Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro

Abstract

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically 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 at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein 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 production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.

A Subset of Osteoblasts Expressing High Endogenous Levels of PPARγ Switches Fate to Adipocytes in the Rat Calvaria Cell Culture Model

“…A combination of limiting dilution single colony assays and replica plating is a useful technique to trap cells at definable developmental stages [24], [25]. In spite of the possibility of a gradient in the proliferation-differentiation sequence from the periphery to the center of developing bone colonies [31], the uniform ALP staining we saw in colonies selected here and the lack of a relationship between the colony size (not shown) and their developmental stage suggest that a development gradient, if present under our culture conditions, was restricted to a narrow range. Using this approach, we found a statistically significant difference in the adipogenic potential amongst colonies defined as being at three development stages: immature, intermediate and mature, but adipocytes were found in all three.…”

Nurse’s A-Phase Material Enhance Adhesion, Growth and Differentiation of Human Bone Marrow-Derived Stromal Mesenchymal Stem Cells

“…The cells were not fully adhered until day 7, when they showed the characteristic flattened polygonal shape and they were metabolically active, displaying higher values of proliferation. It has been demonstrated that levels of Ca concentrations up to 300 mg/L are cytotoxic [25], but lower concentrations are suitable for cell proliferation, differentiation, and extracellular matrix mineralization. The new biomaterial obtained, Nurse’s A-phase, has a high proportion of CaO in its composition, causing the high level of Ca 2+ ion release in the first week.…”

Up-regulation of inhibitors of DNA binding/differentiation gene during alendronate-induced osteoblast differentiation

“…The process of osteoblast differentiation is under various central and local controls including bone morphogenetic proteins (BMP), Indian hedgehog, fibroblast growth factor-2 (FGF2), Wnt, parathyroid hormone, and leptin [6–9]. Further, studies have proposed several possible mechanisms governing bisphosphonate-mediated osteoblast differentiation [10, 11].…”