scholarly journals Cell-layer-associated proteolytic cleavage of the telopeptides of type I collagen in fibroblast culture

1987 ◽  
Vol 245 (3) ◽  
pp. 677-682 ◽  
Author(s):  
J F Bateman ◽  
J J Pillow ◽  
T Mascara ◽  
S Medvedec ◽  
J A M Ramshaw ◽  
...  
Keyword(s):  
Cell Culture ◽  
Proteolytic Activity ◽  
Culture Medium ◽  
Type I Collagen ◽  
Cell Layer ◽  
Specific Inhibitor ◽  
Thrombin Inhibitor ◽  
Human Skin Fibroblast ◽  
Type I ◽  
Medium Ph

In human skin fibroblast cultures a fraction of the procollagen that was processed to collagen and remained in the cell layer was further proteolytically modified by removal of both N- and C-terminal telopeptides. The proteolytic activity was associated with the cell layer, since secreted collagens were found always to contain intact telopeptides. The inclusion of neutral polymers, which caused the accumulation of the collagen in the cell layer [Bateman, Cole, Pillow & Ramshaw (1986) J. Biol. Chem. 261, 4198-4203], made the telopeptide cleavage more apparent in those cells which expressed the proteolytic activity. The extent of this cleavage was variable from cell culture to cell culture and between experiments with the same fibroblast line. The proteolytic activity was pH-dependent; cleavage was greatest at a culture-medium pH of 7.5 and 8.0 and was completely inhibited at a culture-medium pH of 7.0 and 6.5. The activity was significantly inhibited by soybean trypsin inhibitor, an elastase-specific inhibitor (N-acetylalanylalanylprolylvalylchloromethane) and the thrombin inhibitor hirudin. This cell-associated proteolytic activity may play a role in collagen degradation by removing the telopeptides, which are the primary sites of collagen cross-linking, thus destabilizing the collagen matrix sufficiently to render it susceptible to further proteolytic breakdown.

scholarly journals Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Daniel de Melo Pereira ◽  
Matthias Schumacher ◽  
Pamela Habibovic
Keyword(s):  
Cell Culture ◽  
Calcium Phosphate ◽  
Culture Medium ◽  
Type I Collagen ◽  
Type I ◽  
Cell Culture Medium ◽  
Cobalt Ions ◽  
Collagen Membranes ◽  
Human Osteoclasts ◽  
Osteoclast Resorption

Abstract Background Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts. Methods To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium. Results The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded. Conclusions This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Serum enhancement of human endothelial cell attachment to and spreading on collagens I and IV does not require serum fibronectin or vitronectin

1990 ◽  
Vol 95 (2) ◽  
pp. 255-262
Author(s):  
W.D. Norris ◽  
J.G. Steele ◽  
G. Johnson ◽  
P.A. Underwood
Keyword(s):  
Endothelial Cell ◽  
Culture Medium ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Human Endothelial Cell ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
Endothelial Cell Attachment

The initial attachment and spreading of endothelial cells from human umbilical artery onto type I collagen, type IV collagen or gelatin substrata was shown to be enhanced by inclusion of serum in the culture medium. To test whether this serum effect was mediated by adsorption of serum fibronectin or vitronectin onto the collagen, these adhesive glycoproteins were selectively removed from the serum prior to addition to the culture medium. The stimulatory effect of serum on human endothelial cell spreading on collagens I and IV was also observed with serum from which either fibronectin or vitronectin, or both, had been selectively removed. The stimulatory effect for cell spreading on gelatin was diminished by selective removal of serum fibronectin, but unaffected by removal of vitronectin. Human endothelial cell attachment and spreading onto tissue culture plastic was abolished by removal of vitronectin from the serum in the culture medium. These results emphasize that the native structure of collagens is required for serum-enhancement of human endothelial cell attachment and spreading on native collagen types I and IV, and show that on these substrata the stimulated adhesion and spreading are not dependent upon adsorption of serum fibronectin or vitronectin onto the collagen substratum.

The morphology of NCTC cell clone 929 after contact with type I collagen added to culture medium

2014 ◽  
Vol 8 (6) ◽  
pp. 478-487
Author(s):  
Yu. P. Petrov ◽  
Yu. A. Negulyaev ◽  
N. V. Tsupkina
Keyword(s):  
Culture Medium ◽  
Type I Collagen ◽  
Cell Clone ◽  
Type I

Valve interstitial cell culture: Production of mature type I collagen and precise detection

2017 ◽  
Vol 80 (8) ◽  
pp. 936-942 ◽  
Author(s):  
Jana Liskova ◽  
Daniel Hadraba ◽  
Elena Filova ◽  
Miroslav Konarik ◽  
Jan Pirk ◽  
...  
Keyword(s):  
Cell Culture ◽  
Type I Collagen ◽  
Interstitial Cell ◽  
Type I ◽  
Mature Type ◽  
Culture Production

scholarly journals Effects of ECM Proteins and Cationic Polymers on the Adhesion and Proliferation of Rat Islet Cells

2008 ◽  
Vol 2 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Guoping Chen ◽  
Naoki Kawazoe ◽  
Tetsuya Tateishi
Keyword(s):  
Cell Culture ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Islet Cells ◽  
Type Ii Collagen ◽  
Type I ◽  
Cationic Polymers ◽  
Type Iv ◽  
Ecm Proteins ◽  
Cationic Polyelectrolytes

The effects of extracellular matrix (ECM) proteins and cationic polymers on the adhesion and proliferation of rat islet cells, RIN-5F cells, were investigated. ECM proteins of laminin, fibronectin, vitronectin, type I collagen, type II collagen, and type IV collagen, and cationic polyelectrolytes of poly(L-lysine) and poly(allylamine) were coated on the wells of polystyrene cell culture plates. Their effects on the adhesion and proliferation of RIN-5F in serum-free and serum mediums were compared. The cell number on the laminin-coated surface was the highest among the coated surfaces. Laminin promoted cell adhesion more strongly than did the other ECM proteins and cationic polyelectrolytes. Vitronectin, type IV collagen, and poly(L-lysine) showed moderate effects, but type I collagen and type II collagen did not have any effects on adhesion and proliferation compared with the uncoated polystyrene cell culture plate. Fibronectin promoted cell adhesion but not cell proliferation. Cationic poly(allylamine) had an inhibitory effect in serum-free medium and for longterm culture in serum medium. The ECM proteins of laminin, vitronectin, and type IV collagen, and cationic poly(Llysine) will be useful for the surface modification and construction of biomaterials and scaffolds for islet cell culture and tissue engineering.

scholarly journals Cobalt-containing Calcium Phosphate Induces Resorption of Biomineralized Collagen by Human Osteoclasts

2020 ◽  
Author(s):  
Daniel de Melo Pereira ◽  
Matthias Schumacher ◽  
Pamela Habibović
Keyword(s):  
Cell Culture ◽  
Calcium Phosphate ◽  
Calcium Release ◽  
Type I Collagen ◽  
Cell Culture Supernatant ◽  
Type I ◽  
Cobalt Ions ◽  
Collagen Membranes ◽  
Human Osteoclasts ◽  
Osteoclast Resorption

Abstract Background: Biomineralized collagen, consisting of fibrillary type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previous shown to stimulate resorptive activity of osteoclasts.Methods: To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Their morphology was assessed by laser confocal microscopy and their capacity for resorption observed by scanning electron microscopy (SEM), as well as indirectly quantified by calcium release into cell culture supernatant. Results: The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and caused resorption lacunae to form, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.Conclusions: This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, despite the fact that the extent of osteoclast resorption was limited, and the method needs further optimization,. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Stability of the Magnesium Carbonate Apatite/Anionic Collagen Scaffolds: Effect of the Cross-Link Concentration

2011 ◽  
Vol 493-494 ◽  
pp. 844-848 ◽  
Author(s):  
Marcia S. Sader ◽  
Gutemberg Alves ◽  
Racquel Z. LeGeros ◽  
Gloria Dulce de Almeida Soares
Keyword(s):  
Bone Tissue ◽  
Culture Medium ◽  
Type I Collagen ◽  
Type I ◽  
Carbonate Apatite ◽  
Reaction Products ◽  
Collagen Scaffolds ◽  
Freeze Dried ◽  
The Cross

Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fourier-transformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering.

scholarly journals Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density.

1993 ◽  
Vol 41 (6) ◽  
pp. 867-875 ◽  
Author(s):  
F Ruggiero ◽  
B Petit ◽  
M C Ronziere ◽  
J Farjanel ◽  
D J Hartmann ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Cell Layer ◽  
Morphological Characteristics ◽  
Culture Cell ◽  
High Density ◽  
Type I ◽  
Type Ii ◽  
High Density Culture ◽  
Entire Cell ◽  
Fetal Bovine

Fetal bovine chondrocytes isolated from the resting zone of epiphyseal cartilage were maintained in high-density culture for 4 weeks. From Day 2 in culture, the chondrocytes deposited an extracellular matrix composed of Types II, IX, and XI collagen. Types IX and XI collagen were restricted to the pericellular domain from Day 5. By 2 weeks the entire cell layer stained for antibodies to Type II and IX collagens. Type XI could be demonstrated throughout the cell layer by pepsinization of the sections. Results from both rotary shadowing and immunochemistry showed that the fibrils formed in culture were heterotypic, with Type IX collagen arranged along the surface and with Type XI collagen buried in Type II fibrils. Nonspecific Type VI collagen and the glycoproteins tenascin and fibrillin, previously described in cartilaginous tissue, were identified by their ultrastructural characteristics in the cell layer homogenate. Although the cells presented morphological characteristics of chondrocytes and still expressed cartilage-specific collagens, the appearance of Type I collagen in the culture cell layer after 4 weeks of culture demonstrates a partial dedifferentiation of the chondrocytes. The culture system described in this report provides an interesting tool for maintaining chondrocytes in a cartilage-like matrix to study the influence of different physical and chemical factors on the expression and differentiation of the cells.

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