Gadolinium-doped bioglass scaffolds promote osteogenic differentiation of hBMSC via the Akt/GSK3β pathway and facilitate bone repair in vivo

Abstract

Background: Biomaterial-induced osteogenesis is mainly related to hierarchically porous structures and bioactive components. Rare earth elements are well known to promote osteogenesis and stimulate bone repair; however, the underlying biological effects of gadolinium (Gd) element on bone regeneration are not yet known.

Methods: In this study, we successfully fabricated gadolinium-doped bioglass (Gd-BG) scaffolds by combining hollow mesoporous Gd-BG microspheres with chitosan and evaluated in vitro effects and underlying mechanisms with Cell Counting Kit-8, scanning electron microscopy, alkaline phosphatase, Alizarin red staining, and polymerase chain reaction. Cranial defect model of rats was constructed to evaluate their in vivo effects.

Results: The results indicated that Gd-BG scaffolds could promote the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Mechanistically, the Akt/GSK3β signaling pathway was activated by the Gd-BG scaffolds. The enhancing effect of Gd-BG scaffolds on the osteogenic differentiation of hBMSCs was inhibited by the addition of LY294002, an inhibitor of Akt. Moreover, the in vivo cranial defect model of rats indicated that the Gd-BG scaffolds could effectively promote bone regeneration.

Conclusion: Both in vitro and in vivo results suggested that Gd-BG scaffolds have promising applications in bone tissue engineering.

Keywords: Akt/GSK3β pathway; bone regeneration; bone scaffold; gadolinium.

Figure 1

Structural property of Gd-BG scaffold. Notes: (A) SEM image and (B) TEM image of Gd-BGS microspheres; (C) nitrogen adsorption–desorption isotherm, and (D) BJH pore size distribution curve of mesoporous Gd-BGS microspheres. (E) The X-ray diffraction patterns of samples: (I) Gd-BG microspheres and (II) Gd-BG scaffolds. (F) The Fourier transform infrared spectra of samples: (I) Gd-BG microspheres and (II) Gd-BG scaffolds. Abbreviations: BJH, Barrett–Joyner–Halenda; Gd-BG, gadolinium-doped bioglass; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 2

Morphology and chemical composition. Notes: Characterization of Gd-BG scaffolds: (A) low-resolution SEM image; (B) high-resolution SEM image; (C) Ca element distribution map; (D) Si element distribution map; (E) Gd element distribution map; and (F) energy-dispersive X-ray spectrometry pattern. Abbreviations: Gd-BG, gadolinium-doped bioglass; SEM, scanning electron microscopy.

Figure 3

Cell proliferation and adhesion on BG and Gd-BG scaffolds. Notes: (A) Viability of hBMSCs at different concentrations of BG and Gd-BG scaffolds. SEM images showing the attachment of hBMSCs on (B) BG and (C) Gd1/3-BG. Data are presented as mean ± SD from a representative of three separate experiments performed in quadruplicate (*P<0.05). Abbreviations: Gd-BG, gadolinium-doped bioglass; hBMSC, human bone marrow-derived mesenchymal stem cell; SEM, scanning electron microscopy.

Figure 4

Osteogenic differentiation of hBMSCs in BG and Gd-BG dissolution. Notes: (A) Effects of BG and Gd-BG dissolution on the ALP activity of hBMSCs after culturing for 7 days. (B) Osteogenic gene expression of BSP and OCN was detected after hBMSCs were treated with BG and Gd-BG dissolution for 3 days. (C) ALP staining showed osteogenic differentiation of hBMSCs treated with BG and Gd-BG dissolution for 14 days (40×). (D) Alizarin red staining showing the mineralization of hBMSCs treated with BG and Gd-BG dissolution for 21 days. Data are presented as mean ± SD from a representative of three separate experiments performed in quadruplicate (**P<0.01). Abbreviations: Gd-BG, gadolinium-doped bioglass; hBMSC, human bone marrow-derived mesenchymal stem cell.

Figure 5

Activation of Akt/GSK3β pathway by Gd-BG. Notes: (A) Western blotting analysis of p-Akt, total-Akt, p-GSK3β, and total-GSK3β for hBMSCs after treatment with different concentrations of Gd-BG dissolution for 3 hours. GAPDH was used as the loading control. (B) Western blotting analysis of β-catenin for hBMSCs after treatment with different concentrations of Gd-BG dissolution for 24 hours. (C) Western blotting analysis of p-Akt, total-Akt, p-GSK3β, and total-GSK3β for hBMSCs after treatment with BG, Gd1/3-BG dissolution, and Gd1/3-BG dissolution+ LY294002 for 3 hours. (D) Western blotting analysis of β-catenin for hBMSCs after treatment with BG, Gd1/3-BG dissolution, and Gd1/3-BG dissolution+ LY294002 for 24 hours. (E, F) Analysis of protein expression in hBMSCs. (G) Effects of BG, Gd1/3-BG dissolution, and Gd1/3-BG dissolution+ LY294002 on the ALP activity of hBMSCs after culturing for 7 days. (H) ALP staining showed the osteogenic differentiation of hBMSCs after treatment with BG, Gd1/3-BG dissolution, and Gd1/3-BG dissolution+ LY294002 for 14 days (40×). (I) Alizarin red staining showed the mineralization of hBMSCs after treatment with BG, Gd1/3-BG dissolution, and Gd1/3-BG dissolution+ LY294002 for 21 days. Data are presented as mean ± SD from a representative of three separate experiments performed in quadruplicate (**P<0.01). Abbreviations: ARS, Alizarin red staining; Gd-BG, gadolinium-doped bioglass; hBMSC, human bone marrow-derived mesenchymal stem cell.

Figure 6

Micro-CT of rat cranial defects implanted with BG and Gd1/3-BG scaffolds at 8 weeks after implantation. Notes: (A) The images of reconstruction of micro-CT for the bone regeneration of the defect area at week 8. (B, C) BMD and BV/TV in the defects implanted with the Gd1/3-BG and BG scaffolds. Data are presented as mean ± SD from a representative of ten separate experiments (**P<0.01). (D) New bone formation and mineralization measured histomorphometrically by using fluorochrome labeling analysis in rat cranial defects implanted with Gd1/3-BG and BG scaffolds. White arrow indicates the new bone formation. Alizarin red (red) and calcein (green) were intraperitoneally injected at weeks 4 and 6, respectively. Abbreviations: BMD, bone mineral density; BV/TV, bone volume to total bone volume; CT, computed tomography; Gd-BG, gadolinium-doped bioglass.

Figure 7

Histological analysis of cranial bone defects. Notes: (A) H&E, (B) VG, and (C) immunohistochemical staining of OCN in the defects implanted with Gd-BG and BG scaffolds at 8 weeks after implantation. Black stars indicate new bone formation. Green star indicates new collagen formation, and red stars indicate positive staining of OCN. Abbreviation: Gd-BG, gadolinium-doped bioglass; VG, Van Gieson’s.

Figure 8

Gd and Akt/GSK3β signaling pathway, where Gd could activate Akt. Notes: Phosphorylated Akt could prevent GSK3β from forming a complex with β-catenin, resulting in the accumulation of β-catenin. Thus, it could promote osteogenic gene expression and enhance osteogenic differentiation. Abbreviation: Gd, gadolinium.