Biocompatible reinforcement of poly(lactic acid) with graphene nanoplatelets

6 June 2016
Carolina Gonçalves, Artur Pinto, Joaquim Moreira, Inês Gonçalves, Fernão Magalhães, and Ana Machado
Improved mechanical properties of composites produced by melt blending is achieved at low filler loadings.

Graphene—a single layer of carbon atoms arranged in a honeycomb structure—possesses extraordinary mechanical strength and an extremely high surface area.1 In particular, commercially available graphene nanoplatelets (GNPs) have lower costs than single-layer graphene. These GNPs consist of a few stacked graphene layers that have oxygen-containing functional groups at their edges. In addition, GNPs provide a high aspect ratio and can thus form a percolated network in composites. In these networks there is a large interfacial interaction between the platelets and the polymer matrix (mainly at the edges), which results in effective load transfer and increased strength.2 Despite the benefits of using GNPs as fillers within polymer composites, however, previous methods that have been used to incorporate the GNPs are associated with problems of toxicity.

In previous work, for instance, we have demonstrated the potential of GNPs as polymer fillers, i.e., we achieved improvements to the mechanical properties of poly(lactic acid)—PLA—thin films for filler loadings below 1wt%. Moreover, we showed that the GNPs were non-toxic when they were incorporated into the PLA at these low proportions.3, 4 In this earlier work, we used solvent mixing for the GNP incorporation,5 but the use of solvents should generally be avoided because of the toxicity of the residues that remain in the materials (presenting a problem for industrial workers).6 In another approach—used to improve the mechanical properties of ultra-high-molecular-weight polyethylene—the composites were produced by electrostatic deposition of GNPs (at the 1wt% level).2 The resultant composites in this study, however, were toxic to osteoblasts (bone-forming cells) because filler leaching had occurred.2

In this work,7 we have thus investigated the use of melt blending—as a ‘green’ method—to ensure complete embedding of GNPs in a polymer matrix for the production of PLA/GNP composites. For this study, we purchased the PLA (2003D) from NatureWorks and the GNPs from XG Sciences. The GNP grade we obtained (C-750) is a recently available form (which we refer to as GNP-C) and (to our knowledge) our work is the first time it has been studied as a reinforcement filler for PLA. With these materials, we prepared the PLA/GNP-C composites by melt blending, under different conditions, in a Thermo Scientific Haake PolyLab. We then molded the composites into thin sheets (with dimensions of 60 × 15mm and a thickness of 300–500μm) in a hot press (at 190°C for 2 minutes). We also measured the tensile properties of our resultant composites (at room temperature) with the use of a Mecmesin MultiTest-1d motorized test frame. To evaluate the biocompatibility of our materials, we used HFF-1 cells (human fibroblasts) cultured at the surface of the films (with diameter of about 5.5mm). In the last parts of our procedure, the cells were seeded, a resazurin solution (a fluorescent dye) was added, and the mixture was incubated for three hours. We subsequently measured the fluorescence of the mixture (at an excitation wavelength to emission wavelength ratio of 530/590nm) and evaluated the metabolic activity.

Initially, we prepared the PLA/GNP-C blends by mixing at 180°C for 20 minutes and at 50 revolutions per minute (rpm). For these composites (see Figure 1), we obtained maximum Young's modulus and tensile strength values at a loading of 0.25wt%. We also observed a 20% increase in tensile strength, a 12% increase in Young's modulus, and a 16% increase in toughness for the composite with a 0.25wt% filler content compared with the neat PLA. In addition, we found that a decrease in the mechanical performance of the composites occurs when the loading is increased to 0.5wt%. We attribute this change to an increased level of platelet agglomeration, which introduces defects into the polymer matrix. We have also investigated the variation in the mechanical properties of our composites as a function of mixing time and rotation speed (see Figure 2). These results show that we obtained a brittle material at 75rpm, which was probably caused by PLA degradation under high shear. Furthermore, we find that the best processing conditions were mixing for 20 minutes at 50rpm. We believe that shorter mixing times or slower rotation speeds probably provide worse GNP dispersion and thus the poor mechanical properties. We also tested a higher mixing temperature (200°C), but this yielded very brittle materials (probably because of thermo-oxidative degradation of the PLA).


The effect of increasing graphene nanoplatelet (GNP-C) filler content on the mechanical properties of poly(lactic acid)/GNP-C (PLA/GNP-C) composites that were fabricated under the same processing conditions, i.e., melt blending at 180°C and 50 revolutions per minute (rpm) for 20 minutes. Young's modulus (a), tensile strength (b), and toughness (c) results are shown. Error bars represent the standard deviation (SD) for a measurement sample size (n) of 10.


The effect of mixing time and rotation speed on the mechanical properties of PLA/GNP-C composites (with filler content of 0.25wt%) processed at 180°C. Error bars represent the SD of the measurement (for n of 10).

From our scanning electron microscope images (see Figure 3) we measured the density of platelet agglomerates in the composites. We display the cumulative number of agglomerates per unit area, as a function of agglomerate length, for different GNP-C loadings in Figure 3(a). These results show that the composites with loadings of 0.1 and 0.25wt% have similar agglomerate densities, but the composite with a loading of 0.5wt% has a substantially higher concentration of agglomerates (of all sizes).


(a) Cumulative number of GNP agglomerates per unit area, as a function of agglomerate length, for PLA/GNP-C composites (produced at 180°C and 50rpm for 20 minutes) with different loadings. (b) Scanning electron microscope images (5000× magnification) of fracture surfaces within the three different composites.

In the last part of our study, we examined the metabolic activity of our composites. The results shown in Figure 4 indicate that the metabolic activity of the HFF-1 cells on the PLA/GNP-C composite with a 0.25wt% loading never decreased below 97% (expressed as a percentage of the metabolic activity of cells cultured on a PLA surface). Furthermore, we obtained immunocytochemistry images (see Figure 4) that show no morphological differences between the PLA and the PLA/GNP-C composite with 0.25wt% loading.


Top: The metabolic activity of HFF-1 cells (human fibroblasts) that were cultured—in a Dulbecco's Modified Eagle's medium (DMEM+)—on the surface of the PLA/GNP-C composite with a loading of 0.25wt% (produced at 180°C and 50rpm for 20 minutes). The activity (after 24, 48, and 72 hours) is expressed here as a percentage of the metabolic activity of cells cultured in the same way on a PLA surface (PLA =100%). Bottom: Representative immunofluorescence (top row) and phase contrast (bottom row) images of HFF-1 cells obtained after 72 hours. The images were obtained for cells within (left) a DMEM+/Triton mixture, (middle) on a PLA surface, and (right) on the PLA-GNP-C composite with 0.25wt% loading. The cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and phalloidin to highlight the cell nuclei (in blue) and the cytoskeletons (in green), respectively. Scale bars indicate 100μm.

In summary, we have studied a non-toxic and green melt blending approach to achieve the incorporation of graphene nanoplatelets within PLA/GNP composites. We examined the mechanical properties of our composites (with different loading levels) and found an optimum loading of 0.25wt%. At higher loading levels, defects in the matrix (caused by filler agglomerations) caused a decrease in mechanical performance. In addition, we have found that the metabolic activity and morphology of human skin cells (cultured on the surface of the materials) were unaffected by the incorporation of 0.25wt% GNP-C into the PLA. The improved mechanical performance of our composites, at low filler loadings, and their associated biocompatibility means that our approach may have interesting future uses in biomaterial applications. In the next step of our work we will conduct an in vivo evaluation of the biocomposites' biocompatibility.


Authors

Carolina Gonçalves
Network for Competence in Polymers (ARCP), Center of Innovation (UPTEC)

Artur Pinto
University of Porto

Artur Pinto is currently completing his PhD in biomedical engineering within the Faculty of Engineering. He also carries out research in the Laboratory for Process Engineering, Environment, Biotechnology and Energy and the Institute for Research and Innovation in Health (both at the University of Porto).

Joaquim Moreira
University of Porto

Inês Gonçalves
University of Porto

Fernão Magalhães
University of Porto

Ana Machado
Institute of Polymers and Composites and Institute of Nanostructures, Nanomodelling and Nanofabrication (I3N), University of Minho


References

  1. H. Kim, A. A. Abdala and C. W. Macosko, Graphene/polymer nanocomposites, Macromolecules 43, pp. 6515-6530, 2010.

  2. D. Lahiri, R. Dua, C. Zhang, I. de Socarraz-Novoa, A. Bhat and S. Ramaswamy, Graphene nanoplatelet-induced strengthening of ultrahigh molecular weight polyethylene and biocompatibility in vitro, ACS Appl. Mater. Interfaces 4, pp. 2234-2241, 2012.

  3. A. M. Pinto, J. Cabral, D. A. Pacheco Tanaka, A. M. Mendes and F. D. Magalhães, Effect of incorporation of graphene oxide and graphene nanoplatelets on mechanical and gas permeability properties of poly(lactic acid) films, Polym. Int'l 62, pp. 33-40, 2013.

  4. A. M. Pinto, C. Conçalves, D. M. Sousa, A. R. Ferreira, J. A. Moreira, I. C. Conçalves and F. D. Magalhães, Smaller particle size and higher oxidation improves biocompatibility of graphene-based materials, Carbon 99, pp. 318-329, 2016.

  5. A. M. Pinto, I. C. Conçalves, F. M. Gama, A. M. Mendes and F. D. Magalhães, Biocompatibility of poly(lactic acid) with incorporated graphene-based materials, Colloids Surfaces B: Biointerfaces 104, pp. 229-238, 2013.

  6. A. M. Pinto, I. C. Conçalves and F. D. Magalhães, Graphene-based materials biocompatibility: a review, Colloids Surfaces B: Biointerfaces 111, pp. 188-202, 2013.

  7. C. Conçalves, A. Pinto, A. V. Machado, J. Moreira, I. C. Conçalves and F. Magalhães, Biocompatible reinforcement of poly(lactic acid) with graphene nanoplatelets, Polym. Compos., 2016. First published online: 29 April

DOI:  10.2417/spepro.006542



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