Mesenchymal cell polarity and morphogenesis of chick cartilage

Mesenchymal cell polarity was studied in the developing cartilages of chick vertebral bodies and limbs using a silver impregnation technique for the Golgi apparatus. Distinct patterns of cell orientation are apparent in the cells at a number of different stages of morphogenesis. The data support the hypothesis that individual cells are inherently polarized, and that morphogenesis of multicellular patterns results from the coordinated three-dimensional orientation of anisotropic cells.     

In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells

Periosteal cells were enzymatically liberated from the tibiae of young chicks, introduced into cell culture, and allowed to reach confluence. The morphology of the cells gave the impression of a relatively homogeneous population of fibroblast-like cells. These cultured cells did not overtly express osteogenic or chondrogenic properties as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes. The cells were then replated at relatively high density and chronologically evaluated for the differentiation of bone and cartilage. These replated cells formed a multi-layer of fibroblast-like cells, the top portion of which eventually differentiated into bone tissue as evidenced by the presence of mineralization and immunocytochemical reactivity to bone Gla protein- and osteocyte-specific probes. Cells below this distinctive top layer differentiated into chondrocytes, which eventually further developed into hypertrophic chondrocytes as evidenced by their morphology and the presence of immunoreactive type X collagen in the matrix. Mineralization was also observed in the territorial matrix of these hypertrophic chondrocytes, when the culture was augmented with beta-glycerophosphate. Periosteal-derived cells replated at a lower density as controls did not show signs of osteochondrogenic differentiation. These observation suggest that periosteal-derived cells of young chicks contain mesenchymal cells which possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes depending on local environmental or positional cues.     

Calcification of in vitro developed hypertrophic cartilage

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix (C. Tacchetti, R. Quarto, L. Nitsch, D. J. Hartmann, and R. Cancedda, 1987, J. Cell Biol. 105, 999-1006). In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level of alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and 45Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.     

Formation of cartilage in organ-cultured leg buds of chick embryo

When the leg buds from chick embryos at stages 17-23 were cultured as organ cultures in F12 medium, which contained chick serum and an extract of chick embryos, leg cartilage structures developed that included pelvic girdle, femur, tibiofibula, and tarsometatarsus. The proportion of explants with definite distal elements increased when the leg buds from later embryos were used. Supplementation with chick serum was essential. These results suggest that the pattern of formation of limbs in chick embryos can be studied in organ culture.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.