CXCL13 promotes the effect of bone marrow mesenchymal stem cells (MSCs) on tendon-bone healing in rats and in C3HIOT1/2 cells

Abstract

Objectives: Mesenchymal stem cells (MSCs) are potential effective therapy for tissue repair and bone regeneration. In present study, the effects of CXC chemokine ligand-13 (CXCL13) were evaluated on tendon-bone healing of rats.

Methods: Tendon bone healing of the rat model was established and biomechanical testing was performed at 2, 4, 8 weeks after surgery. Murine mesenchymal cell line (C3HIOT1/2 cells) was cultured. The expression of miRNA-23a was detected by real-time PCR. The protein expression of ERK1/2, JNK and p38 was detected by western blotting. MiR-23a mimic and inhibitor were used to overexpress or silence the expression of miR-23a.

Results: MSCs significantly elevated the levels of ultimate load to failure, stiffness and stress in specimens of rats, the effects of which were enhanced by CXCL13. The expression of miR-23a was down-regulated and the protein of ERK1/2 level was up-regulated by CXCL13 treatment in both in vivo and in vitro experiments. ERK1/2 expression was elevated by overexpression of miR-23a and reduced by miR-23a inhibitor.

Conclusions: These findings revealed that CXCL13 promoted the tendon-bone healing in rats with MSCs treatment, and implied that the activation of ERK1/2 via miR-23a was involved in the process of MSCs treated bone regeneration.

Figure 1

The potential of mesenchymal stem cells (MSCs) differentiating into osteoblasts and adipocytes. The Alizarin red staining of calcified nodules and Oil red staining were respectively applied to identify osteoblast differentiation ability (A) and adipogenic differentiation ability (B) of rat MSCs.

Figure 2

Biomechanical assessment of the tendon at the insertion site: (A) Ultimate load to failure; (B) Stiffness; (C) Stress. Data were shown as mean ± SD. * vs. Group1 (Injection with medium), p < 0.05; # vs. Group2 (Injection with MSCs), p < 0.05; and & vs. Group 3 (Injection with CXC chemokine ligand-13 (CXCL13) treated MSCs), p < 0.05. Group 4 is the specimens injected with CXCL13 treated MSCs accompanied by infection with lentiviruses of silencing CXCR5.

Figure 3

The effects of CXCL13 on miR-23a and mitogen-activated protein kinase (MAPK) molecules expression in vivo. (A) The miR-23a expression; (B) The protein expression of ERK1/2, JNK and p38. Data were shown as mean ± SD. * vs. Group1 (Injection with medium), p < 0.05; # vs. Group 2 (Injection with MSCs), p < 0.05; and & vs. Group3 (Injection with CXCL13 treated MSCs), p < 0.05. Group 4 is the specimens injected with CXCL13 treated MSCs accompanied by infection with lentiviruses of silencing CXCR5.

Figure 4

The effects of CXCL13 on miR-23a and MAPK molecules expression in vitro. (A) The miR-23a expression; (B) The protein expression of ERK1/2. Data were shown as mean ± SD; t-test of independent sample was used to analyze the difference between two groups; * considered as significant difference.

Figure 5

The effect of miR-23a on Extracellular signal-Regulated Kinase (ERK)1/2 expression in vitro. The relative miR-23a expression was quantified by real-time PCR (A,C); The p-ERK1/2 and ERK1/2 expression were detected by western blotting (B,D); Data were shown as mean ± SD; t-test of independent sample was used to analyze the difference between two groups; * considered as significant difference.