Chondrogenesis In Cultures Research Articles

Region-Dependent Capacity for Limb Chondrogenesis: Patterns of Chondrogenesis in Cultures from Different Regions of the Developing Chick Wing

The in vitro chondrogenic patterns expressed by mesoderm cells obtained from the proximal, middle, and distal thirds of stage 25 chick embryo wing buds were studied. Region-dependent differences were observed in the patterns of Toluidine Blue-staining extracellular matrix produced. These differences were subsequently shown to be highly reproducible. Chondrogenesis in cultures containing cells from the proximal third of the limb is restricted primarily to the center of the dish and takes the form of small nodules by day 4. These nodules coalesce into a large colony by day 6. In cultures containing similar numbers of cells from the distal third of the limb, aggregates of cells surrounded by metachromatic extracellular matrix materials are detectable somewhat earlier than in those containing cells from the proximal region. A centrally located concentration of matrix material is found in these distal cell cultures, however, a large number of nodules are also scattered throughout the monolayer. The difference between the chondrogenic patterns seen in the proximal and distal cell cultures becomes more pronounced by day 6 of culture. This is due primarily to the absence of demonstrable cartilaginous matrix in the peripheral areas of the proximal cell cultures. Cultures composed of cells derived from the middle third of stage 25 wings exhibit patterns of Chondrogenesis intermediate in appearance between those containing cells from the other two regions. It is unlikely that these readily repeatable differences in chondrogenic patterns can be explained solely by convection currents in the media caused by the seeding and handling of the cultures prior to attachment of the cells. The different chondrogenic patterns might therefore be suggestive of region-dependent differences in the cell surface characteristics of limb mesoderm cells.

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20–21, 23–24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 μg/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20–21, 23–24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20–21 or 23–24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.

Enhancement of Chondrogenesis of Cultured Quail Limb Bud Mesenchymal Cells by Cellophane Films

Cultures of mesenchymal cells dissociated from the hind limb buds of 3.5 day quail embryos (stages 22-23) were used to study the effect of the cellular microenvironment on chondrogenesis. The degree of chondrogenesis in cultures was estimated by two methods : the number of cartilage nodules (characteristic structures formed by chondrocytes) was counted under a phasecontrast microscope, and the amount of toluidine blue absorbed into the extracellular matrix of chondrocytes was measured by spectrophotometry. Aculture technique with cellophane films was devised to establish a suitable microenvironment for culture. When mesenchymal cells were cultured by thistechnique, chondrogenesis was much greater than in usual cultures without cellophane films. Examinations of differences between cultures with and without films indicated that cellophane films promoted differentiation of mesenchymal cells, by the accumulation of some effective factor(s) in culture medium.

Chondrogenesis in cultures of endosteum

In earlier work on the histogenesis of the fowl skeleton (Strangeways and Fell, 1926; Fell, 1928), it was shown that nodules of small-celled, non-ossifying cartilage developed very readily in cultures of limb-bud mesoderm and that in very rare cases (Fell, 1928) the chondroblasts hypertrophied and ossification occurred. More recently (Fell, 1932) similar nodules of small-celled cartilage were found to develop in cultures of early (6-day) embryonic periosteum. Studitsky (1932), using the method of chorio-allantoic grafting, has obtained chondrogenesis in grafts of later (15-19 day) embryonic periosteum, but he does not specify what type of cartilage developed. In previous experiments (Fell, 1932) cultures in vitro of endosteum from the tibiæ of hatched chicks always produced bone or osteoid tissue but chondrogenesis was not observed. In the course of later investigations, however, some striking examples of the formation of hypertrophic cartilage in endosteal cultures were obtained, and the present communication describes the development of such cartilage, and its subsequent direct transformation into osteoid tissue or bone.