Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix

Abstract

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Fetal bone cells for tissue engineering

“…The presence of ascorbic acid and h-glycerophosphate had no effect on proliferation. This observation was previously described for rat calvaria osteoblasts in cultures which maintained the same growth rate when h-glycerophosphate was added to ascorbic acid [28]. Dexamethasone accelerated the proliferation of both fetal and adult bone cells.…”

“…It is also a critical gene involved in bone matrix deposition [56]. Early upregulation of collagen Ia1 confirmed its induction before any other current matrix component and prior to ALP [28,57,58]. Fetal cell gene expression of ALP was augmented due to the presence of dexamethasone, 1a,25-(OH) 2 D 3 , ascorbic acid, and hglycerophosphate.…”

“…Exposing the rat osteosarcoma cell line ROS 17/2.8 to dexamethasone, the rise in alkaline phosphatase mRNA levels could be attributed entirely to an increase in gene transcription [62]. The immortalized human fetal osteoblastic cell line hFOB/ ER9 showed an increase of ALP activity during mineralization [63], whereas in rat fetal calvaria cultures, ALP was expressed maximally during the early stages of differentiation [28]. In our model, ALP activity of fetal bone cells reached a maximum activity after 10 days, when nodule formation started to be markedly enhanced.…”

“…Although its biological function has not been precisely defined, specific interaction with hydroxyapatite was demonstrated [65]. New evidence indicates that osteocalcin participates in the regulation of mineralization and bone turnover [28]. A vitamin D regulatory element was identified in its promoter [66][67][68][69].…”

“…Osteoblast development follows three main phases: proliferation, matrix synthesis, and mineralization [28]. Factors for differentiation of osteoblasts are well described [29,30] and include ascorbic acid, h-glycerophosphate, 1a,25-dihydroxyvitamin D 3 (1a,25-(OH) 2 D 3 ), and dexamethasone.…”