3D printing of PLA:CaP:GO scaffolds for bone tissue applications

Abstract

Graphene oxide (GO) has attracted increasing interest for biomedical applications owing to its outstanding properties such as high specific surface area, ability to bind functional molecules for therapeutic purposes and solubility, together with mechanical resistance and good thermal conductivity. The combination of GO with other biomaterials, such as calcium phosphate (CaP) and biodegradable polymers, presents a promising strategy for bone tissue engineering. Presently, the development of these advanced biomaterials benefits from the use of additive manufacturing techniques, such as 3D printing. In this study, we develop a 3D printed PLA:CaP:GO scaffold for bone tissue engineering. First, GO was characterised alone by XPS to determine its main bond contributions and C : O ratio. Secondly, we determined the GO dose which ensures the absence of toxicity, directly exposed in vitro (human osteoblast-like cells MG-63) and in vivo (zebrafish model). In addition, GO was microinjected in the zebrafish to evaluate its effect on immune cells, quantifying the genetic expression of the main markers. Results indicated that the GO tested (C : O of 2.14, 49.50% oxidised, main bonds: C-OH, C-O-C) in a dose ≤0.25 mg mL^-1 promoted MG63 cells viability percentages above 70%, and in a dose ≤0.10 mg mL^-1 resulted in the absence of toxicity in zebrafish embryos. The immune response evaluation reinforced this result. Finally, the optimised GO dose (0.10 mg mL^-1) was combined with polylactic acid (PLA) and CaP to obtain a 3D printed PLA:CaP:GO scaffold. Physicochemical characterisation (SEM/EDS, XRD, FT-Raman, nano-indentation) was performed and in vivo tests confirmed its biocompatibility, enabling a novel approach for bone tissue-related applications.

Fig. 1. Cell viability of MG-63 after exposure to different concentrations of GO for 24 h. The dotted line indicates the acceptable limit of cytotoxicity according to the UNE-EN-ISO 10993-5:2009 standard. Data are presented as mean ±cumulative error.

Fig. 2. Toxicity response of zebrafish embryos and larvae at different concentrations of GO through two exposition routes: viability rate of embryos at 24 hpe by bath (a), hatching rate during 72 hpe (b), viability rate of larvae exposed to GO by bath for 7 dpe (c) and viability rate of larvae exposed to GO by microinjection for 72 hpe (d). In all cases the results are shown with respect to the control (Ctr).

Fig. 3. Quantification of neutrophil (a and b) and macrophage (c and d) behaviour in response to the injection of different solutions of GO in zebrafish transgenic embryos (3dpf) with fluorescently labelled immune cells, from 2 to 24 hpe. Data are presented as mean ±SEM. Statistically significant differences from the control group are indicated by *, p < 0.05.

Fig. 4. Expression profile of genes related to inflammatory/immune response in zebrafish larvae 24 h after being microinjected with 0.1 mg mL⁻¹ of GO. Data are presented as mean of fold changes, calculated with respect to the basal expression of each gene (CTR), ±standard error of the mean.

Fig. 5. Stereo micrographs in different magnifications (a–c), SEM (d) and corresponding EDS spectrum (e) of the PLA:CaP:GO scaffold with 3.4 wt% of CaP and 0.10 mg mL⁻¹ of GO (0.004 wt%) (d and e).

Fig. 6. XRD diffraction pattern of the 3D printed PLA:CaP:GO scaffolds (a) and FT-Raman spectra showing the characteristic bands for each of the scaffold components (b). In both cases the sample PLA:0CaP:0GO is taken as reference, as well as the GO for FT-Raman. For Raman spectra, the regions associated with GO (1300–1600 cm⁻¹) are highlighted with pointed square, while the band corresponding to CaP is enclosed by a dotted circle.

Fig. 7. Young's modulus and hardness of different PLA:CaP:GO scaffolds. In the bar chart means ±standard errors are represented.

Fig. 8. Toxicity response of zebrafish larvae exposed to extracts obtained from PLA:CaP:GO scaffolds (with 3.4 wt% of CaP and 0.004 wt% of GO) for 30 days: viability rate of WT embryos 7 dpe by bath.