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11 Introduction 3 Recently, nanofibrous materials have been interesting for applications in tissue engineering. They better simulate the structure of fibrous component of natural extracellular matrix than conventional flat or microstructured surfaces and they enable adsorption of cell adhesion mediating molecules in an appropriate spatial conformation. The appropriate spatial conformation enables a good accessibility of active sites on these molecules by adhesion receptors on the cell membrane [1,2]. Most of the clinically used skin substitutes consist of non-resorbable material and allogeneic cells thus they cannot provide permanent coverage due to their final rejection.

The promising approach could be construction of nanofibrous carriers from biodegradable polymers which will be slowly resorbed in organism and finally replaced by regenerated tissue. The attractiveness of the nanofibrous membrane for adhesion and growth of skin cells can be further promoted by coating the membrane with biomolecules normally presented in natural skin (collagen, hyaluronan) or occurring during wound healing (fibrin). The study is focused on evaluation of adhesion and growth of human dermal fibroblasts and human immortal HaCaT keratinocytes on polylactide (PLA) nanofibrous membranes coated with fibrin, collagen and fibronectin.

Materials and methods FIG. Immunofluorescence staining of fibrin structure on PLA membranes (A) and phalloidin staining of F-actin (red) and nucleus staining (Hoechst 33342, blue) in human dermal fibroblasts after 24 hours of cultivation on PLA membranes with immunofluorescence stained fibrin structure (green) (B). Leica TCS SPE DM2500 confocal microscope, obj. 40 oil, bar 50 μm. Download film kutunggu jandamu full movie mp4. We evaluated adhesion, morphology, proliferation, metabolic activity (determined by MTS assay) and viability (determined by Live/dead assay) of dermal human fibroblasts and human immortal HaCaT keratinocytes. We also studied collagen production (real-time PCR, immunofluorescence staining) by fibroblasts stimulated by fibrin structure on PLA nanofibrous membrane.

The PLA membranes were prepared using the novel Nanospider needleless electrospinning technology. We coated the membranes with fibrin, fibrin with fibronectin (cell adhesion-mediating extracellular matrix protein), collagen I or collagen I with fibronectin. Morphology of human dermal fibroblasts (A) and human HaCaT keratinocytes (B) after 3 day-cultivation on pristine PLA membranes, PLA membranes with fibrin and fibronectin, fibrin, collagen and fibronectin or collagen. Standard cell culture polystyrene dish (PS) served as a reference material.

Cells stained with Texas Red C2-Maleimide and Hoechst # Olympus IX 51 microscope, obj. 10 x, DP 70 digital camera, bar 200 μm. Mitochondrial activity of human dermal fibroblasts (A) and human HaCaT keratinocytes (B) determined by MTS assay on day 1, 3 and 7 after cell seeding on pristine PLA membranes, PLA membranes with fibrin and fibronectin, fibrin, collagen and fibronectin or collagen. Standard cell culture polystyrene dish (PS) served as a reference material. Arithmetic means ± S.E.M from 9 measurements made on three independent samples for each experimental group and time interval. Results Results indicate that PLA nanofibrous membrane promoted adhesion and growth of the skin cells.

Fibrin (FIG.1) and collagen structures on PLA membranes further improved adhesion, proliferation and metabolic mitochondrial activity of the skin cells. The human dermal fibroblasts preferentially adhered and were more spread on the membranes coatedwith fibrin, fibrin with attached fibronectin on its surface or collagen I with fibronectin than on the membranes coated only with collagen or on the membranes in pristine form (FIG.2A). Moreover, the metabolic activity of human dermal fibroblasts was the highest on the membranes coated with fibrin or fibrin with fibronectin (FIG.3A). In addition, fibrin structures on PLA membranes stimulate fibroblasts to produce collagen I. The membranes coated with collagen I or collagen I with fibronectin promoted spreading of the HaCaT keratinocytes and increased the cell metabolic activity in comparison with pristine membranes or membranes coated with fibrin or fibrin with fibronectin (FIG.2B, 3B). Viability (determined by a Live/Dead assay) of the fibroblasts and the keratinocytes on the membranes was almost 100% on all samples.

Acknowledgements This study was supported by the Grant Agency of the Charles University in Prague, Czech Republic (GA UK, grant No ) and by the Grant Agency of the Czech Republic (grant No. References [1] Bacakova L, Filova E, Parizek M, Ruml T and Svorcik V. Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. 2011; 29: [2] Parizek M, Douglas TE, Novotna K, et al. Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering. Nanomed; 7: BLACK ORLON AS PROMISING MATERIAL FOR BONE TISSUE ENEGINEERING Martin Parizek¹, Miroslav Vetrik 2, Martin Hruby 2, Vera Lisa¹ And Lucie Bacakova¹ 1 Institute of Physiology, Acad.