Graphene-functionalized thermoplastic biomaterial for 3D printing

Adding functionalization

Poly(ε-caprolactone) (PCL) nanocomposites with reduced graphene oxide (rGO) make fascinating materials for 3D printing. The addition of rGO generates valuable properties such as mechanical strength, electric conductivity, and nanoscale surface roughness, while printability, biocompatibility and biodegradability inherent to PCL are also maintained [1]. Robust and bioactive PCL/rGO scaffolds can be used for in vivo patient-specific bone regeneration [1, 2] or in vitro neural regeneration [3].

Aim

In this study, PCL/rGO granules provided by Nadir Plasma & Polymers were used for 3D printing of multi-layered grids that served as scaffolds for mesenchymal stem cells (MSCs). They were compared to the pure PCL material to find out the beneficial effect of rGO functionalization on PCL thermoplastics. The comparison includes rheological characterization performed to investigate the melting and flow behavior of thermoplastics as well as 3D printing of grids and cell post-seeding on printed grids for evaluation of cell attachment and proliferation.

CELLINK products used

PCL

PCL/rGO.

Experimental setup

Two different thermoplastic materials were chosen for the study: pure PCL (MW = 50 kDa, Nadir Plasma & Polymers) and rGO-functionalized PCL (MW = 50 kDa, Nadir Plasma & Polymers).

For rheological characterization of materials, Discovery HR-2 rheometer (TA Instruments, UK) was used with a Peltier plate and an aluminum 20 mm parallel plate−plate geometry for temperature sweeps from 40°C to 120°C (soak time = 30 s, temperature step = 2°C, strain = 1%, angular frequency = 10 rad∙s-1) and for flow sweeps at optimal printing temperature of 180°C (shear rate = 0.01-500 s-1).

3D printing of two-layer grids (5 cm x 5 cm) was performed using CELLINK’s BIO X equipped with the Thermoplastic Printhead. The optimal printing parameters for the pure PCL: printing temperature = 180°C, printing speed = 4 mm/s, printing pressure = 510 kPa. The optimal printing parameters for the PCL/rGO: printing temperature = 150°C, printing speed = 4 mm/s, printing pressure = 500 kPa.

For cell post-seeding on printed grids, 200 µL of MSC suspension with the concentration of 67k cells/1 mL medium was deposited on cut pieces of grids (approx. 0.5 cm x 0.5 cm) placed into a 48-well plate. A live/dead assay was performed using staining with Calcein AM (Invitrogen eBioscience) and propidium iodide (Sigma-Aldrich) after 7 and 14 days of cell culturing. Images were acquired with a fluorescent microscope (Olympus IX73) utilizing green (FITC) and red (XRED) channels and processed with the image processing program ImageJ.

Rheological characterization

Both PCL-based materials started melting above 40°C as storage modulus values became lower than loss modulus values (Figure 1), however they still maintained too high moduli and viscosity for printing below 120°C.

 

At lower shear rates, pure PCL demonstrated slight viscosity dependence on temperature as viscosity is higher at 120°C than at 180°C, however this dependency is less pronounced for rGO-containing PCL. In general, PCL-based thermoplastics show similar shear thinning behavior at 180°C and shear rates above 100 s-1, with rGO-containing samples having slightly higher viscosity (Figure 2). Moreover, at higher shear rates that correspond to actual flow of thermoplastic melt, the viscosity difference between 120°C and 180°C is not a factor (Figure 3).

3D printing

Though the melting behavior of PCL is not significantly affected by the rGO incorporation, the rGO nanosheets can promote higher material viscosity and faster PCL crystallization upon extrusion [4]. Therefore, we observed higher stringing for the PCL/rGO material in comparison to the pure PCL at increased temperature (180°C). This well-known phenomenon for PCL melt printing was tackled by decreasing printing temperature for rGO modified PCL to 150°C [5], which resulted in smooth high-resolution printing for PCL/rGO material (Figure 4).

Enhanced cell attachment

The results of cell study clearly demonstrate much better cell adhesion to rGO-modified PCL grids than to pure PCL (Figure 5). There were very few cells observed on the pure PCL grids after 7 and 14 days of culturing. In contrast, already after 7 days of culturing, a homogeneous layer of cells completely covered PCL/rGO grids, and after 14 days this layer only became denser. Graphene-containing scaffolds with distinctive nanotopography have been previously shown to influence mechanotransduction of different cell types, which has positive effect on cell adhesion and migration [6].

Conclusion

This study demonstrates that PCL/rGO composites from Nadir Plasma and Polymers are great thermoplastic materials for high-resolution 3D printing of complex structures that enable cell attachment and proliferation. Using CELLINK’s BIO X with the Thermoplastic Printhead allows for smooth 3D printing of the PCL/rGO material at 150°C and a printing pressure around 500 kPa.

 

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References

1. G.F. Caetano, W.Wang, W.-H. Chiang, G. Cooper, C. Diver, J.J. Blaker, M.A. Frade and P. Bártolo, 3D-Printed poly(ɛ-caprolactone)/graphene scaffolds activated with P1-latex protein for bone regeneration. 3D Print. Addit. Manuf. 2018, 5, 127–137.

2. W. Wanga, J.R. Passarini Junior, P. R. Lopes, Nalesso, D. Musson, J. Cornish, F. Mendonça, G. Ferreira Caetano and P. Bártolo. Mater. Sci. Eng. C 2019, 759–770.

3. S. Sánchez-González, N. Diban and A. Urtiaga, Hydrolytic degradation and mechanical stability of poly(ε-caprolactone)/reduced graphene oxide membranes as scaffolds for in vitro neural tissue regeneration. Membranes 2018, 8, 12.

4. I. Castilla-Cortázar, A. Vidaurre, B. Marí and A.J. Campillo-Fernández, Morphology, crystallinity, and molecular weight of poly(ε-caprolactone)/graphene oxide hybrids. Polymers 2019, 11, 1099.

5. R.H.A. Haq, O.M.F. Marwah, M.N.A. Rahman, H.F. Haw, H. Abdullah and S. Ahmad, 3D Printer parameters analysis for PCL/PLA filament wire using Design of Experiment (DOE). IOP Conf. Ser.: Mater. Sci. Eng. 2019, 607, 012001.

6. I. Lasocka, E. Jastrzębska, L. Szulc-Dąbrowska, M. Skibniewski, I. Pasternak, M. Hubalek Kalbacova and E.M Skibniewska, The effects of graphene and mesenchymal stem cells in cutaneous wound healing and their putative action mechanism. Int J Nanomedicine 2019, 14, 2281–2299.