Int J Stem Cells.  2022 May;15(2):195-202. 10.15283/ijsc21120.

Human Umbilical Cord Mesenchymal Stem Cells Improve the Necrosis and Osteocyte Apoptosis in Glucocorticoid-Induced Osteonecrosis of the Femoral Head Model through Reducing the Macrophage Polarization

• Tian G
• Liu C
• Gong Q
• Yu Z
• Wang H
• Zhang D
• Cong H

• KMID: 2529834
• DOI: http://doi.org/10.15283/ijsc21120

Abstract

Background and Objectives
Apoptosis is an outstanding determinant of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been demonstrated to be associated with apoptosis in diseases models. However, the role of hUC-MSCs in GC-induced ONFH via regulating apoptosis still needs further study.
Methods and Results
In the present study, a GC-induced ONFH model was built in vivo through a consecutive injection with lipopolysaccharide (LPS) and methylprednisolone. The necrosis and apoptosis of the femoral head was evaluated by histological and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay. The level of collagen and TRAP positive cells were determined by Masson and TRAP staining, respectively. M1 macrophage polarization was assessed using immunofluorescence assay. The level of proinflammatory cytokines including tumor necrosis factor (TNF)‐α, Interleukin (IL)‐1β and IL-6 of femoral head was determined by enzyme-linked immunosorbent assay (ELISA) kits. The protein expression of AKT, mTOR, p-AKT and p-mTOR was detected using western blot assay. The results showed that hUC-MSCs treatment prominently promoted the GC-induced the decrease of the collagen level and the increase of TRAP positive cells. Besides, hUC-MSCs treatment decreased necrosis and apoptosis, macrophage polarization, the level of TNF‐α, IL‐1β and IL-6, the protein expression of p-AKT and p-mTOR, and the radio of p-AKT to AKT and p-mTOR to mTOR of femoral head in vivo.
Conclusions
Therefore, the present study revealed that hUC-MSCs improved the necrosis and osteocyte apoptosis in GC-induced ONFH model through reducing the macrophage polarization, which was associated with the inhibition of AKT/mTOR signaling pathway.

Keyword

Osteonecrosis of the femoral head (ONFH); Glucocorticoid; Human umbilical cord mesenchymal stem cells; Apoptosis; Macrophage polarization

Figure

• 

  Fig. 1 hUC-MSCs mitigated osteonecrosis and apoptosis in a GC-induced ONFH rat model. (A) Histological analysis was determined by H&E staining (magnification, ×100 or ×400). Scale bar: 50 μm. (B) The apoptosis rate of femoral head tissues was determined by TUNEL (magnification, ×400). Scale bar: 20 μm. (C) The collagen level of femoral head was determined by Masson staining (magnification, ×400). Scale bar: 20 μm. (D) The Number of osteoclasts was detected by TRAP staining (magnification, ×400). Scale bar: 20 μm. The means±SD of six independent samples were shown. *p<0.05 and **p<0.01, compared with control group; #p<0.05 and ##p<0.01, compared with model group.

• 

  Fig. 2 hUC-MSCs reduced M1 polarization in femoral heads. (A) The CD86 and F4/80 of macrophages were stained using immunofluorescence assay (magnification, x400). Scale bar: 20 μm. (B) The CD206 and F4/80 of macrophages were stained using immunofluorescence assay (magnification, x400). Scale bar: 20 μm. The means±SD of six independent samples were shown. *p<0.05 and **p< 0.01, compared with control group; ##p<0.01, compared with model group.

• 

  Fig. 3 hUC-MSCs reduced the level of proinflammatory cytokines in femoral heads. The serum level of TNF‐α (A), IL‐1β (B) and IL-6 (C) was determined by ELISA kits. The means±SD of six independent samples were shown. **p<0.01, compared with control group; ##p<0.01, compared with model group.

• 

  Fig. 4 hUC-MSCs repressed the activation of AKT/mTOR signaling pathway. The protein expression of AKT, mTOR, p-AKT and p-mTOR was detected using western blot. The data were expressed after being normalized to β-actin. The means±SD of six independent samples were shown. *p<0.05 and **p<0.01, compared with control group; ##p<0.01, compared with model group.

Reference

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