Monoclonal antibody 11-fibrau: A useful marker to characterize chondrocyte differentiation stage
ABSTRACT The aim of this study was to determine the feasibility of discriminating between differentiated and dedifferentiated chondrocytes by using the Mab 11-fibrau. Mab 11-fibrau did not bind to differentiated chondrocytes in cartilage of human knee joint, auricle, or nasal septum. During monolayer culture, when cells dedifferentiate, the number of 11-fibrau positive cells gradually increased and reached up to 100% after 4 passages. When differentiated chondrocytes were cultured in alginate, most (90--95%) of the cells remained 11-fibrau negative, in accordance with previous studies demonstrating that differentiated chondrocytes cultured in alginate keep their phenotype. Dedifferentiated (11-fibrau positive) cells were subjected to different redifferentiation regimes. As a well-known fact, cultures in alginate in medium where FCS was replaced by IGF1 and TGF beta 2 results in increased collagen type II formation, indicative for redifferentiation. However, the cells remained 11-fibrau positive, suggesting they are not (yet) fully redifferentiated. On the other hand, when dedifferentiated cells (after 4 passages in monolayer culture) were seeded in a biomaterial and implanted subcutaneously in a nude mouse, the newly formed cartilage matrix contained collagen type II and the 11-fibrau staining on the cells had disappeared. Our results indicate that 11-fibrau may be a reliable and sensitive marker of chondrocyte phenotype.
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- "Cells and histological sections were incubated with either mouse monoclonal antibody against 11-fibrau (Clone D7-FIB; diluted 1:400; Imgen, Netherlands), a marker for fibroblasts , or monoclonal antibody against α-SMA (Clone 1A4; diluted 1:1000; Sigma, St.Louis, Missouri, USA), a marker for smooth muscle cells and pericytes , for two hours. Cells were rinsed in 1×PBS and IHC detection was performed using Link-Label (Biotin-based) Multilink® IHC Detection Kit (Biogenex, San Ramon, CA). "
ABSTRACT: Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.BMC Musculoskeletal Disorders 02/2007; 8:16. DOI:10.1186/1471-2474-8-16 · 1.72 Impact Factor
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ABSTRACT: Tissue engineering is considered to be one of the most innovative approach for tackling many diseases and body parts that need to be replaced. Biopolymeric scaffolds have been utilized in tissue engineering as a technique to confide the desired proliferation of seeded cells in vitro and in vivo into its architecturally porous three-dimensional structures. Novel freeform fabrication methods for tissue engineering polymeric scaffolds have been an interest because of its repeatability and capability of high accuracy in fabrication resolution at the macro and micro scales. A novel multi-nozzle biopolymer deposition system which is capable of extruding biopolymer solutions and living cells for bioactive fabrication of 3D alginate tissue scaffolds is presented. The deposition process is biocompatible and occurs at room temperature and low pressures to reduce damage to cells. Sodium alginate aqueous solution is deposited into calcium chloride solution using 3DD to form hydrogel structures. Feasibility studies showed that the system is capable of extruding Manugel alginate between 0.4% and 3% (w/v). The flow rate, nozzle diameter, and nozzle velocity were studied and a model was developed to design 3D scaffolds with controlled strut diameters (D = 250 - 410 microns) and pore sizes. In addition, rat heart endothelial cells were deposited through the system with alginate to form gel scaffold structures with encapsulated cells in a bioactive fabricated manor. The study showed that the suitable bioactive parameters preferred 1.5% (w/v) sodium alginate with 0.5% (w/v) calcium chloride. Cell viability studies were conducted on the cell encapsulated scaffolds for validating the bioactive freeform fabrication process that sowed viability up to 85%. The bioactive scaffold supported proliferation up to 21 days of incubation time. The elastic modulus was studied over degradation time that showed that the stiffened during the 24 hours due to crosslinking and degraded then on up to 21 days of incubation at 37 oC. The system showed potential use for accurate cell placement in tissue engineering applications and promote regenerative medicine based on CAD systems.
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ABSTRACT: For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.Tissue Engineering 09/2002; 8(4):573-80. DOI:10.1089/107632702760240490 · 4.25 Impact Factor