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Director of Polymer/Composite Materials Laboratory Biocompatibility of Biomaterials Laboratory Materials’ Antibacterial Property Testing Laboratory Assoc. Prof. Dr. Hilal Turkoglu Sasmazel Atilim University, Department of Metallurgical and Materials Engineering E-mail: hilal.sasmazel@atilim.edu.trhilal.sasmazel@atilim.edu.tr Web: www.atilim.edu.tr/~hilal.sasmazelwww.atilim.edu.tr/~hilal.sasmazel biohill.atilim.edu.tr 4th International Conference on Integrative Biology (Integrative Biology 2016) July 18-20, 2016 Berlin, Germany
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1) Management Committee Member and Representative of Turkey in European Cooperation in Science and Technology (COST) Framework Action No: FP 1405, Action Title: Active and intelligent fibre-based packaging - innovation and market introduction (ActInPak), 2015-2019. 2) Management Committee Substitute Member and Representative of Turkey in European Cooperation in Science and Technology (COST) Framework Action No: MP 1206, Action Title: Electrospun Nano- fibres for Bio Inspired Composite Materials and Innovative Industrial Applications, 2013-2017. 3) Management Committee Member and Representative of Turkey in European Cooperation in Science and Technology (COST) Framework Action No: MP 1101, Action Title: Biomedical Applications of Atmospheric Pressure Plasma Technology, 2011-2015. Management Committee Member and Representative of Turkey of European Cooperation in Science and Technology (COST)
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Polymer/Composite Materials Laboratory Biocompatibility of Biomaterials Laboratory Materials’ Antibacterial Property Testing Laboratory biohill.atilim.edu.tr
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DEVELOPMENT OF POLYMERIC AND COMPOSITE MATERIALS PREPARATION Solvent Casting Freeze Drying Electrospinning MODIFICATION Wet Chemistry Atmospheric Pressure Plasma Nozzle Type Dielectric Barrier Discharge
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Developed Composite Materials Development of Materials for Fuel Cell Applications Development of Antibacterial Materials Development of Biomaterials (Dental implants, Maxillofacial implants, etc.) Development of Tissue Scaffolds DEVELOPMENT OF POLYMERIC AND COMPOSITE MATERIALS
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OZAN OZKAN, HILAL TURKOGLU SASMAZEL Department of Metallurgical and Materials Engineering, Atilim University, Ankara, Turkey 4th International Conference on Integrative Biology (Integrative Biology 2016) July 18-20, 2016 Berlin, Germany HYBRID PCL/CHITOSAN SCAFFOLDS WITH MICRO AND MACRO POROSITY
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Aim: Development of hybrid PCL/chitosan polymeric scaffolds Material: Electrospun PCL + solvent casted chitosan freeze dried scaffolds with complex 3D porous structure Methods: Electrospinning Solvent casting Freeze drying techniques SUMMARY
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Introduction Why Poly( ε -caprolactone) (PCL)? Low melting point (60°C) Ease of processability Degradability with non-enzymatic processes (hydrolysis) Good mechanical properties — Hydrophobic structure — Limited bio-interaction — Susceptibility to bacterial biodegradation Why Chitosan? Natural polymer Superior biocompatibility Structural resemblance to the glycosaminoglycan of bone tissue — Low mechanical flexibility — Limited biodegradability properties Why PCL/Chitosan combination? Take advantage of both, Eliminate the disadvantages,...which means... Biocompatibility Biodegradability Processability Mechanical Properties
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Material Fabrication Inovenso NE200, 30 kV Electrospinning Fibrous PCL
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Material Fabrication Inovenso NE200, 30 kV Electrospinning Fibrous PCL 15 wt.% PCL Chloroform/Methanol (75/25 v/v) solvent Stirred 20 mins at 200 rpm and 30°C Electrospun at 20 cm capillary-collector distance and 15 kV applied voltage
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Material Fabrication Inovenso NE200, 30 kV Electrospinning Fibrous PCL Solvent Casting Incorporating Chitosan
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Material Fabrication Inovenso NE200, 30 kV Electrospinning Fibrous PCL Solvent Casting Incorporating Chitosan 1 wt.% Chitosan 0.2M Acetic acid solvent Stirred 1 hour at 400 rpm and 30°C Poured in a Petri dish E-spun PCL immersed Pressure applied
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Material Fabrication Freeze Drying 3D complexity Martin Christ Alpha 2-4 LDplus Inovenso NE200, 30 kV Electrospinning Fibrous PCL Solvent Casting Incorporating Chitosan
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Material Fabrication Freeze Drying 3D complexity Martin Christ Alpha 2-4 LDplus Inovenso NE200, 30 kV Electrospinning Fibrous PCL Solvent Casting Incorporating Chitosan 2 different pre-drying in refrigerator at –20°C for 24 hours in incubator at 40°C for 5 hours 0.015 mbar vacuum –80°C
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Material Characterization Contact Angle Goniometer (Surface Electro Optics Phoenix-300, South Korea): *R+F: Pre-dried in refrigerator at – 20°C for 24 hours, then freeze dried. **I+F: Pre-dried in incubator at 40°C for 5 hours, then freeze dried. Contact Angle (°) Drying timeR+F*I+F** 6h62,63 ± 1,87365,08 ± 1,156 12h71,03 ± 1,98265,35 ± 1,237 18h60,81 ± 2,23460,33 ± 2,927 24h65,23 ± 2,26568,93 ± 2,178 48h63,31 ± 2,10275,73 ± 1,965 Contact Angle Measurements
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Material Characterization SEM (FEI QUANTA 400F Field Emission SEM, USA): REFRIGERATED + FREEZE DRIED * All samples were coated with Au-Pt (~5 nm) 12h24h48h SEM Imaging
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Material Characterization SEM (FEI QUANTA 400F Field Emission SEM, USA): REFRIGERATED + FREEZE DRIED * All samples were coated with Au-Pt (~5 nm) 12h24h48h SEM Imaging The scaffolds refrigerated before freeze drying were generally not very well dried Lost the structural integrity of the electrospun fibers The refrigeration process at -20°C, in which the water inside the structure as well as the refrigerator slowly formed ice crystals which gained volume and put pressure to the surrounding resulting deformations in the structure
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SEM (FEI QUANTA 400F Field Emission SEM, USA): INCUBATED + FREEZE DRIED * All samples were coated with Au-Pt (~5 nm) 12h24h48h Material Characterization SEM Imaging
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SEM (FEI QUANTA 400F Field Emission SEM, USA): INCUBATED + FREEZE DRIED * All samples were coated with Au-Pt (~5 nm) 12h24h48h Material Characterization SEM Imaging The scaffolds prepared by pre-drying with the incubator and then freeze drying were resulted in the desired 3D complex micro/macro porous structures In this route, the incubator cured both polymers, entrapping the water content without any change in the volume, which was then removed during the freeze drying step, resulting with the desired porosity.
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Material Characterization Tensile Test (Zwick/Roell 250kN, Germany): Drying timeYoung’s Modulus (MPa)Yield Strength (MPa) 12h2,68 ± 0,760,19 ± 0,003 24h 3,25 ± 0,780,26 ± 0,002 48h 1,98 ± 0,230,13 ± 0,003 Mechanical Testing Young’s modulus and yield strength of the electrospun PCL is 1.0-3.0 MPa and 0.2 MPa, respectively. Chitosan has much lower mechanical properties than PCL. The 24 hour freeze drying was optimum drying condition for these hybrid scaffolds, since 12 hours and 48 hours drying time provided lower mechanical values, due to the lack of sufficient porosity and over growth of porosity, respectively. The scaffold pre-dried in incubator at 40°C for 5 hours, then freeze dried for 24 hours was selected for further characterization and biocompatibility studies.
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Material Characterization ATR-FTIR Surface Analysis PCL characteristic readings: 1720 cm −1 (carbonyl groups) 2873-2952 cm −1 (CH stretching bonds) 1367-1419 cm −1 (CH 3 group folds) Chitosan characteristic readings: resonance band at 1128-1222 cm −1 1690 cm −1 saccharide structure and carbonyl stretching (amide I)
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Material Characterization PBS Absorption and Shrinkage Tests The scaffold pre-dried in incubator at 40°C for 5 hrs, then freeze dried for 24 hrs, showed that the PBS absorption capacity of the PCL/chitosan scaffold was 67.51% ± 1.37. This indicated that the integration of hydrophilic chitosan into the hydrophobic fibrous PCL increased the fluid uptake capacity. The shrinkage of the PCL/chitosan scaffolds was found to be very low, given that the area of the scaffolds reduced 1.587% ± 0.220 after 24 hours of incubation in PBS and 12 hours of vacuum drying. These results were promising for a tissue engineering scaffold since they indicated that the scaffolds have high enough interconnected porosity to absorb such amount of PBS without showing any significant shrinkage that would cause deformation of the tissue in the healing location.
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Cell culture Attachment MRC5 cells attached rapidly onto both control and the scaffolds within 3 h. 70% attachment for the control, 95% attachment for the scaffolds. Due to the complex micro/macro porosity and the 3D structure, the scaffolds provided a better host for the anchorage dependent MRC5 cells resulting over 25% more cells to attach at the end of 3 h.
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Cell culture MTT Assay 7 days at 48 h intervals. Steady increase without showing any toxicity for fibroblast cells. Significantly better than the TCPS wells (control group). The result of the initial cell attachment performance where the cells attached faster and in greater number resulting an early initiation of the metabolic activity and better overall proliferation of the cells at the end of 7 days. Cell yields at the end of 7 days, proved that the cells favored the structures of the scaffolds at least 1.35 fold, compared to the standard TCPS. The micro pores of the structure served as anchorage points for cells, the macro pores allowed them to grow 3D into the cavities of the scaffold.
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Composite PCL/chitosan scaffold material with complex macro/micro porosity had been developed. Scaffolds were prepared with a three step process: electrospinning the PCL, dipping into a chitosan solution and freeze drying. With the combination of two polymers in the form of layer by layer electrospun mats, the CA can be lowered to the range of 80-85°. The scaffolds prepared by pre-drying with the incubator and then freeze drying were resulted in the desired 3D complex micro/macro porous structures. Summary & Conclusions (1)
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The hybridization of these two polymers with two different fabrication methods, enabled an increase in mechanical properties of the developed scaffold materials, compared to individual polymers. ATR-FTIR result of the scaffolds, pre-dried in the incubator at 40°C for 5 h, then freeze dried for 24 h, showed characteristic readings of both PCL and chitosan. The hybridization supplied the increased swelling capacity and the decreased shrinkage property of the developed scaffolds. Summary & Conclusions (2)
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According to the cell attachment performance study, 25% increase in cell adhesion for the developed PCL/chitosan scaffolds comparing to TCPS control surfaces was obtained. MTT assay proved the impact of the hybrid PCL/chitosan scaffolds for the longer period of cell yield efficiency. Resulting structure: Micro/macro interconnected porosity. Mimicking the natural extracellular matrix of tissues. Enhanced mechanical properties for structural support. Summary & Conclusions (3)
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Highlights Sasmazel Turkoglu, H., Ozkan, O., Advances in Electrospinning of Nanofibers and Their Biomedical Applications, Review, Current Tissue Engineering, 2(2), Pages 91 ‐ 108, 2013.Magnesium is a highly abundant element present in human body, with low density, high specific strength, high electrical conductivity and low toxicity. Manolache S., Sasmazel Turkoglu H., Uygun A., Oksuz L., Plasma Technology, Nanoengineering of Advanced Materials, in Arza Seidel (Ed): Kirk-Othmer Encyclopedia of Chemical Technology, http://dx.doi.org/10.1002/0471238961.plassori.a01, John Wiley & Sons, Ltd., 1 – 27, (on-line 04/13/2012). Patent Application 1 2015: Tissue scaffold with enhanced biocompatibility and mechanical properties and it’s production method Patent Application 2 2015: Antibacterial PCL/Chitosan wound dressing material
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Atilim University, TUB İ TAK (The Scientific and Technological Research Council of Turkey), EU-COST Action MP1101, EU-COST Action MP1206, EU-COST Action FP1405, National and International collaborators, My Research Team. Acknowledgement
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Thank you... Any questions??
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