Conditioned media from PTH-treated osteoblastic cells contain chemotactic factors for bone marrow mesenchymal progenitors.

<p>(A) Migration of freshly flushed rat bone marrow cells toward PTH and conditioned media from UMR 106-01 cells. Bone marrow cells flushed from rat long bones were immediately seeded in the upper chamber of transwell plates. The bottom wells were loaded with media alone (αMEM), media containing 10 nM PTH (PTH), or conditioned media (CM) collected from UMR106-01 cells that had been treated with control (CON) or 10 nM PTH (PTH) for 4 hr. The number of cells that migrated to the bottom wells was counted 24 hr later. αMEM containing 5% FBS (FBS) was used as a positive control for cell migration. *: p<0.05; **: p<0.01 vs. αMEM. (B) Conditioned media from various PTH-treated osteoblastic and osteocytic cells stimulated the migration of either human or rat mesenchymal progenitors (MP) in the Boyden chamber assay. The cells seeded in the upper wells are depicted at the top and conditioned media loaded in the lower wells are shown at the bottom. M: αMEM. ***: p<0.001 vs. αMEM; &: p<0.01; #: p<0.001 vs. CON. (C) Microscopic images of the mesenchymal progenitors that migrated to the lower sides of filters in the assay depicted in B. (D) The migration of mesenchymal progenitors stimulated by conditioned media from PTH-treated osteoblasts is not chemokinesis. Mesenchymal progenitors were suspended in conditioned media harvested from either control or PTH-treated UMR 106-01 cells and seeded in the upper chambers. The lower wells were filled with conditioned media, resulting in a total of 4 combination types. **: p<0.01 vs. CON/CON. (E) Time course of the release of chemotactic factor(s) from osteoblasts after PTH treatment. UMR 106-01 cells were treated with PTH and conditioned media were harvested at indicated time points and loaded in the lower wells for chemotaxis assays. ***: p<0.001 vs. time 0 hr. (F) The dosage effects of PTH on chemotactic factor(s) release from osteoblasts. UMR 106-01 cells were treated with different doses of PTH for 4 hr and then conditioned media were harvested for chemotaxis assays. *: p<0.05; **: p = 0.06; ***: p<0.001 vs. 0 nM.