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Adipose-derived stromal cells (ASCs) are a promising cell source for tissue engineering and regenerative medicine approaches for cartilage replacement. For chondrogenic differentiation, human (h)ASCs were seeded on three-dimensional polyurethane (PU) fibrin composites and induced with a chondrogenic differentiation medium containing TGF-ß3, BMP-6, and IGF-1 in various combinations. In addition, in vitro predifferentiated cell-seeded constructs were implanted into auricular cartilage defects of New Zealand White Rabbits for 4 and 12 weeks. Histological, immunohistochemical, and RT-PCR analyses were performed on the constructs maintained in vitro to determine extracellular matrix (ECM) deposition and expression of specific cartilage markers. Chondrogenic differentiated constructs showed a uniform distribution of cells and ECM proteins. RT-PCR showed increased gene expression of collagen II, collagen X, and aggrecan and nearly stable expression of SOX-9 and collagen I. Rabbit (r)ASC-seeded PU-fibrin composites implanted in ear cartilage defects of New Zealand White Rabbits showed deposition of ECM with structures resembling cartilage lacunae by Alcian blue staining. However, extracellular calcium deposition became detectable over the course of 12 weeks. RT-PCR showed evidence of endochondral ossification during the time course with the expression of specific marker genes (collagen X and RUNX-2). In conclusion, hASCs show chondrogenic differentiation capacity in vitro with the expression of specific marker genes and deposition of cartilage-specific ECM proteins. After implantation of predifferentiated rASC-seeded PU-fibrin scaffolds into a cartilage defect, the constructs undergo the route of endochondral ossification.

Nikolaus Poier,1,2 Johannes Hochstöger,1,2 Stephan Hackenberg,3 Agmal Scherzad,3 Maximilian Bregenzer,3 Dominik Schopper,1,2 Norbert Kleinsasser1,3 1Department of Otorhinolaryngology, Head and Neck Surgery, Kepler University Hospital, Linz 4021, Austria; 2Medical Faculty, Johannes Kepler University Linz, Linz 4040, Austria; 3Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University of Wuerzburg, Würzburg 97080, GermanyCorrespondence: Nikolaus Poier Krankenhausstraße 9, Linz 4021, AustriaTel +43 650 2409889Email nikolaus.poier@kepleruniklinikum.atPurpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated.Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment.Results: NPs showed to be spherical in shape with an average size of 45– 55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 μg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis.Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.Keywords: zinc oxide, nanoparticles, cytotoxicity, genotoxicity, toxicity

Purpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated. Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment. Results: NPs showed to be spherical in shape with an average size of 45 55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 g/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis. Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems. (VLID)5342594 Version of record

Magnetic nanoparticles (NPs), such as very small iron oxide NPs (VSOPs) can be used for targeted drug delivery, cancer treatment or tissue engineering. Another important field of application is the labelling of mesenchymal stem cells to allow in vivo tracking and visualization of transplanted cells using magnetic resonance imaging (MRI). For these NPs, however, various toxic effects, as well as functional impairment of the exposed cells, are described. The present study evaluates the influence of VSOPs on the multilineage differentiation ability and cytokine secretion of human adipose tissue derived stromal cells (hASCs) after long-term exposure. Human ASCs were labelled with VSOPs, and the efficacy of the labelling was documented over 4 weeks in vitro cultivation of the labelled cells. Unlabelled hASCs served as negative controls. Four weeks after labelling, adipogenic and osteogenic differentiation was histologically evaluated and quantified by polymerase chain reaction (PCR). Changes in gene expression of IL-6, IL-8, VEGF and caspase 3 were determined over 4 weeks. Four weeks after the labelling procedure, labelled and unlabelled hASCs did not differ in the gene expression of IL-6, IL-8, VEGF and caspase 3. Furthermore, the labelling procedure had no influence on the multidifferentiation ability of hASC. The percentage of labelled cells decreased during in vitro expansion over 4 weeks. Labelling with VSOPs and long-term intracellular disposition probably have no influence on the physiological functions of hASCs. This could be important for the future in vivo use of iron oxide NPs.