Construction of Bioactive and Reinforced Bioresorbable

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Construction of bioactive and reinforced bioresorbable nanocomposites by reduced nano-graphene oxide carbon dots Nejla B. Erdal, and Minna Hakkarainen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00207 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Construction of Bioactive and Reinforced Bioresorbable Nanocomposites by Reduced Nano-Graphene Oxide Carbon Dots

Nejla B. Erdal and Minna Hakkarainen*

Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, 100 44 Stockholm, Sweden

ACS Paragon Plus Environment

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ABSTRACT Bioactive and reinforced poly(ε-caprolactone) (PCL) films were constructed by incorporation of cellulose derived reduced nano-graphene oxide (r-nGO) carbon nanodots. Two different microwave-assisted reduction routes in superheated water were utilized to obtain r-nGO and rnGO-CA. For the latter a green reducing agent, caffeic acid (CA) was incorporated in the reduction process. The materials were extruded and compression molded to obtain proper dispersion of the carbon nanodots in the polymer matrix. FTIR results revealed favorable interactions between r-nGO-CA and PCL that improved the dispersion of r-nGO-CA. r-nGO and r-nGO-CA endorsed PCL with several advantageous functionalities including improved storage modulus and creep resistance. The considerable increase in storage modulus demonstrates that the carbon nanodots had a significant reinforcing effect on PCL. The PCL films with r-nGO-CA were also evaluated for their osteo-bioactivity and cytocompatibility. Bioactivity was demonstrated by formation of hydroxyapatite (HA) minerals on the surface of r-nGO-CA loaded nanocomposites. At the same time the good cytocompatibility of PCL was retained as demonstrated by the good cell viability to MG63 osteoblast-like cells giving promise for bone tissue engineering applications.

KEYWORDS: aliphatic polyester, polycaprolactone, mineralization, reduced graphene oxide, tissue engineering, mechanical properties


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INTRODUCTION Bone tissue engineering has emerged as a tool for repairing and restoring bone defects after an injury or infection. It involves the use of scaffolds to provide structural support and invoke a biological response for the regeneration of new tissue while the scaffold itself simultaneously degrades or erodes into harmless compounds.1 The developments in tissue engineering have generated a set of scaffolds and implementation strategies.2,3 However there still need for improvements and the goal of producing the ideal scaffold has yet not been accomplished.4–6 The scaffold should be capable of supporting cell attachment, proliferation and differentiation in vitro and may then be transplanted in vivo. Conventional single-component polymers for biomedical applications rarely meet all the criteria in terms of surface functionality and mechanical strength.7 Fillers, additives and reinforcement agents have been pursued to enhance polymer properties and to add certain biological functions and bioactivity.8–13 Poly(ε-caprolactone) (PCL), a Food and Drug Administration (FDA) approved biodegradable and bioresorbable aliphatic polyester is a suitable candidate material for bone tissue engineering applications with regards to its unique chemical properties, hard tissue compatibility and degradation time.14 However, its poor bioactivity and low hydrophilicity has limited its bio- and physiochemical properties, causing a need for modification to add more complex functionalities that can yield both functional and biomechanical stability.15 Graphene derivatives have evolved as a promising class of compounds offering versatility, mechanical stability and interesting optical and electrical properties. Several studies have also demonstrated good biocompatibility and even improved cell proliferation.16,17 Nevertheless, it has poor processability and inappropriate mechanical properties to serve as a scaffold alone.18,19 The highly oxidized form graphene oxide (GO) has shown to create favorable interactions with



several polymer matrices and thereby improving the mechanical properties and osteogenic differentiation, protein adsorption ability, cell adhesion and proliferation of the polymer composites.20–22 These unique properties have attracted researchers to investigate graphene derivatives as a reinforcing agent in biocompatible composite materials. The ability of biobased nano-graphene oxide (nGO) to enhance the mechanical properties of polylactide (PLA) nanocomposites and to promote the bioactivity of PCL films and starch scaffolds was recently reported by our group.23–27 Hydrophilic nGO induced mineralization on the surface of PCL and starch among other advantageous functionalities such as improved thermal stability and good cytocompatibility. However, the addition of nGO compromised the mechanical properties of PCL leading to increased brittleness, (See SI file, Table S1).

Recently, we developed a green pathway to produce reduced nano-graphene oxide (r-nGO) carbon nanodots from cellulose.28 An efficient reduction was shown and further augmented with the inclusion of a non-toxic, environmentally benign reducing agent CA (r-nGO-CA). In vitro cell viability assay indicated no cytotoxicity for r-nGO-CA towards osteoblast-like MG63 cells up to 1000 µg mL-1. Here, we hypothesized that the less hydrophilic r-nGO and r-nGO-CA could have better compatibility with PCL. This could in turn yield a more favorable reinforcement effect, at the same time as the bioactivity of the nanocomposites could be retained to benefit the use in bone tissue engineering applications. In addition, the enhanced thermal stability of the reduced form enables processing by extrusion and compression molding.

EXPERIMENTAL SECTION Materials. Sulfuric acid (H2SO4) (95–98%), nitric acid (HNO3) (70%), α-cellulose, caffeic acid (CA) (≥ 98%), poly(ε-caprolactone) (Mw 70 000-90 000 g/mol), Dulbecco’s modified Eagle’s


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medium, phosphate-buffered saline (10×), and trypsin−EDTA solutions were purchased from Sigma-Aldrich. Standard RC Membrane Spectra/Por® 7 Pre-treated dialysis tubing (MWCO 1 kDa) was obtained from Spectrum Labs. Ethanol (≥ 98%), fetal bovine serum, penicillin−streptomycin, and alamarBlue cell viability reagent were purchased from Fisher. All chemicals were used as received. r-nGO and r-nGO-CA synthesis. r-nGO and r-nGO-CA were obtained through reduction of nGO derived from cellulose via a microwave-assisted process according to our previously reported procedure.28–30 In the synthesis of r-nGO, 100 mg nGO was dispersed in 20 mL deionized water and treated in Milestone UltraWAVE (Milestone Inc) microwave device in dynamic mode with a supreme power of 950 ± 50 W. The temperature was set at 180 °C with a ramp-time of 20 minutes. The temperature was hold at isothermal conditions for 2 hours with a pressure of 40 bar nitrogen. To synthesize r-nGO-CA, nGO was dispersed in deionized water and CA powder (1:3 w:w nGO:CA) was added to the dispersion and mixed. The dispersion was treated in the microwave as above. The obtained black and solid residue was cleaned with a dialysis membrane (MWCO1 kDa) in 500 mL deionized water (changed 2-3 times) for 24-48 hours with stirring. This cleaning step was performed in order to separate r-nGO-CA from excess CA. The dispersions were then freeze-dried to obtain r-nGO and r-nGO-CA and stored for 5 days in a vacuum oven at room temperature to remove excess water. Thorough physicochemical characterization of r-nGO and r-nGO-CA was presented in previous work.28 However, the main characteristics are shortly given here. nGO had a C/O ratio of 1.65 and the formed products rnGO and r-nGO-CA had C/O ratios 2.3 and 4.4, respectively, as determined by X-ray photoelectron microscopy (XPS). r-nGO has more than 50% of the oxygen functionalities in C=O form. r-nGO-CA, that has a much lower oxygen content, has most of the oxygen groups in the



form of OH/C-O-C groups. The size of r-nGO and r-nGO-CA was 70 ± 14 nm and 12 ± 3 nm respectively (measured in an acetone 0.1 mg mL-1 dispersion drop-casted on a TEM grid for TEM observation). Preparation of PCL nanocomposites. 5 g PCL was dissolved in 40 mL chloroform at 45 °C, with a magnetic stirrer. 0, 0.5 and 2 wt % of r-nGO or r-nGO-CA were dispersed in 2 mL ethanol and bath sonicated (Bransonic Ultrasonic cleaners, model 2210, 40 kHz, 130 W at T = 23 °C) for 15 minutes. After sonication the dispersions were poured into the polymer solutions, mixed for 1 minute and solution casted. The solutions were casted on dust-free Petri dishes and dried at room temperature for 3 days, after which the films were kept in a vacuum oven for 24 h at room temperature to evaporate any residual chloroform. Compounding. The films were further processed to obtain a proper dispersion of the nanodots using a mini-extruder, a DSM micro 5 compounder (Co/counter option) with co-rotating intermeshing conical-twin-screw at 100 °C with a screw speed of 100 rpm and a 3 min retention time. TGA analyses revealed that r-nGO and r-nGO-CA were thermally stable up to 120 °C and 300 °C respectively and should not degrade at the processing temperature.28 Film formation. The extruded nanocomposites were then compression molded to 2D films using a Hot Press (Fontijne Presses) at 100°C with a two-cycle method. First, a holding time of 20 s and a pressing force of 100 kN were used followed by a holding time of 3 min and a pressing force of 100 kN. Finally, the pressed films were cooled down to 25 °C by a water cooling system during 10 min. For each film, 3 g of extruded material was placed in a circular-shaped metal frame (Ø 7.5 cm) and covered by two Teflon sheets on each side. Films with a thickness of 0.20 ± 0.05 mm were formed. The films were named according to the percentage of r-nGO and rnGO-CA as follows: PCL0 (pure PCL), PCL0.5 (0.5 wt % of r-nGO), PCL2 (2 wt % of r-nGO), PCL0.5-CA (0.5 wt % of r-nGO-CA), PCL2-CA (2 wt % r-nGO-CA).


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Characterization. Fourier transform infrared spectra (FTIR) were obtained by PerkinElmer Spectrum 2000 FTIR spectrometer (Norwalk, CT) with 16 scans at a resolution of 4 cm-1. The instrument had an attenuated total reflectance (ATR) accessory (golden gate) from Graseby Specac (Kent, United Kingdom). X-ray diffraction (XRD) spectra were collected using the X-ray source CuKR radiation (λ = 0.1541 nm). PANalytical X'Pert PRO diffractometer was used to measure the diffraction at 25 °C with a silicium mono-crystal sample holder. The 2θ angular range was set between 5–40° with a step size of 0.017° for all analyses. An Ultra-High Resolution FE-SEM Hitachi S-4800 scanning electron microscopy (SEM) was utilized to evaluate the surface morphology of the films. The samples were sputter-coated with a 5 nm thick gold/palladium mixture prior to the SEM observations. Energy dispersive X-ray spectroscopy (EDS) spectra were acquired on the same Hitachi S-4800 SEM, equipped with an Oxford Instruments X-MaxN 80 EDS at a voltage of 15kV. The cross-sections were obtained through cryofacturing. Dynamic Mechanical Thermal Analysis (DMTA) was carried out using Q800 TA Instruments in tension film mode. The scan was done from -90 to 40 °C at 5 °C min−1. Specimen dimensions were 2 cm length, 5 mm width and 0.2 mm thickness. Samples were subjected to a cyclic tensile strain with an amplitude of 12% at a frequency of 1 Hz. Rheological properties were investigated using a TA Discovery Hybrid 2 (DHR-2) equipped with thermally regulated stainless steel Peltier plate configuration (Ø 20 mm). An amplitude sweep test was conducted at frequency range 0.01-100 Hz at 100 °C to determine the linear region. After determining the linear viscoelastic region, the tested specimens were subjected to a frequency sweep from 0.01 to 100 Hz at 100 °C to measure the storage and loss modulus. Creep



tests were carried out with a constant stress of 100 Pa for 10 min at 37 °C. The constant stress level applied was chosen in order to work within the linear elastic range behavior. Cell viability test. The indirect cytotoxicity of the PCL0, PCL0.5-CA and PCL2-CA nanocomposites was evaluated using MG63 osteoblast-like cells. For each sample, three parallel tests were carried out. The cells were cultured in complete growth medium (CGM; Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units mL-1 of penicillin, and 100 µg mL-1 of streptomycin) at 37 °C in 5% CO2 and 95% relative humidity. The cell concentration at the bottom of the wells in a 96-well tissueculture polystyrene plate was 8000 cells/well. Nanocomposite samples (0.2 mm in thickness and 2 mg in weight) were sterilized with 70% ethanol, washed three times with PBS buffer, and placed into the wells of a 48-well tissue-culture polystyrene plate. Each sample was immersed in 250 µL of CGM. After 24 h of incubation at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity, 140 µL of extraction medium from each sample was added to each well of a 96-well plate with cell culture. A cell culture in fresh CGM was used as a control. The plate was then further incubated for 24 h at the same conditions. Finally, the extraction media were removed, 100 µL of 1× resazurin (alamarBlue cell viability reagent) solution in PBS was added to each assay well and incubated for approximately 1 hour. A Tecan Infinite 200 PRO multifunctional microplate reader (excitation wavelength = 560 nm; emission wavelength = 590 nm) was used to measure the fluorescence of each well. The cell viability (%) was calculated from 100 × ([Isample] − [Iblanc])/([Inegative]-[Iblanc]), where [Isample] represent the fluorescence intensity values of the wells with cells that were in contact with the extracted liquid from the nanocomposites, [Iblanc] without cells, and [Inegative] cells with fresh CGM. The fluorescence intensity was the average value measured from three wells in parallel for each sample.


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Mineralization Test. The ability of r-nGO-CA to induce mineralization on the surface of the PCL nanocomposites was investigated by immersing the PCL nanocomposite films in Simulated Body Fluid (SBF). A conventional SBF (pH = 7.4) was prepared containing NaCl (7.996 g L-1), NaHCO3 (0.350 g L-1), KCl (0.224 g L-1), K2HPO4·3H2O (0.228 g L-1), MgCl2·6H2O (0.305 g L1

), HCl (1 mol L-1, 40 mL), CaCl2 (0.278 g L-1), Na2SO4 (0.071 g L-1), and

tris(hydroxymethyl)aminomethane (Tris, 6.057 g L-1). 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-1) and mimics the concentrations in human blood plasma.31 The PCL nanocomposites were cut into 0.5 x 0.5 cm2 square films and immersed in SBF and kept at 37 °C. Two specimens for each sample type were immersed and characterized. The SBF was changed every day. After 2 weeks of incubation, all films were taken out and rinsed thoroughly with distilled water to remove any free ions. The films were dried at room temperature before SEM/EDS/XRD characterization.

Results and discussion Recently, we developed a green pathway using microwave technology to produce r-nGO from a widely abundant, environmentally benign and renewable resource; cellulose. In this study two different biobased r-nGO carbon nanodots, r-nGO and r-nGO-CA, were evaluated for their ability to reinforce PCL and to introduce bioactivity by facilitating mineralization.

Morphology of the films and dispersion of r-nGO and r-nGO-CA The PCL nanocomposite films were firstly solution casted to facilitate the feeding to the extruder, then melt mixed by extrusion to improve the dispersion of r-nGO and n-rGO-CA and finally, compression molded to nanocomposite films with two different loadings of nanodots, see Figure 1. The SEM micrographs illustrated clear differences in the morphology of the films containing



carbon nanodots in contrast to neat PCL (PCL0), see Figure 1. The difference was most pronounced for PCL0.5-CA that showed large spherulites with approximate diameters of 100 µm. The enlarged SEM image of PCL2-CA reveals relatively homogeneous dispersion of r-nGO-CA covered by PCL suggesting favorable interactions between the two components. The surface of PCL0.5 and PCL2, on the other hand, seemed to be similar to PCL0 with traces of agglomerated nanodots with an irregular distribution, particularly for PCL2.

Figure 1. Illustration of the processing pathway (top) and SEM images of the surface microstructures for the resulting nanocomposites (bottom).

Chemical characteristics of the nanocomposites The nanocomposites were further analyzed through the FTIR spectra and SEM micrographs of the cross-sections with the highest loading of r-nGO and r-nGO-CA, see Figure 2. The FTIR


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spectra display the characteristics vibrational modes of PCL with typical bands at 3450 cm-1 (OH stretch), 2850 cm-1 and 2950 cm-1 (CH2 stretching vibrations), 1721 cm-1 (C=O vibrations), 1190 cm-1 and 1243 cm-1 (C-O-C symmetric and asymmetric stretches). The FTIR spectra of the PCL nanocomposites were in agreement with the spectrum of PCL where the peaks of the carbon nanodots are masked by the peaks of PCL due to the relatively low content of the nanodots and their small sizes. However, small shifts in band frequencies were registered for PCL0.5, PCL2 and PCL2-CA in contrast to PCL0. PCL0.5 and PCL2 exhibited shift toward higher wavenumbers that would suggest inhibition of the inter/intramolecular hydrogen bonding between the PCL chains. PCL2-CA, on the other hand, displays a slight shift to a lower wavenumber as a consequence of molecular interactions between PCL and r-nGO-CA.24 Favorable molecular interactions between PCL and nanodots would enable an enhanced dispersion of the nanodots as revealed by the SEM images of the cross-section of PCL2-CA.32 Interestingly, the r-nGO-CA nanodots were enwrapped by PCL whereas r-nGO created agglomerates observed on the surface of the films.

These differences might originate from the chemical characteristics of the two different nanostructures, which consequently influences their molecular interactions with PCL. According to our previously reported study demonstrating green reduction of nGO, there is a distinct difference in oxygen functional groups between r-nGO and r-nGO-CA. r-nGO-CA has a lower concentration of oxygen functionalities due to a more efficient reduction but carries mostly COH, C-O-C groups while r-nGO has approximately equal amounts of C=O and C-OH/C-O-C groups.28 This might cause more hydrogen bonding interactions between the C=O of the polymer chains and the more accessible OH groups of r-nGO-CA and explain the redshift observed in



FTIR. In addition, the hydrophilicity of the nanodots is decreased as the reduction processes take place which might improve compatibility with hydrophobic PCL.

Seemingly, the processing resulted in homogeneous nanocomposite films with darkening color as the concentration of the black carbon nanodots is increased, see Figure 2. It was previously found that the nanodots exhibited fluorescence behavior. Subjecting the films to UV light (wavelength ∼250 nm) shows that this property was transferred to the films containing nanodots (Figure 2). rnGO and r-nGO-CA both emitted bright green color. Although r-nGO previously displayed higher fluorescence intensity than r-nGO-CA in water at the same concentration, PCL0.5-CA seemed to exhibit somewhat stronger fluorescence as compared to PCL0.5. This could be correlated to the dispersion since it has previously been reported that aggregation of the fluorescencing component has a negative effect on the fluorescence properties by decreasing the quantum yield.33-35 The same bright green color was not observed in the case of PCL2 and PCL2CA, possibly due to the intense dark color of the films.


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Figure 2. FTIR spectrum of the nanocomposites (left), the image of the nanocomposites in normal light and under UV light at λex = 254 nm (up right) and SEM images of the cross-sections of the nanocomposites (bottom right).

The influence of the carbon nanodots on the PCL morphology was further evaluated by XRD. PCL nanocomposites manifested a typical orthorhombic crystalline unit cell with peaks at 2θ=21.5°, 2θ =22.1° and 2θ =23.8° that were recognized as the [110], [111], and [200] crystallographic planes of PCL, as shown in Figure 3. The diffraction pattern was slightly changed as compared to neat PCL with a shift to lower 2θ for all nanocomposites which suggests a less compact crystalline state, particularly for PCL0.5 and PCL2 that display a more pronounced shift. PCL0.5-CA shows a higher peak intensity indicating a higher degree of crystallinity as indicated by SEM as well.

Figure 3. XRD spectra of the nanocomposites.



Dynamic mechanical thermal analysis Other relevant properties to evaluate considering many bone tissue engineering applications are the dynamic mechanical properties since many living tissues or biological molecules often show a viscoelastic nature in physiological conditions and the general performance of an implanted biomaterial will also depend on its viscoelastic properties.36 Most bone fractures occur under dynamic loading conditions, under which the viscoelasticity of bone may have an evident effect.37 The storage modulus (G’) is useful when determining the load-bearing capability of a composite material38, it is proportional to the energy stored elastically and is reversible.39 The inclusion of the precursor carbon nanodots nGO in the PCL matrix previously resulted in an increased brittleness (See SI file, Table S1). Here, the dynamic mechanical results show a remarkable increment of the storage modulus of the composites after the addition of the reduced carbon nanodots, particularly in the case of PCL2-CA, see Figure 4. The increase in the storage modulus indicates that the addition of carbon nanodots induces reinforcement effects that allow the stress to be transferred from the matrix to the nanodots.40-42

The loss modulus (G’’) on the other hand reflects the materials ability to dissipate energy in the form of heat or molecular rearrangements when there is deformation. Higher loss modulus would indicate a limited molecular mobility and a higher viscosity. As shown in Figure 4 the loss modulus increases with the amount and type of carbon nanodots. The largest increment is seen for PCL2-CA which could be explained by the stronger molecular interactions detected by FTIR that hinder the motions of the polymer chains.43


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It is evident that the position of the peak maximum in the tan δ curves (Figure. 4), which is related to the glass transition temperature (Tg) of the nanocomposites, slightly shifts towards a lower value as compared to neat PCL. In addition, a decrease in the peak intensity is also observed. This elucidates that the molecular mobility of the amorphous PCL chains is constrained in the nanocomposites, in accordance with loss modulus.



Figure 4. DMTA spectra showing the storage modulus (up), loss modulus (middle) and the tan δ curves of the nanocomposites (bottom).

Viscoelastic properties and creep resistance Viscoelastic properties of the carbon nanodots reinforced PCL samples were studied by frequency sweep measurements at 100 °C. The lines show the relationship between the storage


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modulus or elastic response (G’) and the loss modulus or the viscous response (G’’) versus the frequency, see Figure 5. At high frequencies, G’ is larger than G’’ for all nanocomposites showing elastic behavior. As the frequency decreases a cross-over point is observed and the nanocomposites display viscoelastic properties. Highest storage modulus was observed for PCL2CA followed closely by PCL2 confirming the DMTA results.

Figure 5. Dynamic frequency sweeps for the nanocomposites at 100 °C.

The enhanced strength after the incorporation of carbon nanodots was further proved by creep measurements at 37 °C. This is particularly important for load-bearing applications where a load is constantly applied during periods of times. As can be seen in Figure 6 the creep resistance is significantly reduced with the incorporation of r-nGO and r-nGO-CA. This can be explained by the increasing friction and modulus in the solid samples with increasing carbon nanodot content.44 With a larger amount of carbon nanodots, the samples became stiffer and they deformed less under constant applied stress. The most remarkable change is however observed



for PCL2-CA, which displayed the highest increment in creep resistance in contrast to PCL2 possibly due to a prevailing dispersion and interaction.45

Figure 6. Creep behavior of the nanocomposites at 37 °C. Cell viability test It can be concluded that r-nGO-CA superiorly reinforced PCL compared to r-nGO. PCL0.5-CA and PCL2-CA were therefore selected for further characterization and evaluation for their potential use as biomedical materials. One essential property to assess considering biomedical applications is the cell viability. r-nGO-CA was previously shown to exhibit good cell viability up to a concentration of 1000 µg mL-1. In accordance, an indirect cytotoxicity evaluation using MG63 cells, a bone-like cell model,46 showed a high relative cell viability for PCL0.5-CA and PCL2-CA. Low concentration of r-nGO-CA in the films even seemed to nurture the relative cell viability, which was increased to 151% in the case of PCL0.5-CA. As the r-nGO-CA loading increased to 2 wt%, the cell viability was still maintained at a high level (96%).


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Figure 7. Histograms of the relative cell viability of PCL0, PCL0.5-CA and PCL2-CA using MG63 cell line.

Mineralization ability Another key factor especially important for bone tissue engineering applications in addition to the cell viability is the osteo-bioactivity i. e. osteoinductivity and osteoconductivity.47 A materials ability to facilitate mineralization on the material surface is indicative of its osteo-bioactivity. In order to evaluate the affinity of the prepared nanocomposites for biomimetic mineralization, the films were immersed in SBF fluid for 2 weeks in human body temperature at 37 °C.

Figure 8 shows the microstructural evolutions of PCL0, PCL0.5-CA and PCL2-CA during a 14 days’ periods. The surface of PCL0.5-CA and PCL2-CA was heterogeneously nucleated and numerous granule-like, well dispersed and even sized Ca/P crystals were grown on the surface. rnGO-CA provided oxygen functionalities that have been shown to induce mineralization.26 In addition, it can be seen that increasing the loading of r-nGO-CA further enhanced the mineralization as the growth, concentration and size of the formed Ca/P crystals was increased



dramatically. The uniform formation of the Ca/P crystals distributed throughout the surface of PCL2-CA further supports the even distribution of r-nGO-CA domains that acted as nucleation sites for the formation of Ca/P crystals. Evidently, SEM images clearly reveal that PCL alone made no contribution to inducing CaP formation (Figure 8).

EDS confirmed that the crystals on the surface of PCL0.25 and PCL7.5 were mainly composed of Ca and P (Figure 8). Trace amounts of Na and Cl were also detected. Consequently, the adsorption of Ca2+ is enhanced and CaP growth promoted on the r-nGO-CA containing surfaces. The Ca/P ratio that can be calculated via EDS elucidated that Ca/P ratio of PCL0.5-CA and PCL2-CA was 1.82 and 1.70, respectively. The ideal ratio for hydroxyapatite crystals Ca10(PO4)6(OH)2, (HA) which is the main component of bone, is 1.67. Interestingly, this ratio is very close to the Ca/P ratio of the mineralized PCL2-CA surface.


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Figure 8. SEM images of PCL0, PCL0.5-CA and PCL2-CA after 2 weeks of incubation in SBF fluid (up left), EDS spectra of each material and the composition in wt% as determined by EDS (bottom).

To additionally confirm the formation of HA on the nanocomposites surfaces, XRD was carried out (Figure 9). The reflection peaks for PCL; [110], [111] and [200], could be observed for all the nanocomposites. For PCL2-CA new sharp peaks appeared that were assigned to the [211] and [112] reflections of HA.48,49 The [211] plane could also be seen for PCL0.5 however with a lower intensity. Another reflection observed for PCL0.5-CA corresponds to the [020] reflection of CaHPO4 · 2H2O, suggesting slight mineralization or the beginning of a mineralization.50 These result coincided well with the microstructural observations in Figure 8 and further support that rnGO-CA, is an effective additive inducing CaP mineralization.

Figure 9. XRD spectra of PCL0, PCL0.5-CA and PCL2-CA after 2 weeks of incubation in SBF fluid.



Conclusions Bioactive and reinforced polycaprolactone films were created by incorporation of biobased reduced nano-graphene oxide carbon nanodots. Dynamic mechanical thermal analysis and frequency sweep measurements demonstrated that r-nGO and r-nGO-CA served as efficient reinforcement agents improving the storage modulus significantly. A remarkable enhancement in the creep resistance was also registered after addition of carbon nanodots. Cell viability test using MG63 osteoblast-like cells performed on PCL0.5-CA and PCL2-CA showed no cytotoxicity. rnGO-CA was in addition shown to introduce bioactivity by facilitating HA mineralization on PCL surface. The density of the CaP crystals was governed by r-nGO-CA concentration where a Ca/P ratio very close to the Ca/P ratio of the bone could be obtained at the higher r-nGO-CA loading. On the basis of the results, it can be concluded that the reduced carbon nanodots r-nGO and, n-rGO-CA can induce significant reinforcement effects in PCL, in contrast to the precursor nGO, which had a negative influence on the mechanical performance. At the same time the reduced form retained the ability to facilitate bioactivity. These properties were found to be tunable by adjusting the concentration of the green additives, r-nGO-CA or r-nGO.

Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: The supporting information includes tensile testing results of solutions casted PCL- 2wt% nGO and PCL-2wt%-r-nGO-CA.

Acknowledgments


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The Swedish Research Council (VR) is acknowledged for the financial support (Contract Grant No. 2014-4091).

Funding Sources The Swedish Research Council (VR) (Contract Grant No. 2014-4091).

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Table of Contents graphic (TOC) Construction of bioactive and reinforced bioresorbable nanocomposites by reduced nanographene oxide carbon dots

Nejla B. Erdal and Minna Hakkarainen


Construction of Bioactive and Reinforced Bioresorbable

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