Mineralizing Fibroblast-Colony-Forming Assays

Bone formation does not lend itself easily to investigation because bone tissue consists of various cell types embedded in a complex extracellular matrix. These cells interact with each other and with the extracellular matrix, and when cell populations are removed from the network they cease to function normally. In the past, bone cell differentiation was studied using histological methods in either whole embryos or organ cultures. Although this has provided much information regarding the temporal and spatial relationships of the various cells, the complexity of organ culture systems does not easily allow one to investigate the molecular mechanisms involved in bone development and mineralization. Cell culture techniques have given us much information regarding the mechanistic aspects of gene regulation and cell signaling in osteoblastic cells, but isolated osteoblasts do not respond to exogenous agents in a similar manner to that observed in vivo (1 ). Recently a number of in vitro models have been established that re-create discrete elements of the cellular network present in the bone micro-environment. The advantage of these models is that they have reduced complexity compared with organ cultures yet retain osteoblasts and their progenitors at various stages of differentiation, allowing defined aspects of bone formation to be investigated at the cellular and molecular levels. These models are modifications of nodule cultures or fibroblast-colony-forming unit (CFU-f) cultures. In CFU-f cultures, bone marrow cells are cultured at relatively low densities under conditions that allow the individual CFU-f to adhere and proliferate to form colonies.