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Porous PEG-Fibrinogen H ydrogel S caffolds for Tissue E ngineering

Porous PEG-Fibrinogen H ydrogel S caffolds for Tissue E ngineering. Ortal Yom-Tov The Interdepartmental P rogram for Biotechnology Technion Supervisors: Prof. Havazelet Bianco-Peled and Prof. Dror Seliktar. December 2012. Introduction.

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Porous PEG-Fibrinogen H ydrogel S caffolds for Tissue E ngineering

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  1. Porous PEG-Fibrinogen Hydrogel Scaffolds for Tissue Engineering Ortal Yom-Tov The Interdepartmental Program for Biotechnology Technion Supervisors: Prof. Havazelet Bianco-Peled and Prof. DrorSeliktar December 2012

  2. Introduction Tissue engineering is a science of creating new tissues in order to regenerate an organ functionality or to replace damaged organ parts. Required scaffold’s characteristics: • Biocompatibility • Mechanical strength • Porosity • Biodegradability • Enabling cell development and migration

  3. Motivation Designing porous hydrogels with controllable pore size and porosity, which allows for the in vitro encapsulation of cells

  4. System Design PEG polyethylene glycol Fibrinogen PEGylated fibrinogen fragments

  5. Concept Oil-in-water emulsion Oil droplets coated with surfactant layer PEG-fibrinogen polymer solution Encapsulation of cells prior to polymerization PEG-fibrinogen hydrogel Emulsion-templated PF hydrogel

  6. Research Goals • System design - formulations for emulsions and emulsion-templated PF hydrogels will be obtained and manipulated in order to control hydrogel's pore size and porosity. • Structure characterization – the structure of the porous PF hydrogels will be investigated in order to evaluate its relation to the emulsion characteristics. • Physical properties – Young modulus and water weight gain of the resulted emulsion-templated hydrogels will be investigated. • Cellular biocompatibility – cytotoxicity and outgrowth studies will be performed; the relationship between porosity and pore size to cell proliferation will be explored.

  7. System Design Overhead mechanical stirrer Magnetic stirrer Stirring method Surfactant type pluronic®F68 pluronic®F108 75/25 (PF/oil) 95/5 (PF/oil) Emulsion composition 50/50 (PF/oil) 90/10 (PF/oil)

  8. System Design structure characterization Cellular biocompatibility Oil droplets size analysis Determination of oil extraction Hypothesis: structure impacts cellular biocompatibility Cytotoxicity assays Morphology experiments

  9. Oil Extraction Oil extraction versus surfactant conc. - Magnetic stirrer F68 95/5 (PF/oil) F68 90/10 (PF/oil) F108 95/5 (PF/oil) F108 90/10 (PF/oil) Higher surfactant concentrations results in higher amounts of oil extracted

  10. Oil Extraction Oil extraction versus surfactant conc. - Overhead mechanical stirrer F68 95/5 (PF/oil) F68 75/25 (PF/oil) F68 50/50 (PF/oil) Lower oil percentages results in lower amounts of oil extracted

  11. Light Microscopy Images – Magnetic Stirrer As-prepared hydrogels 10% F68 90/10 (PF/oil) 10% F68 95/5 (PF/oil) 10%F108 90/10 (PF/oil) 10%F108 95/5 (PF/oil) Hydrogels submerged in water for 24 hr

  12. Size Analysis Oil droplet diameter versus time - Magnetic stirrer F68 95/5 (PF/oil) F68 90/10 (PF/oil) F108 95/5 (PF/oil) F108 90/10 (PF/oil) The average droplet diameter diminishes with time, which implies that oil from larger droplets diffuse faster

  13. Light Microscopy Images – Overhead Mechanical Stirrer 3%,7%,10% Pluronic® F68 75/25 (PF/oil) 3%,7%,10% Pluronic® F68 95/5 (PF/oil) 3%,7%,10% Pluronic® F68 50/50 (PF/oil) Increasing surfactant concentrations Increasing surfactant concentrations

  14. Size Analysis Oil droplet diameter versus surfactant conc. – Overhead mechanical stirrer F68 95/5 (PF/oil) F68 75/25 (PF/oil) F68 50/50 (PF/oil) Oil droplet diameter decreases with higher surfactant percentages regardless of emulsion composition.

  15. Cytotoxicity Assays HFFs embedded in emulsion solutions composed of 90/10 (DMEM:oil) 5% Pluronic®F68 15% Pluronic®F68 15% Pluronic®F108 5% Pluronic®F108

  16. Cellular Morphometrics Shape index versus time – Magnetic stirrer F68 95/5 (PF/oil) F68 90/10 (PF/oil) F108 95/5 (PF/oil) F108 90/10 (PF/oil) 10%Pluronic®F68 95/5 (PF/oil) exhibits the lowest shape index for t>4. PF-control

  17. Cellular Morphometrics Cell area versus time – Magnetic stirrer F68 95/5 (PF/oil) F68 90/10 (PF/oil) F108 95/5 (PF/oil) F108 90/10 (PF/oil) Cell area increases as a function of time PF-control

  18. Cellular Morphogenesis versus Structure Shape index and cell area at day 7 versus initial oil droplet diameter

  19. Summary and future work • Oil droplet size can be controlled through the stirring method and the surfactant concentration. • A relation between hydrogel structure and cellular morphogenesis exists and needs to be further investigated. • Future work will focus on characterizing the nanostructure of the system and exploring its relationship to cell viability.

  20. Thank you for your attention!

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