Teruko Takano

Studies on Chondrocytes from Mandibular Condylar Cartilage, Nasal Septal Cartilage, and Spheno-occipital Synchondrosis in Culture. I. Morphology, Growth, Glycosaminoglycan Synthesis, and Responsiveness to Bovine Parathyroid Hormone (1-34)

Methods for isolating chondrocytes from the craniofacial complex and culturing them in vitro were established. Chondrocytes which were isolated by collagenase digestion from mandibular condylar cartilage, nasal septal cartilage, and spheno-occipital synchondrosis grew well in vitro. All three types of chondrocytes actively synthesized glycosaminoglycans, a differentiated phenotype of chondrocytes, and responded well to parathyroid hormone. However, some different characteristics were noted among the three types of chondrocytes in culture.

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.

Restoration by cyclic AMP of the differentiated phenotype of chondrocytes from de-differentiated cells pretreated with retinoids

SummaryParathyroid hormone (PTH) increases the cyclic AMP level in rabbit costal chondrocytes in culture. PTH, dibutyryl cyclic AMP (DBcAMP), and 8-bromo cyclic AMP (8-Br cAMP) induce ornithine decarboxylase (ODC) and expression of the differentiated phenotype of chondrocytes in this cell system. On the other hand, retinoids inhibit expression of the differentiated phenotype of chondrocytes. In the present study, the effects of PTH, DBcAMP, and 8-Br cAMP on rabbit costal chondrocytes pretreated with retinoids were examined.PTH did not increase the cellular cyclic AMP level in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for three days, but it did increase the cyclic AMP level four days after removal of retinoids. PTH did not stimulate ODC activity or expression of the differentiated phenotype of chondrocytes in the de-differentiated state. On the other hand, DBcAMP or 8-Br cAMP stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells, as judged by morphological and bistological changes of the cells and increase in glycosaminoglycan synthesis. Cyclic AMP analogues also induced ODC in these cells.

The Effect of Parathyroid Hormone (1-34) on Cyclic AMP Level, Ornithine Decarboxylase Activity, and Glycosaminoglycan Synthesis of Chondrocytes from Mandibular Condylar Cartilage, Nasal Septal Cartilage, and Spheno-occipital Synchondrosis in Culture

Previously, we reported methods for isolating chondrocytes from the craniofacial complex and their culture in vitro. The response of these chondrocyte cultures to bovine parathyroid hormone (1—34) (PTH) has now been investigated. PTH stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype in cultured chondrocytes isolated from mandibular condylar cartilage (MCC), nasal septal cartilage (NSC), and spheno-occipital synchondrosis (SOS). These stimulations of GAG synthesis by PTH were dose-dependent. PTH also increased accumulation of cyclic AMP (cAMP) and the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis. However, PTH did not stimulate DNA synthesis. The increases in the cAMP level, ODC activity, and GAG synthesis after addition of PTH (10-7 mol/L) were greatest in MCCchondrocytes and least in NSC-chondrocytes. The difference in the responses to PTH of these three types of chondrocytes may reflect differences of the characteristics of these cells in vivo.

Restoration by parathyroid hormone and dibutyryl cyclic AMP of expression of the differentiated phenotype of chondrocytes inhibited by a tumor promoter, 12-0-tetradecanoylphorbol-13-acetate

12-0-Tetradecanoylphorbol-13-acetate (TPA) inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture. TPA transformed typical polygonal chondrocytes into multilayered, fibroblastic cells and also inhibited the rate of [35S]sulfate incorporation into glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes. These changes were apparent within 24 h and reached a plateau at 48 h after the addition of TPA. Phorbol didecanoate and phorbol dibenzoate also inhibited sulfation of GAG, even though the effect was weaker than that of TPA. Phorbol diacetate and 4-0-methyl TPA did not inhibit sulfation of GAG. Addition of parathyroid hormone (PTH) or dibutyryl cyclic AMP simultaneously with TPA overcame the inhibition caused by TPA. PTH and dibutyryl cyclic AMP also reversed the inhibition and stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells which had been pretreated for 3 days with TPA. These findings suggest that cyclic AMP plays an important role in the restoration of the differentiated phenotype of chondrocytes in TPA-treated chondrocytes, and that the TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH.

Hydrocortisone stimulation of proliferation and glycosaminoglycan synthesis in rabbit craniofacial chondrocytes in vitro

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a cartilage phenotype, in chondrocytes from mandibular condylar cartilage (MCC), nasal septal cartilage (NSC) and sphenooccipital synchondrosis (SOS). These stimulations were dose- and time-dependent, being maximal 27 h after addition of 10(-7) M hydrocortisone. The maximal induced increase of GAG synthesis was about 100%, 50% and 20% that of non-stimulated MCC, SOS and NSC chondrocytes, respectively. When stained with toluidine blue, all three types of cortisone-treated chondrocytes showed stronger metachromasia than non-treated controls. DNA synthesis was also increased by hydrocortisone, reaching a maximum 20 h after the addition; stimulation was also dose-dependent and maximal at a concentration of 10(-6) M. The maximal increase in DNA synthesis was 200% in NSC chondrocytes, 90% in SOS chondrocytes, and slight in MCC chondrocytes. However, there was no stimulation of DNA synthesis in serum-free medium, in contrast to that of GAG synthesis. These observations suggest that hydrocortisone regulates craniofacial growth by controlling the differentiation of these chondrocytes directly and their proliferation indirectly, and that the difference in their responses to hydrocortisone may reflect different responses in vivo.