Fig. 2. Requirement of HMM HA for osteoclastogenesis suppression. (A) Hyaluronidase treatment abolishes the effect of HA. HA preparation from umbilical cord (1 µg/ml) was digested with 150 U/ml hyaluronidase (HYAL) or pronase for 30 minutes followed by boiling for 10 minutes. Digested and undigested HA was added to BMMs and osteoclastogenesis was induced by culturing the cells with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for 6 days. After TRAP staining, TRAP⁺ MNCs were scored. ^**P<0.01 compared with vehicle-treated control. (B) Stimulatory effects of LMM-HA on BMM osteoclastogenesis. BMMs were cultured for 4.5 days with RANKL (100 ng/ml), M-CSF (30 ng/ml), and indicated concentrations of different sizes of HA. Cells were stained for TRAP and TRAP⁺ MNCs were counted. ^*P<0.05 and ^**P<0.01 compared with vehicle-treated control.