The effect of ascorbic acid on growth and synthesis of matrix components by cultured chick embryo chondrocytes

Abstract

Fifty pglml ascorbic acid was found to accelerate the growth of logarithmically growing chick embryo sternal chondrocytes in cell culture. Differences in the number of cells between control and ascorbate-treated cultures were apparent by the fourth day of treatment when a 58% increase in the number of cells and a 35% increase in protein per culture were observed in ascorbate-treated cultures. Under these conditions, the cell size and protein content per cell was reduced in ascorbate-treated cultures.Collagen synthesis, which was measured by the amount of collagenase-sensitive counts released from 3H-proline labeled protein, was increased by 116% over controls after 24 hours of ascorbate treatment. Observations with scanning and transmission electron microscopy are consistent with the idea that ascorbate has a much greater effect on collagen secretion and demonstrate an ascorbate-mediated change in matrix morphology.35S incorporation into hyaluronidase-sensitive or cetylpyridinium chloride precipitates was accelerated by 57% by 24 hours of ascorbate treatment whereas two, four and six days of treatment caused decreases in this parameter of 19%, 57% and 3596, respectively. This effect was dependent on cell density. 35S incorporation probably reflects synthesis of new glycosaminoglycan because similar results were observed when either 35S or 14C-glucose incorporation was monitored.An effect of ascorbate on both collagen and glycosaminoglycan synthesis is consistent with the possibility that collagen and glycosaminoglycan synthesis can be coregulated under some circumstances.The effect of ascorbate on collagen accumulation is well known. Deficiency of this vitamin results in scurvy, a clinical syndrome characterized by tissue fragility, hemorrhage, impaired wound healing and anemia (Chazan and Mistilis, '63). Cartilage is affected by scurvy. Effects are seen in epiphyseal cartilages of long bones in the guinea pig where there is reduced matrix (Wolbach and Maddock, '52). In addition, in scurvy sulfate incorporation is reduced to one third of normal levels (Reddi and Norstrom, '54).The vitamin is a cofactor for both prolyl and lysyl hydroxylases (Peterkofsky and Udenfriend, '53; Hausman, '67), and 3T3 fibroblasts and chick embryo fibroblasts exhibit a dependence on ascorbate for maximal proline hydroxylation and for optimal collagen secretion (Peterkofsky, '72a). Similarly in chick embryo chondrocytes, Lavietes ('71) has reported increased hydroxylation of proline in the presence of ascorbic acid. Chen and Postlethwait ('70), on the other hand, found that ascorbate did not affect total proline incorporation into the collagen of sponge induced connective tissue growth in vitro but increased the appearance of hydroxyproline, suggesting that the effect of the vitamin in their system was not on collagen synthesis but rather only on proline hydroxylation. Similarly, Peterkofsky (72b) found that the rate of collagen synthesis was not affected by ascorbate in L-929 cells in culture when using an assay which '

Response of engineered cartilage tissue to biochemical agents as studied by proton magnetic resonance microscopy

“…The matrix had comparable levels of GAGs but significantly higher levels of collagen compared with controls both at week 2 (P ϭ 0.005) and week 3 (P ϭ 0.001). These results are consistent with those of other biochemical studies reported in the literature, where it was found that ascorbate is required for the normal hydroxylation and secretion of collagen and results in increased collagen accumulation (14,15). …”

Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture

“…Many investigators have reported that the addition of ascorbic acid and P-glycerophosphate to medium is required for high efficiency calcification. Ascorbic acid may promote cell proliferation (Hajek and Solursh, 1975), and the synthesis of collagen and glycosaminoglycan (Lavietes, 1971;Hajek and Solursh, 1975), whereas f3-glycerophosphate may act as a primary physicochemical factor on hydroxyapatites (Suzuki et al, 1981;Khouja et al, 19901, as an enhancer of alkaline phosphatase, at least in vitro (Maniatopoulos et al, 1988;Khouja et al, 19901, and may be associated with cell dystrophy (Gronowiz et al, 1989;Zimmermann et al, 1991). In the present culture system, matrix calcification may be accelerated more rapidly and with higher efficiency by an addition of P-glycerophosphate.…”

Collagen/Annexin V Interactions Regulate Chondrocyte Mineralization

“…Underhydroxylated collagen ␣ chains cannot form triple helices, resulting in impaired collagen secretion. Therefore, connective tissue cells treated in the presence of ascorbate produce more matrix than cells cultured in the absence of ascorbate (37)(38)(39). In addition, ascorbate has been shown to stimulate proliferation and APase expression and activity in osteoblastic cells and growth plate chondrocytes (23,40,41).…”