Effects of bisphosphonates on different zones of the epiphyseal growth plate of rats

Bisphosphonates (BIS) are indicated for several clinical disorders (e.g., osteoporosis). However, BIS has been associated with osteonecrosis and alterations in osteoclastogenesis and skeletal development. This study aimed to evaluate the effects of BIS (zoledronic acid - ZA and alendronate sodium - AS) on zones of the growth plate of rat femur. Animals (Wistar rats, n = 19) were divided into groups: 1) AS Group: animals received alendronate sodium orally (3 mg/kg per day); 2) ZA Group: ZA was administered intraperitoneally (0.2 mg/kg per week); and 3) Control Group (CG): a vehicle was administered. Animals were euthanized 21 days after the treatment, and femurs were collected for histological analysis. The images of all zones (resting, proliferative, hypertrophic, and calcified) were processed by the Qcapture® software providing a 40 and 400-fold increase. ZA decreased epiphyseal growth plate cell zones (ZA Group vs. CG) in most cases. Likewise, AS diminished the proliferative zone (AS Group vs. CG). Furthermore, ZA increased the calcified zone (ZA Group vs. CG). Previous works demonstrated that BIS decrease the epiphyseal disc. This reduction is probably due to the shortening of the cellular zones that undergoes calcification/ossification. The present results suggest that BIS should be carefully indicated because these drugs might accelerate epiphyseal closure.

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Local Changes to the Distal Femoral Growth Plate Following Injury in Mice

Journal of Biomechanical Engineering ◽

10.1115/1.4036686 ◽

2017 ◽

Vol 139 (7) ◽

Cited By ~ 2

Author(s):

Lauren M. Mangano Drenkard ◽

Meghan E. Kupratis ◽

Katie Li ◽

Louis C. Gerstenfeld ◽

Elise F. Morgan

Keyword(s):

Growth Plate ◽

Skeletal Development ◽

Global Changes ◽

Mineral Density ◽

Injury Site ◽

Hypertrophic Zone ◽

Proliferative Zone ◽

Spatial Changes ◽

Zonal Organization ◽

Growth Plate Injury

Injury to the growth plate is associated with growth disturbances, most notably premature cessation of growth. The goal of this study was to identify spatial changes in the structure and composition of the growth plate in response to injury to provide a foundation for developing therapies that minimize the consequences for skeletal development. We used contrast-enhanced microcomputed tomography (CECT) and histological analyses of a murine model of growth plate injury to quantify changes in the cartilaginous and osseous tissue of the growth plate. To distinguish between local and global changes, the growth plate was divided into regions of interest near to and far from the injury site. We noted increased thickness and CECT attenuation (a measure correlated with glycosaminoglycan (GAG) content) near the injury, and increased tissue mineral density (TMD) of bone bridges within the injury site, compared to outside the injury site and contralateral growth plates. Furthermore, we noted disruption of the normal zonal organization of the physis. The height of the hypertrophic zone was increased at the injury site, and the relative height of the proliferative zone was decreased across the entire injured growth plate. These results indicate that growth plate injury leads to localized disruption of cellular activity and of endochondral ossification. These local changes in tissue structure and composition may contribute to the observed retardation in femur growth. In particular, the changes in proliferative and hypertrophic zone heights seen following injury may impact growth and could be targeted when developing therapies for growth plate injury.

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Ultrastructural analysis of cytochrome oxidase in chick epiphyseal growth plate cartilage.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.9.2841373 ◽

1988 ◽

Vol 36 (9) ◽

pp. 1161-1166 ◽

Cited By ~ 4

Author(s):

T Yamamoto ◽

C V Gay

Keyword(s):

Cytochrome Oxidase ◽

Growth Plate ◽

Oxidative Metabolism ◽

Anaerobic Metabolism ◽

Epiphyseal Growth Plate ◽

Growth Plate Cartilage ◽

Proliferative Zone ◽

Relationship Of ◽

The Relationship ◽

Aerobic And Anaerobic

Histochemical detection of cytochrome oxidase activity in chicken growth plate revealed both positively and negatively stained mitochondria in chondrocytes of all zones, i.e., proliferative, pre-hypertrophic, hypertrophic, and calcifying zones. The proportion of positive to negative cells was lowest in the proliferative zone. As cytodifferentiation progressed, more positively stained cells were present. In positive cells all mitochondria were usually stained, and in negative cells all mitochondria were unstained. A few cells appeared to be in transition and contained both types of mitochondria. The results indicate that chondrocytes utilizing both aerobic and anaerobic metabolism are present in growth plate cartilage and that oxidative metabolism is favored in the more mature cells. The relationship of oxidative metabolism to calcification is discussed.

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IGF and IGF-binding protein expression in the growth plate of normal, dexamethasone-treated and human IGF-II transgenic mice

Journal of Endocrinology ◽

10.1677/joe.0.1750143 ◽

2002 ◽

Vol 175 (1) ◽

pp. 143-153 ◽

Cited By ~ 29

Author(s):

JJ Smink ◽

JG Koster ◽

MG Gresnigt ◽

R Rooman ◽

JA Koedam ◽

...

Keyword(s):

Transgenic Mice ◽

Growth Plate ◽

Growth Retardation ◽

Local Level ◽

Fold Increase ◽

Total Width ◽

Mrna Levels ◽

Growth Plates ◽

Proliferative Zone ◽

Igf I

Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

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Spaceflight and age affect tibial epiphyseal growth plate histomorphometry

Journal of Applied Physiology ◽

10.1152/jappl.1992.73.2.s19 ◽

1992 ◽

Vol 73 (2) ◽

pp. S19-S25 ◽

Cited By ~ 31

Author(s):

D. Montufar-Solis ◽

P. J. Duke ◽

G. Durnova

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Ultrastructural Level ◽

Cell Number ◽

Epiphyseal Growth Plate ◽

Growth Plates ◽

Proliferative Zone ◽

Old Rats ◽

Modifying Effect ◽

Matrix Organization

Growth plate histomorphometry of rats flown aboard the Soviet biosatellite COSMOS 2044, a 14-day spaceflight, was compared with that of control groups. In growth plates of flight animals, there was a significant increase in cell number per column and height of the proliferative zone and a reduction in height and cell number in the hypertrophy/calcification zone. No significant differences were found in matrix organization at the ultrastructural level of flight animals, indicating that although spaceflight continues to affect bone growth of 15-wk-old rats, extracellular matrix is not altered in the same manner as seen previously in younger animals. All groups showed growth plate characteristics attributed to aging: lack of calcification zone, reduced hypertrophy zone, and unraveling of collagen fibrils. Tail-suspended controls did not differ from other controls in any of the parameters measured. Our results suggest that growth plates of older rats are less responsive to unloading by spaceflight or suspension than those of younger rats and provide new evidence about the modifying effect of spaceflight on the growth plate.

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STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2

Development ◽

10.1242/dev.128.11.2119 ◽

2001 ◽

Vol 128 (11) ◽

pp. 2119-2129 ◽

Cited By ~ 4

Author(s):

Malika Sahni ◽

Regina Raz ◽

J. Douglas Coffin ◽

David Levy ◽

Claudio Basilico

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Fgf Signaling ◽

Intramembranous Ossification ◽

Epiphyseal Growth Plate ◽

Growth Plate Chondrocytes ◽

Chondrocyte Proliferation ◽

Transgenic Mouse Line ◽

Proliferative Zone ◽

Significant Correction

Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and we have previously shown that the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. We provide genetic evidence that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. We crossed Stat1−/− mice with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, which affect endochondral and intramembranous ossification. We found that the chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice led to a significant correction of the chondrodysplasic phenotype, but did not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, were restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induced apoptosis in calvarial osteoblasts that was not, however, corrected by the absence of STAT1. Detailed analysis of Stat1−/− growth plates uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, that attenuated as the animals grew older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate.

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Chondroadherin expression changes in skeletal development

Biochemical Journal ◽

10.1042/bj3300549 ◽

1998 ◽

Vol 330 (1) ◽

pp. 549-557 ◽

Cited By ~ 41

Author(s):

Zhenxin SHEN ◽

Svetlana GANTCHEVA ◽

Bengt MÅNSSON ◽

Dick HEINEGÅRD ◽

Yngve SOMMARIN

Keyword(s):

Articular Cartilage ◽

Femoral Head ◽

Growth Plate ◽

Blot Analysis ◽

Skeletal Development ◽

Fold Increase ◽

Long Bone ◽

Rat Tissues ◽

Cell Binding ◽

Head Development

Chondroadherin is a cartilage protein with cell binding properties. The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development. For design of oligonucleotide probes and primers a 1664 bp, full length, rat chondroadherin cDNA was isolated from a rat chondrosarcoma library and sequenced. Northern blot analysis showed chondroadherin mRNA to be present in femoral head and rib cartilage, as well as in tendon. More sensitive reverse-transcriptase PCR additionally identified the mRNA in calvaria, long bone and bone marrow. Localization of chondroadherin by immunocytochemistry in the developing femoral head from postnatal day 14 to day 60 showed presence of the protein in cartilaginous regions. With increasing age a very distinct localization of chondroadherin was seen in the territorial matrix around late proliferative cells in the growth plate as well as in the developing articular cartilage in the maturing femoral head. Localization of chondroadherin mRNA by in situ hybridization was in agreement with immunocytochemistry with strong hybridization signals in late proliferative cells in the growth plate. In the articular cartilage the expression was restricted to cells in the lower regions. A three-fold increase of cartilage chondroadherin content in the growing femoral head was demonstrated by Western blot analysis. The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation.

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Localization of estrogen receptors-alpha and -beta and androgen receptor in the human growth plate at different pubertal stages

Journal of Endocrinology ◽

10.1677/joe.0.1770319 ◽

2003 ◽

Vol 177 (2) ◽

pp. 319-326 ◽

Cited By ~ 82

Author(s):

O Nilsson ◽

D Chrysis ◽

O Pajulo ◽

A Boman ◽

M Holst ◽

...

Keyword(s):

Androgen Receptor ◽

Growth Plate ◽

Pubertal Development ◽

Human Growth ◽

Growth Spurt ◽

Image Analysis System ◽

Epiphyseal Growth Plate ◽

Pubertal Growth ◽

Proliferative Zone ◽

Pubertal Stages

Sex steroids are required for a normal pubertal growth spurt and fusion of the human epiphyseal growth plate. However, the localization of sex steroid receptors in the human pubertal growth plate remains controversial. We have investigated the expression of estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR) in biopsies of proximal tibial growth plates obtained during epiphyseal surgery in 16 boys and eight girls. All pubertal stages were represented (Tanner stages 1-5). ERalpha, ERbeta and AR were visualized with immunohistochemistry and the number of receptor-positive cells was counted using an image analysis system. Percent receptor-positive chondrocytes were assessed in the resting, proliferative and hypertrophic zones and evaluated for sex differences and pubertal trends. Both ERalpha- and ERbeta-positive cells were detected at a greater frequency in the resting and proliferative zones than in the hypertrophic zone (64+/-2%, 64+/-2% compared with 38+/-3% for ERalpha, and 63+/-3%, 66+/-3% compared with 53+/-3% for ERbeta), whereas AR was more abundant in the resting (65+/-3%) and hypertrophic zones (58+/-3%) than in the proliferative zone (41+/-3%). No sex difference in the patterns of expression was detected. For ERalpha and AR, the percentage of receptor-positive cells was similar at all Tanner pubertal stages, whereas ERbeta showed a slight decrease in the proliferative zone during pubertal development (P<0.05). In summary, our findings suggest that ERalpha, ERbeta and AR are expressed in the human growth plate throughout pubertal development, with no difference between the sexes.

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GPR30 Estrogen Receptor Expression in the Growth Plate Declines as Puberty Progresses

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2007-0814 ◽

2007 ◽

Vol 92 (12) ◽

pp. 4873-4877 ◽

Cited By ~ 38

Author(s):

Andrei S. Chagin ◽

Lars Sävendahl

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Receptor Expression ◽

Skeletal Maturation ◽

Tall Stature ◽

Leg Length ◽

Epiphyseal Growth Plate ◽

Growth Plates ◽

Longitudinal Bone Growth ◽

Proliferative Zone

Abstract Objective: Our objective was to study whether GPR30 is expressed in the epiphyseal growth plate and its potential role as a modulator of pubertal growth. Background: Estrogens play a crucial role in the regulation of skeletal maturation and longitudinal bone growth. We have previously shown that both estrogen receptors (ERs) α and β are expressed in the human epiphyseal growth plate. Recently, a membrane-bound ER referred to as GPR30 was discovered, but the role played by this receptor in the regulation of longitudinal bone growth is not yet known. Patients/Methods: Biopsies were collected from the tibial growth plates of 14 boys and seven girls that underwent epiphyseal surgery to arrest longitudinal bone growth. The patients were in different stages of puberty and suffered from inequality in leg length or extreme tall stature. Paraffin-embedded sections of the growth plates were used to detect expression of the GPR30 protein. Results: The highest level of GPR30 expression was observed in hypertrophic chondrocytes, although cells with positive immunostaining were also detected in the resting zone. In contrast, no immunoreactivity was found in the proliferative zone. During pubertal progression there was a clear decline in the level of GPR30 expression in both boys and girls. Conclusions: The novel ER GPR30 is expressed in the human growth plate, and the level of expression declines during pubertal progression. Although a relationship between GPR30 expression and age may underlie the observed pubertal decline in the GPR30 level, our observations suggest that this receptor could be involved in the modulation of longitudinal bone growth during puberty.

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UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis

Biochemical Journal ◽

10.1042/bj3600667 ◽

2001 ◽

Vol 360 (3) ◽

pp. 667-674 ◽

Cited By ~ 15

Author(s):

Cordula MAGEE ◽

Maria NURMINSKAYA ◽

Thomas F. LINSENMAYER

Keyword(s):

Growth Plate ◽

Fold Increase ◽

Subtractive Hybridization ◽

Enzymic Activity ◽

Hypertrophic Chondrocytes ◽

Epiphyseal Growth Plate ◽

Competitive Binding Assay ◽

Hypertrophic Cartilage ◽

Ha Synthase ◽

Cellular Components

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2–3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.

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Effects of Hypophysectomy on the Occurrence and Calcifiability of Matrix Vesicles in the Epiphyseal Growth Plate of Immature Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002579 ◽

1980 ◽

Vol 38 ◽

pp. 578-579

Author(s):

S. I. Coleman ◽

W. J. Dougherty

Keyword(s):

Growth Plate ◽

Matrix Vesicles ◽

Epiphyseal Growth Plate ◽

Hard Tissue ◽

Hormonal Factors ◽

Post Surgery ◽

Mineral Deposition ◽

Thin Sectioning ◽

Integral Role ◽

Cellular Secretion

In the cellular secretion theory of mineral deposition, extracellular matrix vesicles are believed to play an integral role in hard tissue mineralization (1). Membrane limited matrix vesicles arise from the plasma membrane of epiphyseal chondrocytes and tooth odontoblasts by a budding process (2, 3). Nutritional and hormonal factors have been postulated to play essential roles in mineral deposition and apparently have a direct effect on matrix vesicles of calcifying cartilage as concluded by Anderson and Sajdera (4). Immature (75-85 gm) Long-Evans hooded rats were hypophysectomized by the parapharyngeal approach and maintained fourteen (14) days post-surgery. At this time, the animals were anesthetized and perfusion fixed in cacodylate buffered 2.5% glutaraldehyde. The proximal tibias were quickly dissected out and split sagittally. One half was used for light microscopy (LM) and the other for electron microscopy (EM). The halves used for EM were cut into blocks approximately 1×3 mm. The tissue blocks were prepared for ultra-thin sectioning and transmission EM. The tissue was oriented so as to section through the epiphyseal growth plate from the zone of proliferating cartilage on down through the hypertrophic zone and into the initial trabecular bone. Sections were studied stained (double heavy metal) and unstained.

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