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.
- SourceAvailable from: drexel.edu[Show abstract] [Hide abstract]
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.
- [Show abstract] [Hide abstract]
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
- [Show abstract] [Hide abstract]
ABSTRACT: To treat a cartilage defect with tissue-engineering techniques, multiplication of donor cells is essential. However, during this multiplication in monolayer expansion culture chondrocytes will lose their phenotype and produce matrix of inferior quality (dedifferentiation). Dedifferentiation occurs more extensively with low seeding densities and passaging. To obtain cartilage of good quality it is important that the multiplicated cells regain their cartilaginous phenotype (redifferentiation capacity). A "gold standard" for the multiplication of chondrocytes in monolayer, with respect to seeding density and passaging, is lacking. In numerous available studies, various cell densities have been used, making comparison of the results of these studies difficult. Therefore, we performed a comparative study to gain insight concerning the effect of seeding density and passaging on the capacity of cells to redifferentiate. From the resulting data we deduced the seeding density in monolayer culture for which cell expansion is both sufficient and fast, while the cells retain a capacity to redifferentiate. As a guideline we calculated that, at minimum, 20-fold multiplication is needed to fill an average cartilage defect of 4 cm(2) with the amount of donor chondrocytes we obtained. For this study we used isolated ear chondrocytes from five children. Four different seeding densities in monolayer culture were used, ranging from 3500 to 30000 cells/cm(2). The cells were cultured for four passages. The capacity of the expanded chondrocytes to redifferentiate (redifferentiation capacity) was studied after an additional 3-week culture in alginate beads and was assessed by glycosaminoglycan production and immunohistochemical stainings for collagen type I, collagen type II, elastin, and a fibroblast marker (11-fibrau). In general, we found that both passaging and decreasing seeding density yielded an increase in expanded chondrocytes, but at the same time decreased the dedifferentiation capacity. In further analyzing our data according to the proposed guidelines we found that with lower seeding densities sufficient multiplication (20 times) was reached in less time and with less passaging than at higher seeding densities. Importantly, the redifferentiation capacity of these chondrocytes was preserved. It was equal to or even surpassed that of chondrocytes multiplied 20 times at higher seeding densities, which required more time and more passages in monolayer culture. Thus, for cartilage tissue-engineering purposes we propose that expansion culture with low seeding densities is preferable.Tissue Engineering 01/2004; 10(1-2):109-18. DOI:10.1089/107632704322791754 · 4.25 Impact Factor