The effect of TLR-4 on the proliferation and differentiation of bone mesenchymal stem cells and its relationship with the Wnt signal transduction pathway during bone nonunion

Gang Mei; Yan Zhao; Zhenlv Zou; Yongming Liu; Xin Jiang; Yafei Xu; Wende Xiao

doi:10.21037/atm-22-1261

The effect of TLR-4 on the proliferation and differentiation of bone mesenchymal stem cells and its relationship with the Wnt signal transduction pathway during bone nonunion

Ann Transl Med. 2022 Apr;10(8):465. doi: 10.21037/atm-22-1261.

Authors

Gang Mei^#¹, Yan Zhao^#², Zhenlv Zou¹, Yongming Liu¹, Xin Jiang³, Yafei Xu¹, Wende Xiao³

Affiliations

¹ Department of Orthopedics, The Seventh Affiliated Hospital, Southern Medical University, Foshan, China.
² Department of Nephrology, The Sixth People's Hospital of Nanhai District, Foshan, China.
³ Department of Orthopedics, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, China.

^# Contributed equally.

Abstract

Background: Bone nonunion is a special fracture complication that occurs in about 5% to 10% of cases. This type of fracture is difficult to heal, and causes great pain to patients and affects their quality of life. The mechanism of bone nonunion is not clear. In our study, we investigated the influence of Toll-like receptor (TLR)-3, TLR-4, and Wnt signaling pathways on the occurrence of bone nonunion.

Methods: Firstly, we established a Sprague Dawley (SD) rat model of femoral nonunion, and detected the expression levels of TLR-3, TLR-4, β-catenin, nemo-like kinase (NLK), c-Jun N-terminal kinase (JNK), and other proteins during model construction. For in vitro experiments, primary cultured bone mesenchymal stem cells (BMSCs) were divided into 4 groups: lipopolysaccharide (LPS, agonist of TLR-4) group, LPS + CLI095 (inhibitor of TLR-4) group, control group, and LPS + substance P (SP) group. The expression of β-catenin, NLK, JNK, and ALP and the osteogenic differentiation ability of cells were detected during culture.

Results: X-ray and hematoxylin and eosin (HE) staining results confirmed the successful modeling of bone nonunion. During the formation of the bone nonunion model, the expression of TLR-4 showed an upward trend. In vitro experiment results showed that inhibition of TLR-4 expression could enhance the proliferation and differentiation ability of BMSCs. The expression of β-catenin, the core protein of the canonical Wnt signaling pathway, increased rapidly in the first 2 weeks of bone nonunion construction, and decreased after 2 weeks. Non-canonical Wnt signaling pathway proteins NLK and JNK had no change in the first 2 weeks, and showed an upward trend after 2 weeks. In vitro experiment results showed that the expression of β-catenin was dominant in BMSCs with strong proliferation and differentiation ability, while the expression of NLK and JNK was dominant in BMSCs with weak proliferation and differentiation ability. These results suggest that the Wnt signaling pathway may regulate the occurrence of bone nonunion.

Conclusions: TLR-4 inhibits the proliferation and differentiation of BMSCs, and the transformation of the canonical Wnt signaling pathway to the non-canonical Wnt signaling pathway may lead to bone nonunion. Our study may provide new insights into the treatment of bone nonunion.

Keywords: Bone nonunion; Toll-like receptor-4 (TLR-4); Wnt signaling pathway; bone mesenchymal stem cells (BMSCs).