Extrusion-based 3D printing of osteoinductive scaffolds with a spongiosa-inspired structure

Abstract

Critical-sized bone defects resulting from trauma, inflammation, and tumor resections are individual in their size and shape. Implants for the treatment of such defects have to consider biomechanical and biomedical factors, as well as the individual conditions within the implantation site. In this context, 3D printing technologies offer new possibilities to design and produce patient-specific implants reflecting the outer shape and internal structure of the replaced bone tissue. The selection or modification of materials used in 3D printing enables the adaption of the implant, by enhancing the osteoinductive or biomechanical properties. In this study, scaffolds with bone spongiosa-inspired structure for extrusion-based 3D printing were generated. The computer aided design process resulted in an up scaled and simplified version of the bone spongiosa. To enhance the osteoinductive properties of the 3D printed construct, polycaprolactone (PCL) was combined with 20% (wt) calcium phosphate nano powder (CaP). The implants were designed in form of a ring structure and revealed an irregular and interconnected porous structure with a calculated porosity of 35.2% and a compression strength within the range of the natural cancellous bone. The implants were assessed in terms of biocompatibility and osteoinductivity using the osteosarcoma cell line MG63 and patient-derived mesenchymal stem cells in selected experiments. Cell growth and differentiation over 14 days were monitored using confocal laser scanning microscopy, scanning electron microscopy, deoxyribonucleic acid (DNA) quantification, gene expression analysis, and quantitative assessment of calcification. MG63 cells and human mesenchymal stem cells (hMSC) adhered to the printed implants and revealed a typical elongated morphology as indicated by microscopy. Using DNA quantification, no differences for PCL or PCL-CaP in the initial adhesion of MG63 cells were observed, while the PCL-based scaffolds favored cell proliferation in the early phases of culture up to 7 days. In contrast, on PCL-CaP, cell proliferation for MG63 cells was not evident, while data from PCR and the levels of calcification, or alkaline phosphatase activity, indicated osteogenic differentiation within the PCL-CaP constructs over time. For hMSC, the highest levels in the total calcium content were observed for the PCL-CaP constructs, thus underlining the osteoinductive properties.

Keywords: 3D printing; PCL; bone implant; calcium phosphate; critical-sized bone defect; extrusion-based printing; osteogenic differentiation; scaffold.

FIGURE 1

(A) 3D model of the implant with a spongiosa-inspired structure. Different views and angles of the spongiosa-inspired 3D model. The 3D model is shaped cylindrically with 10 mm radius and 10 mm height. The pores with different interconnectivities have a size of 2 mm ± 0.2 mm. Scale bar: 10 mm. (B) Few slices through the 3D model in different heights. The slices show the structure inside the 3D model. Scale bar: 10 mm.

FIGURE 2

Mechanical properties of the printed scaffolds consisting of PCL and PCL-CaP. (A) Different views of the printed constructs. Left: Scale bar: 10 mm, Middle/Right: Scale bar: 8 mm. (B) Left: The mean weight of the scaffolds based on PCL and PCL-CaP, n = 129, Middle: Compression strength measured with an increasing force up to 2,500 N and a speed of 2 mm/s. The statistics were performed with Welch’s t-Test, n = 12. Right: Force-distance-curve for both materials of the printed scaffolds. The saturated grey and light grey curves show the mean trend curves of the tested samples, n = 12.

FIGURE 3

Energy-dispersive x-ray spectroscopy for scaffolds consisting of PCL and PCL-CaP. (A) EDX ratio and atomic percentage for carbon (C), oxygen (O), phosphor (P), and calcium (Ca). The EDX analysis was performed for different regions of the scaffolds, n = 2 scaffolds. (B) Element map for P and Ca for a selected area of the scaffolds, as well as the same region as SEM picture.

FIGURE 4

CLSM (A) and SEM (B) for the MG63 cell-seeded scaffolds. The cells were stained with Phalloidin TRITC (Donzelli et al., 2007) and Hoechst 33342 (blue) to visualize the actin skeleton and the nucleus (A), Scale bar: 100 µm. (B) The surface of the cell-free scaffolds (left) and the cell-laden scaffolds after 7 and 14 days (right), Scale bar: 100 µm.

FIGURE 5

CLSM of hMSC-seeded scaffolds. The hMSC grown on top of the printed scaffolds stained with Phalloidin TRITC (Donzelli et al., 2007) and Hoechst 33342 (blue). Scale bar: 100 µm.

FIGURE 6

DNA Quantification for MG63 cell-seeded constructs on different time points. The MG63 cells show proliferation from day 1 to day 7 at the material PCL. The statistical evaluation was determined using ANOVA with posthoc Tukey test, n = 4 from different passages and 2 technical replicates. Statistical significance level: p < 0.05 (*), p < 0.001 (***), and p < 0.0001 (****).

FIGURE 7

Semi-quantitative gene expression for osteogenesis and adhesion related markers of MG63 cells cultured on printed samples. The statistical evaluation was performed using Welch’s t-test, n = 3 independent experiments. Statistical significance level: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

FIGURE 8

The calcification in MG63 cell-seeded scaffolds using Alizarin Red staining. (A) Light microscopy and morphological appearance of calcified areas over culture time. (B) Quantification of calcification in MG63 cell-seeded scaffolds. Left: Total values for calcification from scaffolds without cells and MG63 cell-seeded constructs, Middle: Cell-based calcification (scaffold values subtracted). The statistical evaluation was performed using ANOVA with posthoc Tukey, n = 3, from three different passages with three technical replicates. ALP activity in cell-seeded construct showed decreasing values over the time. The statistical evaluation was performed using ANOVA with posthoc Tukey with n = 6, from six different passages with three technical replicates. Statistical significance level: p < 0.05 (*), p < 0.001 (***), and p < 0.0001 (****).

FIGURE 9

The calcification in MSC seeded scaffolds using Alizarin Red staining. (A) Light microscopy and morphological appearance of calcified areas over culture time for PCL and PCL-CaP. (B) Quantification of calcification in hMSC-seeded scaffolds on day 14 for different donors. Upper graphs: Total values for calcification from scaffolds without cells and hMSC-seeded constructs in average or depicted for individual donors. Lower graphs: Cell-based calcification (scaffold values subtracted) in hMSC-seeded constructs in average or depicted for individual donors. The statistical evaluation was performed using ANOVA with posthoc Tukey with n = 4 individual donors for each group. The samples were measured in technical duplicates.