We previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of la,25-dihydroxyvitamin D3 and dexamethasone. In this study, we developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells (103-105 cells per well) obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive mononuclear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to la,25-dihydroxyvitamin D3 and dexamethasone. When hemopoletic cells suspended in a collagengel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. All of the colonies consisted of nonspecific esterase-positive cells. The monocyte-depleted population prepared from peripheral blood failed to form colonies, whereas the monocyte-enriched population produced a large number of TRAPase-positive colonies. In addition, alveolar macrophages formed TRAPase-positive colonies most efficiently on the ST2 cell layers in the presence of the two hormones. Salmon 12'I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. When direct contact between the peripheral blood cells and the ST2 cells was inhibited by a collagen-gel sheet, no TRAPasepositive cells were formed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a sulitable microenvironment is provided by bone marrow-derived stromal cells.Osteoclasts are multinucleated cells responsible for bone resorption. It is evident from chicken-quail chimera experiments (1), parabiosis experiments (2, 3), and marrow transplantation studies in osteopetrotic animals (4, 5) that osteoclasts are derived from circulating mononuclear precursors in hemopoietic tissues. However, the nature and the differentiation process of osteoclast precursors are still not known.We recently reported that osteoclast-like multinucleated cells were formed in response to osteotropic hormones in cocultures of mouse spleen cells with osteoblast-rich cell populations freshly isolated from fetal mouse calvaria (6). These multinucleated cells had the typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase (TRAPase), abundant calcitonin receptors, and the ability to form resorption lacunae on dentine slices (6). Then we reported that the two marrow-derived stromal cell lines, MC3T3-G2/ PA6 and ST2, could be substituted for primary osteoblastrich cell populations in inducing osteoclast-like cells in cocultures with spleen cells...

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells.

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.

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Formation of advanced glycation end products (AGEs) in extracellular matrix (ECM) is implicated in the development of chronic diabetic complications. However, the involvement of AGEs in diabetic bone disease has not been well established. We have examined whether AGEs are increased in the bone collagen of streptozotocin-induced diabetic rats in vivo and whether glycation of type I collagen affects the functions of osteoblastic cells in vitro. During 12 weeks of observation, AGEs in collagen extracted from the tibiae of diabetic rats increased in a time-dependent manner and were significantly higher than controls at every time point. In vitro, the incubation of collagen with glucose-6-phosphate resulted in a time-dependent increase of AGEs. When osteoblastic cells isolated from fetal rat calvaria were cultured on AGE-modified type I collagen, it dose-dependently inhibited phenotypic expressions of osteoblasts. Among osteoblastic parameters, nodule formation was the most sensitive, being inhibited by approximately 70% by the glycation of collagen for only 1 week. Alkaline phosphatase activity and osteocalcin secretion were inhibited by 20-30% and 15-70%, respectively, by the glycation of collagen for 1-5 weeks. These results indicate that AGE-modified collagen affects osteoblastic cell differentiation and function in vitro and suggest that similar changes occurring in vivo may contribute to diabetic osteopenia.

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