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The aim of this study was to examine the influence of matrix elasticity on the maintenance of the chondrogenic phenotype of chondrocytes cultured in monolayer. We used a two-dimensional culturing system in which polyacrylamide gels with different concentrations of bis-acrylamide were coated with collagen type I. Matrices with a Young's modulus of 4, 10, 40, and 100 kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on these matrices at a low density for 7 days. The proliferation of cells was analyzed by 5-Bromo-2′-deoxy-uridine incorporation. Maintenance of the chondrogenic phenotype was analyzed by measuring collagen type I, type II, and aggrecan gene expression, immunofluorescence staining for collagen type II, and phalloidin staining for actin filaments. Cellular proliferation and actin organization were decreased on matrices of 4 kPa compared with stiffer substrates. The differentiated phenotype of the chondrocytes grown on matrices of 4 kPa was stabilized, indicated by higher collagen type II and aggrecan, and lower collagen type I expression. These findings indicate that chondrocytes sense the elasticity of the matrix and might be used for the design of scaffolds with mechanical properties specifically tailored to support the chondrogenic phenotype in tissue engineering applications.

Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro . Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.