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. Dror Seliktar 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. 1.Biocompatibility 2.Mechanical strength 3.Porosity 4.Biodegradability 5.Enabling cell development and migration Required scaffold’s characteristics:

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

4. System Design Fibrinogen PEG polyethylene glycol PEGylated fibrinogen fragments

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

6. Research Goals 1.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. 2.Structure characterization – the structure of the porous PF hydrogels will be investigated in order to evaluate its relation to the emulsion characteristics. 3. Physical properties – Young modulus and water weight gain of the resulted emulsion-templated hydrogels will be investigated. 4.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 pluronic ® F108pluronic ® F68 75/25 (PF/oil) 50/50 (PF/oil) 90/10 (PF/oil) 95/5 (PF/oil) Overhead mechanical stirrer Magnetic stirrer Stirring method Surfactant type Emulsion composition

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

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

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

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

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

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

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

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

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

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

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!