Ascorbic acid induction of chondrocyte maturation

Leboy, Phoebe S.; Sullivan, T.A.; Menko, A. Sue; Enomoto, Motomi

doi:10.1016/0169-6009(92)90744-x

Cited by 6 publications

(1 citation statement)

References 8 publications

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“…Primary cultures of early hypertrophic chondrocytes were established from 17-day-old embryonic chick sterna as described previously [10,11]. Chondrocytes from the cephalic part of chick sterna were used in the examination of type X collagen mRNA levels.…”

Section: Methodsmentioning

confidence: 99%

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

et al. 2006

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Objective: Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods:Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA.Results: Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion:These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression.

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Section: Methodsmentioning

confidence: 99%

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

et al. 2006

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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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