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

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.

Role of the Neurologic System in Fracture Healing: An Extensive Review

“…In addition to CGRP, SP has been implicated in aspects of bone healing, including angiogenesis. Models of fracture nonunion have revealed a substantial reduction in the mRNA expression of both CGRP and SP [ 40 ]. Both CGRP and SP are increased in the first 24 h after fracture [ 41 ].…”

The role of toll-like receptors in orchestrating osteogenic differentiation of mesenchymal stromal cells and osteoimmunology

“…This was confirmed by Mei et al, the 10 ng/mL LPS group of m-BMSCs demonstrated the lowest expression of ALP, Col I, and RUNX2, as well as the least osteogenesis. The downregulated expression levels of β-catenin and c-Myc, core proteins of the canonical Wnt signaling pathway were also observed, indicating the positive effects of the Wnt signaling pathway on the proliferation and differentiation of BMSCs ( Mei et al, 2022 ). He and others showed contradictory results.…”

Stromal vascular fraction gel promoted wound healing and peripheral nerve repair in diabetic rats via TLRs/MyD88/NF-κB signaling pathway

“…14 A recent study indicates that TLR4 inhibits the proliferation and differentiation of bone mesenchymal stem cells. 15 However, the effect of SVF-gel on diabetes ulcers and its potential mechanism are not clear. In this study, we constructed a diabetic ulcer rat model to investigate the mechanism of SVF-gel on the diabetic ulcers and peripheral nerves, in order to provide new ideas for the clinical treatment of diabetic ulcers and peripheral nerve regeneration.…”