Changes in the endogenous BMP expression during redifferentiation of chondrocytes in 3D cultures.

Engineering cartilage tissue is challenging, mainly because chondrocytes lose their differentiated phenotype when cultured in monolayer. The aim of this study was to analyse the influence of 3D-culture conditions on the redifferentiation of chondrocytes, devoting special attention to BMPs. Dedifferentiated chondrocytes were seeded onto two different scaffolds (Bio-Gide and HYAFF-11) and were then cultured for 38 days. Every week, samples were taken for gene expression analysis and immunohistochemistry. In both scaffolds an increasing differentiation was observed caused by an increase in Col2 and a reduction in Col1 expression. The various BMPs were regulated, albeit differently by the changing culture conditions. While GDF-5 and BMP-4 expression increased in the monolayer culture in comparison with native cartilage and decreased again in the 3D culture, the BMP-2 and BMP-6 expression decreased dramatically in the monolayer as well as in the 3D culture. BMP-7 was not detectable in any probe. Scaffold cultivation appears to stimulate the induction of redifferentiation, but is not sufficient to induce expression of BMP-2 -6 or -7. Since, in comparison to cartilage development, there is a lack of surrounding signal centres, external stimuli seem to be required to obtain complete redifferentiation. Our data indicate that a combination of BMP-2, -6 and -7 may be promising for this purpose.

Alterations in CD44 isoforms and HAS expression in human articular chondrocytes during the de- and re-differentiation processes.

The purpose of this study was to investigate the expression of different CD44 and hyaluronan synthase isoforms in cartilage, their alterations during the chondrocyte dedifferentiation process in monolayer culture and during the redifferentiation process on 3D scaffolds. Chondrocytes isolated from human articular cartilage were cultured as a monolayer for up to 36 days and were seeded on two different 3D scaffolds (HYAFF 11 and Bio-Gide). Expression levels of CD44s, CD44-lt, CD44-st, HAS1, HAS2, HAS3 and UDPGD were determined by real-time RT-PCR at different time points. At the protein level CD44 and CD90 were analyzed by flow cytometry. HAS2 was found to be the predominantly expressed hyaluronan synthase in chondrocytes and was not subjected to any regulation during the dedifferentiation process. CD44s, CD44-lt, CD44-st and UDPGD, however, were upregulated immediately after cell isolation. In addition, a high cell density was found to significantly increase CD44-st and CD44-lt expression. Redifferentiation on 3D scaffolds reversed the increase of the CD44 expression. Our data point out that CD44 expression does not correlate with matrix assembly in chondrocytes and that CD44 has a regulatory function in chondrocytes, not necessarily on differentiation, but probably on proliferation.

An in vivo mouse model for human cartilage regeneration.

Cartilage regeneration methods have been examined in various animal models. The major limitation of those studies is the biological difference between human and animal cartilage. We propose an in vivo model for human chondrocytes in a human cartilage defect environment. Human full-thickness (2-4 mm) articular cartilage discs (diameter 10 mm) attached to 3-6 mm subchondral bone, were obtained from human femur heads. Chondral defects (diameter 4 mm) were set within the cartilage disc without violating the subchondral bone. Human chondrocytes were isolated, cultivated for three passages and then suspended at a concentration of 10(7) cells/ml. The defect was completely filled with the cell suspension (approximately 30 microl) and then covered with a thin sheet of human periosteum, fixed with fibrin sealant. Discs were implanted subcutaneously in the backs of nude mice for 5 and 8 weeks. Controls were uncovered discs filled with cell suspension and covered discs without cells. Histological evaluation revealed a gradient of differentiation from the cartilage lateral side to the centre of the defect. A proteoglycan-rich matrix was formed with some chondron-like structures at the border of native cartilage, whereas fibrous tissue was built in the centre of the defect. After 8 weeks the areas of differentiating cells enlarged compared to 5 weeks, indicating the progress of cartilage repair. The control discs without cells or cover showed no chondrogenesis. Interestingly, uncovered discs filled with cells showed comparable areas of differentiating cells at the defect surface but lack of fibrous tissue in the middle. The histological results were supported by MRI measurement.

Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.