Multiscale characterization of a chimeric biomimetic polypeptide for stem cells scaffolding

Transcription

1 Multiscale characterization of a chimeric biomimetic polypeptide for stem cells scaffolding Massimo Vassalli Institute of Biophysics (IBF) National Research Council (CNR) Genova Italy 03 / 02 / 2012

2 Summary Biomaterials for tissue engineering and regenerative medicine [Digression on force curves] REC: a protein inspired chimeric system with potentially interesting features Conclusions and perspectives

3 Tissue engineering Combining cells with scaffolding materials to generate functional tissue constructs describes tissue engineering at its most basic level. Understanding and manipulating the complex relationship between the cells and the scaffolding materials, however, represents the great challenge for tissue engineers. Extracted from the mission statement of the McGowan Institute for Regenerative Medicine University of Pittsburgh Medical Center

4 Biomaterials A biomaterial is material, to be implanted in the body or that contacts body fluids outside the body, that serves as mechanical scaffold or supporting structure for growing cells and tissues. It can also be engineered to modify the cellular response (through specific adhesion molecules) and to change (through the release of growth factors or degradation of a polymer) both its chemical and mechanical properties with time. Key factors for a biomaterial are: Biocompatibility Resorbability Mechanical properties

5 Role of mechanics D.E. Discher, P. Janmey, Y-L. Wang. Tissue cells feel and respond to the stiffness of their substrate..science 310: (2005)

6 Li, H., & Cao, Y. Protein mechanics: from single molecules to functional biomaterials. Accounts of chemical research, 43(10), 2010

7 Polymer stretching Worm-like chain model

8 Protein stretching

9 Tip & Substrate adhesion Random adsorption Chemical functionalization Chemical adhesion Protein tag Cys - Au Random adsorption

10 Tip & Substrate adhesion A.Imparato, F.Sbrana, M.Vassalli, Reconstructing the free-energy landscape of a polyprotein by single-molecule experiments, EPL (2008) 82:58006

11 Filtering graph Nothing N-M Bad A N curves Anything else B M Something M-A Interesting C Single Titin

12 Substrate and tip effect Titin (C/B+C) Bad curves (A/M) Biolever CSG Biolever CSG AU Glass SH HOPG Mica AU Glass SH HOPG Mica Preliminary

13 Substrate and tip effect Titin (C/B+C) Bad curves (A/M) Biolever CSG Biolever CSG AU Glass SH HOPG Mica AU Glass SH HOPG Mica Preliminary

14 Zero background technology

15 REC What's REC Single molecule biomechanics Aggregation properties Cytocompatibility From aggregates to fibers From fibers to materials

16 Chimera The Chimera was a monstrous fire-breathing female creature composed of the parts of multiple animals: upon the body of a lioness with a tail that ended in a snake's head, the head of a goat arose on her back at the center of her spine.

17 Biomimicry Innovation driven by Nature Haeshin Lee, Norbert F. Scherer, and Phillip B. Messersmith Single-molecule mechanics of mussel adhesion PNAS (35) ;

18 Resilin Elastin Collagen A.Bracalello, V.Santopietro, M.Vassalli, G.Marletta, R.DelGaudio, B.Bochicchio, A.Pepe, Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide, Biomacromolecules (2011) 12:

19 Single molecule biomechanics

20 Mechanical cycles

21

22 REC vs Ovalbumin

23 Aggregation: optical imaging

24 Aggregation: AFM imaging

25 Aggregation: AFM imaging

26 Aggregation: AFM imaging

27 Aggregation: AFM imaging

28 Cytocompatibility

29 Cytocompatibility REC Polystyrene

30 From aggregates to fibers Time laps AFM images of t0 t1 REC at 50 C for 7 days (t0-t7) t2 t3

31 From aggregates to fibers Time laps AFM images of t5 REC at 50 C for 7 days (t0-t7) t7

32 Fibers elasticity Left panel: panel single AFM image taken in non-contact mode in water, clearly showing the presence of polypeptide fibers. fibers Right panel: panel zoom of the dashed region in the left panel. The white line indicates the fiber profile identified by the software procedure.

33 Young's modulus Distribution of the Young's modulus estimated for a set of 88 fibrils measured by AFM microscopy. The estimated Young's modulus of REC polypeptide is 0.1 3MP. This result is in agreement with a representation of the polymer as a series of three springs, in which the whole elasticity is closer to that of the softer components.

34 From fibers to material Self aggregation properties are exploited by chemical cross-linking strategies based on the added lysine residues (K) Glutaraldehyde (GTA) homobifunctional cross-linker hexamethylene diisocyanate (HDI) F.Sbrana, M.Lorusso, C.Canale, B.Bochicchio, M.Vassalli, Effect of chemical cross-linking on the mechanical properties of elastomeric peptides studied by single molecule force spectroscopy, J.Biomech. (2011) 44:

35 Preliminary results

36 Conclusions REC is a chimeric biomimetic polypeptide showing interesting properties as a potential material for stem cells scaffolding applications. The system was tested with a reductionist approach and the Atomic Force Microscope was intensively adopted to characterize single molecules, aggregates and fibers. Preliminary results indicate a good biocompatibility of the compound and the cross-linking strategies implemented are providing relevant results towards a three-dimensional structure.

37 Acknowledgements Brigida Bochicchio Chemistry department University of Basilicata Giovanni Marletta Chemistry dept. PRIN 2008 University of Catania Gabriela Ciapetti, Nicola Baldini Orthopedic hospital Rizzoli (Bologna, Italy) Italian Institute of Technology

38 Questions?