Comparison of morphological effects of PGE2 and TGFβ on osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

Abstract

RANKL, in the presence of M-CSF, induces the development and fusion of TRAP+ osteoclasts in mouse bone marrow cultures at 3–5 days. Early during culture (day 3), most cells are small (up to six nuclei). At lower cell densities, these osteoclasts exhibit a rounded morphology with cytoplasm extending around the cells but, at higher densities, this changes to a stellate morphology with the cytoplasm being retracted around the nuclei with numerous localised cytoplasmic extensions. Under optimal conditions, osteoclast fusion results in conglomerates of many cells, which become large cytoplasmic masses on day 4. PGE2 and TGFβ have both been shown to increase osteoclast development in this model and their effects on the morphology of osteoclasts during fusion and differentiation have been compared under all these conditions. PGE2 or TGFβ increase osteoclast numbers and size and also the number of nuclei, indicating increased osteoclast development and fusion. TGFβ increases the size of rounded osteoclasts (with respect to the number of nuclei) more than PGE2, suggesting that TGFβ increases cytoplasmic extension. TGFβ increases the size and number of nuclei in stellate cells but particularly increases the number and length of the cytoplasmic extensions, in contrast to PGE2. Fusion of these extensions with other osteoclasts results in large networks of interconnected cells. On day 4, spreading cells develop but these are still interconnected by cytoplasmic links, a phenomenon not seen in control wells or after treatment with PGE2. TGFβ is more effective than PGE2 in increasing fusion in the formation of cell conglomerates and cytoplasmic masses. PGE2 decreases overall cell density resulting in additional indirect effects on osteoclast numbers and morphology. However, PGE2 particularly promotes the formation of large mature spreading osteoclasts later during culture.