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ABSTRACT: 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.
Journal of Cellular Biochemistry 10/1997; 66(3):394-403. · 2.87 Impact Factor
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ABSTRACT: During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.
Journal of Biological Chemistry 10/1994; 269(36):22500-6. · 4.77 Impact Factor
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ABSTRACT: Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10(-8) M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well as osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10(-8) M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.
Developmental Biology 02/1994; 161(1):218-28. · 4.07 Impact Factor
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Bone and Mineral 06/1992; 17(2):242-6.
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ABSTRACT: Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10-5M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well us osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10-5M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.
Developmental Biology.
