Fabrication and properties of PLA/β-TCP scaffolds using liquid crystal display (LCD) photocuring 3D printing for bone tissue engineering

Abstract

Introduction: Bone defects remain a thorny challenge that clinicians have to face. At present, scaffolds prepared by 3D printing are increasingly used in the field of bone tissue repair. Polylactic acid (PLA) has good thermoplasticity, processability, biocompatibility, and biodegradability, but the PLA is brittle and has poor osteogenic performance. Beta-tricalcium phosphate (β-TCP) has good mechanical properties and osteogenic induction properties, which can make up for the drawbacks of PLA. Methods: In this study, photocurable biodegradable polylactic acid (bio-PLA) was utilized as the raw material to prepare PLA/β-TCP slurries with varying β-TCP contents (β-TCP dosage at 0%, 10%, 20%, 30%, 35% of the PLA dosage, respectively). The PLA/β-TCP scaffolds were fabricated using liquid crystal display (LCD) light-curing 3D printing technology. The characterization of the scaffolds was assessed, and the biological activity of the scaffold with the optimal compressive strength was evaluated. The biocompatibility of the scaffold was assessed through CCK-8 assays, hemocompatibility assay and live-dead staining experiments. The osteogenic differentiation capacity of the scaffold on MC3T3-E1 cells was evaluated through alizarin red staining, alkaline phosphatase (ALP) detection, immunofluorescence experiments, and RT-qPCR assays. Results: The prepared scaffold possesses a three-dimensional network structure, and with an increase in the quantity of β-TCP, more β-TCP particles adhere to the scaffold surface. The compressive strength of PLA/β-TCP scaffolds exhibits a trend of initial increase followed by decrease with an increasing amount of β-TCP, reaching a maximum value of 52.1 MPa at a 10% β-TCP content. Degradation rate curve results indicate that with the passage of time, the degradation rate of the scaffold gradually increases, and the pH of the scaffold during degradation shows an alkaline tendency. Additionally, Live/dead staining and blood compatibility experiments suggest that the prepared PLA/β-TCP scaffold demonstrates excellent biocompatibility. CCK-8 experiments indicate that the PLA/β-TCP group promotes cell proliferation, and the prepared PLA/β-TCP scaffold exhibits a significant ability to enhance the osteogenic differentiation of MC3T3-E1 cells in vitro. Discussion: 3D printed LCD photocuring PLA/β-TCP scaffolds could improve surface bioactivity and lead to better osteogenesis, which may provide a unique strategy for developing bioactive implants in orthopedic applications.

Keywords: 3D printed scaffolds; beta-tricalcium phosphate; bone tissue engineering; liquid crystal display; polylactic acid.

SCHEME 1

Schematic illustration of the PLA/β-TCP scaffolds using LCD photocuring 3D printing in bone tissue engineering. With the help of a computer and a display screen, ultraviolet light passes through the transparent area to illuminate the photosensitive resin in the resin tank. The PLA/β-TCP resin material is exposed and solidified layer by layer into a 3D scaffold.

FIGURE 1

Rheological curve of PLA/β-TCP slurry.

FIGURE 2

Morphology of the 3D-printed scaffolds. (A) 3D printed PLA/β-TCP scaffolds with different proportions. (B) SEM images of the 3D-printed scaffolds, respectively. Scale bar = 400 μm, 200 μm and 20 μm. (C) The three-dimensional morphology of the scaffold fiber surface and the surface roughness of the selected area Ra (mean arithmetic height) (μm).

FIGURE 3

Mechanical performance of the 3D-printed scaffolds. (A) SEM morphology of the fractured section of the different 3D-printed scaffold under compression. (B) FTIR spectra of 3D-printed PLA/30%β-TCP scaffold and PLA/30%β-TCP scaffold (KH-550). (C) The compression strength of the different 3D-printed scaffolds.

FIGURE 4

The degradation of the scaffold. (A)Weight of scaffolds after degradation in vitro. (B) pH value in the SBF.

FIGURE 5

Biocompatibility of the scaffolds in vitro. (A) Fluorescence images of MC3T3-E1 cells cultured on PLA and PLA/β-TCP scaffolds for days 1, 3, and 5, respectively. Scale bar = 100 μm. (B) CCK-8 assay of the different scaffolds for days 1, 3, and 5. (C) Hemolysis test in vitro. Data are presented as mean ± SD (n = 3), *p < 0.05, **p < 0.01.

FIGURE 6

Osteogenic differentiation of the scaffolds in vitro. (A) The ALP staining of the MC3T3-E1 cells was co-cultured with the scaffolds. Scale bar = 100 μm. (B) Alizarin red staining of the MC3T3-E1 cells co-cultured with the scaffolds. Scale bar = 100 μm. (C) ALP activity of the scaffolds. (D) Alizarin red S quantitation of MC3T3-E1. (E) Relative mRNA expression of the osteogenic-related genes (BMP-2, OCN, Runx-2, and COL-1) of the MC3T3-E1 cocultured with the scaffolds. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

FIGURE 7

Immunofluorescence staining of osteogenic factors. (A) The image of immunofluorescence staining of Runx-2. Scale bar = 100 μm. (B) Average fluorescence intensity of Runx-2. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

FIGURE 8

ELISA assay detects the effects of different scaffolds on BMP-2 expression. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.