Muramyl Dipeptide Enhances Lipopolysaccharide-Induced Osteoclast Formation and Bone Resorption through Increased RANKL Expression in Stromal Cells

Figure 6

Effects of muramyl dipeptide (MDP) on parathyroid hormone- (PTH-) induced osteoclast formation in vivo. (a) Histological sections of sutures of calvariae were obtained from mice after 5 days of daily supracalvarial administration with PBS (A), 1 μg/day PTH (B), 100 μg/day MDP (C), 10 μg/day PTH (D), or 1 μg/day PTH and 100 μg/day MDP (E). Sections were stained with tartrate-resistant acid phosphatase (TRAP) staining and counterstained with hematoxylin. Cells that stained red are considered to be TRAP-positive. Scale bars = 50 μm. (b) Number of TRAP-positive cells with three or more nuclei in the calvariae (; ). (c) Histological sections of calvariae were obtained from mice after 5 days of daily supracalvarial administration with PBS (A), 1 μg/day PTH (B), 100 μg/day MDP (C), 10 μg/day PTH (D), or 1 μg/day PTH and 100 μg/day MDP (E). Sections were stained with tartrate-resistant acid phosphatase (TRAP) staining and counterstained with hematoxylin. Cells that stained red are considered to be TRAP-positive. Scale bars = 100 μm. (d) The percentage of bone/marrow interface covered by osteoclasts was histomorphometrically determined in specimens (; ). (e) TRAP and cathepsin K mRNA levels in mouse calvariae were detected using real-time RT-PCR. Total RNA from mouse calvariae was isolated after 5 days of daily supracalvarial injections, as in (a). RNA levels for TRAP and cathepsin K were normalized to those of GAPDH. Results are expressed as the mean ± SD (; ; ). (f) Expression levels of RANKL mRNA in mouse calvariae in vivo. Total RNA from mouse calvariae was isolated after 5 days of daily supracalvarial injections, as in (a). mRNA levels for RANKL were normalized to those of GAPDH. Results are expressed as the mean ± SD (; ; ). Differences were determined using Scheffe’s test.

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