Phenotype stability under hypoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under hypoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Confocal microscopic observations showed positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that more than 80% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§)PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control, (#) PEMs <i>versus</i> collagen. (§,* and #: <i>p</i><0.05 and §§§ and ***: <i>p</i><0.001).</p

Morphological aspect of differentiated cell.

<p>Optical phase contrast microscopy visualization of differentiated cells seeded on type I collagen (A, B) and polyelectrolyte multilayer films (PEMs) (C, D) until confluence under normoxic (A, C) and hypoxic (B, D) environment. Objective×20, scale bar 55 µm. The morphological examination of the confluent cells showed cobblestone shape (A, C) in normoxia and a spindle like (B, D) shape in hypoxia.</p

Phenotype stability under normoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under normoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Microscopical observations show positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that about 90% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§) PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control. (§ and *: <i>p</i><0.05, §§ and **: <i>p</i><0.01, and *** <i>p</i><0.001).</p

Vascular cell phenotype characterization.

<p>The endothelial cell were characterized by the expression of specific markers: CD31 (A–D) and von Willebrand Factor (E–H) and the smooth muscle cells by the expression of contractile markers: α- Smooth Muscle Actin (α-SMA: E–H), Smooth Muscle Myosin Heavy Chain (SM-MHC: I–L) and Calponin (M–P). Images were obtained by confocal microscopy observation at cell confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)) and cultivated under normoxic and hypoxic conditions. Objective×40, NA = 0.8, scale bars 75 µm. The figure showed the positive expression of specific ECs markers for cell differentiated under normoxic environment and positive expression of specific contractile SMCs markers for cell differentiated under hypoxic environment.</p

Chemically Detachable Polyelectrolyte Multilayer Platform for Cell Sheet Engineering

Human gingival fibroblasts (HGFs) cell sheets have a potential use for in vivo wound healing due to the ability of HGFs to adopt a contractile phenotype which is typically expressed during extracellular matrix tissue remodeling. For this purpose, we developed a chemically detachable platform based on poly­(allylamine hydrochloride)/poly­(styrene sulfonate) multilayer film built on a sacrificial precursor film which served as a substrate for HGF cell layer formation. The sacrificial precursor film, based on disulfide-containing polycation and polyanion, is degradable under mild conditions compatible for cell sheet detachment. Cellular viability and cell phenotype analysis of HGF show that the designed platform promotes cell phenotype switch into contractile phenotype, maintained after cell sheet lift-off. This contractile phenotype is acquired by fibroblasts during in vivo wound healing and tissue remodeling. HGFs cell sheet fragments, obtained by this detachment process, could be cultured later on showing a good retention of the typical spindle-shape of differentiated cells after 10 days of culture. HGFs cell sheets have great potential applications as autologous substrates for tissue repair and cellular synthetic platforms for research on connective tissue diseases or evaluation of novel therapeutic agents