[Impacts of osteoclast-like cells cultured on bone wafer and their sub-cellular structures on osteoblast]

To investigate the effects of osteoclast-like cells (OLC) and its sub-cellular structures on the osteoblast (OB) differentiation and function.

Spleen cells from C57 mice administrated with 5-fluorouracil were induced by IL-3, 6 and granulocyte-macrophage colony stimulating factor (GM-CSF), and 1alpha, 25-(OH)(2)D(3) to obtain massive OLCs. These OLC cells were cultured in culture fluid and on bone wafers (called bolcs). Osteoblasts were cultured and added with NaF, OLCs of two kinds, culture fluid free of OLC, and sub-unit structures such as nucleus, mitochondria, and cytoplasma from OLCs for 5 days. The proliferation rate of OBs was measured by MTT method and the alkaline phophatase (ALP) activity was measured by PNPP method. Immunochemistry was used to detect the core-binding factor alpha1 (Cbfalpha1) in the OBs, Enzyme linked immunosorbent assay was used to measure the osteocalcin.

The OB number was lower in the OLC (1.288 +/- 0.039), OLC cytoplasm (1.138 +/- 0.024), 50% OLC culture fluid (1.203 +/- 0.033), 50% OLC culture medium of OLCs cultured on bone wafer (1.128 +/- 0.028) in comparison with the pure OB group (1.393 +/- 0.016, all P < 0.05). The increase functions of OBs by OLC cultured on bone wafer and their nucleus and mitochondria were all more significant than those of the OLCs not cultured on bone wafer. The ALP activity was increased in the NaF (1.027 +/- 0.024), OCL cytoplasm (1.850 +/- 0.033), 50% OLC medium (2.074 +/- 0.065), 50% OLC medium of OLCs cultured on bone wafer (1.718 +/- 0.048), and mitochondria and cytoplasm of the OLC cultured on bone wafer groups (1.246 +/- 0.037, all P < 0.05). NaF (0.0825 +/- 0.0025), OLCs (0.0775 +/- 0.0025), nucleus (0.0775 +/- 0.0025), mitochondria (0.0875 +/- 0.0025), and cytoplasm of OLCs (0.1100 +/- 0.0007), 50% OLC medium (0.0900 +/- 0.0000), 50% OLC medium of OLCs cultured on bone wafer (0.1200 +/- 0.0041), OLCs cultured on bone wafer and nucleus, mitochondria, and cytoplasm of OLCs cultured on bone wafer all significantly increase the oeteocalcin activity of OBs (0.525 +/- 0.0063, all P < 0.05). NaF (57.6% +/- 2.6%), OLC cytoplasm (45.3% +/- 4.7%), 50% OLC medium (46.6% +/- 3.3%), 50% medium of OLCs cultured on bone wafer (54.0% +/- 2.1%), OLCs cultured on bone wafer (44.8% +/- 3.0%), and cytoplasm of OLCs cultured on bone wafer (48.7% +/- 3.5%) all significantly increased the Cbfalpha1 protein in the OBs (32.8% +/- 4.5%, all P < 0.05).

The sub-cellular elements of OLC and the supernatant of OLC culture media free of OLC promote the functions of OB, especially the OLCs cultured on bone wafer.