Mutations in fam20b and xylt1 Reveal That Cartilage Matrix Controls Timing of Endochondral Ossification by Inhibiting Chondrocyte Maturation

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be transplanted into tissue defect site with no artificial scaffold. Importantly, most bone formation in the developing process or fracture healing proceeds via endochondral ossification. Accordingly, this present study investigated whether C-MSCs generated with chondro-inductive medium (CIM) can induce successful bone regeneration and assessed its healing process. Human bone marrow-derived MSCs were cultured with xeno-free/serum-free (XF) growth medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The cell clumps, i.e., C-MSCs, were maintained in XF-CIM. C-MSCs generated with XF-CIM showed enlarged round cells, cartilage matrix, and hypertrophic chondrocytes genes elevation in vitro. Transplantation of C-MSCs generated with XF-CIM induced successful bone regeneration in the SCID mouse calvaria defect model. Immunofluorescence staining for human-specific vimentin demonstrated that donor human and host mouse cells cooperatively contributed the bone formation. Besides, the replacement of the cartilage matrix into bone was observed in the early period. These findings suggested that cartilaginous C-MSCs generated with XF-CIM can induce bone regeneration via endochondral ossification.

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Skeletal Malformations Caused by Overexpression of Cbfa1 or Its Dominant Negative Form in Chondrocytes

The Journal of Cell Biology ◽

10.1083/jcb.153.1.87 ◽

2001 ◽

Vol 153 (1) ◽

pp. 87-100 ◽

Cited By ~ 267

Author(s):

Chisato Ueta ◽

Masahiro Iwamoto ◽

Naoko Kanatani ◽

Carolina Yoshida ◽

Yang Liu ◽

...

Keyword(s):

Transgenic Mice ◽

Endochondral Ossification ◽

Skeletal Development ◽

Type Ii Collagen ◽

Dominant Negative ◽

Chondrocyte Maturation ◽

Skeletal Malformations ◽

Dominant Negative Form ◽

Cellular Phenotypes

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor α1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.

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Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611771114 ◽

2017 ◽

Vol 114 (10) ◽

pp. 2556-2561 ◽

Cited By ~ 19

Author(s):

Johnathan J. Ng ◽

Yiyong Wei ◽

Bin Zhou ◽

Jonathan Bernhard ◽

Samuel Robinson ◽

...

Keyword(s):

Articular Cartilage ◽

Endochondral Ossification ◽

Human Articular Cartilage ◽

Control Groups ◽

Human Cartilage ◽

Chondrocyte Maturation ◽

Self Assembling ◽

Spatiotemporal Regulation

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage.

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Runx2 Regulates Endochondral Ossification in Condyle during Mandibular Advancement

Journal of Dental Research ◽

10.1177/154405910508400211 ◽

2005 ◽

Vol 84 (2) ◽

pp. 166-171 ◽

Cited By ~ 37

Author(s):

G.H. Tang ◽

A.B.M. Rabie

Keyword(s):

Endochondral Ossification ◽

Mandibular Condyle ◽

Mandibular Advancement ◽

New Bone Formation ◽

Sprague Dawley ◽

Condylar Cartilage ◽

Runx2 Expression ◽

Chondrocyte Maturation ◽

Sprague Dawley Rats ◽

Functional Appliances

Runx2 is a transcription factor prerequisite for chondrocyte maturation and osteoblast differentiation. We tested the hypothesis that Runx2 is responsible for signaling chondrocyte maturation and endochondral ossification in the condyle during mandibular advancement. Fifty 35-day-old Sprague-Dawley rats were fitted with functional appliances for 3, 7, 14, 21, and 30 days. Experimental animals with 50 matched controls were labeled with bromodeoxyuridine for evaluation of the invasion of chondroclasts and osteoblasts into condylar cartilage. Mandibular advancement elicited Runx2 expression in condylar cartilage, and subsequently led to an expansion of type X collagen domain in the hypertrophic layer. Stronger Runx2 mRNA signals in subchondral bone corresponded with the increase in the recruitment of osteoblasts and chondroclasts, which preceded the increase of new bone formation in the condyle. Thus, Runx2 mediates chondrocyte terminal maturation and endochondral ossification in the mandibular condyle in response to mandibular advancement.

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Sox9 Family Members Negatively Regulate Maturation and Calcification of Chondrocytes through Up-Regulation of Parathyroid Hormone–related Protein

Molecular Biology of the Cell ◽

10.1091/mbc.e09-03-0227 ◽

2009 ◽

Vol 20 (21) ◽

pp. 4541-4551 ◽

Cited By ~ 48

Author(s):

Katsuhiko Amano ◽

Kenji Hata ◽

Atsushi Sugita ◽

Yoko Takigawa ◽

Koichiro Ono ◽

...

Keyword(s):

Parathyroid Hormone ◽

Endochondral Ossification ◽

Family Members ◽

Chondrocyte Differentiation ◽

Related Protein ◽

Primary Chondrocytes ◽

Chondrocyte Maturation ◽

Parathyroid Hormone Related Protein ◽

Pthrp Expression ◽

Late Stages

Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. However, the molecular mechanisms underlying the regulation of chondrocyte maturation and calcification by Sox9 remain unknown. In this study, we attempted to clarify roles of Sox9 in the late stages of chondrocyte differentiation. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone–related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling.

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Distinct requirements of wls , wnt9a , wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

Developmental Biology ◽

10.1016/j.ydbio.2016.11.016 ◽

2017 ◽

Vol 421 (2) ◽

pp. 219-232 ◽

Cited By ~ 13

Author(s):

Irving TC Ling ◽

Lucie Rochard ◽

Eric C. Liao

Keyword(s):

Endochondral Ossification ◽

Chondrocyte Maturation

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Effect of Diet on Longitudinal Bone Growth and Osteochondrosis in Swine

Veterinary Pathology ◽

10.1177/030098588702400202 ◽

1987 ◽

Vol 24 (2) ◽

pp. 109-117 ◽

Cited By ~ 18

Author(s):

J. C. Woodard ◽

H. N. Becker ◽

P. W. Poulos

Keyword(s):

Articular Cartilage ◽

Growth Plate ◽

Radial Growth ◽

Endochondral Ossification ◽

Bone Growth ◽

Cartilage Matrix ◽

Protein Diet ◽

Cell Production ◽

Proliferative Zone ◽

Daily Rate

Weanling gilts were fed either a 12% or 16% protein diet for 10 weeks. Animals fed the 12% protein diet had reduced body weights and reduced longitudinal bone growth as measured in the distal radial growth plate. There was no difference in the growth plate widths between the two animal groups, but there was a significant reduction in the daily rate of cell production in the proliferative zone of animals fed the 12% protein diet. No effect of diet on the rate of expansion of the epiphysis at the articular-epiphyseal junction of the distal femur or humerus could be detected. All animals in both groups had morphologic cartilage lesions consistent with early changes associated with osteochondrosis (OCD), and there was no difference in the lesion morphology between the dietary groups. Areas of disorderly endochondral ossification in the radial growth plate were associated with perpendicular growth cartilage infractions. Growth plate lesions were characterized by increased widths of the maturing cartilage zone without increased width of the proliferative zone or an increase in the daily rate of cell production. Focal growth plate lesions developed because of a transitory inhibition of cartilage mineralization and resorption. Disorderly foci of endochondral ossification beneath articular cartilage were characterized by an area of chondrocyte necrosis which prevented normal cartilage matrix mineralization. Lamellae of cartilage necrosis were also present within the reserve zone of the articular cartilage. These were associated with abnormalities of the cartilage canal vessels, and chondrocyte necrosis was considered to precede degenerative changes in articular cartilage matrix. Since similar changes were rarely seen in the distal metacarpal (tarsal) bones of the dew claws, endogenous trauma was considered an important factor in lesion pathogenesis. The possibility that a basic cartilage matrix abnormality might represent the primary cause of OCD is discussed.

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Simultaneous differentiation of articular and transient cartilage: WNT-BMP interplay and its therapeutic implication

The International Journal of Developmental Biology ◽

10.1387/ijdb.190149ab ◽

2020 ◽

Vol 64 (1-2-3) ◽

pp. 203-211

Author(s):

Tathagata Biswas ◽

Akrit P. Jaswal ◽

Upendra S. Yadav ◽

Amitabha Bandyopadhyay

Keyword(s):

Tissue Engineering ◽

Developmental Biology ◽

Endochondral Ossification ◽

Recent Literature ◽

New Paradigm ◽

Treatment Of Osteoporosis ◽

Chondrocyte Maturation ◽

Different Types ◽

Insight Into ◽

And Cartilage

Limb skeleton forms through the process of endochondral ossification. This process of osteogenesis proceeds through an intermediate cartilage template and involves several stages of chondrocyte maturation and eventual bone formation. During the process of endochondral ossification, interplay between BMP and WNT signaling regulate simultaneous differentiation of articular and transient cartilage. In this review, we focus on the recent literature which explores the simultaneous differentiation of these two different types of cartilage. We discuss a new paradigm of developmental biology-inspired tissue engineering of bone and cartilage grafts and provide novel insight into treatment of osteoporosis.

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TBX1 Regulates Chondrocyte Maturation in the Spheno-occipital Synchondrosis

Journal of Dental Research ◽

10.1177/0022034520925080 ◽

2020 ◽

Vol 99 (10) ◽

pp. 1182-1191 ◽

Cited By ~ 1

Author(s):

N. Funato ◽

D. Srivastava ◽

S. Shibata ◽

H. Yanagisawa

Keyword(s):

Digeorge Syndrome ◽

Endochondral Ossification ◽

Transcriptional Activity ◽

Ectopic Expression ◽

Chondrocyte Differentiation ◽

Craniofacial Skeleton ◽

Chondrocyte Maturation ◽

Expression Of Genes ◽

Growth Center ◽

Deficient Mice

The synchondrosis in the cranial base is an important growth center for the craniofacial region. Abnormalities in the synchondroses affect the development of adjacent regions, including the craniofacial skeleton. Here, we report that the transcription factor TBX1, the candidate gene for DiGeorge syndrome, is expressed in mesoderm-derived chondrocytes and plays an essential and specific role in spheno-occipital synchondrosis development by inhibiting the expression of genes involved in chondrocyte hypertrophy and osteogenesis. In Tbx1-deficient mice, the spheno-occipital synchondrosis was completely mineralized at birth. TBX1 interacts with RUNX2, a master molecule of osteoblastogenesis and a regulator of chondrocyte maturation, and suppresses its transcriptional activity. Indeed, deleting Tbx1 triggers accelerated mineralization due to accelerated chondrocyte differentiation, which is associated with ectopic expression of downstream targets of RUNX2 in the spheno-occipital synchondrosis. These findings reveal that TBX1 acts as a regulator of chondrocyte maturation and osteogenesis during the spheno-occipital synchondrosis development. Thus, the tight regulation of endochondral ossification by TBX1 is crucial for the normal progression of chondrocyte differentiation in the spheno-occipital synchondrosis.

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Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3656-3668.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3656-3668 ◽

Cited By ~ 28

Author(s):

Blanca Scheijen ◽

Marieke Bronk ◽

Tiffany van der Meer ◽

René Bernards

Keyword(s):

Bone Formation ◽

Endochondral Ossification ◽

Bone Growth ◽

Late Phase ◽

Type Ii Collagen ◽

Endochondral Bone Formation ◽

Nodule Formation ◽

Chondrocyte Differentiation ◽

Endochondral Bone ◽

Chondrocyte Maturation

ABSTRACT Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.

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