Carboxyterminal telopeptide of type I collagen, ICTP, as a marker of matrix degradation in neonatal mouse calvarial bones, in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 407-411 ◽  
Author(s):  
Östen Ljunggren ◽  
Sverker Ljunghall
Keyword(s):  
Parathyroid Hormone ◽  
Bone Resorption ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Neonatal Mouse ◽  
Type I ◽  
Matrix Degradation ◽  
Dose Responses ◽  
Carboxyterminal Telopeptide

Bone resorption, in vitro, is often measured as the release of prelabelled45Ca from neonatal mouse calvarial bones, or from fetal rat long bones. In this report we describe a technique to measure the breakdown of bone-matrix, in vitro. We also describe a new way to dissect neonatal mouse calvarial bones, in order to obtain large amounts of bone samples. Twelve bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 culture medium in serum-free conditions in 0.5 cm2 multiwell culture dishes. Matrix degradation after treatment with parathyroid hormone was assessed by measuring the amount of carboxyterminal telopeptide of type I collagen (ICTP) by RIA. The data on matrix degradation was compared to the release of prelabelled45Ca from neonatal mouse calvarial bones. We found that the dose-responses for parathyroid hormone-induced release of prelabelled45Ca and ICTP were identical. In conclusion: RIA-analysis of the ICTP-release is an easy and accurate method to measure degradation of bone-matrix, in vitro. Furthermore, the new dissection technique, described in this report, makes it easy to obtain large amounts of bone samples and thus to perform extensive experiments, e.g. dose-responses for agents that enhance bone resorption.

scholarly journals Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Endocrinology ◽  
2001 ◽  
Vol 142 (12) ◽  
pp. 5371-5378 ◽  
Author(s):  
Vilhelmiina Parikka ◽  
Petri Lehenkari ◽  
Mirja-Liisa Sassi ◽  
Jussi Halleen ◽  
Juha Risteli ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Organic Matrix ◽  
Cysteine Proteases ◽  
Bone Collagen ◽  
Type I ◽  
Matrix Degradation ◽  
Carboxyl Terminal

Abstract Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.

DDR2 induces linear invadosome to promote angiogenesis in a fibrillar type I collagen context

2020 ◽  
Author(s):  
Aya Abou Hammoud ◽  
Sébastien Marais ◽  
Nathalie Allain ◽  
Zakaria Ezzoukhry ◽  
Violaine Moreau ◽  
...  
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Molecular Mechanisms ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Type I ◽  
Matrix Degradation ◽  
The Matrix ◽  
Discoidin Domain Receptor 2 ◽  
Collagen Receptor

AbstractTo generate new vessels, endothelial cells (ECs) form invadosomes, which are actin-based microdomains with a proteolytic activity that degrade the basement membrane. We previously demonstrated that ECs form linear invadosomes in fibrillar type I collagen context. In this study, we aim to investigate the molecular mechanisms by which ECs guides angiogenesis in a fibrillar type I collagen context. We found that Discoidin Domain Receptor 2 (DDR2) is the collagen receptor tyrosine kinase required to form linear invadosomes in ECs. We further demonstrated that it acts in synergy with VEGF to promote extracellular matrix degradation. We highlighted the involvement of an interaction between DDR2 and the matrix metalloproteinase MMP14 in this process. Finally, using in vitro and ex-vivo angiogenesis assays, we demonstrated a pro-angiogenic function of DDR2 in a collagen-rich microenvironment. This study allows us to propose DDR2-dependent linear invadosomes as targets to modulate angiogenesis.

scholarly journals Fusion Potential of Human Osteoclasts In Vitro Reflects Age, Menopause, and In Vivo Bone Resorption Levels of Their Donors—A Possible Involvement of DC-STAMP

2020 ◽  
Vol 21 (17) ◽  
pp. 6368
Author(s):  
Anaïs M. J. Møller ◽  
Jean-Marie Delaissé ◽  
Jacob B. Olesen ◽  
Luisa M. Canto ◽  
Silvia R. Rogatto ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Type I Collagen ◽  
Transmembrane Protein ◽  
Type I ◽  
Contributing Factors ◽  
Expression Levels ◽  
Procollagen Type ◽  
Age Related

It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14+ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.

scholarly journals Control of Bone Matrix Properties by Osteocytes

2021 ◽  
Vol 11 ◽  
Author(s):  
Amy Creecy ◽  
John G. Damrath ◽  
Joseph M. Wallace
Keyword(s):  
Type I Collagen ◽  
Bone Matrix ◽  
Type I ◽  
Bone Homeostasis ◽  
Surrounding Matrix ◽  
Matrix Properties ◽  
The Matrix ◽  
Exciting Potential

Osteocytes make up 90–95% of the cellular content of bone and form a rich dendritic network with a vastly greater surface area than either osteoblasts or osteoclasts. Osteocytes are well positioned to play a role in bone homeostasis by interacting directly with the matrix; however, the ability for these cells to modify bone matrix remains incompletely understood. With techniques for examining the nano- and microstructure of bone matrix components including hydroxyapatite and type I collagen becoming more widespread, there is great potential to uncover novel roles for the osteocyte in maintaining bone quality. In this review, we begin with an overview of osteocyte biology and the lacunar–canalicular system. Next, we describe recent findings from in vitro models of osteocytes, focusing on the transitions in cellular phenotype as they mature. Finally, we describe historical and current research on matrix alteration by osteocytes in vivo, focusing on the exciting potential for osteocytes to directly form, degrade, and modify the mineral and collagen in their surrounding matrix.

scholarly journals Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

2021 ◽  
Vol 43 (3) ◽  
pp. 1451-1459
Author(s):  
Kengo Kato ◽  
Manami Ozaki ◽  
Kumiko Nakai ◽  
Maki Nagasaki ◽  
Junya Nakajima ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Type I Collagen ◽  
Inflammatory Diseases ◽  
Bone Matrix ◽  
Nodule Formation ◽  
Type I ◽  
Alizarin Red ◽  
Odontogenic Infections ◽  
Low Concentrations

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

scholarly journals The effects of serine proteinase inhibitors on bone resorption in vitro

2003 ◽  
Vol 178 (3) ◽  
pp. 437-447 ◽  
Author(s):  
A Tumber ◽  
S Papaioannou ◽  
J Breckon ◽  
MC Meikle ◽  
JJ Reynolds ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Plasminogen Activator ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Serine Proteinase ◽  
Tissue Type ◽  
Type I ◽  
Type Plasminogen Activator ◽  
Primary Mouse ◽  
Sites Of Action

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%.Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.

scholarly journals The Glucagon-Like Peptide-1 Receptor Agonist Exendin-4 Inhibits Lipopolysaccharide-Induced Osteoclast Formation and Bone Resorption via Inhibition of TNF-αExpression in Macrophages

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Wei-Ren Shen ◽  
Keisuke Kimura ◽  
Masahiko Ishida ◽  
Haruki Sugisawa ◽  
Akiko Kishikawa ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Mrna Expression ◽  
Receptor Agonist ◽  
Type I Collagen ◽  
Osteoclast Formation ◽  
Type I ◽  
Receptor Agonists ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) receptor agonists are an effective treatment approach for type 2 diabetes. Recently, anti-inflammatory effects of GLP-1 receptor agonists have also been reported. Lipopolysaccharide (LPS) induces inflammation and osteoclast formation. In this study, we investigated the effect of exendin-4, a widely used GLP-1 receptor agonist, in LPS-induced osteoclast formation and bone resorption. LPS with or without exendin-4 was administered on mouse calvariae by daily subcutaneous injection. The number of osteoclasts, the ratio of bone resorption pits, and the level of C-terminal cross-linked telopeptide of type I collagen (CTX) were significantly lower in LPS- and exendin-4-coadministered mice than in mice administered with LPS alone. RANKL and TNF-αmRNA expression levels were lower in the exendin-4- and LPS-coadministered group than in the LPS-administered group. Ourin vitroresults showed no direct effects of exendin-4 on RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation, or LPS-induced RANKL expression in stromal cells. Conversely, TNF-αmRNA expression was inhibited in the exendin-4- and LPS-cotreated macrophages compared with cells treated with LPS alone. These results indicate that the GLP-1 receptor agonist exendin-4 may inhibit LPS-induced osteoclast formation and bone resorption by inhibiting LPS-induced TNF-αproduction in macrophages.

scholarly journals Mechanisms balancing skeletal matrix synthesis and degradation

2002 ◽  
Vol 364 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Harry C. BLAIR ◽  
Mone ZAIDI ◽  
Paul H. SCHLESINGER
Keyword(s):  
Parathyroid Hormone ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Type Ii Collagen ◽  
Type I ◽  
Parathyroid Hormone Related Protein ◽  
Bone Degradation ◽  
The Matrix ◽  
Acid Secreting ◽  
Matrix Removal

Bone is regulated by evolutionarily conserved signals that balance continuous differentiation of bone matrix-producing cells against apoptosis and matrix removal. This is continued from embryogenesis, where the skeleton differentiates as a solid mass and is shaped into separate bones by cell death and proteolysis. The two major tissues of the skeleton are avascular cartilage, with an extracellular matrix based on type II collagen and hydrophilic proteoglycans, and bone, a stronger and lighter material based on oriented type I collagen and hydroxyapatite. Both differentiate from the same mesenchymal stem cells. This differentiation is regulated by a family of related signals centred on bone morphogenic proteins. Fibroblast growth factors, Indian hedgehog and parathyroid hormone-related protein are important in determining the type of matrix and the relation of skeletal and non-skeletal structures. Removal of mineralized matrix involves apoptosis of matrix cells and differentiation of acid-secreting cells (osteoclasts) from macrophage precursors. Key regulators of matrix removal are signals in the tumour-necrosis-factor family. Osteoclasts dissolve bone by isolating a region of the matrix and secreting HCl and proteinases at that site. Successive cycles of removal and replacement allow growth, repair and remodelling. The signals for bone turnover are predominantly cell-membrane-associated, allowing very specific spatial regulation. In addition to its support function, bone is a reservoir of Ca2+, PO3-4 and OH−. Secondary modulation of mineral secretion and bone degradation are mediated by humoral signals, including parathyroid hormone and vitamin D, as well as the cytokines that also regulate the underlying cell differentiation.

Commitment of the teratocarcinoma-derived mesodermal clone C1 towards terminal osteogenic differentiation

1993 ◽  
Vol 106 (2) ◽  
pp. 503-511 ◽  
Author(s):  
A. Poliard ◽  
D. Lamblin ◽  
P.J. Marie ◽  
M.H. Buc-Caron ◽  
O. Kellermann
Keyword(s):  
Ascorbic Acid ◽  
Osteogenic Differentiation ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Type I ◽  
Adenovirus E1a ◽  
Matrix Deposition ◽  
Bone Matrix Proteins ◽  
Kinetics Of

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)

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