Malaval L, Liu F, Roche P, Aubin J E
INSERM U403, Hôpital Edouard Herriot, 69437 Lyon, France.
J Cell Biochem. 1999 Sep 15;74(4):616-27.
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.
以极低密度接种的胎鼠颅骨细胞可形成离散的集落，其中一些是来自单个骨祖细胞的骨集落（结节），这些骨祖细胞会分裂并分化。我们分析了从最初的单个细胞到完全矿化状态的骨集落中细胞增殖与组织特异性分化标志物获得之间的关系。完全形成的结节的大小分布是单峰的，这表明骨祖细胞增殖与分化之间的耦合受随机因素控制，但围绕一个最佳值分布，该最佳值对应于集落大小/分裂潜能的峰值。集落生长的动力学分析表明，在发育中的集落中首次出现形态学上分化的成骨细胞之前，骨祖细胞经历9 - 10次群体倍增。双重免疫标记显示，这些增殖细胞表达骨标志物梯度，从集落周边增殖性碱性磷酸酶阴性细胞，到集落中心有丝分裂后产生骨钙素的成骨细胞。细胞分裂与骨钙素表达之间存在反比关系，骨钙素仅限于晚期、BrdU阴性的成骨细胞表达，而所有其他标志物的表达在增殖停止前获得，但并非同时获得。骨唾液蛋白表达是双相的，在一些早期碱性磷酸酶阴性细胞中可检测到，之后在晚期前成骨细胞（BrdU阳性）和成骨细胞（BrdU阴性、骨钙素阳性）中再次检测到。在晚期高度矿化的结节中，最老/最成熟的细胞中检测不到骨钙素和骨唾液蛋白的染色。我们的观察结果支持这样一种观点，即源自单个骨祖细胞并最终包含矿化基质产生的骨结节“组织样”结构，以增殖/成熟序列概括了成骨细胞分化途径的连续阶段。了解骨结节内发生的增殖/分化动力学的复杂性，将有助于对成骨细胞分化过程中组织特异性标志物表达进行定性和/或定量解释。
