Functionalization of 3D printed poly(lactic acid)/graphene oxide/β-tricalcium phosphate (PLA/GO/TCP) scaffolds for bone tissue regeneration application

Abstract

The challenge of bone tissue regeneration implies the use of new advanced technologies for the manufacture of polymeric matrices, with 3D printing technology being a suitable option for tissue engineering due to its low processing cost, its simple operation and the wide use of biomaterials in biomedicine. Among the biopolymers used to obtain porous scaffolds, poly(lactic acid) (PLA) stands out due its mechanical and biodegradability properties, although its low bioactivity to promote bone regeneration is a great challenge. In this research, a 3D scaffold based on PLA reinforced with bioceramics such as graphene oxide (GO) and β-tricalcium phosphate (TCP) was designed and characterized by FTIR, XRD, DSC, SEM and mechanical tests. The in vitro biocompatibility, viability, and cell proliferation of the poly-l-lysine (POLYL) functionalized scaffold were investigated using Wharton Jelly mesenchymal stem cells (hWJ-MSCs) and confirmed by XPS. The incorporation of GO/TCP bioceramics into the PLA polymer matrix increased the mechanical strength and provided a thermal barrier during the fusion treatments that the polymeric material undergoes during its manufacturing. The results show that the functionalization of the scaffold with POLYL allows improving the cell adhesion, proliferation and differentiation of hWJ-MSCs. The resulting scaffold PLA/GO/TCP/POLYL exhibits enhanced structural integrity and osteogenic cues, rendering it a promising candidate for biomedical applications.

Fig. 1. PLA (16% w/v), PLA/GO (GO, 0.1% w/w), PLA/TCP (TCP, 5% w/w), PLA/GO/TCP (GO/TCP, 0.1/4.9% w/w) polymeric nanocomposites before extrusion (a); PLA and polymer filament after extrusion (b); 3D scaffolding (c) and flexural strength testing (d).

Fig. 2. FTIR spectra for PLA pellets and 3D scaffolds PLA, PLA/GO, PLA/TCP and PLA/GO/TCP (a), TCP characteristic bands are evident in PLA/TCP (b) and PLA/GO/TCP (c); XRD of GO/TCP sintered for one hour, PLA pellets and PLA, PLA/GO, PLA/TCP and PLA/GO/TCP 3D scaffolds (d).

Fig. 3. Differential scanning calorimetry (DSC) of PLA pellets and PLA, PLA/GO, PLA/TCP and PLA/GO/TCP 3D scaffolds (a); Flexure–strain curves corresponding to PLA, PLA/GO, PLA/TCP and PLA/GO/TCP 3D scaffolds (b).

Fig. 4. Photographic image and energy dispersive X-ray (EDS) spectra of the surface of 3D scaffolds PLA (a), PLA/GO (b), PLA/TCP (c) and PLA/GO/TCP (d).

Fig. 5. SEM of three-dimensional PLA, PLA/GO, PLA/TCP and PLA/GO/TCP 3D scaffolds after degradation treatment with 0.5 M NaOH for 24 and 96 h.

Fig. 6. Hydrolysis catalyzed by an alkaline catalyst of polyester.

Fig. 7. Cell adhesion images (a) and cell adhesion count (b) at 24, 48 and 72 h of hWJ-MSCs-GFP seeded on PLA, PLA/GO, PLA/TCP, and PLA/GO/TCP. Scale bar 500 μm. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Fig. 8. XPS spectra of PLA/GO/TCP 3D scaffold for the elements C, O and N, untreated (a) and surface modified by POLYL (b).

Fig. 9. Viability images (a) and cell proliferation by resazurin assay (b) at 1, 3, 5, 7 and 14 days of hWJ-MSCs on PLA, PLA/GO/TCP and PCL/GO/TCP/POLYL. Scale bar 100 μm, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Fig. 10. The mineralization of the scaffolds was observed through Alizarin Red S staining. PLA, PLA/GO/TCP, and PLA/GO/TCO/POLYL scaffolds were seeded with hWJ-MSCs and cultured in a supplemented DMEM medium and an osteogenic differentiation medium. After 7 and 14, these were stained. Scale bar in black, bottom right = 1 cm (a). Illustrates the gene expression of SPP1, RUNX2, and BMP2 of hWJ-MSCs seeded on PLA, PLA/GO/TCP, and PLA/GO/TCO/POLYL with supplemented DMEM medium and osteogenic differentiation medium at 7 and 14 days (b).

Fig. 11. Confocal microscopy images of hWJ-MSCs seeded on PLA/GO/TCP (a) and PLA/GO/TCP/POLYL (b) at 48 h.