Ultrastructure and Cytochemical Detection of Alkaline Phosphatase in Long-Term Cultures of Osteoblast-like Cells from Rat Calvaria

Abstract

Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation.

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

“…Isolated primary cultures from rat calvaria (Ecarot-Charrier et al 1983;Binderman et al 1989) and the rat osteosarcoma cell line ROS 17/2.8 (Majeska et al 1980) are well-established bone cell cultures which are often used for in vitro studies of osteogenesis. Up to now, differentiation markers for cultured osteoblastic cells have been restricted to alkaline phosphatase (Majeska and Rodan 1982;Herbert et al 1997), osteonectin, osteocalcin, bone sialoprotein and osteopontin (Collin et al 1992;Nanci et al 1996;Moursi et al 1997), all of which occur over a considerably large span of the osteoblastic-osteocytic differentiation period (Bruder et al 1997). According to the differentiation properties of the respective cell culture system, E11 was detected abundantly in our relatively well osteogenically differentiated rat calvaria cultures (see Figs.…”

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent

“…Consistent with the findings of an earlier study on the proliferation and differentiation of osteoprogenitor cells in the presence or absence of dex [32], CFU-O were not detectable beyond 18-20 population doublings. It is also of interest that in the hematopoietic system dex in combination with erythropoietin and stem cell factor was able to induce erythroid progenitors to undergo a similar number of cell divisions (15)(16)(17)(18)(19)(20)(21)(22), corresponding to a 10 5 -10 6 -fold amplification before a loss in proliferation and differentiation capacity was measured [43]. Dex acts directly on human erythroid progenitors, maintaining their colony-forming capacity and delaying their differentiation, with the glucocorticoid receptor acting as a key regulator for the decision between self-renewal and differentiation [43,44].…”

“…Osteoblast activities including bone matrix synthesis and mineralization are regulated by various local and systemic factors. Osteoblasts express alkaline phosphatase (ALP) and synthesize type I collagen and noncollagenous proteins such as osteopontin, osteonectin, osteocalcin, bone sialoprotein, and bone morphogenetic histological, ultrastructural, and immunohistochemical characteristics of woven bone when grown in the presence of ascorbic acid and organic phosphate [11,[20][21][22]. These nodules are thought to represent the end stage of the proliferation-differentiation sequence of individual osteoprogenitor cells present in the input cell population.…”

Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral,Pocillopora damicornis

“…As demonstrated by the progress achieved by using in vitro models of mammalian ossification, long-term primary cell cultures provide innovative tools to investigate skeleton formation at the cellular level (10,11). In these models, the nature of the cells involved and the mechanisms of their cooperation in the regulation of calcification can be explored.…”