for C4 is observed at 78.80 ppm. The three peaks ranging from 73.30-71.65 ppm are attributed to C3, C2 and C5 of the anhydroglucose units. C6 peak is at 60.55 ppm. These results are supported by the literature.

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As expected, the

unreacted formic acid was detected in ST-FA at 163.12 ppm. In addition, a clear peak at 162.07 ppm is present in the

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C NMR spectrum of ST-FA, which is assigned to the ester

bonds of starch formate. Accordingly, C5 shifted from 71.65 to 68.50 and C6 varied from 60.55 to 62.63 ppm due to the o-formylation esterification at position C6 (Figure 3). These results fully correlate with the work of Divers et al.49 Three signals for C1 are detectable, according to starch structure; they are suggested to be the main C1 in starch chain; terminal C1; and branching C1.48 It is thus concluded that intramolecular bond breakdown also took


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place during stir-dissolving and esterification reaction. According to Divers et al

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destructuration and hydrolysis occurred during o-formylation of starch in formic acid. Formic acid acted both as an efficient reactant and as a powerful destructuring agent.

Figure 3. 13C NMR spectra of Hylon ST and ST-FA.

Fiber morphology To examine the morphology of the prepared ST-nGO nanofibers, SEM and TEM were employed. Figure 4 shows the SEM images of the prepared electrospun STnGO nanofibers with different concentration of nGO. The nanofibers were all successfully prepared through electrospinning of ST-FA solution and nGO blended ST-FA solutions. Xu et al. have reported the preparation of electrospun starch acetate nanofibers using formic acid/water as solvent. Starch-acetate agglomerations were inevitably shown in the reported SEM images, suggesting poor electrospinnability.50 SEM images in Figure 4 displaying the starch-formate nanofibers prepared here, show that the fibers are smaller in diameter and more stable. Although the diameter distribution of the neat ST nanofiber (Figure 4a) is relatively wide (20~140 nm), the gradual addition of nGO to ST-FA solution narrowed the size distribution of the fiber diameter (Figure 4b, c and d). The fiber size of ST-nGO is as small as 30 to 50 nm, which is similar to the size dimensions of the fibrous structure of the natural ECMs. In addition the fiber diameter decreased as a function of nGO addition. Besides


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of the fiber phase, starch microspheres as defects were seen through all the samples (Figure 4a1, b1, c1 and d1), the size of which became smaller and more uniform but with higher frequency as more nGO was added. It was assumed that the high viscosity of the ST-FA (25 wt%) solution impedes the continuous flow through the capillary tip. Irregular clots accumulated on the tip and dropped down on the collector to form the starch microspheres. Thus neat ST-FA itself had poor electrospinnability.

Figure 4. SEM images and fiber size distribution of ST (a1, a2 and a3), ST-nGO1 (b1, b2 and b3), ST-nGO2.5 (c1, c2 and c3) and ST-nGO5 (d1, d2 and d3) fibers.


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There are three influencing factors of nGO that affected the morphology of the obtained fibers. Firstly, the viscosity of ST-FA solution decreased after addition of nGO to the ST-FA solution because nGO interacted with starch chains through hydrogen bonding,33 which probably broke some of the strong intermolecular bonding between starch chains, rendering the ST-FA/nGO solutions less viscous and more spinnable at the same starch concentration. Meanwhile, higher nGO concentration also lead to shorter starch chains (as described by 1H NMR, where more terminal C6 -OH were detected), which should additionally reduce the viscosity of the starch-FA solutions. Secondly, nGO reduced the reactivity between starch and formic acid. Higher concentration of nGO lead to lower starch formate substitution, thus producing less water, which is a poor-volatility solvent for electrospinning. Thirdly, ST-nGO has higher starch fraction and lower starch-formate fraction, and formic acid dissolves starch better than starch-formate due to closer polarity. Therefore, nGO is a beneficial additive for electrospinning ST-FA. TEM images in Figure 5 depict an even closer look at the starch-formate fibers. Clear beads were shown to string through the ST fibers (Figure 5a). Phase separation was also detected in the fiber phase (imbedded picture in Figure 5a), which could be due to mixed phases of starch and starch-formate (inserted scheme in Figure 5a). The bead-like structure turned gradually to spindle-like structure (Figure 5b and c) and finally fine fibers without beads were obtained when the concentration of nGO was increased to 5% (nGO/starch, wt/wt, Figure 5d). nGO has been shown to be a good compatibilizer for starch/polylactide composites due to the amphiphilic structure.33 The improved electrospinnability could be due to the ability of nGO to act as a compatibilizer between starch and starch-formate. Clusters of nGO nanodots were detected in the ST-nGO5 fiber sample (Figure 5d and Figure 6a), implying some agglomeration of nGO during jet continuous spinning. This could be related to the fact that more starch microspheres were obtained during electrospinning of ST-nGO5 (Figure 4d1):


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nGO agglomeration sprayed out from the tip, followed by a droplet of starch solution which together formed the starch microspheres (inserted scheme in Figure 5d). In the formed fiber, nGO interacted with starch fibers by attachment onto fiber surface (Figure 6b and 6c). The similar phenomenon was reported before that nGO can form strong hydrogen bonding with multiple hydroxyl groups of starch.

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The presence of both fiber phase and starch

microspheres suggests a multijet mechanism during the electrospinning process, working on a) less rich nGO solution which tends to form fibers and b) high rich nGO solution which tends to form agglomerates and particle clusters. nGO, thus, benefits the ST-FA electrospinnability and higher concentration of nGO leads to thinner and more uniform fibers. 5 wt% of nGO may cause agglomeration and the cutout of continuous jet, forming large amount of starch microspheres.

Figure 5. TEM images of ST, ST-nGO1, ST-nGO2.5 and ST-nGO5 fibers.


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Figure 6. TEM images of nGO clusters and ST-nGO5 fibers.

After the morphological study, surface properties, thermal stability, cell viability and bioactivity of the prepared nanofibers were investigated by water contact angle measurement, FTIR imaging, MTT assay and mineralization test in SBF. Surface property As is well known, surface wettability is vital to cell attachment and proliferation. The wettability of ST, ST-nGO2.5 and ST-nGO5 fibers was influenced by both hydrophilicity (chemical properties of the substrate) and surface roughness. The wettability of fibers was depicted in Figure 7 in the form of water contact angle, and surface roughness was visualized by three-dimensional IR imaging of -OH functional groups at around 3407 cm-1 (OH of starch). A decreasing trend in contact angle was observed as nGO loading increased in the ST-FA solutions. Starch is hydrophilic in nature. Starch-formate, however, is less hydrophilic due to the ester structure. nGO loaded ST fibers contained less starch-formate than the neat ST fiber, which thus lead to higher hydrophilicity. nGO is also full of -COOH functional groups on its edges, which can additionally increase the hydrophilicity. Contact angle is also closely related to the surface roughness. As indicated by the -OH IR imaging (Figure 7a2, b2 and c2), the landscape of starch -OH transferred from mountainous to plain


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filed, suggest smoother surface for the ST-nGO fiber-mats. This probably further benefits and reduces the water contact angle.

Figure 7. Water contact angle measurements of ST, ST-nGO2.5 and ST-nGO5 nanofibers (a1, b1 and c1); Three-dimensional images illustrating the hydroxyl of starch component single peak absorbance for ST, ST-nGO2.5 and ST-nGO5 (a2, b2 and c2) fibers.

Thermal stability analysis Graphene (oxide) sheets have been widely reported to enhance the thermal stability of starch matrix.51-53 Same was observed in our previous work where nGO was blended in PLA/starch composites

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and in freeze-dried porous starch scaffolds 38.

SI Figure 4 shows the DTG curves of neat ST fibers and nGO containing fibers. ST fiber experienced two major degradation stages after initiation of decomposition, slow mass loss was observed in the beginning followed by a sharper mass loss. It should be attributed to the different stability of starch and starch-formate in the ST fibers. On the other hand the nGO loaded starch fibers have only one decomposition phase. More stable chain integration was achieved due to hydrogen bonding interactions between nGO and matrix components, where nGO could act as a compatibilizer between starch and starch-formate. Usually, the better the


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interactions are between the nanoparticle and the matrix, the higher the thermal stability of the composite. This is verified in our study showing that increased nGO content enhanced the thermal stability of ST fibers (SI Figure 4b) as illustrated by the higher decomposition temperature. Cell viability test. Favorable cell adhesion on the material surface is considered as the prerequisite to the subsequent cell activity. The 24 h cell viability on the surface of ST fibers and nGO loaded ST fibers was evaluated by MTT assay using osteoblast MG63 cell lines. Optical images of MG63 cells after 7 days of culture were also taken. As shown by Figure 8a, the ST fibers displayed superior cell viability (the relative cell viability reached 158% compare to the negative control). This is reasonable since starch is an energy storage material and can provide energy, in the form of glucose sugar, for the cells proliferation. The cell viability of ST-nGO1 and ST-nGO2.5 shows the same high level as was observed for ST fibers. Previous studies suggest that nGO, under a certain concentration (lower than 1 mg/mL in solution), has no cytotoxicity to MG63 cells.38 However, Figure 8a shows dramatic reduction of the cell viability on the surface of ST-nGO5 fibers with the highest nGO content. The small traces of FA in all the ST-nGO fibers might have negative effect on biological response. However, plain ST fibers which contained the largest amount of FA (among STnGO fibers, SI Figure 3) displayed superior cell viability (158%). Therefore the negative influence of small amount of FA to biology response is considered weak in this study. Meanwhile, to evaluate the long term cell viability, the morphology of the cultured MG63 cells after 7 days of incubation with fibers was examined under light microscopy in Figure 8b. Starch nanofibers are not expected to be detected at this scale. In the case of ST and STnGO1; the cells attached well and exhibited normal morphology on both surfaces. The cells also distributed uniformly, revealing no specific detrimental area. MG63 cells were densely arranged on ST-nGO2.5. However, some dead opaque cells with round shape were among the


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detected cells, suggesting slight cytotoxicity. Lastly, on ST-nGO5, blur patterns on the upper side of the image, surrounded by dead cells, appeared and are suspected to be the degraded ST-nGO5 fibers. MG63 cells tended to avoid this area, suggesting there might be harmful substance around this area. This could be correlated to large amount of migrated nGO caused by the higher concentration of nGO in combination with faster degradation of ST-nGO5 with the smallest fiber diameter and biggest surface to volume ratio as shown by the SEM images in Figure 4. TEM (Figure 5d) also suggests that in ST-nGO5, nGO was partly spun into clusters, which could result in too high local concentrations that are detrimental to the cells. Indeed, it has been reported that GO has a dose-dependent effect on A549 cells,54 HeLa cells,55 human fibroblast cells,56 human erythrocytes and skin fibroblasts,57 and higher concentration of GO generally leads to reduction of cell viability. ST fibers are, thus, beneficial for the growth of MG63 cells, but higher loading amount of nGO in ST fibers might be harmful to MG63 cell viability on the fiber surfaces. ST nanofibers underwent degradation during 7 day cell culture.


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Figure 8. Relative cell viability of ST, ST-nGO1, ST-nGO2.5 and ST-nGO5 fibers under MTT assay (a); cell morphology after 7 days’ culture on ST, ST-nGO1, ST-nGO2.5 and STnGO5 fibers under optical microscopy (b).

To visualize the morphology of the nanofibers after 7 days of cell culture, the glass covers with ST-nGO2.5 fibers were examined by SEM (Figure 9). Traces of starch nanofibers can still be detected on the glass covers (Figure 9a, b and c). Marks from the cells were also shown in Figure 9a, suggesting that MG63 cells attached on the fiber surface during cell culture. In addition, ST-nGO2.5 has larger diameter than originally but the density decreased compared to the starting ST-nGO2.5 fibers (SEM images in Figure 4c). It is well known that starch itself is not stable in water. Starch-formate, on the contrary, has lower amount of hydrophilic -OH, and exhibits better water stability. Salt ions from cell culture medium are another factor that could promote the dissociation of the fibers during the cell culture. These salts around the fibers were clearly seen in Figure 9. The results in Figure 8b and 9 indicate that starch fibers got swollen and partially dissolved/degraded during the cell culture.

Figure 9. SEM images of ST-nGO2.5 fibers taken after 7 days’ of cell culture (a, b and c). The fiber trace of c1 is emphasized by blue color in c2.


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Biomineralization in SBF. GO has the ability to promote hydroxyapatite mineralization. Our previous study also proved that nGO, after fabrication in PCL films and starch scaffolds, can induce mineralization of CaP on the surface of these composites.37-38 In order to explore whether nGO could further endorse bioactivity to starch nanofibers and to explore the potential of the prepared starch nanofibers as bone tissue scaffolds, mineralization test was executed at the same time as the degradation behavior was investigated. Plain starch nanofibers on foil were incubated in SBF at 37°C for 1 week. SEM/EDS was employed to examine the starch fibers and the formed minerals (Figure 10). Neat ST fibers displayed a dissociation of the fiber phase (Figure 10 a1). Instead of a cylindrical fiber, a crossed network pattern was shown on the aluminum surface. EDS (Figure 10 a2) illustrates only the breakdown of the starch matrix and no mineralization. This is correlated with previous reports stating that neat starch could not induce CaP mineralization.38, 58 Interestingly, some of the larger ST-nGO1 fibers were still detectable in Figure 10b, indicating a higher aqueous stability. Spherical CaP minerals were also observed attached on the ST-nGO1 fiber surface. Higher amount of nGO further enhanced the amount of CaP minerals formed on the surface of ST-nGO2.5 and ST-nGO5. A uniform distribution of the minerals was seen on ST-nGO2.5 fibers, while CaP clusters were shown on ST-nGO5. The reason could be due to the CaP mineralization on the starch microspheres or nGO agglomerates which were illustrated in Figure 4d1 (showing the similar pattern density) and Figure 5d. The results, thus, indicate that the degradation of starch fibers and the mineralization of CaP were integrated and occurred simultaneously.


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SEM images in Figure 11 provide further evidence for this assumption. The degraded fiber phase and the formed CaP mineral phase were both observed in the images of ST-nGO2.5 fiber samples (Figure 11 a+b). a1 and a2 illustrate a loss of structural integrity for the fibers. Starch fiber transformed to a porous network (a1) and finally to spherical particles (a2). On the other hand, CaP spherical crystals grew along the starch fiber to form a fibrous net pattern (b1 and b2). Thinner fibers dissociated earlier than fibers with larger diameter in SBF. nGO was thus gradually exposed and released during the fiber degradation. Meanwhile, -COOH groups of nGO functioned as anchor sites inducing CaP mineralization on the remaining starch fiber template and on the starch microspheres. Starch nanofiber/CaP hybrid composites were thus derived. The formation of natural bone involves a process where organic matrices (e.g. collagen) control the CaP mineralization. The bone itself is an organic/inorganic hybrid composite. Therefore, our ST-nGO nanofibers seem capable of mimicking artificial extracellular matrices (ECMs) to regulate CaP crystallization and to form hybrid structure similar to those of natural bone.


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Figure 10. SEM image and EDS of ST (a1 and a2), ST-nGO1 (b1 and b2), ST-nGO2.5 (c1 and c2) and ST-nGO5 (d1 and d2) fibers after 1 week of mineralization.


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Figure 11. SEM images of ST-nGO2.5 fibers on glass cover substrate after 1 week mineralization (a+b), degraded fiber phase (a1 and a2) and the mineralized CaP phase (b1 and b2)

CONCLUSIONS Starch nanofibers with incorporated nGO were successfully prepared by electrospinning. Formic acid functioned both as an esterification reagent and efficient solvent for starch electrospinning. The reactivity was positively correlated to starch concentration and temperature, while it had negative relation to nGO concentration. Incorporation of nGO improved the electrospinnability of starch; it decreased the fiber diameter and narrowed the fiber diameter distribution. In addition, nGO improved the hydrophilicity and thermal stability


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of the fibers. Cell viability test confirmed that when the loading amount of nGO was equal or lower than 2.5 wt%, ST-nGO fibers exhibited good biocompatibility to MG 63 cells. In addition, nGO could induce starch nanofibers bioactivity by promoting CaP mineralization in SBF solution. Meanwhile, the biodegradable starch nanofibers underwent a simultaneous dissociation and mineralization process during 1 week of cell culture and mineralization test. ST-nGO nanofibers, thus, seem as good analogs of ECM and have potential to serve as functional scaffolds in bone tissue engineering applications.

ASSOCIATE CONTENT Supporting Information Supporting information contains 1H NMR of Hylon ST+FA, ST-FA+FA, 15% concentration of ST-FA, 25% ST-FA dissolved at 60°C, ST-nGO2.5 fibers and ST-nGO5 fibers and DTG

traces for ST, ST-nGO1, ST-nGO2.5 and ST-nGO5.

Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

ACKNOWLEDGEMENTS: The authors gratefully appreciate the China Scholarship Council (CSC).

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Table of Contents Graphic Starch Derived Nano-graphene Oxide Paves the Way for Electrospinnable and Bioactive Starch Scaffolds for Bone Tissue Engineering Duo Wu, Archana Samanta, Rajiv Srivastava, Minna Hakkarainen


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Table of Contents Graphic 88x35mm (300 x 300 DPI)


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Starch-Derived Nanographene Oxide Paves the Way for

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