Fabrication and evaluation of 3D printed BCP scaffolds reinforced with ZrO2 for bone tissue applications

Abstract

Fused deposition modeling (FDM) is a promising 3D printing and manufacturing step to create well interconnected porous scaffold designs from the computer-aided design (CAD) models for the next generation of bone scaffolds. The purpose of this study was to fabricate and evaluate a new biphasic calcium phosphate (BCP) scaffold reinforced with zirconia (ZrO₂ ) by a FDM system for bone tissue engineering. The 3D slurry foams with blending agents were successfully fabricated by a FDM system. Blending materials were then removed after the sintering process at high temperature to obtain a targeted BCP/ZrO₂ scaffold with the desired pore characteristics, porosity, and dimension. Morphology of the sintered scaffold was investigated with SEM/EDS mapping. A cell proliferation test was carried out and evaluated with osteosarcoma MG-63 cells. Mechanical testing and cell proliferation evaluation demonstrated that 90% BCP and 10% ZrO₂ scaffold had a significant effect on the mechanical properties maintaining a structure compared that of only 100% BCP with no ZrO₂ . Additionally, differentiation studies of human mesenchymal stem cells (hMSCs) on BCP/ZrO₂ scaffolds in static and dynamic culture conditions showed increased expression of bone morphogenic protein-2 (BMP-2) when cultured on BCP/ZrO₂ scaffolds under dynamic conditions compared to on BCP control scaffolds. The manufacturing of BCP/ZrO₂ scaffolds through this innovative technique of a FDM may provide applications for various types of tissue regeneration, including bone and cartilage.

Keywords: 3D printing; biphasic calcium phosphate; bone tissue engineering; scaffold; zirconia.

Figure 1

FDM 3D printing setup. (a) FDM system, (b) deposition heads, (c) temperature controller, and (d) pressure controller.

Figure 2

CAD drawing and 3D printed BCP scaffolds. (a) Pro-E model of the fabricated slurry foams, showing alternating lattice structures, producing fully interconnected square pores. (b) BCP slurry foam from top after printing and curing (c) BCP/ZrO₂ slurry foam from bottom after printing and curing. Final dimension of the printed scaffolds was at 8.5 × 8.5 × 3.2 mm.

Figure 3

Weight loss and shrinkage results of BCP and BCP/ZrO₂ scaffolds after sintering process. (a) Calculated weight loss using the weight ratio between BCP and BCP/ZrO₂ scaffolds, depicting a 4.4% greter weight loss in BCP/ZrO₂ scaffolds. (n=4) (b) Shrinkage of the scaffolds using volume ratios showed a 4.5% difference between BCP and BCP/ZrO₂ scaffolds. (n=4)

Figure 4

SEM images of BCP/ZrO₂ scaffold. Left image shows pore size of approximately 350 μm and printed lattice width of 500 μm. Right side depicts a granular surface of the 3D printed scaffold after printing and sintering.

Figure 5

Ouantitative analysis results of BCP scaffold using EDS on SEM. (a) Image of surface area used to analyze the scaffolds for their composition. Table results show the percentage of oxygen, phosphate, calcium, and gold (from sputtering). (b) Graph shows that majority of the surface composition of the scaffold is made up of phosphate and calcium. (c) Mapping image of phosphate. (d) Mapping image of calcium.

Figure 6

Quantitative analysis results of BCP/ZrO₂ scaffold using EDS on SEM. (a) Image of surface area used to analyze the scaffolds for their composition. Table results show the percentage of oxygen, phosphate, calcium, zirconia, and gold (from sputtering). (b) Graph shows that majority of the surface composition of the scaffold is made up of phosphate, calcium, and zirconia.(c) Mapping image of phosphate. (d) Mapping image of calcium. (e) Mapping image of zirconia.

Figure 7

Stress-strain curves of BCP and BCP/ZrO₂ scaffolds. The stress-strain curve was used to calculate the compressive strength of the scaffolds. Results indicate that the compressive strength for BCP/ZrO₂ scaffolds (red) is greater compared to BCP scaffolds (black).

Figure 8

Cell proliferation results on the BCP and BCP/ZrO₂ scaffolds using MG63 cells for 7 days. Statistical difference between OD levels was observed between the BCP and BCP/ZrO₂ scaffolds over the 7 days, excluding day 1. However, cells seeded on BCP scaffolds (black) and BCP/ZrO₂ scaffolds (red) showed similar cell proliferation after 7 days, indicating no adverse effects on cell viability due to the addition of ZrO₂. (*p<0.05 and **p<0.01, compared with a BCP/ZrO₂ scaffold, n=4)

Figure 9

Figure 9a. Fluorescent viability imaging of hMSCs on BCP and BCP/ZrO₂ scaffolds. Cells were labeled with Ethidium Homodimer (green, live) and Calcein AM (red, dead). Top row shows imaged taken at 2.5x, and rows 2–4 show images at 10x. Figure 9b. Quantification of hMSCs viability on BCP scaffolds on Day 21 under static and dynamic conditions. Fluorescence images of cells on scaffolds were counted (n=3), yielding percent viable (green) and percent non-viable cells (red) of total cells counted. Results indicate significantly greater viability of cells compared to non-viable cells in all groups on day 21 (p<0.05).

Figure 10

Osteogenic gene expressions of hMSCs on BCP and BCP/ZrO₂ scaffolds. Groups with the same letters indicate no statistical difference between groups for that time point, with p<0.05. Results indicate significantly greater expression of BMP-2 in BCP/ZrO₂ compared to BCP scaffolds on Day 21, but dynamic culture did not affect BMP-2 gene expression.