Biofunctional magnesium-coated Ti6Al4V scaffolds promote autophagy-dependent apoptosis in osteosarcoma by activating the AMPK/mTOR/ULK1 signaling pathway

Abstract

The recurrence of osteosarcoma (OS) after reconstruction using Ti6Al4V prostheses remains a major problem in the surgical treatment of OS. Modification of the surfaces of Ti6Al4V prostheses with antitumor functions is an important strategy for improving therapeutic outcomes. Magnesium (Mg) coating has been shown to be multifunctional: it exhibits osteogenic and angiogenic properties and the potential to inhibit OS. In this study, we determined the proper concentration of released Mg²⁺ with respect to OS inhibition and biosafety and evaluated the anti-OS effects of Mg-coated Ti6Al4V scaffolds. We found that the release of Mg²⁺ during short-term and long-term degradation could significantly inhibit the proliferation and migration of HOS and 143B cells. Increased cell apoptosis and excessive autophagy were also observed, and further evidence of AMPK/mTOR/ULK1 signaling pathway activation was obtained both in vitro and in vivo, which suggested that the biofunctional scaffolds induce OS inhibition. Our study demonstrates the ability of an Mg coating to inhibit OS and may contribute to the further application of Mg-coated Ti6Al4V prostheses.

Keywords: Antitumour; Apoptosis; Autophagy; Coating; Magnesium.

Graphical abstract

Fig. 1

Effects of Mg²⁺release on the viability and apoptosis of OS cells. (A) Mg²⁺ release during the biodegradation of Mg metal. (B) Viabilities of HOS and 143B cells cultured in conditioned medium containing different concentrations of Mg²⁺ ions. (C) Apoptosis rates of HOS and 143B cells were analyzed by flow cytometry following Annexin V-PE/PI staining. n = 3; ∗p < 0.05; ∗∗p < 0.01. (D) TUNEL staining of HOS and 143B cells. TUNEL-positive cells are stained green.

Fig. 2

Progressive Mg²⁺release during surface degradation of the Mg-coated Ti6Al4V scaffolds. (A) Surface morphology was examined by SEM (3000x), and the elemental composition of the Mg-coated Ti6Al4V scaffolds before degradation was analyzed by EDS. (B) The different surface morphologies of Mg-coated Ti6Al4V scaffolds during immersion were examined by SEM. (C) Mg²⁺ release and variations in pH after immersion of the Mg-coated Ti6Al4V scaffolds.

Fig. 3

Effects of the Mg-coated Ti6Al4V scaffolds on the proliferation, migration, and invasion of 143B and HOS cells. (A) Schematic diagram showing OS cell coculture with the scaffolds. (B) CCK-8 assay analysis of 143B and HOS cell proliferation after 0, 24, 48, 72, and 96 h. (C) Live/dead viabilities of 143B and HOS cells cocultured for 48 h (live cells are stained green, and dead cells are stained red). (D) Images and (E) colony numbers were obtained from the plate cloning assay after 14 days. n = 3; ∗∗p < 0.01. (F) Images of wound healing and (G) invasion assays with 143B and HOS cells cocultured in the different groups. (H) The rates of wound repair and (I) numbers of migrated cells were analyzed. n = 3; ∗p < 0.05; ∗∗p < 0.01.

Fig. 4

The Mg-coated Ti6Al4V scaffolds increased the apoptosis of 143B and HOS cells. (A) Results from the flow cytometric analysis of cell apoptosis following Annexin V-PE/PI staining. (B) The proportions of apoptotic cells were determined. n = 3; ∗∗p < 0.01. (C) Caspase-3 activity in 143B and HOS cells was detected after 48 h n = 3; ∗∗p < 0.01. (D) Western blot analysis of Bcl-2, PARP, and cleaved PARP levels in 143B and HOS cells after 48 h. (E) The relative expression levels were examined by greyscale analysis. n = 3; ∗p < 0.05; ∗∗p < 0.01.

Fig. 5

The Mg-coated Ti6Al4V scaffolds induced an increased level of autophagy in apoptotic 143B and HOS cells. (A) Fluorescent inverted microscopy analysis of LC3 puncta in 143B and HOS cells cocultured for 48 h (red; the cells were counterstained with DAPI to show nuclei in blue). (B) Transmission electron microscopy of autophagosomes in 143B and HOS cells. (C) Fluorescent inverted microscopy of GFP-mCherry-LC3 adenovirus-transfected 143B and HOS cells after 48 h. (D) The relative LC3B and p62 expression levels were detected by Western blot analysis and determined by greyscale analysis. n = 3; ∗p < 0.05; ∗∗p < 0.01.

Fig. 6

The inhibition of autophagy alleviated 143B and HOS cell apoptosis induced by the Mg-coated Ti6Al4V scaffolds. (A) 143B and HOS cells were cocultured with the Mg-coated Ti6Al4V scaffolds (MgB) in the presence or absence of 10 mM 3-MA or 20 μM CQ; pM and inhibitor groups without scaffolds were established as controls. LC3 puncta formation was captured by fluorescence inverted microscopy. (B) TEM images of autophagosomes in 143B and HOS cells. (C) Autophagic flux detection in 143B and HOS cells. (D) Flow cytometry analysis of 143B and HOS cell apoptosis after coculture. n = 3; ∗p < 0.05; ∗∗p < 0.01.

Fig. 7

The Mg-coated Ti6Al4V scaffolds induced autophagy-dependent apoptosis in 143B and HOS cells through the AMPK/mTOR/ULK1 signaling pathway. (A) Western blot analysis of phosphorylated AMPK, mTOR, and ULK1 in response to different treatments. (B) Western blot analysis of phosphorylated mTOR and ULK1 in the MgB group after treatment with compound C. The relative phosphorylated protein expression level was quantified. n = 3; ∗∗p < 0.01. (C) LC3 puncta formation in the MgB group with or without AMPK inhibition.

Fig. 8

Anti-OS effects of the Mg-coated Ti6Al4V scaffolds in vivo. (A) Tumor growth in tumor-bearing mice treated with different extracts. (B) Images and calculated volumes of harvested tumors after 30 days. n = 3; ∗∗p < 0.01. (C) TUNEL staining (apoptotic cells shown in red) and histological IF staining of Bcl-2, p62, LC3, p-AMPK, p-mTOR, and p-ULK1 in tumor sections (red indicates positive expression; the nuclei were stained blue by DAPI). (D) HE staining of tumor tissues.