摘要：

Tissue engineering (TE) of cartilage holds the promise to overcome the problems arising from the loss or failure of tissue. The optimization of the in vitro culture conditions is a very important step in the process of TE. The goal of this thesis was to study different factors affecting the development of in vitro engineered cartilage.Chondrocytes were seeded on biodegradable scaffolds. Then the cell-polymer constructs were cultivated over 4 weeks to develop a cartilage-like tissue. After that, the cell-polymer construct was biochemically and histologically investigated. The effects of exogenously applied interleukin-4 (IL-4) and growth factor binding proteins (IGFBPs) on the development of the engineered cartilage were analyzed; investigations into the mechanism of action of IL-4 were conducted. Furthermore, the potential of new copolymer scaffolds to generate cartilage-like tissue was evaluated. An experiment was designed to monitor the development of in vitro engineered cartilage for 16 months with a special focus on a possible calcification of the engineered cartilage.IL-4 was investigated in order to evaluate its potential to improve the quality of engineered cartilage. IL-4 was previously shown to inhibit degradation of GAG, a major component of cartilage ECM, though mechanisms of action are still controversially discussed. IL-4 had positive effects on ECM contents, and GAG distribution of the engineered cartilage. IL-4 increases the GAG content may be by inhibition of GAG degradation or increased GAG subtype synthesis. In order to elucidate the respective contributions, RT-PCR was employed to investigate the expression of (1) matrix metalloproteinases MMP-1, -3 and -13; (2) tissue inhibitor of metalloproteinases-1 (TIMP-1); and (3) aggrecan and biglycan. Our results suggest that IL-4 increases GAG fractions in engineered cartilage at least in part by decreasing MMP-13 expression. IGFBPs are a family of six or more related proteins that affect the growth rate of cartilage, have a high affinity for IGF-I and modulate its actions. The hypothesis that IGFBP-4 inhibits the growth of cartilage in absence of IGF-I was evaluated in a well-established 3-D culture system. IGFBP-4 in the absence of exogenous IGF-I, had significant inhibitory effects on the growth rate and ECM of the engineered cartilage compared to control constructs in a dose-dependent manner. IGFBP-4 in the presence of IGF-I (50 ng/ml) had significant inhibitory effects at all molar ratios (1:0.5 - 1:100; IGF-I: IGFBP-4), as compared to constructs treated only with IGF-I. In contrast, compared to the control constructs, the combinations showed no inhibitory effects on the engineered constructs at low IGFBP-4 concentrations (1:3), demonstrating that IGF-I can overcome the inhibitory effects of IGFBP-4.The hypothesis that IGFBP-5 may function as a growth factor in cartilage, as for bone tissue, was evaluated. The effects of IGFBP-5 on in vitro engineered cartilage were investigated in the presence and absence of exogenous IGF-I. The results demonstrated that exogenous IGFBP-5 in absence of IGF-I clearly stimulated the growth of cartilaginous tissue constructs. The collagen per ww of the constructs was slightly increased. Histological cross-sections revealed a more coherent tissue formation after application of IGFBP-5, as compared to control constructs; GAG was more evenly distributed throughout the sections in IGFBP-5 constructs. In combinations with IGF-I, IGFBP-5 generally significantly reduced the effects of IGF-I at all molar ratios investigated.Recently, a new copolymer scaffold has been developed consisting of hyaluronic acid, gelatin, and collagen. In order to improve mechanical stability of scaffolds made from natural polymers, chemical crosslinking (CL) can be employed. However, as chemical CL processes may lead to cell toxic effects or impairment of tissue development, such scaffolds have to be carefully investigated in cell culture. Scaffolds made from different derivatives of this copolymer, that is non-CL, genipin-, EDC- and glutaraldehyde-CL,

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