Characterization and osteogenic evaluation of mesoporous magnesium-calcium silicate/polycaprolactone/polybutylene succinate composite scaffolds fabricated by rapid prototyping

Abstract

The properties of scaffolds for bone tissue engineering, including their biocompatibility, highly interconnected porosity, and mechanical integrity, are critical for promoting cell adhesion, proliferation, and osteoinduction. We used various physical and biological assays to obtain in vitro confirmation that the proposed composite scaffolds are potentially suitable for applications to bone tissue engineering. The proposed new composite scaffolds, which we fabricated by a rapid prototyping technique, were composed of mesoporous magnesium-calcium silicate (m_MCS), polycaprolactone (PCL), and polybutylene succinate (PBSu). We systematically evaluated the characteristics of the composite scaffolds, such as the hydrophilicity and bioactivity. We also investigated the proliferation and osteogenic differentiation of human mesenchymal stem cells (MSCs) scaffolded on the m_MCS/PCL/PBSu composite. Our results showed that, compared to the m_MCS/PCL scaffold, the m_MCS/PCL/PBSu scaffold has improved water absorption, in vitro degradability, biocompatibility, and bioactivity in simulated body fluid, while its mechanical strength is reduced. Moreover, the results of the cytotoxicity tests specified in ISO 10993-12 and ISO 10993-5 clearly indicate that the m_MCS/PCL scaffold is not toxic to cells. In addition, we obtained significant increases in initial cell attachment and improvements to the osteogenic MSC differentiation by replacing the m_MCS/PCL scaffold with the m_MCS/PCL/PBSu scaffold. Our results indicate that the m_MCS/PCL/PBSu scaffold achieves enhanced bioactivity, degradability, cytocompatibility, and osteogenesis. As such, this scaffold is a potentially promising candidate for use in stem cell-based bone tissue engineering.

Fig. 1. Photographs (A) and SEM images (B) of m_MCS/PCL and m_MCS/PCL/PBSu scaffolds.

Fig. 2. XRD patterns of m_MCS (A), PCL, PBSu (B), m_MCS/PCL composite, and m_MCS/PCL/PBSu composite (C).

Fig. 3. Contact angle (A) and compressive modulus (B) of each scaffold (n = 5, *p < 0.05).

Fig. 4. Water absorptivity (A), weight loss (B) and pH level (C) of the solution for m_MCS/PCL and m_MCS/PCL/PBSu scaffolds after the samples had been immersed in DPBS for different times (1, 3, 4, 14, 21, and 28 days).

Fig. 5. Variation in the concentrations of Ca, P (A), Mg, Si (B) ions in SBF solution after immersing the m_MCS/PCL and m_MCS/PCL/PBSu scaffolds for different length of time.

Fig. 6. Cytotoxicity evaluation of composite scaffold with L929 mouse fibroblast-like cells in comparison with the negative (DMEM alone and PCL extracts) and positive (diluted phenol) controls after 24 h (n = 6, *p < 0.05).

Fig. 7. SEM images (A) and fluorescence microscope images (B) of MSCs cultured on the two types of scaffolds from day 7. The nuclei are colored blue (DAPI) and the F-actin is colored red (rhodamine phalloidin), (×100, scale bar = (A) 500 μm and (B) 100 μm, respectively).

Fig. 8. Live-dead cell staining images of MSCs cultured on each scaffold on day 7. Living cells were detected as green fluorescence and dead cells were detected as red fluorescence (×100, scale bar = 100 μm).

Fig. 9. DNA content reflecting the proliferation of MSCs cultured in osteogenic media on the two types of scaffolds for up to 21 days (n = 6, *p < 0.05).

Fig. 10. Normalized ALP activity of MSCs on each scaffold for up to 21 days (n = 6, *p < 0.05).

Fig. 11. Expression levels of the osteogenic-markers, such as RUNX2, BSP, and OPN, in MSCs cultured on each scaffold on day 21.