Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro

Fetal rat calvaria cells plated at very low density generate discrete colon ies, some of which are bone colonies (nodules) from individual osteoprogeni tors that divide and differentiate. We have analyzed the relationship betwe en cell proliferation and acquisition of tissue-specific differentiation ma rkers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodul es is unimodal, suggesting that the coupling between proliferation and diff erentiation of osteoprogenitor cells is governed by a stochastic element, b ut distributed around an optimum, corresponding to the peak colony size/div ision potential. Kinetic analysis of colony growth showed that osteoprogeni tors undergo 9-10 population doublings before the appearance of the first m orphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts a t the centers. An inverse relationship exists between cell division and exp ression of osteocalcin, the latter being restricted to late-stage, BrdU-neg ative osteoblasts, while the expression of all other markers is acquired be fore the cessation of proliferation, but not concomitantly. Bone sialoprote in expression is biphasic, detectable in some of the early, alkaline phosph atase-negative cells, and again later in both late preosteoblast (BrdU-posi tive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-s tage, heavily mineralized nodules, staining for osteocalcin and bone sialop rotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix p roduction, recapitulates successive stages of the osteoblast differentiatio n pathway, in a proliferation/maturation sequence. Understanding the comple xity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of t issue-specific marker expression during osteoblastic differentiation. (C) 1 999 Wiley-Liss, Inc.