Properties of chick embryo chondrocytes grown in serum-free medium.

Glaser, J H; Conrad, H. Edward

doi:10.1016/s0021-9258(17)39794-6

Cited by 51 publications

(1 citation statement)

References 35 publications

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“…Under these circumstances, precise conditions for any particular cell type cannot easily be established. There are several reports on monolayer culture of chondrocytes in chemically defined media (Choi et al, 1980;Rifas et ai., 1982;Schwartz and Sugumaran, 1983;Jennings and Ham, 1983;Glaser and Conrad, 1984;Malemud and Papay, 1986). Such culture systems may be beneficial for detailed studies on chondrocyte dedifferentiation (for review, see Benya and Brown, 1986) but they will not easily allow direct insight into the mechanisms of terminal differentiation of cartilage cells.…”

Section: Discussionmentioning

confidence: 99%

Induction and prevention of chondrocyte hypertrophy in culture.

Brückner

Hörler

Mendler

et al. 1989

The Journal of Cell Biology

155

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Abstract. Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification.At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability.Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.

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Section: Discussionmentioning

confidence: 99%

Induction and prevention of chondrocyte hypertrophy in culture.

Brückner

Hörler

Mendler

et al. 1989

The Journal of Cell Biology

155

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Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid

et al. 1997

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In serum-containing medium, ascorbic acid induces maturation of prehypertrophic chick embryo sternal chondrocytes. Recently, cultured chondrocytes have also been reported to undergo maturation in the presence of bone morphogenetic proteins or in serum-free medium supplemented with thyroxine. In the present study, we have examined the combined effect of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocytes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels within 24-72 h, and simultaneous exposure to both ascorbate and BMP yielded enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and BMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent serum-free cultures treated for 48 h with BMP+ascorbate were equivalent to those reached in serum-containing medium only after confluence. While ascorbate addition was required for maximal alkaline phosphatase activity, it did not induce a rapid increase in type X collagen mRNA. In contrast, BMP added to serum-free medium induced a three- to fourfold increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required. Addition of thyroid hormone to serum-free medium was required for maximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chondrocytes, which do not undergo hypertrophy during embryonic development. These results indicate that ascorbate+BMP in serum-free culture induces rapid chondrocyte maturation of prehypertrophic chondrocytes. The mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction.

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Modulation of chondrocyte proliferation by ascorbic acid and BMP-2

et al. 1998

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Chondrocytes show an unusual ability to thrive under serum-free conditions as long as insulin, thyroxine, and cysteine are present. Studies with sternal chondrocytes from chick embryos indicate that thymidine incorporation in chondrocytes cultured under serum-free conditions is 30-50% of that seen with fetal bovine serum (FBS). In contrast, skin fibroblast proliferation in serum-free culture is <5% of that seen with serum. Addition of 30-50 microM ascorbic acid to serum-free medium stimulates chondrocyte proliferation 4-5x, resulting in levels of thymidine incorporation higher than that seen with 10% serum. Three to five hours of ascorbate exposure is sufficient to stimulate proliferation, with maximal stimulation seen after 12-15 h. Bromo-deoxyuridine (BrdU) labelling indicated that approximately 25% of chondrocytes transit S phase during a 4-h period (16-20 h after ascorbate). Once maximal stimulation is reached, the proliferation rate remains fairly constant over at least 40 h. Ascorbate therefore increases the steady-state level of chondrocytes in the cycle. Because the stimulation of chondrocyte proliferation was greater than the net increase in cell numbers, we examined the level of apoptosis. Nuclear morphology, terminal uridine nucleotide end-labelling (TUNEL) assay, and 7-AAD/Hoechst dye FACS analyses all indicated that approximately 15% of the ascorbate-treated chondrocytes were undergoing apoptosis, while only 5% of the control chondrocytes were apoptotic. When prehypertrophic chondrocytes from the cephalic region of embryonic sternae were stimulated to undergo hypertrophy with rhBMP-2 + ascorbate, levels of apoptosis were similar to that seen with ascorbate alone. In contrast, treatment of caudal chondrocytes with BMP plus ascorbate does not induce hypertrophy, and the proportion of apoptotic cells was less than that seen with ascorbate alone. These results imply that in chondrocytes the transition to hypertrophy is associated with a decreased number of proliferating cells and a relatively high level of apoptosis.

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Properties of chick embryo chondrocytes grown in serum-free medium.

Cited by 51 publications

References 35 publications

Induction and prevention of chondrocyte hypertrophy in culture.

Induction and prevention of chondrocyte hypertrophy in culture.

Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid

Modulation of chondrocyte proliferation by ascorbic acid and BMP-2

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