The isolation of newly formed bone associated cells in vitro: preliminary observations on origin and morphology

Agar-medium suspensions were used to culture guinea-pig femoral bone fragments which had been physically depleted of marrow. A sequential study of the histological events occurring within the regenerating shaft, in the agar wholemount preparations and on the underlying coverglass allowed (1) the localization of the early regenerative cells within the endosteal crevices and their rapid proliferation to fill and 'encapsulate' the explant; (2) the detection of residual marrow granulocytic and mononuclear precursors amidst the regenerative non-hemopoietic cells within the endosteal crevices of bone; (3) a study of the development of the adherent cells on the coverglasses. Cumulative labeling studies indicated that almost all the migrating cells were newly formed cells with a small fraction (less than 5%) within the endosteal crevices observed as unlabeled cells after 5 days of culture. The adherent mononuclear cells were derived directly from the regenerating bone fragment and showed similar morphology and proliferative characteristics (97% cumulative labeling index) to the bone associated cells. A unique feature of these monolayers was the formation of an extensive network of extracellular material between cells. These observations have not been described in monolayers derived from marrow suspensions and appear to be a characteristic of the monolayers derived from regenerating bone fragments.