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. 2020 Apr:238:119828.
doi: 10.1016/j.biomaterials.2020.119828. Epub 2020 Jan 31.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model

Li-Zhen Zheng  1 , Jia-Li Wang  2 , Jian-Kun Xu  1 , Xiao-Tian Zhang  3 , Bao-Yi Liu  4 , Le Huang  1 , Ri Zhang  1 , Hai-Yue Zu  1 , Xuan He  1 , Jie Mi  1 , Qian-Qian Pang  1 , Xin-Luan Wang  5 , Ye-Chun Ruan  3 , De-Wei Zhao  6 , Ling Qin  7
Affiliations

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model

Li-Zhen Zheng et al. Biomaterials. 2020 Apr.

Abstract

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

Keywords: Corticosteroids; Magnesium; Osteonecrosis; Preclinical studies; Vitamin C.

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Conflict of interest statement

Declaration of competing interest All authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Effect of Mg and VC on osteoblast cell line MC3T3-E1. (A) Representative live/dead cell staining images. (B) and (C) MTT cell viability assays. (**p < 0.01, n = 8). (D) ALP activity and calcium nodules staining showed the effect of Mg and VC on osteoblast differentiation affected by MPS. (E) VC and Mg effect on ALP activity and calcium deposition affected by LPS. (F) qPCR results about SP7, Runx2, OPN, OCN and IGF-1 mRNA expression 24 h after LPS/MPS treatment. (G) IF staining showed anti-inflammation effect of Mg on LPS treated MC3T3-E1. (*p < 0.05, **p < 0.01, ****p < 0.0001, n = 4).
Fig. 2
Fig. 2
Effect of Mg and VC on osteoclast cell line. (A) Quantitative of TRAP staining showed differentiated osteoclast number in Mg and VC treated groups, and qPCR showed Cathepsin K mRNA expression in each group. (**p < 0.01, n = 4). (B) Quantitative of TRAP staining showed the inhibiting effect of Mg on differentiated osteoclast number at both LPS and RANKL induced conditions, and qPCR showed the inhibiting effect of Mg on NFATc1 and Cathepsin K mRNA expression at both LPS and RANKL induced conditions. (*p < 0.05, **p < 0.01, n = 4). (C) Pit formation assay showed the inhibiting effect of Mg on the function of osteoclasts at both LPS and RANKL induced conditions. (**p < 0.01, n = 4). (D) IF staining and WB showed the inhibiting effect of Mg on NF-κB p65 nuclear translocation at both LPS and RANKL induced conditions. (*p < 0.05, n = 4).
Fig. 3
Fig. 3
Mg inhibits osteoclast differentiation through regulating Ca signal. (A) Mg blocks LPS-induced intracellular Ca2+ increase in RAW264.7 cells. A1-A2) Representative Fura-2 fluorescence images (A1) and corresponding time-course traces (A2) of intracellular Ca2+ measurement in RAW264.7 cells before (a) and after adding LPS (100 ng/ml, b) and subsequently MgCl2 (10 mM, c) into a normal bath solution. White bar, 30 μm. Pseudo-colors from purple/blue to red indicate Fura-2340/380 nm ratio from low to high. A3-A5) Representative time-course traces of cells pretreated with Ca2+-free (A3), MgCl2 (10 mM, A4), or NMDG-Cl (20 mM, a non-permeable cation control, A5) before the addition of LPS. A6) Summary of LPS-induced intracellular Ca2+ change in RAW264.7 cells under different conditions. (B) Mg blocks RANKL-induced intracellular Ca2+ increase in RAW264.7 cells. B1–B2) Representative Fura-2 fluorescence images (B1) and corresponding time-course traces (B2) of intracellular Ca2+ measurement in RAW264.7 cells before (a) and after adding RANKL (50 ng/ml, b) and subsequently MgCl2 (10 mM, c) into a normal bath solution. White bar, 30 μm. Pseudo-colors from purple/blue to red indicate Fura-2340/380 nm ratio from low to high. B3–B5) Representative time-course traces of cells pretreated with Ca2+-free (B3), MgCl2 (10 mM, B4), or NMDG-Cl (20 mM, a non-permeable cation control, B5) before the addition of RANKL. B6) Summary of RANKL-induced intracellular Ca2+ change in RAW264.7 cells under different conditions. ***p < 0.001 by t-test; n is shown for each group. (C) Illustration of the pathway of Mg in inhibiting LPS/RANKL induced osteoclast differentiation.
Fig. 4
Fig. 4
Effect of Mg and VC on prevention of SAON and suppress the apoptosis of osteocytes. (A) Representative H&E staining images showed ON, indicated by the empty lacunae (black arrow) in trabecular bone or pyknotic nucleus of osteocytes (blue arrow). (B) The incidence of SAON in each group. All the SAON model rats developed ON at 2 weeks after induction. The incidence of SAON decreased at 6 weeks after induction, suggested progressive repair after week 2. VC further significantly decreased the incidence SAON at 6 weeks after induction. (*p < 0.05, **p < 0.01; Chi-square test, n = 8; osteonecrosis+: with osteonecrosis detected by histology; Osteonecrosis-: without osteonecrosis detected by histology) (C) Representative TUNEL staining images showed apoptosis of osteocytes. (D) Apoptosis rate indicated by percentage of TUNEL-positive osteocytes. (*p < 0.05, **p < 0.01; one-way ANOVA; aa: p < 0.01, for Mg factor; bb: p < 0.01, for VC factor, two-way ANOVA; n = 8).
Fig. 5
Fig. 5
Effect of Mg and VC on bone metabolism in vivo. (A) Representative TRAP staining images showed osteoclasts (red arrow). (B) Histomorphometry results showed osteoclast number (N.Oc/B.Pm), osteoblast surface (Ob. S/BS) and bone marrow fat area fraction in each group. (*p < 0.05, **p < 0.01; a: p < 0.05, aa: p < 0.01 for Mg factor; bb: p < 0.01 for VC factor; two-way ANOVA; n = 6). (C) bone formation marker PINP and (D) bone resorption marker CTX (*p < 0.05, **p < 0.01, two-way ANOVA; n = 6). (E) Representative 3D images of micro-CT based trabecular bone of proximal tibia at 6 weeks after SAON induction. (B) Quantitative analysis of the bone mineral density (BMD), bone volume fraction (BV/TV), connective density (Conn. D.), trabecular number (Tb. N), trabecular thickness (Tb. Th) and the structure model index (SMI) in each group. (*p < 0.05, **p < 0.01; a: p < 0.05 for Mg factor; bb: p < 0.01 for VC, two-way ANOVA; n = 8).
Fig. 6
Fig. 6
Ex vivo cell culture. (A) The Bone marrow MSCs of rats at 2 weeks after SAON induction from every group were cultured ex vivo, and then differentiated in osteogenic induction medium. Representative images of Alizarin red staining and qPCR of ALP and COL I A1 showed the potential of osteogenic differentiation of MSCs in each group. (B) The bone marrow macrophages of rats 2 weeks after SAON induction from every group were cultured ex vivo and differentiated in osteoclastic induction medium. Quantitative analysis of TRAP staining images about osteoclast number and size and qPCR of Cathepsin K expression showed the osteoclast differentiation potential of BMMs in each group (*p < 0.05, **p < 0.01, vs. SAON; #: p < 0.05, vs. VC, n = 4).
Fig. 7
Fig. 7
Mg promotes angiogenesis and reduces vessel leakage. (A) Representative 3D images of micro-CT based vessel architecture and CD31 IHC staining images in proximal tibia and distal tibia 2 weeks after SAON induction in each group. Histogram showed the subtotal volume of the small (<100 μm), medium (100–200 μm) and large-sized (>200 μm) blood vessels/perfused microfil in each group in proximal (above) and distal tibia (below) (*p < 0.05, **p < 0.01, n = 4). (B) Effect of Mg on endothelial cell migration. (C) Effect of Mg on endothelial cell tube formation. (*p < 0.05, **p < 0.01, n = 4) (D) Transwell permeability assay showed the effect of Mg on endothelial cell leakage induced by LPS. (E) Co-IF-staining of ZO-1 and NF-κB p65 showed the coincidental effects of Mg on protecting the integrity of endothelial cell monolayer (showed by ZO-1 staining) and reducing the inflammation (showed by NF-κB p65 staining) induced by LPS. (*p < 0.05, **p < 0.01, n = 4).
Fig. 8
Fig. 8
In vivo anti-inflammation, anti-oxidative and anabolic effect and pathways of Mg & VC in preventing SAON. (A) Representative IHC staining images and quantification for TNFα expression in bone marrow. (B) Representative IHC staining images and quantification for 8-Oxo-dG expression in bone marrow. (C) Representative IHC staining images and quantification for IGF-1 expression in bone marrow. (*p < 0.05, **p < 0.01, n = 6).
Scheme 1
Scheme 1
Illustration of the pathway of Mg and VC in attenuating SAON. CS has its anti-anabolic effect: to negative regulate bone formation and increase apoptosis in bone; and negative regulate angiogenesis. LPS induced inflammation, mimic inflammatory diseases, decreases bone formation and increases apoptosis and bone resorption in bone event; and increases vessel permeability and leakage. Both bone matrix degeneration and ischemia induce SAON. CS also induces oxidative stress, which promotes cell death. There is a mutual activation loop among anti-anabolic, inflammation and oxidative stress. CS treatment could induce Mg deficiency. Mg deficiency is associated with inflammation, oxidative stress, and anabolic defect. Mg supplementation can maintain the serum Mg level at physiological range. Mg has anti-inflammatory effect and anabolic effect (e.g. through IGF-1 pathway). VC is an antioxidant and can induce osteogenesis. The combination of Mg and VC exerts synergistic effect for attenuating SAON.
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