Growth and Differentiation of Isolated Calvarium Cells in a Serum-Free Medium

Burks, James K.; Peck, William A.

doi:10.1016/s0074-7696(08)60616-6

Cited by 15 publications

(7 citation statements)

References 38 publications

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“…Studies on primary cultures of bone cells, prepared from embryonic rat calvaria, suggested the existence of a heterogeneous cell population, which consists of osteoblast-like, osteoclast-like, osteocytes, osteoprogenitors, and fibroblast-like cells (Chen et al, 1977;Wong, 1979). That these cells are actually bone cells is supported by the fact that most of the cells showed positive histochemical staining for alkaline phosphatase (Chen et al, 1977;Burks and Peck, 1979;Frish, Szydel, and Eilam, unpublished observations). This property had been considered as indicative of osteoblasts (Peck et al, 1973;1977), as well as changes in notion can be derived from the studies showing increased cyclic AMP production in response to parathyroid hormone (F'TH) and calcitonin (Peck et al, 1973(Peck et al, : 1977, as well as changes in Ca++ fluxes in response to calcitonin, FTH, and metabolites of vitamin D (Eilam et al, 1980a,b;Dziak and Stern, 1975;Dziak, 1978).…”

Section: Discussionmentioning

confidence: 91%

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Eilam

Szydel

1981

Journal Cellular Physiology

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Primary cultures of bone cells and skin fibroblasts were examined for their Ca++ content, intracellular distribution and Ca++ fluxes. Kinetic analysis of 45Ca++ efflux curves indicated the presence of three exchangeable Ca++ compartments which turned over at different rates: a "very fast turnover" (S1), a "fast turnover" (S2, and a "slow turnover" Ca++ pool (S3). S1 was taken to represent extracellular membrane-bound Ca++, S2 represented cytosolic Ca++, and S3 was taken to represent Ca++ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca++ as compared with cultured fibroblasts. Most of this extra Ca++ was localized in the "slow turnover" intracellular Ca++ pool (S3). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca++: (1) In both types of cells serum caused an increase in the amount of Ca++ in the "very fast turnover" Ca++ pool, and an increase in the rate constant of 45Ca++ efflux from this pool, indicating a decrease in the strength of Ca++ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca++ in the "slow turnover" Ca++ pool (S3), an increase in the rates of Ca++ efflux from the cells to the medium, and from S3 to S2, as well as a decrease in the rate of influx into S3. (3) In bone cells the amount of Ca++ in S3 remained high in "serum activated" cells, the rate of efflux from S3 to S2 increased, and the rate of influx into S3 also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca++ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca++ in bone cells and fibroblasts, in parallel with measurements of 3H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca++ which was followed by a biphasic increase lasting until cell division.

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Section: Discussionmentioning

confidence: 91%

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Eilam

Szydel

1981

Journal Cellular Physiology

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“…All culture media were supplemented with 50 µg/ml ascorbate. To increase plating efficiency, during cell isolation and inoculation, no ascorbate was used (Burks and Peck, 1979). No antibiotics, or antimycotics, nor b-glycerophosphate were added.…”

Section: Methodsmentioning

confidence: 99%

Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures

Gerber¹,

Gwynn²,

Alini³

et al. 2005

eCM

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The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically.The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

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“…We have accumulated evidence that cells in this system, perhaps the fetal chondrocranial cells, elaborate a growthpromoting factor (or factors), and the evidence has been the subject of preliminary reports (8,9) . Perhaps cells of fetal cartilage origin thrive in the absence of added serum or growth factors by virtue of their capacity to elaborate an autostimulatory substance.…”

Section: Proteoglycan Labeling In Primary and Secondary Culturesmentioning

confidence: 99%

“…Successful culture, however, required an initial period of exposure to a serum-supplemented incubation medium . We have found that cells isolated from the calvaria of rat fetuses proliferate and specialize when cultured in a medium which is serum-free and, at least initially, chemically defined (7,8) . Although these cells possessed morphological and biochemical characteristics reminiscent of osteoblasts (40,41), subsequent studies suggested that chondrocytes, presumably derived from the chondrocranium, grew and matured in this system (8) .…”

mentioning

confidence: 99%

Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

Rifas¹,

Uitto²,

Memoli³

et al. 1982

The Journal of Cell Biology

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Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.

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Growth and Differentiation of Isolated Calvarium Cells in a Serum-Free Medium

Cited by 15 publications

References 38 publications

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts

Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures

Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

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