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Electrospinning of poly-vinylalcohol-polycaprolactone composites scaffolds for Tissue Engineering

Authors S. U. Maheshwari S. V. Kumar (&) Department of Nanoscience Karunya University,

N. Nagiah T. S. Uma Bioproducts Laboratory, Biomaterials Division,

Central Leather Research Institute, Adyar, Chennai, India

Presented By Kaushik suneet Mtech Industrial Biotechnology 150946005

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Contents• Introduction• Experimental part• Materials • Polymer solution preparation• Fabrication of pure PVA, PCL nanofibrous mats

and PVA/PCL bilayer nanofibrous membranes• Characterization• Results and discussions• References

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Introduction• The Prefix nano is given to polymer fiber materials whose diameters are

reduced from micrometers to submicrons or nanometers.• The application of nanofibers -membranes for gas filtration ,water

purification, tissue engineering scaffolds, sound absorption mats, and transdermal drug delivery patches.

• Electrospinning is a cost effective and elegant method to produce aligned or• random nanofibers depending upon the application . A very high strength• electric field ([1 kV/cm) is applied to the droplet • of a fluid which may be a melt or solution coming• out from the tip of a needle, which acts as one of• the electrodes.The charged droplet overcomes the• surface tension and whichfurther splits into fine• jets of the fiber accelerating towards the counter • Electrode leading to the formation of continuous • fibers

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• As the fibers travel towards the target the solvent evaporates forming a series of web like fine fibres

• PCL is an aliphatic polyester that has been used as biomaterial• It appears as semicrystalline polyester and is highly soluble in a wide

range of organic solvents• Due to its compatibility with many types of polymers ,PCL has been

commonly used for different biomedical applications• the limitations to the tissue regeneration using PCL scaffolds are the

hydrophobic property, which may affect the cell adhesion and its rate of degradation.

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• Polyvinyl alcohol (PVA) is a semicrystalline hydrophilic polymer soluble in water

• Has excellent chemical resistance, biocompatibility, physical properties, and biodegradability of PVA have led to the development of various commercial products based on this polymer.

• PVA is a inert and nontoxic polymer.• It has high water permeability and can be easily processed. PVA

readily reacts with different cross-linking agents to form a gel.• Hence, it is used in many biomedical and pharmaceutical applications,

due to its advantages such as nontoxic, non-carcinogenic, and bioadhesive characteristics with the ease of processing

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Experimental Part• Materials• Poly (vinyl alcohol) (PVA, MW 1, 15,000, 98 % hydrolyzed• Polycaprolactone (PCL, Mw 80,000, Merck, India) was used as raw material

for the present study. De-ionized water, Chloroform, and N–N Dimethylformamide (DMF) were used as solvents.

• Polymer solution preparation• PVA was dissolved in de-ionized water at • 70 C with constant stirring for 1 h using• a magnetic stirrer. The prepared solution• was cooled and used for electrospinning.

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• PCL was dissolved in combination solvent system of chloroform and DMF in the ratio of 4:1. The solution was vigorously stirred with a magnetic stirrer for 3 h. The following polymer concentrations which were prepared for 10 mL in appropriate solvents for PVA and PCL were 7.0, 8.0, and 10.0 wt%.

• Fabrication of pure PVA, PCL nanofibrous mats and PVA/PCL bilayer• nanofibrous membranes• electrospinning setup was used• A 10-mL glass syringe with a stainless-steel needle (18 Gauge) was

used to create drop of polymer solution.• A high-voltage power supply with maximum. voltage of 50 kV was

used as voltage source for electrospinning. • The polymer solution loaded in the syringe was ejected by a syringe

pump

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• Aluminium foil was pasted over the grounded copper plate electrode to collect the deposited nanofibers. The working parameters such as

• applied voltage (9 and 12 kV), concentration of polymer solution, and tip-tocollector distance (10 cm) were optimized to obtain uniform nanofibers of PVA

• To obtain PCL nanofibers the applied voltage was varied from 10 to 15 kV by keeping the Tip-collector distance constant at 10 cm

• The PVA/PCL bilayer was prepared by depositing PCL nanofibers over PVA nanofibrous membranes. The polymer solutions of PVA and PCL prepared in appropriate solvents were taken in separate 5 mL standard syringes attached to an 18-gauge blunted needle.

• The polymer solution drop was created, using a syringe pump, at the rate of 5 lL/min for PVA and PCL solution. A copper, grounded collector was used to collect the electrospun nanofibers at a distance of 15 cm from the needle. The applied voltage was maintained at 15 kV.

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• Characterizations• Scanning electron microscopy• The aluminum substrates coated with the electrospun fibers were mounted

under a scanning electron microscope • The samples were coated with platinum before SEM analysis and the

average fiber diameter (AFD) was estimated based on the micrographs taken at the higher magnification. Fibers were electrospun with

• different concentrations and by varying different process parameters.• X-ray diffraction• XRD patterns have been recorded using a X-ray diffractometer with Cu Kß

radiation (k = 1.5418 A ° ) at 40 kV and 30 mA in the 2h range of 0–90 with scan speed of 10/min.

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• FTIR spectroscopy• Chemical characteristics of PVA/PCL nanofibrous bilayer membrane

were determined using peeled fibrous membranes using a FTIR, IR Affinity 1. Typically, 21 scans were signal-averaged to reduce spectral noise.

• Thermal analysis• Differential scanning calorimetry (DSC) analysis of the fiber was

performed from 0 to 350 C at 10 C/min

• Viscosity and porosity is also measured

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Biocompatibility of the electrospun fibers

NIH 3T3 fibroblast cell suspension was slowly dispersed over the top surface of nanofiber-coated cover slips placed in a 24-well culture plate and maintained in DMEM with 10 % fetal calf serum supplemented with penicillin (120 units/mL), streptomycin (75 mg/mL), gentamycin (160 mg/mL), and amphotericin B (3 mg/mL) at 378 C, humidified with 5 % CO2.

Cells cultured in a blank well were used as a control. Seeded cells were cultured for 72 h and the medium was changed every day. The number of viable cell in the scaffold was determined using Q5 MTT [3-(4,

• 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide] assay.• The supernatant of each well was replaced with MTT diluted in serum-free

medium and the plates incubated at 37 C for 3 h. The medium was aspirated and the formazan needles were dissolved in 500 lL of dimethyl sulfoxide (DMSO), and then kept at room

• temperature for a few minutes to ensure that all the crystals were dissolved. Finally, absorbance was measured at 630 nm using UV spectrophotometer

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Results• Morphological studies of pure PVA nanofibers• Effect of polymer concentration• The fiber diameter in the electrospinning process depends on polymer

concentration.• Increase in solution concentration >increase in solution viscosity.• Usually, high-viscosity polymer solution ejects with extreme difficulty from the

needle to form nanofibers• . Therefore, appropriate solution concentration becomes one of the key

parameters to optimize the final electrospinning fibers

• Fig. 1 SEM microstructures of pure PVA nanofibers fabricated at a concentration of a 7.0, b 8.0, andc 10.0 wt%. Applied voltage is kept at 12 KV, flow rate at 6 lL min-1 and working distance at 10 cm

• is clearly observed that the AFD increases with increase in solution viscosity.

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• Effect of applied voltage• In the process of electrospinning, the applied electrical voltage affects

the jet stability and the fiber morphology to a remarkable degree.• An increase in the applied voltage causes high deposition rate due to

large amount of mass flow from the needle tip.

• Fig. 2 SEM images of 8.0 wt% and 10 wt% pure PVA nanofibers under different applied voltages. Feedrate 6 lL min-1, working distance 10 cm. a 8 wt% at 9 KV, b 8 wt% at 12 KV, c and d 10 wt% at 9 KVand 12 KV

• Decrease in fiber diameter is observed with increase in applied voltage

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Morphological studies of pure PCL nanofibers

• Effect of polymer concentration

• Fig. 3 SEM microstructures of pure PCL nanofibers fabricated at a concentration of a 7.0, b 8.0, and c 10.0 wt%. Applied voltage is 10 KV, flow rate 5 lL min-1, tip-collector distance is 10 cm

• As the solution concentration was increased to 8 and 10 t%,uniform beadless nanofibers were obtained

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• Effect of applied voltage

• Fig. 4 SEM images of 8.0 wt% and 10 wt% pure PCL nanofibers under different applied voltages. Feed rate 5 lL min-1, working distance 10 cm. a 7 wt% at 10 KV, b 7wt% at 15 KV, c, d 8 wt% at 10 KV and 15 KV, e, f 10 wt% at 10 KV and 15 KV.

• Decrease in fiber diameter is observed with increase in applied voltage for both polymer solution concentration of 8 and 10 wt% spun under flow rate of 5 lL min-1 and tip-collector distance of 10 cm.

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Morphological studies of PVA/PCL bilayers• Different bilayer samples were formed by spinning PCL (8 wt%) over

PVA(8 wt%) nano-membranes keeping the spinning parameters as 5 lL min-1 flowrate, 15 kV applied voltage, and tip-collector distance of 15 cm.

• The bilayer samples (A, B, and C) have varying thickness of PCL fibers based on their rate of deposition. The average fiber diameter (AFD) for bilayer samples A, B, and C is 203, 252, and 244 nm

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• XRD spectra• The diffraction pattern for PVA nanofibers shows typical peak at 2teta

= 19.82 depicting the semicrystalline behavior of PVA. For the PCL nanofibers

• very sharp diffraction peaks were observed• around 2teta = 23.8 and 21.4, respectively.• The prominent peaks for PVA-PCl bilayer• nanofibers occur nearly• at 19.30–20.89, 21.19–21.97, and• 23.23–23.45. All the corresponding• peaks are sharp, indicating semi crystalline• behaviour

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• Thermal analysis• Differential scanning calorimetric analysis of different electrospun

samples of (PVA-PCL) bilayer nanofibers are shown in Fig. Tm of PVA and PCL were found to be around 223 and 62^ C..

• All the bilayer samples show appropriate thermal stability as the degradation temperature shifts towards higher temperature range as compared with pure PVA sample.

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• Porosity• The PCL nanofibrous membrane showed enhanced porosity of 88 %.

The individual PVA scaffold was found have a porosity of 80 %. Porosity of bilayer membranes was 77, 89, and 78 % for samples A, B, and C, respectively. Enhanced porosity of the scaffolds may improve hydrophilicity of scaffold which is important for tissue engineering scaffold and in turn would enhance the cell viability

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• Biocompatibility• Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay showed 60–90 %

viability of NIH 3T3 cells after 72 h of culture on a pure PVA; PCL and PVA/PCL [A and B] bilayer fiber films (n=3) wrapped cover slips

• when compared• Best result was obtained for PVA/PCL sample [B] indicating that appropriate

thickness of PCL polymer overPVA membranes triggers better cell adhesion growth.

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Conclusion• The PVA/PCL bilayer membranes exhibited increased porosity in the

range of 75–90 %. It was observed that the PVA/ PCL bilayer membrane was more favorable for the cell culture than the pure PVAand PCL membrane.

• Cells cultured on the PVA/PCL bilayer membrane had good spreading and adhesion than on the pure PVA and PCL membrane indicating that

• presence of PCL layer over PVA membranes enhances the overall hydrophilicity of bilayer membranes providing suitable atmosphere for the cells to adhere and grow.

• The PVA/PCL bilayer membrane can be considered as a suitable biomaterial for cell culture applications

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References• 1. Gibson PW, Lee C, Ko F, Reneker D (2007) Application of nanofiber

technology to nonwoven• thermal insulation. J Eng Fiber Fabr 2:32–40• 2. Huang ZMH, Zhang YZ, Kotaki M, Ramakrishna S (2003) A review on

polymer nanofibers by• electrospinning and their applications in nanocomposites. Compos Sci Technol

63:2223–2253• 3. Gilbert W (1628) De Magnete, Magneticisque Corporibus, et de Magno

Magnete Tellure (on the• magnet and magnetic bodies, and on that great magnet the earth). Peter

Short, London• 4. Doshi J, Reneker DH (1995) Electrospinning process and applications of

electrospun fibers. J Electrostat• 35:151–160

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Thank you