Development of reliable screening systems for drugs which regulate bone resorption : In vitro assay systems for osteoclast formation and function.

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Using a co-culture system of mouse osteoblastic cells and bone marrow cells, we have established reliable assay systems for examining osteoclast development and function. In a assay system for osteoclast development, chronological changes in phenotypic expression by postmitotic osteoclast precursors were examined during their differentiation into osteoclasts. The mechanism of action of bone resorption-inhibiting agents such as calcitonin and bisphosphonates in bone resorption was also examined in the present study using those assay systems.(1) Characteristics of postmitotic precursor cells that differentiate into osteoclastsCharacteristics of osteoclast precursors that differentiate into osteoclasts were examined using a co-culture system of mouse osteoblastic cells and bone marrow cells. Postmitotic osteoclast precursors were mononuclear cells which expressed macrophage-associated phenotypes such as nonspecific esterase (NSE), Mac-1 and Mac-2. Some of the macrophage-associated phenoty … More pes in osteoclast precursors disappeared rapidly during their differentiation into osteoclasts.(2) Establishment of assay systems for examining osteoclast functionWe have previously established a method for preparing a large number of functionally active osteoclasts from cocultures of mouse osteoblastic cells and bone marrow cells. To examine effects of bone resorption-regulatory factors on osteoclast function, we have developed assay systems for pit formation and actin ring formation using osteoclasts formed in vitro. We have shown that osteclast function is regulated by several signaling patheays mediated by cyclic AMP dependentprotein kinase, tyrosine kiases, phosphatidylinositol-3 kinase, and small GTP binding protein, rho.Effects of calcitonin and bisphosphonates on osteoclastic bone resorptionThe mechanism of inhibitory action of calcitonin and bisphosphonates on bone resorption was examined in established assay systems as described above. Both calcitonin and bisphosphonates inhibited pit forming activity of osteoclasts placed on dentine slices. Only polarized osteoclasts having ruffled borders incorporated bisphosphonates, which inhibited tyrosine phosphatases and disrupted actin rings of osteoclasts. Calcitonin also induced marked changes in osteoclast morphology including disruption of actin rings. This effect was observed in both polarized and non-polarized osteoclasts. Thus, both calcitonin and bisphosphonates suppress osteoclast function, but their mechanisms of action are quite different from each other. Less