Fabrication of an electrospun nanofibrous scaffold for use in the field of tissue engineering

1. Fabrication of an electrospun nanofibrous scaffold for use in the field of tissue engineering Tyler Crawford Shannon Daily

2. Purpose: To create a polycaprolactone mesh which enables cell activity and seeks to eventually provide an application in the field of tissue engineering toward biomimetic skin graft.

3. The Extracellular Matrix (ECM) • ECM - main structural tissue of skin • Helps skin renew and generate • Provides signals to intercellular pathways • Engineered ECMs are known as scaffolds

4. Electrospinning • Ability to create scaffolds • Mimic the ECM (size and porosity) • High surface to volume ratio • Easy to vary mechanical and biological properties through changing materials • Flexible- allows cells to manipulate their environment

5. Polycaprolactone (PCL) • Biocompatible polymer • Biodegradable at a rate that allows increased cell growth and stability • Easy to manipulate • Relatively low melting point - easy to use

6. Polycaprolactone • Clinically safe (FDA approval) • Proven to have potential for scaffolds in relation to tissue regeneration • Has created scaffolds w/ ideal conditions • High porosities • Large amounts of surface areas

7. Additional Biochemical Material • Adding another biochemical can: • Increase stress resistance • Provide better adhesion of cells to the final scaffold • Increase the potential for cell proliferation • Biochemical should • Be a component of skin naturally • Must be able to be combined in a solution to be electrospun

8. Chitosan (CHT) • Natural polymer that exhibits biocompatible and biodegradable qualities • Cellular binding capabilities • Anti-bacterial properties • High viscosity which limits electrospinning

9. Experimental Design Procedure: • Create control meshes of pure PCL • Solution= PCL and acetic acid (solvent) • Electrospin • Starting parameters: 15 wt.% concentration, 20 cm from tip of syringe to collector plate, & 20 kV

10. Procedure continued: • Vary voltage to create 9 meshes • 3 Voltages- 3 trials for each • 20 kV • 15 kV • 25 kV • Examine mesh using Scanning Electron Microscope (SEM) • Culture fibroblast cells onto mesh

11. Procedure continued: • Observing cells • Inverted light microscope • Analyze cell growth • Cell counts in cells per unit area (mm2) • Means and standard deviations • ANOVA (Analysis of Variance) tests

12. Procedure continued: • Create solutions of PCL and chitosan • Electrospin • Vary concentration of chitosan to PCL • .5% CHT • 1% CHT • 2% CHT • Total of 9 meshes (3 trials of each concentration)

13. Procedure continued: • Analyze with SEM • Culture fibroblast cells and seed into meshes created • Determine cell density • Analyze with means, standard deviations, and ANOVA tests

14. Data and Analysis: • Data obtained: • Fiber diameter and pore diameter of mesh • Cell density amounts • Analysis includes: • Means* • Standard Deviations* • ANOVA tests • 3 comparisons *5-7 measurements/areas for these methods

15. Data: Creating Solutions

16. Data continued: • 15 wt.% solution created • 17 g. acetic acid, 3 g. PCL • Electrospun • 5 mL syringe with bevel tip • Flow rate: .02?? • Mesh created within 2 hrs.

17. Data continued:

18. Progress • Background Research • Experimental Design • ISEF (International Science and Engineering Fair) Forms • Started solutions • Just began spinning

19. References • Akhyari, P., Kamiya, H., Haverich, A., Karck, M., & Lichtenberg, A. (2008). Myocardial tissue engineering: The extracellular matrix. European Journal of Cardio-Thoracic Surgery, 34, 229-241. doi: 10.1016/j.ejcts.2008.03.062 • Bhardwaj, N. & Kundu, S. C. (2010). Electrospinning: A fascinating fiber fabrication technique. Biotechnology Advances, 28, 325-347. doi: 10.1016/j.biotechadv.2010.01.004 • Chong, E.J., Phan, T.T., Lim, I.J., Zhang, Y.Z., Bay, B.H., Ramakrishna, S., & Lim, C.T. (2007). Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomaterialia, 3, 321-330. doi: 10.1016/j.actbio.2007.01.002 • Geng, X., Kwon, O-H., & Jang, J. (2005). Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials, 26, 5427-5432. • Han, J., Branford-White, C.J., & Zhu, L.M. (2010). Preparation of poly(є-caprolactone)/poly(trimethylene carbonate) blend nanofibers by electrospinning. Carbohydrate Polymers, 79, 214-218. doi: 10.1016/j.carbpol.2009.07.052 • Homayoni, H., Ravandi, S.A.H., & Valizadeh, M. (2009). Electrospinning of chitosan nanofibers: Processing optimization. Carbohydrate Polymers, 77, 656-661. • Lowery, J.L., Datta, N., & Rutledge, G.C. (2010). Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly(є-caprolactone) fibrous mats. Biomaterials, 31, 491-504. doi: 10.1016/j.biomaterials.2009.09.072 • Nisbet, D.R., Forsythe, J.S., Shen, W., Finkelstein, D.I., & Horne, M.K. (2009). A review of the cellular response on electrospun nanofibers for tissue engineering. Journal of Biomaterials Application, 24, 7-29. • Pham, Q.P., Sharama, V., & Mikos, A.G. (2006). Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Engineering, 12,1197-1211. • Shevchenko, R.V., James, S.L., & James, S.E. (2010). A review of tissue-engineered skin bioconstructs available for skin reconstruction. Journal of the Royal Society Interface, 7, 229-258. doi: 10.1098/rsif.2009.0403 • Sill, T.J., & von Recum, H.A. (2008). Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials, 29, 1989-2006. doi: 10.1016/j.biomaterials.2008.01.011 • Woodruff, M.A., & Hutmacher, D.W. (in press). The return of a forgotten polymer- Polycaprolactone in the 21st century. Progress in Polymer Science. doi: 10.1016/j.progpolymsci.2010.04.002

20. THE END ANY QUESTIONS?