?Fig.6,6, which was consistent with the International Society for Cellular Therapy (Krampera et al. and the nature of the chemical bonds between atoms were evaluated using Fourier transform infrared spectroscopy (FTIR) spectrum. Characterization of the seeded cells was morphologically evaluated by scanning electron microscopy and by flow cytometry for the expression of the stem cell surface markers. The differentiation potential was verified after chondrogenic induction by analyzing the expression of chondrogenic marker genes using real-time (RT PCR). Current study suggest significant potential for the use of ADSCs with the nanofibrous scaffolds in improving the osteoarthritis pathology. in rabbits. It has been mentioned that CS with a variety of delivery materials such as alginate, hydroxyapatite, hyaluronic acid, and growth factors have a potential application in orthopedic tissue engineering (Li et al. 2005; Yamane et al. 2005; Hsieh et al. 2005). Interestingly, it has been reported that CS blended with poly (vinyl alcohol) (PVA) have good mechanical and chemical characteristics (Charernsriwilaiwat et al. 2010). PVA is usually a water-soluble synthetic resin that produced via polymerization of vinyl acetate monomer. PVA was used in controlled release systems and due to its biocompatible nature; it has a variety of biomedical uses (Soppimath et al. 2000). Water-soluble polymers including polysaccharides (such as alginate) as well as synthetic polymers such as [Poly (ethylene oxide), PEO], [Poly (vinyl alcohol), PVA], [Poly (vinyl pyrrolidine, PVP] are known to be more biocompatible than other organic-soluble polymers. The electrospinning process which of relatively low cost and low toxicity, is an interesting approach for regenerative medicine requirements (Jimmy and Kandasubramanian 2020; Krishnan et al. 2013). There is another important factor in tissue engineering which is the scaffold fabrication method. Recently researcher focused on the electrospinning for the manufacture of nanofibrous scaffolds that are suitable for the 3D cell cultures for tissue regeneration (Li et al. 2002). Continuous nanofibers in electrospinning are formed due to the electrostatic Coulombic repulsive forces applied throughout elongation of the viscoelastic solution as it strengthens to form a fiber. Electrospinning is a simple method to produce nanofibers that is similar to the collagen part of the extracellular matrix (ECM). Fibers produced by this method have the features of large surface-to-volume ratio, and high porosity that are needed for tissue engineering, by which nanofibers allow better cellular spreading, attachment and supply efficient nutrient to the cells (Hezma et al. 2017; El-Rafei 2015; El-Rafei et al. 2017). The aim of the current study was to establish suitable physiologically and biochemically relevant microenvironment allowing ADSCs proliferation and differentiation into chondrocyte-like cells using CS/PVA nanofiber scaffolds. Methods Preparation of CS/PVA solutions Various combinations of the factors that control the quality of the electrospun fibers (e.g., composition of the electrospinning L-Ascorbyl 6-palmitate solution and its viscosity, applied voltage, and distance between collector and nozzle) were investigated by try-and-error method. The reported conditions are the optimal ones that gave fibers a homogeneous structure and high quality. Fibers were prepared by the dissolution of chitosan (medium molecular weight, deacetylated chitin, poly (D-glucosamine), Aldrich) in 2% acetic acid solution for 2C3?h at room temperature until the formation of a clear solution. PVA (common molecular weight?=?124,000, 87C89% hydrolyzed, Sigma-Aldrich) was gradually added to the chitosan solution at 75??5?C while stirring for an additional 2?h in order to enhance the dissolution of the PVA crystals. After complete dissolution, the prepared solution was stirred overnight in a magnetic stirrer at room temperature to obtain homogeneous solution. The CS/PVA nanofibrous mat L-Ascorbyl 6-palmitate was prepared using electrospinning apparatus (NaBond Company, China). The solution was transferred into a 10?ml plastic syringe equipped with a metallic capillary nozzle connected to a high power supply. The voltage was adjusted at 25?kV. The inner diameter of the used nozzle was 0.49?mm and its height from the collector was set at 10?cm. The selected flow rate was 0.7?mL/h. The electrospun CLDN5 fibers were collected on an aluminum foil collector. Then, the electrospun mat was collected, dried for 24?h then stored for further characterization. Characterization of the CS/PVA The microstructure of as-spun nano fibers was examined using Field Emission Scanning Electron Microscopy (FE-SEM) (Philips XL30, Netherlands).The Fourier transform infrared spectroscopy (FTIR) spectrum of nanofibrous mats was recorded using a Vertex 70 spectrometer (Bruker Optiks, L-Ascorbyl 6-palmitate Germany). The nanofibers were mixed with KBr powder, at a weight ratio of 1/100 nanofiber/KBr, then pressed to form a disc. The spectrum was in the spectral range of 4000-400?cm??1 with spectral resolution of 2.0?cm??1 and scan velocity of 2?mm??1. The viscosity of the blend solutions CS/PVA was measured using a rotating Viscometer (Brookfield viscometer DV-E, USA). Isolation of adipose tissue Mesenchymal stem cells (ADSCs) ADSCs were obtained from freshly isolated subcutaneous fat from healthy.
