The Chondroblast and the Chondrocyte

227

128

Abstract. As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen m R N A occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II coUagen m R N A occurs which parallels the progressive accumulation of cartilage matrix by ceils. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen m R N A are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. T HE onset of cartilage differentiation in the developinglimb is characterized by a transient cellular condensation or aggregation process in which prechondrogenic mesenchymal cells become closely juxtaposed to one another before initiating cartilage matrix deposition. During this process, a cell-cell interaction, cell shape change, or some other event occurs which is necessary to trigger the chondrogenic differentiation of the cells (15). The critical condensation process may be initiated, at least in part, by a progressive decrease in the accumulation of extracellular hyaluronate (20,35). Fibronectin and type I collagen have been implicated in the cell-cell interaction occurring during condensation (7, 21, 34). Prostaglandin-mediated elevations in cAMP levels during condensation are involved in regulating chondrogenesis (3, 6, 11, 14, 16-18, 32, 33). A change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration also plays an important role in the process (2, 41).Although considerable insight has been gained into the extracellular influences and intracellular regulatory molecules that are involved in regulating limb chondrogenesis, virtually nothing is known about the molecular mechanisms by which these factors directly influence the changes in gene activity that occur during cartilage differentiation. Prechondrogenic limb mesenchymal cells synthesize type I collagen (36). As the cells differentiate into chondrocytes, they initiate the synthesis of cartilage-characteristic type II collagen and cease synthesizing type I collagen (36). Thus the conversion of mesenchymal chondrogenic progenitor cells into chondrocytes i...

Section: T He Onset Of Cartilage Differentiation In the Developingmentioning

confidence: 99%

Collagen gene expression during limb cartilage differentiation.

Kosher¹,

Kulyk²,

Gay³

1986

227

128

“…URING the organogenesis of the chick embryo tibiae, mesenchymal cells developing into hypertrophic chondrocytes pass through at least three distinct differentiation stages: (a) committed mesenchymal cells (20); (b) proliferating (stage I) chondrocytes (18,20); and (c) hypertrophic (stage II) chondrocytes (6,19,24). Chondrocytes from Hamburger and Hamilton stage 28-30 (17) chick embryo tibiae dedifferentiate when cultured on plastic dishes.…”

mentioning

confidence: 99%

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

Quarto

Dozin

Tacchetti

et al. 1990

Abstract. Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of ,,o13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while ,x,50% of them showed positivity for type II collagen. (6,19,24). Chondrocytes from Hamburger and Hamilton stage 28-30 (17) chick embryo tibiae dedifferentiate when cultured on plastic dishes. These cells, as well as dedifferentiated chondrocytes from another source (4), maintain the dedifferentiated phenotype as long as the conditions for the cell attachment to the plastic are kept. When the same cells are transferred into suspension culture, they resume the chondrocytic phenotype and mature to hypertrophic chondrocytes through a transient stage of cell aggregation (7). This process is accompanied by an increase in the length of the cell cycle and in the number of quiescent and degenerating cells (16), as well as by a modulation in the transcriptional activity of the collagen genes (9). We have recently reported the identification, purification, and characterization of a novel, low molecular weight protein, named Ch21, expressed and secreted by the in vitro differentiating chondrocytes at a late stage of development (11). When dedifferentiated cells are cultured in suspension in the constant presence of ascorbic acid, they organize their own extracellular matrix and develop into a tissue closely resembling calcifying hypertrophic cartilage (28,29).The heterogeneity of the dedifferentiated cell population did not allow us to definitely state whether the cells followed successive stages of differentiation or the culture conditions made a selection of cell subpopulations. To answer this important question we selected clones of dedifferentiated cells to follow the respective behavior of each clone in terms of morphology and collagen phenotype in the different culture conditions. We show that up to 45 % of the cell populations obtained from single cloned dedifferentiated cells are still able to differentiate to hypertrophic chondrocytes and to reconstitute hyp...

“…After birth, similar events take place in the long bone growth plate and at the bone fracture sites. Bone formation and remodeling have been extensively investigated, starting from pioneering work describing the morphological and biochemical changes occurring during early bone formation to more recent studies aimed at the elucidations of the cellular and molecular mechanisms involved (7,22,34) . It is widely agreed that cells present in a continuous collar surrounding, but separated from the cartilage rudiment, give rise to osteoblasts, i.e., cells responsible for the synthesis and mineralization of the osteoid extracellular matrix .…”

mentioning

confidence: 99%

Hypertrophic chondrocytes undergo further differentiation in culture

Cancedda

Gentili

Manduca

et al. 1992

190

Abstract. Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae . Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda . 1986. J. Cell Biol. 102 :2310-2317, when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized . Type II and X collagen synthesis was halted and replaced by type I collagen synthesis . In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain oNG bone organogenesis occurs in the embryo by endochondral ossification from undifferentiated mesenchyme. During the early stages of development, mesenchymal cells in the limb buds condense to form a core of differentiated chondrocytes ; osteogenesis starts at the periphery ofthe cartilage core, which is subsequently invaded by blood vessels and replaced by bone marrow and trabecular bone. After birth, similar events take place in the long bone growth plate and at the bone fracture sites. Bone formation and remodeling have been extensively investigated, starting from pioneering work describing the morphological and biochemical changes occurring during early bone formation to more recent studies aimed at the elucidations of the cellular and molecular mechanisms involved (7,22,34) . It is widely agreed that cells present in a continuous collar surrounding, but separated from the cartilage rudiment, give rise to osteoblasts, i.e., cells responsible for the synthesis and mineralization of the osteoid extracellular matrix . In the past, occasionally and recently more frequently, it has been postulated thatgrowth platehypertrophic chondrocytes might also contribute to the formation of a bone matrix, since in some organ culturesthese cells start to express bone markers . During culture of mouse mandibular condyles, the expression of type I collagen, osteonectin, alkaline phosphatase, osteopontin, and osteocalcin by mature chondrocytes was detected by in situ hybridization (38). A morphological study © The Rockefeller University Press, 0021-9525/92/04/427/9 $2 .00 The Journal of Cell Biology, Volume 117, Number 2, April 1992 427-435 collagenous domains, and is glycosylated . The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture . We have defined this additional differentiation stage as an osteoblast-like stage . Calcium deposition in the extracellular matrix occurred regardless of the addition of ß glycerophosphate to the culture medium . Comparable results were obtained both when the cells were plated at low density and when they were alrea...