1. Peptide Surface Modification of Methacrylamide Chitosan for Neural Tissue Engineering Applications -from Journal of Biomedical Materials Research 2006 Laura M. Y. Yu,1,2 Karineh Kazazian,1 Molly S. Shoichet1–3 1Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, Canada M5S 3E5 2Institute of Biomaterials and Biomedical Engineering, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, RM 514 Toronto, Ontario, Canada M5S 3E1 3Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 Ingólfur Magnússon 28th of January 2014

2. Introduction The aim of this study was: – Synthesize and characterize cell-adhesive and biodegradable chitosan scaffolds – Synthesize water soluble methacryl chitosan (MC) – Synthesize Thiolated methacrylamide chitosan (TMC) by coupling thioglycolic acid (TGA) to MC – Crosslinking of methacryl groups in TMC by the crosslinking initiators ammonium persulfate (PS) and sodium metabisulfite – Couple maleimide-terminated cell adhesive peptides, mi-GDPGYIGSR and mi-GQASSIKVAV to TMC scaffold to promote cell adhesion

4. Materials and Methods Materials: – Chitosan (Sigma, 190-230 kDa, 88 % deactylated), Ellmans reagent, APS and SMBS (crosslinking initators), TGA, EDC, sulfo-NHS – Maleimide peptides sequences were synthesized by solid state peptide synthesis on PEG-PS resin, based on Fmoc chemistry usinga peptide synthesizer Degree of methacrylamidation found by NMR Thiol groups present on TMC determined by Ellmans Method Scanning electron microscopy (SEM) Cell penetration assay Degration of chitosan scaffolds by lysozyme Mass analyzis

5. Results

6. Proton NMR of Chitosan

7. Proton NMR of Methacrylchitosan

8. Degree of substitution For MC: 23 % Thiol groups in 1:1 blend of TMC and MC was 45.7 µmol/g – It´s likely that TGA also reacts to through methacryl group through a Michael-type addition reaction – Oxidation of thiol groups?

9. Mass spectra of Maleidmie peptides – mi-GQAASIKVAV

10. Mass spectra of Maleidmie peptides – mi-GDPGYIGSR

11. SEM The lower concentration used of APS/SMBS initiators gave MC scaffold with larger pore size and formed a less dense porous network than higher concentration. This ability gave better cell penetration, facilitating neurite penetration (neurite outgrowth)

12. SEM of SCG ganglion on MC scaffold This chitosan scaffold was crosslinked by 3 % APS/SMBS Neurites extending from the ganglion were able to penetrate the chitosan scaffold

13. SEM of SCG ganglion on MC scaffold This chitosan scaffold was crosslinked by 7 % APS/SMBS Neurites extending from the ganglion were unable to penetrate the chitosan scaffold

14. Response of SCG neurons on the surface of scaffold a) Unmodified MC scaffold b) Peptide modified TMC scaffold. SCG neurons adhere end extended neurites on the peptide modified scaffold

15. Adherence and neurite properties of the scaffolds

16. Degradation of the MC scaffold in the presence of lysozyme

17. Conclusion Authors conclude that a novel derivative of chitosan can be transformed into a scaffold that is biodegradable, allows neurite penetration into it´s matrix and can be modified chemically to improve cell adhesion Interesting article Was oxidation of the sulfhydryl groups a problem? It would be useful to see the supplementary data, to see if there was done more characterization of the compounds

18. Thank you for listening Questions?