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ECM - main structural tissue of skin › Helps skin renew and generate › Provides signals to intercellular pathways Main components › Glycoproteins (such as collagen) › Proteoglycans › Hyaluronic Acid Engineered ECMs are known as scaffolds
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Ability to create scaffolds › Mimic the ECM in size and porosity › Have high surface to volume ratio Easy to vary mechanical and biological properties through changing materials Flexible- allows cells to manipulate their environment
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Biocompatible polymer Biodegradable at a slow enough rate to allow increased cell growth and stability Easy to manipulate Relatively low melting point - easy to use
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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
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Much research has shown that 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
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Natural polymer that exhibits biocompatible and biodegradable qualities Cellular binding capabilities Anti-bacterial properties High viscosity which limits electrospinning
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Good for health reasons (low toxicity, immunogenic) Low cost – easily obtained Poor spinnability - possibly fixed with addition of a synthetic polymer
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To create an optimal nanofibrous mesh consisting of PCL and another biological polymer (Chitosan/Alginate) 1) To determine which mesh best cultures cells on the meshes 2)
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By combining PCL with another biological polymer, an electrospun mesh can be created that mimics the ECM, exhibits optimal biocompatibility, and encourages cell attachment and proliferation
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Independent Variables Analyzing method of meshes (AFM in HCI vs SEM in AOS) Type of cell used (Drosophila in HCI vs Fibroblast in AOS) Dependent Variables: Microscope image produced (Resolution, magnification) Cell growth Controlled Variables Material spun - PCL Spinning conditions of PCL mesh (Applied voltage, flow rate, tip-to- collector distance, concentration)
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AOS: Spin PCL; vary spinning conditions HCI: Replicate spinning conditions AOS: Observe mesh under SEM and grow Fibroblast cells on PCL mesh HCI: Observe mesh under AFM and grow Drosophila cells on PCL mesh
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Baseline can be set for comparison of images › For the Second stage: microscope used › For the Third stage: cells used Control image- image X
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Independent Variables Secondary material used when spinning meshes Conditions for spinning (Applied voltage, flow rate, tip-to- collector distance, concentration) Dependent Variables: Quality of nanofibers (Porosity, diameter, number of beadings) Controlled Variables Primary material used when spinning meshes – PCL Method of combining chemicals (chemically) Mesh shown in Image X (used for comparing other meshes)
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AOS: Spin PCL-Chitosan mesh; vary spinning conditions HCI: Spin PCL-Alginate mesh; vary spinning conditions AOS: Observe under SEM, compare with Image X HCI: Observe under AFM, compare with Image X
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Independent Variables Different types of meshes produced PCL-Chitosan mesh in AOS PCL-Alginate mesh in HCI Dependent Variables: Rate of cell growth (Cell size, number of cells) Overall cell adhesion to mesh Controlled Variables Conditions for cell growth (Cell characteristics, surrounding conditions)
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HCI: Attach Drosophila cells to every PCL-Alginate mesh AOS: Attach Fibroblast cells to every PCL-Chitosan mesh Observe meshes under inverted light microscope to see if cells successfully attached to meshes
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Compare number of cells per unit area for each mesh and cell sizes Derive rate of cell growth from comparing initial state of cells and final state of cells
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Triplicates created Conclusion can be drawn based on rate of cell growth measured
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November/ December AOS: Start electrospinning pure PCL mesh begin finding parameters; analyze mesh HCI: Look into research for cells (Drosophila vs Fibroblast) and other areas January AOS: Attach cells on pure PCL mesh; analyze cells HCI: Replicate parameters from AOS counterparts to spin PCL mesh
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February AOS: Start electrospinning solutions of Chitosan and PCL HCI: Replicate parameters from AOS counterparts to spin PCL mesh; analyze mesh March AOS: Vary spinning conditions, scan meshes HCI: Attach cells on pure PCL mesh and analyze cell growth
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April/May AOS: Grow cell culture on PCL-Chitosan meshes; Analyze cells HCI: Start electrospinning PCL-Alginate mesh; Vary spinning conditions June/July AOS: Compare images HCI: Grow cell culture on PCL-Alginate meshes; Analyze cells
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August HCI: Compare images AOS and HCI: Come together to compare results on both sides (PCL-Chitosan mesh vs PCL-Alginate mesh)
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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.
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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.
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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 21 st century. Progress in Polymer Science. doi: 10.1016/j.progpolymsci.2010.04.002
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