Cartilage-Specific Matrix and Integrin Expression in Three-Dimensional Articular Chondrocyte Cultures Overexpressing Human Interleukin-10

Interleukin (IL)-10 overexpression inhibits joint inflammation, however the effect of high local concentrations of IL-10 on chondrocyte homeostasis remains unclear. The aim of this study was to determine the effects of IL-10 overexpression on cartilage matrix production in three-dimensional (3D) chondrocyte cultures. Human articular chondrocytes were transduced with adenoviral vectors alone (adv/empty) or by vectors either overexpressing enhanced green fluorescence protein (adv/EGFP) or human IL-10 (adv/hIL-10) before their transfer to a 3D culture system. Non-transduced chondrocytes were used as controls. The expression of IL-10 or EGFP was confirmed using ELISA or flow cytometry. Chondrocytes synthesis of collagen types II and I, aggrecan, fibronectin and β1-integrin was determined over a period of 14 days post transduction using flow cytometry or immunohistochemistry. adv/EGFP or adv/IL-10 transduced chondrocytes expressed EGFP or secreted IL-10 detectable over the 2 weeks culture period. No suppression of collagen type II, aggrecan or β1-integrin synthesis by IL-10 overexpression was found and the deposition of collagen type I and fibronectin remained unaffected compared to the controls. IL-10 overexpression does not impair key features of chondrocytes differentiated phenotype (e.g. collagen type II and aggrecan expression) suggesting the potential use of IL-10 for gene therapeutic approaches in the joint.

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Effects of Low Concentration BMP-7 on Human Osteoarthritic Chondrocytes: Comparison of Different Applications

Journal of Biomaterials Applications ◽

10.1177/0885328210388439 ◽

2010 ◽

Vol 26 (7) ◽

pp. 845-859 ◽

Cited By ~ 7

Author(s):

Karsten Gavenis ◽

Nicole Heussen ◽

Bernhard Schmidt-Rohlfing

Keyword(s):

Collagen Type ◽

Articular Chondrocytes ◽

3D Culture ◽

Cartilage Tissue ◽

Type I ◽

Type Ii ◽

Collagen Type Ii ◽

Low Concentration ◽

Osteoarthritic Chondrocytes ◽

Extracellular Matrix Components

While BMP-7 (OP-1) is one of the most potent growth factors in cartilage tissue engineering, the effects of exogenous low concentration BMP-7 on osteoarthritic chondrocytes are still unknown. Human osteoarthritic chondrocytes obtained from the femoral condyles of 10 patients were grown either in monolayer or in 3D collagen type-I gel culture in vitro. The growth factor was either given as a single dose of 50 ng/mL, a repeated dose, or continuously released from PGLA microspheres. Matrix formation was monitored by immunohistochemical staining and real-time PCR. In contrast to monolayer culture, the differentiated phenotype was prevailed in 3D culture. Collagen type-II protein production in the 3D group with a continuous BMP-7 release was enhanced in comparison to all other groups. Gene expression of collagen type-II and aggrecan was elevated in all treatment groups, with the highest extent in the BMP-7 microsphere group. In summary, treatment of articular chondrocytes with a low dose of BMP-7 leads to an elevated production of extracellular matrix components. This effect is further increased when BMP-7 is given repeatedly or continuously, which proved to be the most effective form of application.

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Maintenance of chondrocyte phenotype during expansion on PLLA microtopographies

Journal of Tissue Engineering ◽

10.1177/2041731418789829 ◽

2018 ◽

Vol 9 ◽

pp. 204173141878982 ◽

Cited By ~ 6

Author(s):

Elisa Costa ◽

Cristina González-García ◽

José Luis Gómez Ribelles ◽

Manuel Salmerón-Sánchez

Keyword(s):

Lactic Acid ◽

Type I Collagen ◽

Collagen Type ◽

Articular Chondrocytes ◽

Collagen Type I ◽

Cell Phenotype ◽

Type I ◽

Type Ii ◽

Collagen Type Ii ◽

Chondrocyte Phenotype

Articular chondrocytes are difficult to grow, as they lose their characteristic phenotype following expansion on standard tissue culture plates. Here, we show that culturing them on surfaces of poly(L-lactic acid) of well-defined microtopography allows expansion and maintenance of characteristic chondrogenic markers. We investigated the dynamics of human chondrocyte dedifferentiation on the different poly(L-lactic acid) microtopographies by the expression of collagen type I, collagen type II and aggrecan at different culture times. When seeded on poly(L-lactic acid), chondrocytes maintained their characteristic hyaline phenotype up to 7 days, which allowed to expand the initial cell population approximately six times without cell dedifferentiation. Maintenance of cell phenotype was afterwards correlated to cell adhesion on the different substrates. Chondrocytes adhesion occurs via the α5 β1 integrin on poly(L-lactic acid), suggesting cell–fibronectin interactions. However, α2 β1 integrin is mainly expressed on the control substrate after 1 day of culture, and the characteristic chondrocytic markers are lost (collagen type II expression is overcome by the synthesis of collagen type I). Expanding chondrocytes on poly(L-lactic acid) might be an effective solution to prevent dedifferentiation and improving the number of cells needed for autologous chondrocyte transplantation.

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Collagen Expression in Tissue Engineered Cartilage of Aged Human Articular Chondrocytes in a Rotating Bioreactor

The International Journal of Artificial Organs ◽

10.1177/039139880302600407 ◽

2003 ◽

Vol 26 (4) ◽

pp. 319-330 ◽

Cited By ~ 34

Author(s):

S. Marlovits ◽

B. Tichy ◽

M. Truppe ◽

D. Gruber ◽

W. Schlegel

Keyword(s):

Electron Microscopy ◽

Collagen Type ◽

Articular Chondrocytes ◽

Three Dimensional ◽

Collagen Type I ◽

Human Articular Chondrocytes ◽

Type I ◽

Transmission Electron ◽

Low Shear Stress ◽

Engineered Cartilage

This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel. Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks. Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy. During serial monolayer cultures chondrocytes dedifferentiated to a “fibroblast-like” structure and produced predominantly collagen type I. When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time. Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected. Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils. This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.

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Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.1160/vcot-06-07-0061 ◽

2007 ◽

Vol 20 (03) ◽

pp. 185-191 ◽

Cited By ~ 18

Author(s):

A. O. Oshin ◽

E. Caporali ◽

C. R. Byron ◽

A. A. Stewart ◽

M. C. Stewart

Keyword(s):

Articular Cartilage ◽

Collagen Type ◽

Articular Chondrocytes ◽

Soluble Form ◽

Mrna Levels ◽

Type Ii ◽

Collagen Type Ii ◽

Endogenous Expression ◽

Chondrocytic Phenotype

SummaryArticular chondrocytes are phenotypically unique cells that are responsible for the maintenance of articular cartilage. The articular chondrocytic phenotype is influenced by a range of soluble factors. In particular, members of the bone morphogenetic protein (BMP) family support the articular chondrocytic phenotype and stimulate synthesis of cartilaginous matrix. This study was carried out to determine the importance of BMPs in supporting the differentiated phenotype of articular chondrocytes in vitro. Exogenous BMP-2 supported expression of collagen type II and aggrecan in monolayer chondrocyte cultures, slowing the dedifferentiation process that occurs under these conditions. In contrast, BMP-2 had little effect on expression of these genes in three-dimensional aggregate cultures. Endogenous BMP-2 expression was lost in monolayer cultures, coincident with the down-regulation of collagen type II and aggrecan mRNAs, whereas BMP-2 mRNA levels were stable in aggregate cultures. Antagonism of endogenous BMP activity in aggregate cultures by Noggin or a soluble form of the BMP receptor resulted in reduced expression of collagen type II and aggrecan mRNAs, reduced collagen type II protein and sulfated glycosaminoglycan (GAG) deposition into the aggregate matrices and reduced secretion of GAGs into the culture media. These results indicate that endogenous BMPs are required for maintenance of the differentiated articular chondrocytic phenotype in vitro. These findings are of importance to cell-based strategies designed to repair articular cartilage. Articular chondrocytes require conditions that will support endogenous expression of BMPs to maintain the specialized phenotype of these cells.

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Limb bud mesenchyme cultured under tensile strain remodel collagen type I tubes to produce fibrillar collagen type II

Biorheology ◽

10.3233/bir-2009-0553 ◽

2009 ◽

Vol 46 (6) ◽

pp. 439-450 ◽

Cited By ~ 1

Author(s):

Jennifer R. Amos ◽

Shigeng Li ◽

Michael Yost ◽

Harry Phloen ◽

Jay D. Potts

Keyword(s):

Tensile Strain ◽

Collagen Type ◽

Collagen Type I ◽

Limb Bud ◽

Type I ◽

Fibrillar Collagen ◽

Type Ii ◽

Collagen Type Ii

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Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin

Experimental Biology and Medicine ◽

10.1258/ebm.2011.011183 ◽

2011 ◽

Vol 236 (11) ◽

pp. 1333-1341 ◽

Cited By ~ 48

Author(s):

Giuseppe Musumeci ◽

Debora Lo Furno ◽

Carla Loreto ◽

Rosario Giuffrida ◽

Silvia Caggia ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Collagen Type ◽

Three Dimensional ◽

3D Culture ◽

Collagen Type I ◽

Type I ◽

Human Mscs ◽

Alternative Source

The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

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Limb bud mesenchyme cultured under tensile strain remodel collagen type I tubes to produce fibrillar collagen type II

Biorheology ◽

10.3233/bir-2010-0571 ◽

2010 ◽

Vol 47 (2) ◽

pp. 163-163

Author(s):

Jennifer R. Amos ◽

Shigeng Li ◽

Michael Yost ◽

Harry Phloen ◽

Jay D. Potts

Keyword(s):

Tensile Strain ◽

Collagen Type ◽

Collagen Type I ◽

Limb Bud ◽

Type I ◽

Fibrillar Collagen ◽

Type Ii ◽

Collagen Type Ii

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Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

The Journal of Cell Biology ◽

10.1083/jcb.138.5.1159 ◽

1997 ◽

Vol 138 (5) ◽

pp. 1159-1167 ◽

Cited By ~ 76

Author(s):

Lisbet Camper ◽

Dick Heinegård ◽

Evy Lundgren-Åkerlund

Keyword(s):

Cell Adhesion ◽

Matrix Protein ◽

Collagen Type ◽

Human Fibroblasts ◽

Cartilage Matrix ◽

Affinity Column ◽

Integrin Subunit ◽

Β1 Integrin ◽

Type Ii ◽

Collagen Type Ii

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg2+ or Mn2+ but not Ca2+. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ∼110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.

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Enhanced Early Tissue Regeneration after Matrix-Assisted Autologous Mesenchymal Stem Cell Transplantation in Full Thickness Chondral Defects in a Minipig Model

Cell Transplantation ◽

10.3727/096368909x471297 ◽

2009 ◽

Vol 18 (8) ◽

pp. 923-932 ◽

Cited By ~ 59

Author(s):

Martin Jung ◽

Balazs Kaszap ◽

Anna Redöhl ◽

Eric Steck ◽

Steffen Breusch ◽

...

Keyword(s):

Articular Cartilage ◽

Collagen Type ◽

Type I ◽

Type Ii ◽

Collagen Type Ii ◽

Histomorphometric Analysis ◽

Tissue Quality ◽

Repair Tissue ◽

Adult Mesenchymal Stem Cells ◽

Defect Area

Adult mesenchymal stem cells (MSCs) are an attractive cell source for new treatment strategies in regenerative medicine. This study investigated the potential effect of matrix assisted MSC transplantation for articular cartilage regeneration in a large-animal model 8 weeks postoperatively. MSCs from bone marrow aspirates of eight Goettingen minipigs were isolated and expanded prior to surgery. Articular cartilage defects of 5.4 mm were created bilaterally in the medial patellar groove without penetrating the subchondral bone plate. Defects were either left empty ( n = 4), covered with a collagen type I/III membrane ( n = 6) or additionally treated with autologous MSC transplantation (2 × 106; n = 6). After 8 weeks animals were euthanized and the defect area was assessed for its gross appearance. Histomorphological analysis of the repair tissue included semiquantitative scoring (O'Driscoll score) and quantitative histomorphometric analysis for its glycosaminoglycan (GAG) and collagen type II content. All membranes were found to cover the defect area 8 weeks postoperatively. Median defect filling was 115.8% (membrane), 117.8% (empty), and 100.4% (MSC), respectively (not significant). Histomorphological scoring revealed significantly higher values in MSC-treated defects (median 16.5) when compared to membrane treatment (median 9.5) or empty defects (median 11.5; p = 0.015 and p = 0.038). Histomorphometric analysis showed larger GAG/collagen type II-positive areas in the MSC-treated group (median 24.6%/29.5% of regeneration tissue) compared to 13.6%/33.1% (empty defects) and 1.7%/6.2% (membrane group; p = 0.066). Cell distribution was more homogeneous in MSC compared to membrane-only group, where cells were found mainly near the subchondral zone. In conclusion, autologous matrix-assisted MSC transplantation significantly increased the histomorphological repair tissue quality during early articular cartilage defect repair and resulted in higher GAG/collagen type II-positive cross-sectional areas of the regenerated tissue.

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