scholarly journals Effects of Extracellular Matrix on the Morphology and Behaviour of Rabbit Auricular Chondrocytes in Culture

2005 ◽  
Vol 2005 (4) ◽  
pp. 364-373 ◽  
Author(s):  
Vega Villar-Suárez ◽  
B. Colaço ◽  
I. Calles-Venal ◽  
I. G. Bravo ◽  
J. G. Fernández-Álvarez ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type Ii Collagen ◽  
Cell Phenotype ◽  
Nodule Formation ◽  
Type Ii ◽  
Protein Extract ◽  
Positive Effects ◽  
Reticular Pattern ◽  
Chondrocyte Cultures ◽  
Plastic Surfaces

Isolated chondrocytes dedifferentiate to a fibroblast-like shape on plastic substrata and proliferate extensively, but rarely form nodules. However, when dissociation is not complete and some cartilage remnants are included in the culture, proliferation decreases and cells grow in a reticular pattern with numerous nodules, which occasionally form small cartilage-like fragments. In an attempt to reproduce this stable chondrogenic state, we added a cartilage protein extract, a sugar extract, and hyaluronan to the medium of previously dedifferentiated chondrocytes. When protein extract was added, many cartilaginous nodules appeared. Hyaluronan produced changes in cell phenotype and behaviour, but not nodule formation. Protein extract has positive effects on the differentiation of previously proliferated chondrocytes and permits nodule formation and the extensive production of type-II collagen. A comparison with incompletely dissociated chondrocyte cultures suggests that the presence of some living cells anchored to their natural extracellular matrix provides some important additional factors for the phenotypical stability of chondrocytes on plastic surfaces. In order to elucidate if it is possible that the incidence of apoptosis is related to the results, we also characterized the molecular traits of apoptosis.

scholarly journals Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study

2020 ◽  
Vol 2020 ◽  
pp. 1-16 ◽  
Author(s):  
Christel Henrionnet ◽  
Léa Pourchet ◽  
Paul Neybecker ◽  
Océane Messaoudi ◽  
Pierre Gillet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Cell Encapsulation ◽  
Type Ii Collagen ◽  
Mitochondrial Activity ◽  
Type Ii ◽  
3D Bioprinting ◽  
Matrix Synthesis ◽  
M Cell ◽  
Low Concentration

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10×10×4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

Effects of bio-mimic collagen II-g-hyaluronic acid copolymers on chondrocyte maintenance

2019 ◽  
Vol 34 (4-5) ◽  
pp. 373-385
Author(s):  
Kuan Wei Lee ◽  
Tang-Ching Kuan ◽  
Ming Wei Lee ◽  
Chen Show Yang ◽  
Lain-Chyr Hwang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Hyaluronic Acid ◽  
Specific Binding ◽  
Type Ii Collagen ◽  
Polymerase Chain Reaction Analysis ◽  
Collagen Fibrils ◽  
Type Ii ◽  
Human Cartilage

Extracellular matrix has an important part of the role in tissue engineering and regenerative medicine, so it is necessary to understand the various interactions between cells and extracellular matrix. Type II collagen and hyaluronic acid are the major structural components of the extracellular matrix of articular cartilage, and they are involved in fibril formation, entanglement and binding. The aim of this study was to prepare type II collagen fibrils with surface grafted with hyaluronic acid modified at the reducing end. The topographic pattern of type II collagen fibrils showed a significant change after the surface coupling of hyaluronic acid according to atomic force microscopy scanning. The presence of hyaluronic acid on the type II collagen fibrillar surface was confirmed by the specific binding of nanogold labelled with lectin. No significant increase in cell proliferation was detected by a WST-1 assay. According to histochemical examination, the maintenance of the round shape of chondrocytes and increased glycosaminoglycan secretion revealed that these cell pellets with Col II- g-hyaluronic acid molecules contained un-dedifferentiated chondrocytes in vitro. In the mixture with the 220-kDa Col II- g-hyaluronic acid copolymer, the expression of type II collagen and aggrecan genes in chondrocytes increased as demonstrated by real-time polymerase chain reaction analysis. Experimental results show that the amount of hyaluronic acid added during culturing of chondrocytes can maintain the functionality of chondrocytes and thus allow for increased cell proliferation that is suitable for tissue repair of human cartilage.

Facilitating In Vivo Articular Cartilage Repair by Tissue-Engineered Cartilage Grafts Produced From Auricular Chondrocytes

2017 ◽  
Vol 46 (3) ◽  
pp. 713-727 ◽  
Author(s):  
Chin-Chean Wong ◽  
Chih-Hwa Chen ◽  
Li-Hsuan Chiu ◽  
Yang-Hwei Tsuang ◽  
Meng-Yi Bai ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Articular Cartilage ◽  
Cartilage Repair ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Articular Cartilage Repair ◽  
3 Dimensional ◽  
Cell Conversion

Background: Insufficient cell numbers still present a challenge for articular cartilage repair. Converting heterotopic auricular chondrocytes by extracellular matrix may be the solution. Hypothesis: Specific extracellular matrix may convert the phenotype of auricular chondrocytes toward articular cartilage for repair. Study Design: Controlled laboratory study. Methods: For in vitro study, rabbit auricular chondrocytes were cultured in monolayer for several passages until reaching status of dedifferentiation. Later, they were transferred to chondrogenic type II collagen (Col II)–coated plates for further cell conversion. Articular chondrogenic profiles, such as glycosaminoglycan deposition, articular chondrogenic gene, and protein expression, were evaluated after 14-day cultivation. Furthermore, 3-dimensional constructs were fabricated using Col II hydrogel-associated auricular chondrocytes, and their histological and biomechanical properties were analyzed. For in vivo study, focal osteochondral defects were created in the rabbit knee joints, and auricular Col II constructs were implanted for repair. Results: The auricular chondrocytes converted by a 2-step protocol expressed specific profiles of chondrogenic molecules associated with articular chondrocytes. The histological and biomechanical features of converted auricular chondrocytes became similar to those of articular chondrocytes when cultivated with Col II 3-dimensional scaffolds. In an in vivo animal model of osteochondral defects, the treated group (auricular Col II) showed better cartilage repair than did the control groups (sham, auricular cells, and Col II). Histological analyses revealed that cartilage repair was achieved in the treated groups with abundant type II collagen and glycosaminoglycans syntheses rather than elastin expression. Conclusion: The study confirmed the feasibility of applying heterotopic chondrocytes for cartilage repair via extracellular matrix–induced cell conversion. Clinical Relevance: This study proposes a feasible methodology to convert heterotopic auricular chondrocytes for articular cartilage repair, which may serve as potential alternative sources for cartilage repair.

scholarly journals MouseSnailFamily Transcription Repressors Regulate Chondrocyte, Extracellular Matrix, Type II Collagen, and Aggrecan

2003 ◽  
Vol 278 (43) ◽  
pp. 41862-41870 ◽  
Author(s):  
Kenji Seki ◽  
Toshihiko Fujimori ◽  
Pierre Savagner ◽  
Akiko Hata ◽  
Tomonao Aikawa ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Matrix Type ◽  
Transcription Repressors

Recombinant human type II collagen as a material for cartilage tissue engineering

2008 ◽  
Vol 31 (11) ◽  
pp. 960-969 ◽  
Author(s):  
H.J. Pulkkinen ◽  
V. Tiitu ◽  
P. Valonen ◽  
E.-R. Hämäläinen ◽  
M.J. Lammi ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Confocal Microscopy ◽  
Cartilage Tissue Engineering ◽  
Type Ii Collagen ◽  
Cartilage Tissue ◽  
Homogeneous Distribution ◽  
Type Ii ◽  
Human Type ◽  
Cultivation Period

Purpose Collagen type II is the major component of cartilage and would be an optimal scaffold material for reconstruction of injured cartilage tissue. In this study, the feasibility of recombinant human type II collagen gel as a 3-dimensional culture system for bovine chondrocytes was evaluated in vitro. Methods Bovine chondrocytes (4x106 cells) were seeded within collagen gels and cultivated for up to 4 weeks. The gels were investigated with confocal microscopy, histology, and biochemical assays. Results Confocal microscopy revealed that the cells maintained their viability during the entire cultivation period. The chondrocytes were evenly distributed inside the gels, and the number of cells and the amount of the extracellular matrix increased during cultivation. The chondrocytes maintained their round phenotype during the 4-week cultivation period. The glycosaminoglycan levels of the tissue increased during the experiment. The relative levels of aggrecan and type II collagen mRNA measured with realtime polymerase chain reaction (PCR) showed an increase at 1 week. Conclusion Our results imply that recombinant human type II collagen is a promising biomaterial for cartilage tissue engineering, allowing homogeneous distribution in the gel and biosynthesis of extracellular matrix components.

Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF

2001 ◽  
Vol 281 (4) ◽  
pp. C1291-C1299 ◽  
Author(s):  
Brant E. Isakson ◽  
Richard L. Lubman ◽  
Gregory J. Seedorf ◽  
Scott Boitano
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Type I Collagen ◽  
Cultured Cells ◽  
Cell Phenotype ◽  
Alveolar Type ◽  
Specific Cell ◽  
Specific Marker ◽  
Type I ◽  
Type Ii

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca2+ changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca2+ signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca2+ signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.

scholarly journals Functional Lateral Shift of the Mandible Effects on the Expression of ECM in Rat Temporomandibular Cartilage

2009 ◽  
Vol 79 (4) ◽  
pp. 652-659 ◽  
Author(s):  
Tanapan Wattanachai ◽  
Ikuo Yonemitsu ◽  
Sawa Kaneko ◽  
Kunimichi Soma
Keyword(s):  
Extracellular Matrix ◽  
Experimental Period ◽  
Type Ii Collagen ◽  
Lateral Shift ◽  
Control Group ◽  
Type Ii ◽  
Early Puberty ◽  
Condylar Cartilage ◽  
Cartilage Extracellular Matrix ◽  
Extracellular Matrix Components

Abstract Objective: To test the hypothesis that the effects of mechanical stress from a functional lateral shift of the mandible have no effect on the expression of two main condylar cartilage extracellular matrix components, type II collagen and aggrecan, in rats from early puberty to young adulthood. Materials and Methods: Functional lateral shift of the mandible was induced in experimental groups of 5-week-old male Wistar rats, using guiding appliances. The rats were sacrificed at 3, 7, 14, and 28 days post appliance attachment. The condyles were immunohistochemically evaluated for type II collagen and aggrecan (the immunoreactive areas were quantified). Results: As compared with the control group, on the contralateral condyles, the immunoreactivity of the experimental groups was significantly increased from 7 to 14 days. While on the ipsilateral condyles, the immunoreactive areas were significantly decreased throughout the experimental period. Conclusion: A functional lateral shift of the mandible modulated the condylar cartilage extracellular matrix differently on each side of the condyle, which affected condylar morphology, growth, biomechanical properties, and even the susceptibility of the condylar cartilage to pathogenesis.

Bone growth retardation in mouse embryos expressing human collagenase 1

2007 ◽  
Vol 293 (4) ◽  
pp. C1209-C1215 ◽  
Author(s):  
Kazushi Imai ◽  
Seema S. Dalal ◽  
John Hambor ◽  
Peter Mitchell ◽  
Yasunori Okada ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Retardation ◽  
Bone Growth ◽  
Type Ii Collagen ◽  
Cellular Growth ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Simultaneous Increase ◽  
Integrin Subunit ◽  
Type Ii ◽  
Matrix Metalloproteinase 1

Cellular growth and differentiation are readouts of multiple signaling pathways from the intercellular and/or extracellular milieu. The extracellular matrix through the activation of cellular receptors transmits these signals. Therefore, extracellular matrix proteolysis could affect cell fate in a variety of biological events. However, the biological consequence of inadequate extracellular matrix degradation in vivo is not clear. We developed a mouse model expressing human collagenase (matrix metalloproteinase-1, MMP-1) under the control of Col2a1 promoter. The mice showed significant growth retardation during embryogenesis and a loss of the demarcation of zonal structure and columnar array of the cartilage. Immunological examination revealed increased degradation of type II collagen and upregulation of fibronectin and α5-integrin subunit in the transgenic cartilage. The resting zone and proliferating zone of the growth plate cartilage exhibited a simultaneous increase in bromodeoxyuridine (BrdU)-incorporated proliferating cells and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling-positive apoptotic cells, respectively. Chondrocyte differentiation was not disturbed in the transgenic mice as evidenced by normal expression of the Ihh and type X collagen expression. These data demonstrate that type II collagen proteolysis is an important determinant for the skeletal outgrowth through modulation of chondrocyte survival and cartilagenous growth.

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