Macromolecular organization of chicken type X collagen in vitro.

The macromolecular structure of type X collagen in the matrices of primary cultures of chick hypertrophic chondrocytes was initially investigated using immunoelectron microscopy. Type X collagen was observed to assemble into a matlike structure with-in the matrix elaborated by hypertrophic chondrocytes. The process of self assembly was investigated at the molecular level using purified chick type X collagen and rotary-shadowing EM. It was shown that under neutral conditions at 34 degrees C, individual type X collagen molecules associate rapidly into multimeric clusters via their carboxy-terminal globular domains forming structures with a central nodule of carboxy-terminal domains and the triple helices radiating outwards. Prolonged incubation resulted in the formation of a regular hexagonal lattice by lateral association of the juxtaposed triple-helical domains from adjacent multimeric clusters. This extended lattice may play an important role in modifying the cartilage matrix for subsequent events occurring in endochondral bone formation.

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c-fos expression precedes osteogenic differentiation of cartilage cells in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.111.3.1313 ◽

1990 ◽

Vol 111 (3) ◽

pp. 1313-1323 ◽

Cited By ~ 50

Author(s):

E I Closs ◽

A B Murray ◽

J Schmidt ◽

A Schön ◽

V Erfle ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Osteoblastic Differentiation ◽

Hypertrophic Chondrocytes ◽

Endochondral Bone Formation ◽

In Vitro Cultivation ◽

Endochondral Bone ◽

Expression Of Genes ◽

Cartilage Cells ◽

Fos Expression

We have investigated the temporal pattern of expression of c-fos in cartilage cells in mouse mandibular condyles. During in vitro cultivation, the progenitor cells in this organ differentiate to osteoblasts, and hypertrophic chondrocytes start to show features indicative of osteogenic differentiation. Prior to these processes we observed two distinct patterns of c-fos expression. High, transient c-fos expression was found in the entire tissue within 30 min of culture. This type of c-fos expression appeared to result from mechanical forces applied during dissection. The second type of c-fos expression appeared in individual cells in the zone of hypertrophic chondrocytes. A varying number of formerly quiescent chondrocytes expressed high levels of c-fos mRNA after between 30 min and 10 d in culture, with a peak in the number of cells between days 1 and 3. c-fos expression in these cartilage cells was followed by DNA replication and expression of genes typifying osteoblastic differentiation. After 7 d in culture, groups of cells with the typical ultrastructural features of osteoblasts, and surrounded by an osteoid-like matrix, were observed in single chondrocyte-type lacunae, suggesting division of chondrocytes and differentiation to osteoblasts. The data suggest that c-fos may play a crucial role in the perturbation of determined pathways of skeletoblast differentiation and in the regulation of endochondral bone formation.

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Domains of type X collagen: alteration of cartilage matrix by fibril association and proteoglycan accumulation.

The Journal of Cell Biology ◽

10.1083/jcb.117.3.687 ◽

1992 ◽

Vol 117 (3) ◽

pp. 687-694 ◽

Cited By ~ 25

Author(s):

Q Chen ◽

C Linsenmayer ◽

H Gu ◽

T M Schmid ◽

T F Linsenmayer

Keyword(s):

Endochondral Bone Formation ◽

Type X Collagen ◽

Endochondral Bone ◽

Marrow Cavity ◽

Hypertrophic Cartilage ◽

Component Type ◽

The Matrix ◽

Collagenous Domain

During endochondral bone formation, hypertrophic cartilage is replaced by bone or by a marrow cavity. The matrix of hypertrophic cartilage contains at least one tissue-specific component, type X collagen. Structurally type X collagen contains both a collagenous domain and a COOH-terminal non-collagenous one. However, the function(s) of this molecule have remained largely speculative. To examine the behavior and functions of type X collagen within hypertrophic cartilage, we (Chen, Q., E. Gibney, J. M. Fitch, C. Linsenmayer, T. M. Schmid, and T. F. Linsenmayer. 1990. Proc. Natl. Acad. Sci. USA. 87:8046-8050) recently devised an in vitro system in which exogenous type X collagen rapidly (15 min to several hours) moves into non-hypertrophic cartilage. There the molecule becomes associated with preexisting cartilage collagen fibrils. In the present investigation, we find that the isolated collagenous domain of type X collagen is sufficient for its association with fibrils. Furthermore, when non-hypertrophic cartilage is incubated for a longer time (overnight) with "intact" type X collagen, the molecule is found both in the matrix and inside of the chondrocytes. The properties of the matrix of such type X collagen-infiltrated cartilage become altered. Such changes include: (a) antigenic masking of type X collagen by proteoglycans; (b) loss of the permissiveness for further infiltration by type X collagen; and (c) enhanced accumulation of proteoglycans. Some of these changes are dependent on the presence of the COOH-terminal non-collagenous domain of the molecule. In fact, the isolated collagenous domain of type X collagen appears to exert an opposite effect on proteoglycan accumulation, producing a net decrease in their accumulation, particularly of the light form(s) of proteoglycans. Certain of these matrix alterations are similar to ones that have been observed to occur in vivo. This suggests that within hypertrophic cartilage type X collagen has regulatory as well as structural functions, and that these functions are achieved specifically by its two different domains.

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Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation

Endocrinology ◽

10.1210/en.2015-1473 ◽

2016 ◽

Vol 157 (1) ◽

pp. 127-140 ◽

Cited By ~ 11

Author(s):

Shaohong Cheng ◽

Weirong Xing ◽

Sheila Pourteymoor ◽

Jan Schulte ◽

Subburaman Mohan

Keyword(s):

Bone Formation ◽

Growth Plate ◽

Prolyl Hydroxylase ◽

Conditional Knockout ◽

Hypertrophic Chondrocytes ◽

Endochondral Bone Formation ◽

Bone Surface ◽

Endochondral Bone ◽

Prolyl Hydroxylase Domain

Abstract The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

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Alterations of chondroitin sulfate synthesized by chick embryo cartilage cultured in the presence of 6-aminonicotinamide

Development ◽

10.1242/dev.59.1.207 ◽

1980 ◽

Vol 59 (1) ◽

pp. 207-216

Author(s):

Robert E. Seegmiller ◽

Allen L. Horwitz ◽

Albert Dorfman

Keyword(s):

Chondroitin Sulfate ◽

Anaerobic Metabolism ◽

Major Constituent ◽

Endochondral Bone Formation ◽

Gel Chromatography ◽

Chick Embryos ◽

Endochondral Bone ◽

Cartilage Development ◽

Integral Role

Treatment of day-4 chick embryos with 6-aminonicotinamide (6-AN) impairs limb chondrogenesis and produces micromelia. Interference with limb cartilage development may be related to decreased NAD-dependent synthesis of ATP due to the fact that chondrogenesis is dependent upon anaerobic metabolism. To better understand the effect of 6-AN on chondrogenesis, isolated cartilage epiphyses from day-11 chick embryos were treated in vitro. Sulfate incorporation into total glycosaminoglycans of treated epiphyses was 30 % of control. Incorporation of [3H]glucosamine was normal. Fractionation by gel chromatography showed that 40 % of the glycosaminoglycans synthesized by treated cells had a molecular weight of less than 15000 compared with 5 % of that of the control. A decrease in amount of chondroitin 6-sulfate, an increase of chondroitin 4-sulfate and no change in amount of unsulfated polysaccharide were observed. These results suggest that, upon exposure to 6-AN, chondrocytes produce shorter than normal chondroitin sulfate chains that are preferentially sulfated in the 4 position. Since endochondral bone formation plays an integral role in growth and development of the limb, a defect in production of chondroitin sulfate, a major constituent of cartilage matrix, appears to be involved in 6-AN-induced micromelia.

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Fetal Jaw Movement Affects Condylar Cartilage Development

Journal of Dental Research ◽

10.1177/154405910508400514 ◽

2005 ◽

Vol 84 (5) ◽

pp. 474-479 ◽

Cited By ~ 30

Author(s):

H. Habib ◽

T. Hatta ◽

J. Udagawa ◽

L. Zhang ◽

Y. Yoshimura ◽

...

Keyword(s):

Bone Formation ◽

Mandibular Condyle ◽

Hypertrophic Chondrocytes ◽

Endochondral Bone Formation ◽

Condylar Cartilage ◽

Mechanical Factor ◽

Endochondral Bone ◽

Jaw Movement ◽

Cartilage Development ◽

Number Of Cells

Using a mouse exo utero system to examine the effects of fetal jaw movement on the development of condylar cartilage, we assessed the effects of restraint of the animals’ mouths from opening, by suture, at embryonic day (E)15.5. We hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle. Condylar cartilage was reduced in size, and the bone-cartilage margin was ill-defined in the sutured group at E18.5. Volume, total number of cells, and number of 5-bromo-2′-deoxyuridine-positive cells in the mesenchymal zone were lower in the sutured group than in the non-sutured group at E16.5 and E18.5. Hypertrophic chondrocytes were larger, whereas fewer apoptotic chondrocytes and osteoclasts were observed in the hypertrophic zone in the sutured group at E18.5. Analysis of our data revealed that restricted fetal TMJ movement influences the process of endochondral bone formation of condylar cartilage.

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FGFR1 signaling in hypertrophic chondrocytes is attenuated by the Ras-GAP neurofibromin during endochondral bone formation

Human Molecular Genetics ◽

10.1093/hmg/ddv019 ◽

2015 ◽

Vol 24 (9) ◽

pp. 2552-2564 ◽

Cited By ~ 16

Author(s):

Matthew R. Karolak ◽

Xiangli Yang ◽

Florent Elefteriou

Keyword(s):

Bone Formation ◽

Hypertrophic Chondrocytes ◽

Endochondral Bone Formation ◽

Endochondral Bone

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Effects of in vitro chondrogenic priming time of bone-marrow-derived mesenchymal stromal cells on in vivo endochondral bone formation

Acta Biomaterialia ◽

10.1016/j.actbio.2014.11.029 ◽

2015 ◽

Vol 13 ◽

pp. 254-265 ◽

Cited By ~ 25

Author(s):

Wanxun Yang ◽

Sanne K. Both ◽

Gerjo J.V.M. van Osch ◽

Yining Wang ◽

John A. Jansen ◽

...

Keyword(s):

Bone Marrow ◽

Bone Formation ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Endochondral Bone Formation ◽

Endochondral Bone

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Identifying Candidate Mechanoregulators of Skeletal Development

ASME 2009 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2009-206246 ◽

2009 ◽

Author(s):

Niamh C. Nowlan ◽

Patrick J. Prendergast ◽

Shahragim Tajbakhsh ◽

Paula Murphy

Keyword(s):

Skeletal Muscle ◽

Endochondral Ossification ◽

Muscle Development ◽

Skeletal Development ◽

Endochondral Bone Formation ◽

Mechanical Forces ◽

Endochondral Bone ◽

Mutant Strains ◽

The Relationship

Studying the relationship between mechanical forces and skeletal development can provide vital clues to the mechanoregulation of skeletogenesis, providing important information to tissue engineers hoping to create functional cartilage or bone in vitro. Many studies of the mechanoregulation of skeletal development have focused on the chick embryo e.g., [1, 2]. However, as no endochondral ossification takes place in the embryonic chick long bones [1], mammalian systems must be used to examine the effect of mechanical forces on endochondral bone formation. Mouse mutant strains exist in which muscle development is affected, providing models with which to examine skeletogenesis in the absence of skeletal muscle contractions. One such strain is Pax3sp/sp [3], also known as splotch. The splotch mutant lacks the transcription factor Pax3, which prevents the migration of muscle pre-cursor cells into the limb buds, resulting in a complete absence of skeletal muscle.

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Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.

The Journal of Cell Biology ◽

10.1083/jcb.129.3.881 ◽

1995 ◽

Vol 129 (3) ◽

pp. 881-892 ◽

Cited By ~ 125

Author(s):

D Aeschlimann ◽

O Kaupp ◽

M Paulsson

Keyword(s):

Tissue Transglutaminase ◽

Physiological Mechanism ◽

Cross Linking ◽

Endochondral Bone Formation ◽

Cross Link ◽

Endochondral Bone ◽

Tracheal Cartilage ◽

Matrix Stabilization ◽

The Matrix

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.

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