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1.
Injury ; 43(3): 259-65, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21696723

ABSTRACT

Articular cartilage is easily injured but difficult to repair and cell therapies are proposed as tools to regenerate the defects in the tissue. Both differentiated chondrocytes and adult mesenchymal stem cells (MSCs) are regarded as cells potentially able to restore a functional cartilage. However, it is a complex process from the cell level to the tissue end product, during which growth factors play important roles from cell proliferation, extracellular matrix synthesis, maintenance of the phenotype to induction of MSCs towards chondrogenesis. Members of the TGF-ß superfamily, are especially important in fulfilling these roles. Depending on the cell type chosen to restore cartilage, the effect of growth factors will vary. In this review, the roles of these factors in the maintenance of the chondrocyte phenotype are discussed and compared with those of factors involved in the repair of cartilage defects, using chondrocytes or adult mesenchymal stem cells.


Subject(s)
Adult Stem Cells/metabolism , Cartilage, Articular/growth & development , Chondrocytes/metabolism , Chondrogenesis , Mesenchymal Stem Cells/metabolism , Transforming Growth Factor beta/metabolism , Cartilage, Articular/injuries , Cartilage, Articular/metabolism , Cell Proliferation , Chondrogenesis/physiology , Female , Humans , Male , Phenotype , Tissue Engineering , Tissue Scaffolds
2.
Biotechnol Lett ; 33(10): 2091-101, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21660579

ABSTRACT

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-ß3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-ß3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.


Subject(s)
Bone Marrow Cells/metabolism , Chondrogenesis/physiology , Collagen/biosynthesis , Mesenchymal Stem Cells/metabolism , Adult , Aged , Bone Marrow Cells/drug effects , Bone Morphogenetic Protein 2/pharmacology , Cell Proliferation/drug effects , Chondrogenesis/drug effects , Collagen/genetics , Collagen Type II/genetics , Collagen Type II/metabolism , Fibroblast Growth Factor 2/pharmacology , Gene Expression/drug effects , Humans , Immunohistochemistry , Mesenchymal Stem Cells/drug effects , Middle Aged , Protein Isoforms , Transforming Growth Factor beta3/pharmacology
3.
Biomed Mater Eng ; 20(3): 145-58, 2010.
Article in English | MEDLINE | ID: mdl-20930322

ABSTRACT

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-ß family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-ß3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-ß3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-ß3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.


Subject(s)
Adipose Tissue/cytology , Bone Marrow Cells/cytology , Chondrocytes/cytology , Chondrocytes/physiology , Chondrogenesis/physiology , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Adult , Aged , Cell Culture Techniques/methods , Cell Differentiation , Cells, Cultured , Female , Humans , Male , Middle Aged
4.
Tissue Eng Part A ; 15(6): 1233-45, 2009 Jun.
Article in English | MEDLINE | ID: mdl-18950259

ABSTRACT

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.


Subject(s)
Cartilage, Articular/cytology , Chondrocytes/cytology , Chondrocytes/drug effects , Collagen Type II/pharmacology , Collagen Type I/pharmacology , Tissue Scaffolds/chemistry , Aggrecans/metabolism , Animals , Biocompatible Materials/metabolism , Biomarkers/metabolism , Cattle , Cell Dedifferentiation/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Shape/drug effects , Cells, Cultured , Chondrocytes/enzymology , Collagen Type I/ultrastructure , Collagen Type II/ultrastructure , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gels , Gene Expression Regulation/drug effects , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Phenotype
5.
J Biomed Mater Res B Appl Biomater ; 81(1): 153-61, 2007 Apr.
Article in English | MEDLINE | ID: mdl-16924608

ABSTRACT

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering. Hard (ceramic substitute) and soft (collagen sponge) biomaterials were seeded respectively, on one side of the scaffold, with human fibroblasts and bovine chondrocytes labelled with carbocyanine dyes (DiD and DiR). The mean penetration depth for DiR labelled fibroblasts and chondrocytes in the two scaffolds, around 270 m, was greater than for DiD (136-218 microm) labelled cells. These depths were independent of cell origin but were influenced by the nature of the scaffolds. Collagen sponge is transparent in contrast to ceramic substitutes where measurements could only be made in opened macropores. Besides the limits of the equipment, the limits of the supports were diffusion for collagen sponges and transmission for ceramic substitutes. Confocal microscopy techniques could thus be used to address the question of cell colonization of porous biomaterials in a noninvasive manner.


Subject(s)
Biocompatible Materials/standards , Bone Substitutes/standards , Carbocyanines/analysis , Ceramics/standards , Collagen/standards , Materials Testing/methods , Microscopy, Confocal/methods , Animals , Cartilage/cytology , Cattle , Chondrocytes/chemistry , Chondrocytes/cytology , Chondrocytes/physiology , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/physiology , Fluorescent Dyes/analysis , Hardness , Humans
6.
Proteomics ; 6(8): 2609-25, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16526084

ABSTRACT

In this report we present a catalogue of 162 proteins (including isoforms and variants) identified in a prototype of proteomic map of breast cancer cells. This work represents the prosecution of previous studies describing the protein complement of breast cancer cells of the line 8701-BC, which has been well characterized for several parameters, providing to be a useful model for the study of breast cancer-associated candidate biomarkers. In particular, 110 spots were identified ex novo by PMF, or validated following previous gel matching identification method; 30 were identified by N-terminal microsequencing and the remaining by gel matching with maps available from our former work. As a consequence of the expanded number of proteins, we have updated our previous classification extending the number of protein groups from 4 to 13. In order to facilitate comparative proteome studies of different kinds of breast cancers, in this report we provide the whole complement of proteins so far identified and grouped into the new classification. A consistent number of them were not described before in other proteomic maps of breast cancer cells or tissues, and therefore they represent a valuable contribution for breast cancer protein databases and for future application in basic and clinical researches.


Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Proteomics/methods , Biomarkers, Tumor , Cell Line, Tumor , Cytoskeleton/metabolism , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/chemistry , Humans , Oxidation-Reduction , Proteome , RNA/chemistry , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
Biomaterials ; 27(1): 79-90, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16026827

ABSTRACT

This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2 x 10(6) chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.


Subject(s)
Biocompatible Materials/chemistry , Cell Culture Techniques/methods , Chondrocytes/cytology , Animals , Cartilage/metabolism , Cattle , Cell Culture Techniques/instrumentation , Cell Proliferation , Cells, Cultured , Chondrocytes/metabolism , Collagen/metabolism , Collagen Type I , Collagen Type II/metabolism , Collagenases/biosynthesis , Culture Media/chemistry , DNA/chemistry , Gene Expression Regulation , Immunohistochemistry , Integrin alpha Chains/metabolism , Integrins/metabolism , Matrix Metalloproteinase 1/biosynthesis , Matrix Metalloproteinase 13 , Phenotype , Proteoglycans/chemistry , RNA/chemistry , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tissue Engineering , Up-Regulation , Wound Healing
8.
Bull Acad Natl Med ; 190(7): 1399-1408; discussion 1408-9, 1475-7, 2006 Oct.
Article in French | MEDLINE | ID: mdl-17450676

ABSTRACT

Joint cartilage has a poor intrinsic ability to heal. Common surgical treatments for traumatic lesions, after debridement of the chondral defect, include stimulation of subchondral bone (microfracture), perichondrial or periosteal grafting, and mosaicplasty (osteochondral cylinder transplantation). Autologous chondrocyte transplantation (ACT) was the first application of cell therapy to orthopaedic surgery. Despite promising results, several groups have tested tissue engineering protocols based on ex vivo colonization of biodegradable polymer matrices that are subsequently transplanted to the target site. Tissue engineering as a treatment for osteoarthritis is even more challenging. Transplantation of genetically modified cells is an interesting concept, based on the production of therapeutic proteins directly at the target site.


Subject(s)
Biocompatible Materials , Bone Transplantation , Cartilage Diseases/surgery , Cartilage, Articular/transplantation , Chondrocytes/transplantation , Osteoarthritis/surgery , Tissue Engineering , Cartilage, Articular/injuries , Chondrocytes/metabolism , Debridement , Humans , Phenotype , Transplantation, Autologous
9.
Methods Mol Med ; 100: 165-82, 2004.
Article in English | MEDLINE | ID: mdl-15280595

ABSTRACT

Changes in chondrocyte metabolism in vitro using different support systems and under different culture conditions were studied with a proteomic approach. Qualitative and quantitative modifications in the synthesis of chondrocyte proteins were investigated using two-dimensional (2D) gel electrophoresis. This technique provided a simple way to visualize the most abundant chondrocyte proteins. Proteins were identified after in-gel proteolysis with trypsin and matrix-assisted laser desorption ionization-time of flight mass spectrometry, using peptide mass fingerprinting. Tryptic peptide masses were measured and matched against a computer-generated list from the simulated trypsin proteolysis of a protein database (SwissProt).


Subject(s)
Chondrocytes/metabolism , Proteins/analysis , Proteomics/methods , Animals , Cattle , Cells, Cultured , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Humans , Peptide Mapping/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
Proteomics ; 4(3): 849-60, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14997505

ABSTRACT

We have previously described the occurrence, in breast and colon cancer extra-cellular matrix, of an oncofoetal form of collagen, OF/LB, able to induce an increase in cell proliferation and motility in the breast cancer cell line 8701-BC. It also caused an increased amount of type V collagen which appears to exert an anti-proliferative effect on the same cells. The aim of the present study was to investigate, at the proteomic level, the effect of OF/LB and type V collagens used as substrates for neoplastic cell growth. Due to the complexity of a whole proteomic profile, a subset of significant protein classes was used to assess variations in protein expression levels. For this study we adopted a multivariate statistical procedure that allows a global view of the variations induced by different growth conditions, when several variables have to be analyzed simultaneously. The results of this research indicate that in response to different growth substrates, chaperons and heat shock proteins contributed most to the dissimilarity in levels of expression of the selected protein spots. Moreover, we observed that different isoforms of the same protein showed independent levels of expression from one another in relation to the different collagen treatments.


Subject(s)
Breast Neoplasms/metabolism , Collagen/chemistry , Proteome , Proteomics/methods , Breast Neoplasms/pathology , Cell Division , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/metabolism , Hot Temperature , Humans , Image Processing, Computer-Assisted , Multivariate Analysis , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
11.
Biomaterials ; 24(5): 851-61, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12485803

ABSTRACT

This report completes a previous study on the growth and metabolism of fetal bovine epiphyseal chondrocytes cultured, within native or cross-linked collagen sponges carried out without the addition of fresh ascorbate. At low initial cell density (2.3 x 10(6)cells/cm(3)) cell proliferation and a low matrix deposition were observed, whereas at high initial cell density (2.3 x 10(7)cells/cm(3)) there was an absence of cell proliferation, but the deposition of a cartilage-like matrix was measured. In both cases, only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. In this report, we observed, after 1 month in culture with ascorbate, in both type of scaffolds and initial cell densities, an increase in cell proliferation (2-fold) and in expression of genes encoding for collagen types I, II, X and MMP-2 and -13, but no change in the level of matrix deposition (collagen and GAG). With regard to the proteins present, the main differences with or without ascorbate concerned the increase of neosynthesised type I collagen (up to 35% of the total collagen deposited in the sponge) and of the MMP-2 active form. In conclusion, these results show that ascorbate is an important factor to consider when preparing cartilage constructs for its action on chondrocyte phenotype modulation and proliferation.


Subject(s)
Ascorbic Acid/pharmacology , Chondrocytes/cytology , Chondrocytes/physiology , Extracellular Matrix Proteins/genetics , Gene Expression Regulation/drug effects , Animals , Base Sequence , Cattle , Cell Division/drug effects , Cells, Cultured , Chondrocytes/drug effects , Collagen/genetics , DNA Primers , Gelatinases/genetics , Gene Expression Regulation, Enzymologic/drug effects , Glycosaminoglycans/genetics , Immunohistochemistry , Kinetics , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
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