Biofabrication using recombinant spider silk proteins as a biomaterial

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Engineering Conferences International ECI Digital Archives Biofabrication for Hierarchical in Vitro Tissue Models Proceedings 6-7-2017 Biofabrication using recombinant spider silk proteins as a

1 Engineering Conferences International ECI Digital Archives Biofabrication for Hierarchical in Vitro Tissue Models Proceedings Biofabrication using recombinant spider silk proteins as a biomaterial Tamara B. Aigner Department for Biomaterials, University of Bayreuth, Germany, tamara.aigner@bm.uni-bayreuth.de Elise K. DeSimone Department for Biomaterials, University of Bayreuth, Germany Thomas Scheibel Department for Biomaterials, University of Bayreuth, Germany Follow this and additional works at: Part of the Engineering Commons Recommended Citation Tamara B. Aigner, Elise K. DeSimone, and Thomas Scheibel, "Biofabrication using recombinant spider silk proteins as a biomaterial" in "Biofabrication for Hierarchical in Vitro Tissue Models", Jürgen Groll (University of Würzburg, Germany) Jos Malda (University Medical Centre Utrecht, The Netherlands) Eds, ECI Symposium Series, (2017). This Abstract and Presentation is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Biofabrication for Hierarchical in Vitro Tissue Models by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.

2 Biofabrication using recombinant spider silk proteins as a biomaterial Aigner T.B., DeSimone E.K., Scheibel T. Department for Biomaterials, University of Bayreuth, Germany

Material Stiffness (GPa) Strength (GPa) Extensibility (%) Toughness (MJm -3 ) Araneus diadematus dragline silk 6 0.

3 Why spider silk? Image: E. Doblhofer no toxicity no immuno-reactivity slow biodegradation good mechanical properties interesting material for biomedical applications Material Stiffness (GPa) Strength (GPa) Extensibility (%) Toughness (MJm -3 ) Araneus diadematus dragline silk Bombyx mori cocoon silk Nylon fiber Kevlar 49 fiber High-tensile steel J. M. Gosline, P. A. Guerette, C. S. Ortlepp, K. N. Savage, J Exp Biol 1999, 202, J. G. Hardy, T. R. Scheibel, J Polym Sci Pol Chem 2009, 47, /06/17

4 Spider silk architecture A. diadematus dragline core-shell structure Heidebrecht, A. & Scheibel, T. (2013). Adv. Appl. Microbiol. 82, crystalline regions in amorphous matrix 3 20/06/17

5 allowing outstanding toughness Strength [MPa] Stiffness [GPa] Extensibility [%] Yield point softening Toughness [MJ/m 3 ] stiffening Modified after: Heidebrecht, A. 4 6/20/2017

6 Restrictions? spiders are cannibals silk quality depends on nutrition male spiders are hard to raise farming of spiders is NOT feasible! use a biotechnological approach for silk production J. M. Gosline, P. A. Guerette, C. S. Ortlepp, K. N. Savage, J Exp Biol 1999, 202, /20/2017

7 Engineered spider silk origin major ampullate silk Araneus diadematus main components A. diadematus fibroins: ADF3 & ADF4 M. Heim, D. Keerl, T. Scheibel, Angew Chem Int Edit 2009, 48, /06/17

Engineered spider silk ADF4 C-module repetitive domain = template

diadematus fibroin 4) eadf3 N1L(AQ) 12 NR3 (engineered A.

Scheibel, Angew Chem Int Edit 2009, 48, 3584-3596 D. Huemmerich, C.

8 Engineered spider silk ADF4 C-module repetitive domain = template AQ-module ADF3 16x 12x + N- & C-Termini eadf4(c16) (engineered A. diadematus fibroin 4) eadf3 N1L(AQ) 12 NR3 (engineered A. diadematus fibroin 3) M. Heim, D. Keerl, T. Scheibel, Angew Chem Int Edit 2009, 48, D. Huemmerich, C. W. Helsen, S. Quedzuweit, J. Oschmann, R. Rudolph, T. Scheibel, Biochemistry-Us 2004, 43, /06/17

9 Biotechnological silk production genetic information plasmid with genetic information protein fermentation E. coli D. Huemmerich, C. W. Helsen, S. Quedzuweit, J. Oschmann, R. Rudolph, T. Scheibel, Biochemistry-Us 2004, 43, /06/17

10 Engineered spider silk morphologies Image by: K.Schacht processing steps J. G. Hardy, T. R. Scheibel, J Polym Sci Pol Chem 2009, 47, /06/17

11 Spider silk fibers 40 µm Image by: A. Heidebrecht

12 Preparation of spinning dope classical spinning dope biomimetic spinning dope dialysis: PEG protein solution dialysis: phosphate buffer no phase separation phase separation fiber spinning fiber spinning Heidebrecht, A.; Eisoldt, L.; Diehl, J.; Schmidt, A.; Geffers, M.; Lang, G.; Scheibel, T., Adv Mater 2015, 27, /06/17

Lang post-stretching Heidebrecht, A.; Eisoldt, L.; Diehl, J.; Schmidt, A.; Geffers, M.; Lang, G.

13 Wet-spun silk fibers natural fiber synthetic fiber 5 µm 40 µm natural fiber synthetic fiber filament double single surface smooth smooth diameter 4-8 µm µm Image by: G. Lang post-stretching Heidebrecht, A.; Eisoldt, L.; Diehl, J.; Schmidt, A.; Geffers, M.; Lang, G.; Scheibel, T., Adv Mater 2015, 27, 2189 Keerl, D. & Scheibel, T. (2012). Bioinspired, Biomimetic Nanobiomater. 1, /06/17

parallel to fiber axis IR beam perpendicular

recombinant fibers are aligned along fiber

competes with that of natural ones Modified

14 absorbance / a.u. Polarized FTIR and tensile testing IR beam parallel to fiber axis IR beam perpendicular to fiber axis natural dragline silk classic spinning dope biomim. spinning dope poly-ala stretches in recombinant fibers are aligned along fiber axis toughness of biomimetic spider silk competes with that of natural ones Modified after: Heidebrecht, A.; Eisoldt, L.; Diehl, J.; Schmidt, A.; Geffers, M.; Lang, G.; Scheibel, T., Adv Mater 2015, 27, /06/17

15 Spider silk non-wovens Image by: E. DeSimone

Preparation of non-wovens e-spinning syringe with

canula contacted collector plate 1 µm Image by: G.

Lang, C. Mauerer, J. Wickinghoff, M. Weber, S.

16 Preparation of non-wovens e-spinning syringe with eadf4(c16) solution non-woven fiber mat contacted canula contacted collector plate 1 µm Image by: G. Lang Image by: E. DeSimone A. Leal-Egana, G. Lang, C. Mauerer, J. Wickinghoff, M. Weber, S. Geimer, T. Scheibel, Adv Eng Mater 2012, 14, B67-B /06/17

Non-woven diameter Parameters: 6 % w/v 8 % w/v

distance needle diameter humidity temperature

concentration increases fiber diameter larger

JoVE 2013, e50492 A. Leal-Egana, G. Lang, C.

17 Non-woven diameter Parameters: 6 % w/v 8 % w/v 10 % w/v concentration applied voltage spinning distance needle diameter humidity temperature Balb/3T3 fibroblasts Increasing protein concentration increases fiber diameter larger fiber diameter promote cell proliferation G. Lang, S. Jokisch, T. Scheibel, Journal of visualized experiments : JoVE 2013, e50492 A. Leal-Egana, G. Lang, C. Mauerer, J. Wickinghoff, M. Weber, S. Geimer, T. Scheibel, Adv Eng Mater 2012, 14, B67-B /06/17

Cell interaction with non-wovens SEM & TEM:

18 Cell interaction with non-wovens SEM & TEM: Balb/3T3 fibroblasts 5 µm 10µm 10 µm 4 µm 500 nm 4µm fibroblasts spread on and migrate into non-woven meshes A. Leal-Egana, G. Lang, C. Mauerer, J. Wickinghoff, M. Weber, S. Geimer, T. Scheibel, Adv Eng Mater 2012, 14, B67-B /06/17

19 Spider silk hydrogels Image by: K. Schacht/T. Jüngst

20 Hydrogel preparation highly concentrated eadf4(c16) solution cast in well plates gelation cell seeding hydrogel Modified after: Schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54, /06/17

Muller, A. Schmidt, S. Neubauer, H. Kessler, A. Leal-Egana, T.

21 Spider silk variation Balb/3T3 eadf4(c16) genetic modification eadf4(c16)-rgd weak cell adhesion on eadf4(c16) hydrogels introduction of adhesion motif RGD leads to enhanced cell adhesion S. Wohlrab, S. Muller, A. Schmidt, S. Neubauer, H. Kessler, A. Leal-Egana, T. Scheibel, Biomaterials 2012, 33, 6650 Schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54, /06/17

22 Different cell lines on spider silk hydrogels cell adhesion on eadf4(c16)-rgd hydrogels was significantly improved also on other cell types Modified after:schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54,

Hydrogel preparation hydrogel gelation highly concentrated eadf4(c16) solution gelation kinetics secondary protein structure rheology gelation stress-strain viscosity + cell

23 Hydrogel preparation hydrogel gelation highly concentrated eadf4(c16) solution gelation kinetics secondary protein structure rheology gelation stress-strain viscosity + cell culture medium hydrogel + cell culture medium Modified after: Schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54, /06/17

24 Gelation kinetics and secondary structure Increase in the speed of nucleation and growth phase Increase in the speed of crosslinking FTIR measurements revealed no effects of the media on secondary structure content DeSimone, E.; Schacht, K.; Scheibel, T.; Mater Lett 2016, 183,

25 Rheology stress-strain Bivalent cations can reduce the electrostatic repulsion between protein chains leading to an increase in stiffness DeSimone, E.; Schacht, K.; Scheibel, T.; Mater Lett 2016, 183,

26 Rheology shear-rate dependent viscosity High shear rates disrupt ionic binding Shear thinning behaviour is observed Shear thinning properties protect cells from shear forces DeSimone, E.; Schacht, K.; Scheibel, T.; Mater Lett 2016, 183,

Hydrogel preparation + printing gelation hydrogel highly concentrated eadf4(c16) solution 3D printing by robotic dispensing + fibroblasts gelation + fibroblasts hydrogel + encapsulated

27 Hydrogel preparation + printing gelation hydrogel highly concentrated eadf4(c16) solution 3D printing by robotic dispensing + fibroblasts gelation + fibroblasts hydrogel + encapsulated cells Cell viability: ~70% 3 mm Cell viability: ~97% Modified after: Schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54, /06/17

Cells on & encapsulated in printed constructs human

eadf4(c16) 7 d 200 µm fibroblasts adhere well on printed

disturb cell-material interaction fibroblasts

Modified after: Schacht, K.; Jüngst, T.; Schweinlin, M.

28 Cells on & encapsulated in printed constructs human fibroblasts live/dead staining eadf4(c16)-rgd 2 d eadf4(c16) 7 d 200 µm fibroblasts adhere well on printed eadf4(c16)-rgd hydrogels robotic dispensing doesn t disturb cell-material interaction fibroblasts encapsulated in eadf4(c16) hydrogels survive 7 d in situ Modified after: Schacht, K.; Jüngst, T.; Schweinlin, M.; Ewald, A.; Groll, J.; Scheibel, T., Angewandte Chemie 2015, 54, /06/17

29 Summary non-wovens engineered spider silk protein 10µm tissue engineering wound dressing 40 µm hydrogels fibers tissue engineering surture material 28 20/06/17

30 Thank you for your attention SCHE 603/4-4 U /6-2

SPIDER CAPTURE SILK: A hierarchical chain model REPORT. Ramnath Ramachandran (Ram)

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