3D printed bioabsorbable composite scaffolds of poly (lactic acid)-tricalcium phosphate-ceria with osteogenic property for bone regeneration

Abstract

The fabrication of customized implants by additive manufacturing has allowed continued development of the personalized medicine field. Herein, a 3D-printed bioabsorbable poly (lactic acid) (PLA)- β-tricalcium phosphate (TCP) (10 wt %) composite has been modified with CeO₂ nanoparticles (CeNPs) (1, 5 and 10 wt %) for bone repair. The filaments were prepared by melt extrusion and used to print porous scaffolds. The nanocomposite scaffolds possessed precise structure with fine print resolution, a homogenous distribution of TCP and CeNP components, and mechanical properties appropriate for bone tissue engineering applications. Cell proliferation assays using osteoblast cultures confirmed the cytocompatibility of the composites. In addition, the presence of CeNPs enhanced the proliferation and differentiation of mesenchymal stem cells; thereby, increasing alkaline phosphatase (ALP) activity, calcium deposition and bone-related gene expression. Results from this study have shown that the 3D printed PLA-TCP-10%CeO₂ composite scaffold could be used as an alternative polymeric implant for bone tissue engineering applications: avoiding additional/revision surgeries and accelerating the regenerative process.

Keywords: Additive manufacturing; Biomaterial; Bone tissue engineering; Ceria nanoparticle; Polymer-matrix composites.

Graphical abstract

Scheme 1

Experimental procedure to obtain PLA-TCP-CeO₂ scaffolds and their potential osteogenic mechanism. PLA, TCP and CeNPs powders are poured into a twin-screw extruder at 175 °C. The filament is extruded with a constant diameter of 1.75 mm, immersed in a water bath for cooling, and collected as a spool. The filament spool is then used to print the scaffolds by additive manufacturing. The interaction of cerium oxide nanoparticles with mesenchymal stem cells activates the Smad-dependent BMP signaling pathway, promoting osteogenesis and bone healing.

Fig. 1

(a) TEM image showing the size of the CeNPs with 18 ± 5 nm. (b) Zeta potential measurement of the CeNPs displaying value of 24.4 mV. (c) Ce 3d XPS spectra of the CeNPs presenting 13.70 % of Ce³⁺ and 86.30 % of Ce⁴⁺. (d) DSC curves obtained in the second heating, (e) calculated degree of crystallinity from DSC results (compared to the PLA-TCP matrix unless identified with bars, N = 3; *: p <0.05; **: p <0.01; ***: p <0.005). (f) TGA and (g) DTG curves of the PLA and PLA-TCP samples. (h) TGA and (i) DTG curves of the samples containing CeNPs. Steady-state rheology measured from the filaments at (j-k) 175 °C and (l) 165 °C. Dynamic-state rheology measured from the filaments at (m-n) 175 °C and (o) 165 °C.

Fig. 2

(a) Photo of the scaffolds. (b) Cross-sectional SEM-EDS and (c) top view SEM of the PLA-TCP-10CeO₂ scaffold. (d) XRD of the CeNPs powder, TCP powder and the PLA, PLA-TCP and PLA-TCP-10CeO₂ scaffolds. (e) Ce 3d XPS spectra of the PLA-TCP-10CeO₂ scaffold showing the presence of 30.39 % of Ce³⁺. (f) Elastic modulus extracted from the linear region of the stress-strain curves in compression mode (N = 5). (g) Protein adsorption to scaffold samples (N=3). Degradation in (h) PBS, (i) PBS containing proteinase and (j) culture medium, as a function of time (N=3). The data are expressed as mean ± standard deviation and compared to the PLA-TCP matrix unless identified with bars. (*: p <0.05; **: p <0.01; ***: p <0.005).

Fig. 3

(a) SEM images of the osteoblasts adhered to the scaffold surface after 1 day of cell culture. Cytotoxicity evaluation after 1 and 7 days of (b) osteoblast culture and (c) MSCs culture on the scaffolds. The data are expressed as mean ± standard deviation and compared to the PLA-TCP matrix at the same time point unless identified with bars. (N = 3; *: p <0.05; **: p <0.01; ***: p <0.005). (d) Representative fluorescence micrographs after 7 days of MSCs culture on the scaffolds, showing substantial cell proliferation for all samples. Alexa Fluor 488 Phalloidin was used to stain the cytoskeleton of cells in red, and DAPI nuclei staining appear in blue.

Fig. 4

Mesenchymal stem cell differentiation using an osteogenic medium. (a) Alizarin red S (ARS) staining and (b) alkaline phosphatase (ALP) staining after 28 days of MSCs culture on the scaffolds. (c) ARS quantification and (d) ALP activity after 14 and 28 days. RT-qPCR results after 28 days of MSCs culture on the scaffolds. Expression level of (e) BMP2, (f) ALPL, (g) RUNX2, (h) OCN, (i) OPN and (j) COL1A1. The data are expressed as mean ± standard deviation and compared to the PLA-TCP matrix at the same time point. (N = 3; *: p <0.05; **: p <0.01; ***: p <0.005).