engleski

Lipopolysaccharide induces increased bone resorption by stimulating homing of osteoclast progenitors to the periosteal bone surface

Lipopolysaccharide (LPS) from gram-negative bacteria causes chronic inflammation and subsequent bone loss, and is involved in the pathogenesis of several bacterially induced bone diseases. We investigated the effects of LPS on bone metabolism and osteoclast differentiation from hematopoietic cells. C57BL/6 mice were injected during 4 weeks in a dose of 10 µ ; g LPS/g body weight and sacrificed at different time-points. Cells from different tissue sources (bone marrow, homogenized bone shafts, spleen and peripheral blood) were cultured with RANKL (40 ng/mL) and M-CSF (15 ng/mL) to stimulate osteoclast differentiation. Osteoclasts (OCL) were identified as TRAP positive multinucleated cells with three or more nuclei per cell. Femoral sections (5 µ ; m) were stained with Goldner-trichrome and TRAP staining. Microtomography (µ ; CT) was performed by SkyScan1172 high resolution micro-CT. OCL progenitors were characterized by flow cytometry as a population negative for lymphoid markers (B220, CD3, NK1.1) and positive for CD115 and/or CD117, within both CD11b negative/low and CD11b positive populations. Gene expression analysis of OCL differentiation genes was performed by qPCR. Three weeks after LPS stimulation, the number of OCL differentiated from cells extracted from bone shafts (504.8± ; 74.28) was higher compared with control mice (383.3± ; 30.48 ; p=0.0025). This was in correlation to the decrease in bone volume and trabecular thickness detected by µ ; CT. Femoral sections showed that LPS treatment altered bone metabolism in vivo by inducing increased osteoresorption in bone cortex starting from the periosteal bone surface. This was confirmed by gene expression analysis of bone shafts, showing increased expression of OCL differentiation genes including RANK and cFms. Flow cytometry indicated that enhanced bone resorption starting at the periosteal surface may be caused by homing of peripheral OCL progenitors, since we found approx. 2-3 fold increase of OCL progenitor cell populations in peripheral blood and spleen 10 days after LPS treatment. This was confirmed by increase in number of differentiated OCL from both blood (181± ; 32.16) and spleen (299.25± ; 61.37) compared to controls (blood 43.8± ; 25.7 ; spleen 189± ; 54.88). LPS administration stimulates homing of OCL progenitors to periosteal bone surface and supports osteoclast differentiation. Our further aim is to identify factors induced by LPS that mediate this osteoclastogenic effect and cause enhanced bone resorption.

lipopolysaccharide; osteoclasts; resorption; inflammation

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