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1.
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
2.
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
3.
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
4.
Biochim Biophys Acta ; 1746(1): 55-64, 2005 Oct 30.
Article in English | MEDLINE | ID: mdl-16198011

ABSTRACT

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.


Subject(s)
Chondrocytes/metabolism , Collagen Type I/metabolism , Collagenases/metabolism , Integrin alpha1beta1/metabolism , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/pharmacology , Cell Line , Chondrocytes/drug effects , Collagenases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Integrin alpha1beta1/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Matrix Metalloproteinase 13 , Mice , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/metabolism , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
5.
Biorheology ; 41(3-4): 535-42, 2004.
Article in English | MEDLINE | ID: mdl-15299284

ABSTRACT

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.


Subject(s)
Cartilage, Articular , Chondrocytes/metabolism , Collagen/biosynthesis , Osteoarthritis/metabolism , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Extracellular Matrix/metabolism , Humans , Procollagen/metabolism , Transforming Growth Factor beta/pharmacology
6.
FEBS Lett ; 545(2-3): 115-9, 2003 Jun 19.
Article in English | MEDLINE | ID: mdl-12804760

ABSTRACT

Type II collagen is the major protein of cartilage and is synthesized as a procollagen in two forms (IIA and IIB), generated by differential splicing of the gene primary transcript. Previous studies have indicated that only type IIB is expressed in differentiated chondrocytes. Here, we examined the effects of bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 on the expression of IIA and IIB forms expressed in de-differentiated chondrocytes grown in monolayer. Our results demonstrate that BMP-2 favors expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These observations reveal the specific capability of BMP-2 to reverse the de-differentiation state of chondrocytes.


Subject(s)
Alternative Splicing/genetics , Bone Morphogenetic Proteins/metabolism , Chondrocytes/metabolism , Gene Expression Regulation, Developmental , Procollagen/metabolism , RNA Precursors/metabolism , Transforming Growth Factor beta/metabolism , Animals , Bone Morphogenetic Protein 2 , Cell Differentiation/genetics , Cells, Cultured , Chondrocytes/cytology , Mice , Procollagen/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Precursors/genetics
7.
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
8.
J Biol Chem ; 277(37): 33545-58, 2002 Sep 13.
Article in English | MEDLINE | ID: mdl-12082094

ABSTRACT

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.


Subject(s)
Activin Receptors, Type I/physiology , Chondrocytes/physiology , DNA-Binding Proteins/physiology , Osteoblasts/physiology , Proteins , Receptors, Transforming Growth Factor beta/physiology , Trans-Activators/physiology , Transforming Growth Factor beta , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation , Cell Line , Chondrocytes/drug effects , Collagen Type II/genetics , Humans , Hypertrophy , Mice , Osteoblasts/drug effects , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type I , Smad Proteins , Smad1 Protein
9.
Wilehm Roux Arch Dev Biol ; 192(5): 205-215, 1983 Sep.
Article in English | MEDLINE | ID: mdl-28305506

ABSTRACT

Collagen types I and III were purified from the skin of 3-or 7-week-old chickens, collagen type IV from bovine skin or EHS mouse tumour, fibronectin from human serum, and laminin from EHS mouse tumour. Antibodies were produced in rabbits or sheep, and used in indirect immunofluorescence on frozen sections of 9-to 16-day-old normal or mutant (scaleless) chick-embryo foot skin. In normal scale-forming skin and inscaleless skin, the distribution of anti-laminin and anti-type IV collagen label was uniform along the dermal-epidermal junction and showed no stage-related variations, except for fluorescent granules located in the dermis of early scale rudiments. By contrast, in normal scale-forming skin, the density of anti-types I and III label decreased in the dermis within scale rudiments, whereas it gradually increased in interscale skin. Conversely, anti-fibronectin label accumulated at a higher density within scale rudiments than in interscale skin. In the dermis of thescaleless mutant, anti-types I and III label and antifibronectin label were distributed evenly: the density of anti-collagen label increased with age, while that of antifibronectin decreased and almost completely vanished in 16-day-old skin, except around blood vessels. The microheterogeneous distribution of some extracellular matrix components, namely interstitial collagen types I and III and fibronectin, is interpreted as part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of scales. The even distribution of these components in mutantscaleless skin is in agreement with this view. Basement membrane constituents laminin and type-IV collagen do not appear to be part of the dermal morphogenetic message.

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