Customized Design 3D Printed PLGA/Calcium Sulfate Scaffold Enhances Mechanical and Biological Properties for Bone Regeneration

Abstract

Polylactic glycolic acid copolymer (PLGA) has been widely used in tissue engineering due to its good biocompatibility and degradation properties. However, the mismatched mechanical and unsatisfactory biological properties of PLGA limit further application in bone tissue engineering. Calcium sulfate (CaSO₄) is one of the most promising bone repair materials due to its non-immunogenicity, well biocompatibility, and excellent bone conductivity. In this study, aiming at the shortcomings of activity-lack and low mechanical of PLGA in bone tissue engineering, customized-designed 3D porous PLGA/CaSO₄ scaffolds were prepared by 3D printing. We first studied the physical properties of PLGA/CaSO₄ scaffolds and the results showed that CaSO₄ improved the mechanical properties of PLGA scaffolds. In vitro experiments showed that PLGA/CaSO₄ scaffold exhibited good biocompatibility. Moreover, the addition of CaSO₄ could significantly improve the migration and osteogenic differentiation of MC3T3-E1 cells in the PLGA/CaSO₄ scaffolds, and the PLGA/CaSO₄ scaffolds made with 20 wt.% CaSO₄ exhibited the best osteogenesis properties. Therefore, calcium sulfate was added to PLGA could lead to customized 3D printed scaffolds for enhanced mechanical properties and biological properties. The customized 3D-printed PLGA/CaSO₄ scaffold shows great potential for precisely repairing irregular load-bearing bone defects.

Keywords: 3D printing scaffold; biological properties; bone defect; calcium sulfate; mechanical properties; polylactic glycolic acid copolymer.

SCHEME 1

Schematic illustration of the fabrication process for PLGA/CaSO₄ scaffolds. The rabbit radius was first scanned by micro-CT and a bone tissue model was constructed by 3D reconstruction. Then the scaffold was designed using CAD software, and the CAD data was transferred to a 3D printer to fabricate PLGA/CaSO₄ scaffolds. The PLGA/CaSO₄ scaffolds could promote cell proliferation, migration, and osteogenesis.

FIGURE 1

SEM morphology of CaSO₄ particles.

FIGURE 2

Characterization of the scaffolds. (A) Representative pictures of 3D printed PLGA, PLGA/10%CaSO₄, PLGA/20%CaSO₄, and PLGA/30%CaSO₄ scaffolds. Scale bar = 5 mm. (B) SEM images of the side of the different scaffolds. Scale bar = 200 and 100 μm, respectively. (C) Water contact angle images of different scaffolds. (D) Contact angle (degree) of PLGA, PLGA/10%CaSO₄, PLGA/20%CaSO₄, and PLGA/30%CaSO₄ scaffolds, respectively. (E) The pore size of each scaffold. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Mechanical performance of the 3D printed scaffolds. (A) The stress-strain curves. (B) The compress stress. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Degradation in vitro.

FIGURE 5

In vitro biocompatibility. (A) Representative fluorescence images for HUVEC cells cultured with different scaffolds. Live cells were stained by calcein-AM (green color). Scale bar = 200 μm. (B) CCK-8 assay for HUVECs cultured with the PLGA, PLGA/10%CaSO₄, PLGA/20%CaSO₄, and PLGA/30%CaSO₄ scaffolds, respectively. (C) In vitro hemolysis of different scaffolds and Hemolytic rate (%). Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

In vitro cell migration. (A) In vitro wound healing assay of MC3T3-E1 cell. Scale bar = 100 μm. (B) Transwell assay of MC3T3-E1 cells. Scale bar = 100 μm. (C) Cell migration rate (%) of cells. (D) Cell migration number of MC3T3-E1 cells per field. (E) Absorbance at 590 nm. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

MC3T3-E1 cytoskeleton in the scaffolds for 24 h. Scale bar = 50 μm.

FIGURE 8

In vitro osteogenesis capability of the scaffolds. (A) ALP staining. Scale bar = 200 μm. (B) Staining area of Alizarin red. Scale bar = 200 μm. (C) The ALP activity of the MC3T3-E1 cells co-cultured with the scaffolds on days 7 and 14. ALP level was significantly high in the PLGA/20%CaSO₄ scaffolds compared to the other scaffolds. (D–H) Relative mRNA expression of the osteogenic genes (OPN, OCN, RUNX-2, Collagen I, and BMP-2). Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.