Factors secreted from the stem cells of human exfoliated deciduous teeth inhibit osteoclastogenesis through the activation of the endogenous antioxidant system

Abstract

Objectives

Systemic administration of conditioned medium (CM) from stem cells derived from human exfoliated deciduous teeth (SHED-CM) in mouse models of rheumatoid arthritis, osteoporosis, and osteoarthritis suppresses excessive osteoclast activity and restores bone integrity. However, the mechanism through which SHED-CM regulates osteoclastogenesis remains largely unknown. In the present study, we examined the anti-osteoclastogenic mechanism of SHED-CM in vitro.

Methods

Bone marrow macrophages and RAW264.7 cells were treated with receptor activator of nuclear factor kappa-Β ligand (RANKL) in the presence of SHED-CM or CM from bone marrow mesenchymal stem cells (BMSC-CM). Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase staining, actin ring formation, and expression of osteoclast-specific markers. RANKL-induced reactive oxygen species (ROS) production was analyzed as a critical mediator of osteoclastogenesis. The activation of endogenous antioxidant gene expression was examined using reverse transcription quantitative PCR. Liquid chromatography with tandem mass spectrometry (LC-MS) was used to identify proteins enriched in SHED-CM, and neutralizing antibodies were used to evaluate their functional roles.

Results

Compared to BMSC-CM, SHED-CM effectively inhibited RANKL-induced early osteoclast differentiation and late maturation. Notably, SHED-CM but not BMSC-CM suppressed RANKL-induced ROS production. SHED-CM increased the expression of genes encoding antioxidant enzymes. The LC-MS analysis identified seven proteins uniquely enriched in SHED-CM that activated the endogenous antioxidant system. Neutralizing antibodies against some of these proteins restore RANKL-induced ROS production and osteoclast differentiation.

Conclusions

SHED-CM inhibited osteoclastogenesis, partially through the activation of multiple antioxidant enzymes in osteoclast precursors, highlighting its potential for treating bone-destructive diseases.