The potency of culture-expanded nasal septum chondrocytes for tissue engineering of cartilage.

Tissue engineering techniques to create extra autologous cartilage for reconstructive surgery receive more and more scientific and industrial attention. The objective of this experimental study was to assess the use of in vitro multiplied chondrocytes of the nasal septum for generation of cartilage grafts using tissue engineering techniques. Cells isolated from a biopsy of septal cartilage of rabbits and humans were expanded in culture to get a sufficient number of cells to engineer a cartilage graft. The drawback of the expansion procedure is that the cells lose their cartilaginous phenotype (dedifferentiation). We studied a method to reverse the dedifferentiation of expanded cells to stimulate them to produce cartilage matrix of good quality. Rabbit chondrocytes showed reversion of dedifferentiation (redifferentiation) when fetal calf serum was replaced by the growth factors IGF1 and TGFbeta2. This was expressed by increased glycosaminoglycan synthesis and increased numbers of collagen type II-producing cells. The redifferentiation capacity of septal cartilage cells of young rabbits was higher than that of adult rabbits. In human chondrocytes from the nasal septum redifferentiation could also be induced by replacement of serum with IGF1 and TGFbeta2. This method, however, was less efficient than in rabbits. Chondrocytes of older patients (>40 years old) were no longer sensitive to the growth factor treatment. In conclusion, our study demonstrates a method to regain cartilage phenotype in multiplied cells of nasal septum cartilage needed for tissue engineering of new cartilage. These results are promising for this technique to generate cartilage grafts for facial plastic surgery of the nasal septum.

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction.

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.

Monoclonal antibody 11-fibrau: a useful marker to characterize chondrocyte differentiation stage.

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.

Chondrogenic potential of in vitro multiplied rabbit perichondrium cells cultured in alginate beads in defined medium.

Perichondrium has a chondrogenic capacity and is therefore a candidate tissue for engineering of cartilage in vitro. Donor age and culture conditions probably influence chondrogenesis. The aim of this study was to compare the chondrogenic capacity of ear and nasal perichondrium from young and adult rabbits, using serum containing and serum-free culture conditions. This study demonstrates that more than 1 million cells can be generated out of 1 cm(2) of perichondrium tissue in 3-5 weeks of culture, irrespective of age. Culturing of these cells in alginate in medium with 2, 10, or 20% fetal calf serum did result in the production of small amounts of glycosaminoglycan, but no collagen type II was demonstrated. When serum was replaced however by insulin-like growth factor-1 (IGF-1) (10 ng/mL) plus transforming growth factor-beta2 (TGF-beta2) (10 ng/mL) an increased glycosaminoglycan production and induction of collagen type II was found, especially in cells isolated from perichondrium of the ear. Cells derived from perichondrium of young rabbits showed larger chondrogenic potential than cells from perichondrium of adult rabbits. Moreover, stimulation of both glycosaminoglycan synthesis and collagen type II production was about five times higher in cells isolated from the ear perichondrium of young rabbits than of adult rabbits. We conclude that young auricular perichondrium seems a useful source of cells for tissue engineering of cartilage when cultured in serum-free medium in combination with IG-F1 and TGF-beta2.

The role of trabecular demineralized bone in combination with perichondrium in the generation of cartilage grafts.

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic effect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the effect of IGF1, TGFbeta2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an effect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no effect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis.

Effect of transforming growth factor-beta on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix.

The effects of transforming growth factor-beta (TGF-beta) on proteoglycan synthesis of chondrocytes are controversial. The hypothesis that the differential effect of TGF-beta is related to the differentiation stage of the chondrocytes is investigated in this study. Rabbit auricular chondrocytes were cultured in alginate. When seeded in alginate immediately after isolation, cells keep their cartilaginous phenotype. When cells are first cultured in monolayer, they lose their cartilaginous phenotype and become dedifferentiated. We used three different cell populations: (1) Differentiated cells (P0: immediately after isolation); (2) partially (de)differentiated cells (P1: after one passage in monolayer); (3) dedifferentiated cells (P4: after four passages in monolayer). Cells were characterized by morphology using electron microscopy, amount of proteoglycans using the Farndale assay and type of collagen produced using immunohistochemistry. The effects of addition of 10 ng/ml TGF-beta2 for 7 days to P0, P1 and P4 cells were compared. TGF-beta was added either directly from the start of the alginate culture, or after a preculture period of three weeks in alginate. The amount of proteoglycans was increased in all chondrocyte populations when TGF-beta was added immediately after seeding in alginate, indicating that the effect of TGF-beta on proteoglycan synthesis does not depend on the differentiation stage of cells. After preculture in alginate, stimulation of proteoglycan synthesis (as measured by amount of proteoglycans and 35S-sulfate incorporation) had vanished. This effect was independent of differentiation stage . A dose-response experiment with TGF-beta (1, 10, 50 ng/ml) confirmed this differentiation-stage-independent effect of TGF-beta on proteoglycan synthesis. Stimulation by TGF-beta can be retained after enzymatic digestion of the pericellular matrix and reseeding of the cells in alginate, indicating the importance of pericellular matrix for the effect of TGF-beta on matrix synthesis. Alkaline phosphatase (ALP) activity was largely inhibited by TGF-beta in P0 chondrocytes, either with or without preculture in alginate. After culturing in monolayer, ALP activity was not substantially changed by TGF-beta. This indicates that the effect of TGF-beta on ALP activity, in contrast to the effect on proteoglycan synthesis, does depend on the differentiation stage of the cells. Furthermore, the fact that ALP synthesis in P0 cells is still inhibited by TGF-beta after preculture indicates that these cells remain responsive to TGF-beta. This provides additional evidence for the importance of the pericellular matrix for regulation of the effect of TGF-beta on proteoglycan synthesis. The results indicate that, in pathological cartilage, matrix depletion might be the trigger for increased matrix synthesis in reaction to TGF-beta, suggesting an important role for TGF-beta in cartilage repair.