Gold nanoparticles-loaded hydroxyapatite composites guide osteogenic differentiation of human mesenchymal stem cells through Wnt/β-catenin signaling pathway

Abstract

Background: Precise control and induction of the differentiation of stem cells to osteoblasts by artificial biomaterials are a promising strategy for rapid bone regeneration and reconstruction.

Purpose: In this study, gold nanoparticles (AuNPs)-loaded hydroxyapatite (HA-Au) nanocomposites were designed to guide the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) through the synergistic effects of both AuNPs and HA.

Materials and methods: The HA-Au nanoparticles were synthesized and characterized by several analytical techniques. Cell viability and proliferation of hMSCs were characterized by CCK-8 test. Cellular uptake of nanoparticles was observed by transmission electron microscope. For the evaluation of osteogenic differentiation, alkaline phosphatase (ALP) activity and staining, Alizarin red staining, and a quantitative real-time polymerase chain reaction (RT-PCR) analysis were performed. In order to examine specific signaling pathways, RT-PCR and Western blotting assay were performed.

Results: The results confirmed the successful synthesis of HA-Au nanocomposites. The HA-Au nanoparticles showed good cytocompatibility and internalized into hMSCs at the studied concentrations. The increased level of ALP production, deposition of calcium mineralization, as well as the expression of typical osteogenic genes, indicated the enhancement of osteogenic differentiation of hMSCs. Moreover, the incorporation of Au could activate the Wnt/β-catenin signaling pathway, which seemed to be the molecular mechanism underlying the osteoinductive capability of HA-Au nanoparticles.

Conclusion: The HA-Au nanoparticles exerted a synergistic effect on accelerating osteogenic differentiation of hMSCs, suggesting they may be potential candidates for bone repair and regeneration.

Keywords: bone regeneration; gold nanoparticles; hydroxyapatite; nanocomposites; osteoblast differentiation.

Scheme 1

Schematical illustration showing the possible molecular mechanism for enhanced osteogenesis by HA-Au nanoparticles by activation of Wnt/β-catenin signaling pathway as well as internalization into the hMSCs. Abbreviations: HA-Au, gold nanoparticles-loaded hydroxyapatite; hMSCs, human bone marrow-derived mesenchymal stem cells.

Figure 1

TEM images of (A) HA, (B) HA-Au1, and (C) HA-Au2 (scale bar, 50 nm). (D) XRD, (E) FT-IR, (F) XPS analysis of HA, HA-Au1, and HA-Au2. (G) The high-resolution spectra of Au4f for HA-Au1 and HA-Au. Abbreviations: TEM, transmission electron microscope; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite; XRD, X-ray powder diffractometry; FT-IR, fourier-transform infrared spectroscopy; XPS, X-ray photoelectron spectroscopy.

Figure 2

(A) Cell viability of hMSCs incubated with PBS (control), HA or HA-Au in growth medium at concentrations of 10, 50, 100, and 200 μg/mL for 2 days. (B) Cell proliferation of hMSCs incubated with PBS (control), HA or HA-Au in osteogenic induction medium at the concentration of 100 μg/mL for 3 and 7 days (*p<0.05, comparison between control group and other groups). Abbreviations: hMSCs, human bone marrow-derived mesenchymal stem cells; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite.

Figure 3

TEM images of HA (A, C) and HA-Au (B, D)-internalized hMSCs show that the particles can be uptaken by cells (black dots in white boxed areas in A and B (scale bar, 1 μm), with corresponding amplified images of C and D (scale bar, 500 nm). Abbreviations: TEM, transmission electron microscope; hMSCs, human bone marrow-derived mesenchymal stem cells; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite.

Figure 4

ALP staining (A) and ALP activity assay (B) of hMSCs after incubation with PBS (control), HA or HA-Au in osteogenic induction medium at the concentration of 100 μg/mL for 3, 7, and 14 days (scale bar: 100 μm. *p<0.05, **p<0.01, comparison between control group and other groups. ⁺p<0.05, ⁺⁺p<0.01, comparison between HA group and HA-Au group). Abbreviations: ALP, alkaline phosphatase; hMSCs, human bone marrow-derived mesenchymal stem cells; PBS, phosphate buffer saline; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite.

Figure 5

ARS staining (A) and calcium deposition assay (B) of hMSCs after incubation with PBS (control), HA or HA-Au in osteogenic induction medium at the concentration of 100 μg/mL for 14 and 21 days. (scale bar: 100 μm. *p<0.05, **p<0.01, comparison between control group and other groups. ⁺p<0.05, ⁺⁺p<0.01, comparison between HA group and HA-Au group). Abbreviations: ARS, Alizarin Red S; hMSCs, human bone marrow-derived mesenchymal stem cells; PBS, phosphate buffer saline; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite.

Figure 6

The expression of osteogenic differentiation specific genes in hMSCs after incubation with PBS (control), HA or HA-Au in osteogenic induction medium at the concentration of 100 μg/mL for 3 and 7 days: ALP (A), Runx2 (B), COL1 (C), BSP (D), OCN (E) and, OPN (F). (*p<0.05, **p<0.01, comparison between control group and other groups. ⁺p<0.05, ⁺⁺p<0.01, comparison between HA group and HA-Au group). Abbreviations: hMSCs, human bone marrow-derived mesenchymal stem cells; PBS, phosphate buffer saline; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; COL1, collagen typeⅠ; BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin.

Figure 7

Western blot and RT-PCR results showed that HA-Au increased the expression of β-catenin and β-catenin target gene axin-2 in hMSCs (A–D). Meanwhile, β-catenin and axin-2 expression levels were significantly reduced when ICG-001 was added (E–H). (*p<0.05, **p<0.01, comparison between control group and other groups). Abbreviations: RT-PCR, real-time polymerase chain reaction; hMSCs, human bone marrow-derived mesenchymal stem cells; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite.

Figure 8

Wnt/β-catenin inhibitor reversed effects of HA-Au on osteogenic differentiation of hMSCs. hMSCs osteogenic differentiation was assessed by ALP staining (A, B) and ARS staining (C, D) after culture for 7 and 21 days, respectively. The mRNA levels of Runx2 (E), OCN (F) and OPN (G) were determined by RT-PCR. (*p<0.05, **p<0.01, comparison between the control group and other groups). Abbreviations: hMSCs, human bone marrow-derived mesenchymal stem cells; HA, hydroxyapatite; HA-Au, gold nanoparticles-loaded hydroxyapatite; ALP, alkaline phosphatase; ARS, Alizarin Red S; Runx2, runt-related transcription factor 2; OCN, osteocalcin; OPN, osteopontin; RT-PCR, real-time polymerase chain reaction.