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.

Flavonoids of Herba Epimedii stimulate osteogenic differentiation and suppress adipogenic differentiation of primary mesenchymal stem cells via estrogen receptor pathway

“…Components of HETF extracts were identified by HPLC fingerprinting with chemical standards isolated by our previous publication (Cheng et al, 2006(Cheng et al, , 2007. Osteoblasts progress through a three-stage process of differentiation, i.e., proliferation, matrix maturation, and matrix mineralization (Malaval et al, 1999). In the present study, 0.006-6 mg/mL HETF significantly enhanced the proliferation of MSCs from 17% to 22% (Figure 2).…”

The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

“…To investigate the relationship between intracellular calcium phosphate accumulations and extracellular bone apatite formation, we cultured mouse calvarial osteoblasts and marrow stromal cells (MSC) in osteogenic medium according to standard in vitro methods for bone-like nodule formation (13). We have previously demonstrated by multivariate analyses of microRaman spectra that such live, unprocessed nodules possess important characteristics of native bone, including the presence of a complex combination of mineral and matrix environments (14).…”

Knock‐in of nuclear localised β‐galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar

“…At this stage, Ptprv expression was barely detectable by Northern blot. At day 5 of culture, these cells express several markers of the osteoblastic lineage including Bone sialoprotein, and Osteopontin (Malaval et al, 1999). At this stage of the culture Ptprv mRNA was readily detectable.…”

“…1B). At day 0 of culture, these cells express only markers of undifferentiated mesenchymal cells such as ␣1(I)-collagen (Malaval et al, 1999). At this stage, Ptprv expression was barely detectable by Northern blot.…”