High-dose PMA with RANKL and MCSF induces THP­1 cell differentiation into human functional osteoclasts in vitro.

Osteoclasts are large multinuclear cells, which serve role in erosive bone disease. However, it is not possible to separate osteoclasts from cortical bone in order to culture the cells for further experiments. Therefore, a human osteoclast model is required to investigate the underlying mechanism of bone destruction. The most commonly­used osteoclast model is the RAW264.7 cell line, a murine mononuclear macrophage cell line; however, there exists no reliable osteoclast model using a human cell line. The aim of the present study was to establish a functional osteoclast model using the THP­1 cell line. Suspended THP­1 cells were stimulated for 2 days with 5 or 100 ng/ml phorbol­12 myristate­13 acetate (PMA) in order to induce the cells to differentiate into adherent macrophages. A 10­day stimulation with 50 ng/ml receptor activator of nuclear factor κ­B ligand (RANKL) and macrophage colony­stimulating factor (MCSF) was performed in order to induce macrophage differentiation into osteoclasts. Treatment with high­dose PMA with RANKL and MCSF enabled the THP­1 cells to form tartrate­resistant acid phosphatase­positive osteoclasts, which were able absorb bone in a bone resorption test. Treatment with low­dose PMA with RANKL and MCSF failed to induce THP­1 cell differentiation into osteoclasts. PMA alone, or a combination of RANKL and MCSF alone, is insufficient to stimulate THP­1 cell differentiation into osteoclasts. In the present study, a reliable human osteoclast model was established using the THP­1 cell line. This osteoclast model may provide a useful tool for further studies.

TLR4 drives the pathogenesis of acquired cholesteatoma by promoting local inflammation and bone destruction.

Acquired cholesteatoma is a chronic inflammatory disease characterized by both hyperkeratinized squamous epithelial overgrowth and bone destruction. Toll-like receptor (TLR) activation and subsequent inflammatory cytokine production are closely associated with inflammatory bone disease. However, the expression and function of TLRs in cholesteatoma remain unclear.We observed inflammatory cell infiltration of the matrix and prematrix of human acquired cholesteatoma, as well as dramatically increased expression of TLR4 and the pro-inflammatory cytokines TNF-α and IL-1ß. TLR2 exhibited an up-regulation that was not statistically significant. TLR4 expression in human acquired cholesteatoma correlated with disease severity; the number of TLR4-positive cells increased with an increased degree of cholesteatoma, invasion, bone destruction, and hearing loss. Moreover, TLR4 deficiency was protective against experimental acquired cholesteatoma-driven bone destruction and hearing loss, as it reduced local TNF-α and IL-1ß expression and impaired osteoclast formation by decreasing expression of the osteoclast effectors receptor activator of nuclear factor (NF)-κB ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP). TLR2 deficiency did not relieve disease severity, inflammatory responses, or osteoclast formation. Moreover, neither TLR2 nor TLR4 deficiency had an effect on antimicrobial peptides, inducible iNOS,BD-2 expression or bacterial clearance. Therefore, TLR4 may promote cholesteatoma-induced bone destruction and deafness by enhancing inflammatory responses and osteoclastogenesis.