Objective: Low-molecular-weight hyaluronic acid (LMW-HA) enhances osteoclast differentiation in vitro. However, whether LMW-HA promotes bone resorption in vivo remains unclear. This study aimed to identify the effect of LMW-HA on osteoclast formation and bone resorption in vivo and explore the underlying molecular mechanism.

Methods: Phosphate-buffered saline (PBS), high-dose lipopolysaccharide (LPS), low-dose LPS, low-dose LPS + LMW-HA, and LMW-HA were subcutaneously administered into the calvariae in mice. Micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) staining, and enzyme-linked immunosorbent assay (ELISA) for C-telopeptide of type I collagen (CTX-I) and TRAP-5b were conducted to analyze osteoclast formation and bone destruction. Serum levels of inflammatory cytokines were measured using ELISA. Real-time quantitative reverse transcription PCR (Polymerase Chain Reaction) was performed to evaluate cathepsin K (CTSK), matrix metallopeptidase 9 (MMP-9), TRAP, toll-like receptor-4 (TLR-4), interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) in peritoneal murine macrophages (PMMs), receptor activator of nuclear factor kappa-B (RANKL), and bone marrow stromal cells (BMSCs). Immunohistochemistry was used to evaluate the infiltration of F4/80 positive cells into the skin. Finally, we evaluated whether LMW-HA-enhanced osteoclast formation was inhibited by the TLR-4 inhibitor (Resatorvid, TAK-242).

Results: Co-injection of LMW-HA and low-dose LPS significantly boosted skull destruction and increased the number of osteoclasts. The expression levels of osteoclast genes (TRAP, CTSK, MMP-9) expression, CTX-I, TRAP-5b, and inflammatory cytokines in the low-dose LPS + LMW-HA group were significantly higher than those in the low-dose LPS or LMW-HA groups (p < 0.01). Moreover, RANKL, TNF-α, IL-1β, IL-6, and TLR-4 mRNA expression and inflammatory cytokines (TNF-α, IL-1β, IL-6) levels in serum were significantly higher in the group administered with LPS + LMW-HA than those in the group administered low-dose LPS (p < 0.01). Furthermore, LMW-HA could promote infiltration of F4/80 positive cells into the skin and elevate TNF-α, IL-1β, and IL-6 mRNA expressions in PMMs and RANKL mRNA expression in BMSCs. TAK-242 reduced osteoclast formation enhanced by LMW-HA in vivo.

Conclusions: Our findings indicate that LMW-HA increases the secretion of inflammatory cytokines and exacerbates LPS-induced osteolysis in vivo. However, further studies are required to elucidate this potential mechanism.