Immobilization of bioactive vascular endothelial growth factor onto Ca-deficient hydroxyapatite-coated Mg by covalent bonding using polydopamine

Abstract

Background: Bone tissue engineering (BTE) is considered a promising technology for repairing bone defects. Mg²⁺ promotes osteogenesis, which makes Mg-based scaffolds popular for research on orthopedic implant materials. Angiogenesis plays an important role in the process of bone tissue repair and regeneration, and it is one of the important problems in BTE urgently needs to be solved.

Methods: Mg was firstly coated with Ca-deficient hydroxyapatite (CDHA) via hydrothermal treatment, and polydopamine (DOPA) was then used as the connecting medium to immobilize vascular endothelial growth factor (VEGF) on the CDHA coating. The physicochemical properties of the coatings were characterized by SEM, EDS, XPS, FTIR and immersion experiment in SBF. The ahesion, proliferation, and angiogenesis potential of the coatings were determined in vitro.

Results: The composite coating significantly improved the corrosion resistance of Mg and prohibited excessively high local alkalinity. VEGF could be firmly immobilized on Mg via polydopamine. The CCK-8, live/dead staining and adhesion test results showed that the VEGF-DOPA-CDHA coating exhibited excellent biocompatibility and could significantly improve the adhesion and proliferation of MC3T3-E1 cells on Mg. Microtubule formation, immunofluorescence and Quantitative Real-Time PCR (qRT-PCR) experiments showed that VEGF immobilized on Mg still possessed bioactivity in promoting the differentiation of rat mesenchymal stem cells into endothelial cells.

Conclusion: In this study, we enabled the angiogenic biological activity of Mg by immobilizing VEGF on Mg. Mg was successfully coated with a functional VEGF-DOPA-CDHA composite coating. The CDHA coating significantly increased the corrosion resistance of Mg and prohibited the negative effect of excessively high local alkalinity on the biological activity of VEGF. As an intermediate layer, the DOPA coating protects Mg, and DOPA provides a binding site for VEGF so that VEGF can be firmly immobilized on Mg and give Mg angiogenic bioactivity during the initial period of implantation.

The translational potential of this article: The treatment of large bone defect is still one of the orthopedic trauma diseases that are difficult to be completely treated in clinic. The development of tissue engineering technology provides a new option for the treatment of large bone defects. The regeneration of blood vessels is of great significance for the repair of bone defects. In this study, VEGF was connected on the surface of degradable magnesium by covalent bonding. Vascular biofunctionalized magnesium scaffolds are expected to regenerate bone tissue with blood transport and be used in the clinical treatment of large bone defects.

Keywords: Angiogenesis; Bone tissue engineering; Magnesium; VEGF.

Fig. 1

Schematic illustration. Mg was coated with CDHA, DOPA-CDHA and VEGF-DOPA-CDHA via a layer-by-layer assembly method.

Fig. 2

Surface morphologies and elemental components of different coatings. (a) CDHA-Mg, (b)DOPA-CDHA-Mg, (c) VEGF-DOPA-CDHA-Mg.

Fig. 3

FTIR (a) and XPS (b) survey spectra of CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg.

Fig. 4

(a) immunofluorescence detection of VEGF on CDHA-Mg, (b) immunofluorescence detection of VEGF on VEGF-DOPA-CDHA-Mg, (c) the amount of VEGF in PBS before and after 3 h immersion as determined by ELISA.

Fig. 5

Surface morphologies of CDHA-Mg (a), DOPA-CDHA-Mg (b) and VEGF-DOPA-CDHA-Mg (c) after immersion in SBF for 14 days; (d) The concentration of released Mg²⁺ from CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg during immerison in PBS for 14 days; (e) pH variations of CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg immersed in the SBF; in vitro kinetics of the VEGF release from VEGF-DOPA-CDHA-Mg.

Fig. 6

a) Measurement of MC3T3-E1 cell proliferation by the CCK-8 assay after 1, 4, and 7 days of incubation on the surfaces of CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg. For each group, n ​= ​3; asterisks (∗) indicate statistical significance, p ​< ​0.05; live/dead staining of MC3T-E1 cells cultured on the surfaces of CDHA-Mg (b), DOPA-CDHA-Mg (c) and VEGF-DOPA-CDHA-Mg (d) at day 7.

Fig. 7

Fluorescence images of actin (red) and nuclear (blue) staining of MC3T3-E1 cells cultured on CDHA-Mg (a), DOPA-CDHA-Mg (b) and VEGF-DOPA-CDHA-Mg (c) after 24 ​h; SEM micrographs of MC3T3-E1 cells cultured on CDHA-Mg (d), DOPA-CDHA-Mg (e) and VEGF-DOPA-CDHA-Mg (f) after 7 days.

Fig. 8

Immunofluorescence staining of CD31 expression (a, c, e) and vWF expression (b, d, f) in rBMSCs cultured on CDHA-Mg (a, b); DOPA-CDHA-Mg (c, d) and VEGF-DOPA-CDHA-Mg (e, f) after 2 weeks.

Fig. 9

In vitro microtubule formation of rBMSCs cultured on CDHA-Mg (a), DOPA-CDHA-Mg (b) and VEGF-DOPA-CDHA-Mg (c) after 2 weeks; Immunofluorescence staining of alpha-smag expression (d, e, f) in rBMSCs cultured on CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg after 2 weeks; qRT-PCR analyses of the CD31 (g) and vWF (h) expression levels in rBMSCs cultured on CDHA-Mg, DOPA-CDHA-Mg and VEGF-DOPA-CDHA-Mg for 7 and 14 days, n ​= ​3; asterisks (∗) indicate statistical significance, p ​< ​0.05.