Tissue Engineering: Scaffold Materials

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1. Tissue Engineering: Scaffold Materials By: Elaheh Entezar-Almahdi 1

2. References: Hollander AP, Hatton PV. Biopolymer methods in tissue engineering: Springer; 2004. Shoichet MS, Hubbell JA. Polymers for tissue engineering. Journal of Biomaterials Science, Polymer Edition. 1998;9(5):405-6. Yang S, Leong K-F, Du Z, Chua C-K. The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue engineering. 2001;7(6):679-89. Shoichet MS. Polymer scaffolds for biomaterials applications. Macromolecules. 2009;43(2):581-91. Araci IE, Brisk P. Recent developments in microfluidic large scale integration. Current opinion in biotechnology. 2014;25:60-8. Coluccino L, Stagnaro P, Vassalli M, Scaglione S. Bioactive TGF- β1/ HA alginate-based scaffolds for osteochondral tissue repair: design, realization and multilevel characterization. Journal of applied biomaterials & functional materials. 2016;14(1). 2

3. Characterizing Regenerative Medicine Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. (Mayo Clinic) Tools and Procedures ( Biofabrication or Additive Manufacturing ) of Regenerative Medicine Tissue Engineering: Tissue Repair/Replacement and Lab Grown Organs Technologies Stem cells Natural and Synthetic Scaffolds 3-D Printing and Chip Technologies 3

4. Regenerative Medicine 1. Artificial organs 4

5. Regenerative Medicine 2. Tissue Engineering and Biomaterials 5

6. The Beginning… Joseph Vacanti ( Harvard Stem Cell Institute) And Robert Langer (MIT ) 1993 6

7. Regenerative medicine pioneer Dr. Ali Khademhosseini (Wyss Institute at Harvard) Organs in the lab Lab on a chip 7

8. Process of Tissue Engineering 8

9. 3 Tools for Tissue Engineering Cells Living part of tissue Produces protein and provides function of cells Gives tissue reparative properties Scaffold Provides structural support and shape to construct Provides place for cell attachment and growth Usually biodegradable and biocompatible Cell Signaling Signals that tell the cell what to do Proteins or Mechanical Stimulation 9

10. Stem Cells Stem cells are cells that: (1) can self-renew (2) have the potential to differentiate along one or two lineages . Totipotent  Can produce all cell types Pluripotent  Can produce most cell types Multipotent  Can produce more than one cell type Unipotent  Can produce one cell type 10

11. Cell Sources Autologous: Come from the person that needs the new cells . Allogeneic: Come from a body from the same species . Xenogenic : Come from a different species then the organism they’re going into . Isogenic ( Syngenic ): Come from identical twins. 11

12. Scaffolds Scaffolds are 3-dimensional materials constructed in order to provide structure to a developing tissue and to allow cells to adhere, proliferate, differentiate and most importantly, secrete extracellular matrix (ECM) (Leong MF. et al., 2009). Many different materials have been investigated in order to construct scaffolds such as polymers (PLA, PGA, PCL, PEG), bioactive ceramics (HA, TCP) as well as natural polymers (Collagen, GAGs, Chitosan). 12

13. Scaffold Construction Requirements Scaffolds should provide void volume for vascularization, new tissue formation and remodeling so as to facilitate host tissue integration upon implantation . Should have high porosity and have suitable pore sizes, and the pores should be interconnected. the biomaterials should also be degradable upon implantation at a rate matching that of the new matrix production by the developing tissue. The biomaterials used to fabricate the scaffolds need to be compatible with the cellular components of the engineered tissues and endogenous cells in host tissue. Scaffolds provide mechanical and shape stability to the tissue defect. 13

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16. Scaffolds Synthesis Nanofiber self-assembly : Molecular self-assembly is one of the few methods for creating biomaterials with properties similar in scale and chemistry to that of the natural in vivo extracellular matrix (ECM), a crucial step toward tissue engineering of complex tissues. Moreover, these hydrogel scaffolds have shown superiority in in vivo toxicology and biocompatibility compared to traditional macro scaffolds and animal-derived materials (Cassidy JW, 2014). 16

17. Scaffolds Synthesis Textile technologies: These techniques include all the approaches that have been successfully employed for the preparation of non-woven meshes of different polymers. In particular, non-woven polyglycolide structures have been tested for tissue engineering applications: such fibrous structures have been found useful to grow different types of cells. The principal drawbacks are related to the difficulties in obtaining high porosity and regular pore size( Ekevall E, 2004). 17

18. Scaffolds Synthesis Freeze- drying : First , a synthetic polymer is dissolved into a suitable solvent (e.g. polylactic acid in dichloromethane) then water is added to the polymeric solution and the two liquids are mixed in order to obtain an emulsion. Before the two phases can separate, the emulsion is cast into a mold and quickly frozen by means of immersion into liquid nitrogen. The frozen emulsion is subsequently freeze-dried to remove the dispersed water and the solvent, thus leaving a solidified, porous polymeric structure ( Haugh MG, 2010). 18

19. Scaffolds Synthesis CAD/CAM (3D-Printing): 19

20. Scaffolds Synthesis Electrospinning : A highly versatile technique that can be used to produce continuous fibers from submicrometer to nanometer diameters. In a typical electrospinning set-up, a solution is fed through a spinneret and a high voltage is applied to the tip. The buildup of electrostatic repulsion within the charged solution, causes it to eject a thin fibrous stream. A mounted collector plate or rod with an opposite or grounded charge draws in the continuous fibers, which arrive to form a highly porous network. The primary advantages of this technique are its simplicity and ease of variation. At a laboratory level, a typical electrospinning set-up only requires a high voltage power supply (up to 30 kV), a syringe, a flat tip needle and a conducting collector. For these reasons, electrospinning has become a common method of scaffold manufacture in many labs. By modifying variables such as the distance to collector, magnitude of applied voltage, or solution flow rate researchers can dramatically change the overall scaffold architecture ( Lannutti J, et al., 2007 ). 20

21. Electrospinning 21

22. Poly- α - hydroxy acid 22 Methods: Drying Extrusion Knitting Gamma sterilization

23. Poly- α - hydroxy acid Extensive research has been performed in developing a full range of PLGA polymers. Both L- and DL- lactides have been used for co-polymerization. The ratio of glycolide to lactide at different compositions allows control of the degree of crystallinity of the polymers. When the crystalline PGA is co-polymerized with PLA, the degree of crystallinity is reduced and as a result this leads to increases in rates of hydration and hydrolysis. In general, the higher the content of glycolide , the quicker the rate of degradation. However, an exception to this rule is the 50:50 ratio of PGA: PLA, which exhibits the fastest degradation. 23

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25. Fibrin microbeads (FMB) Fibrinogen exerts adhesive effects on cultured fibroblasts and other cells. Specifically , fibrin( ogen ) and its various lytic fragments (e.g., FPA, FPB, fragments D and E) were shown to be chemotactic to macrophages, human fibroblasts, and endothelial cells Micro-carrier beads made of some plastic polymers or glass provide cells with a surface area on the order of 104 cm2/L for cell attachment, which is one order of magnitude larger than the area available with stack plates or multi-tray cell-culture facilities microparticles from plasma proteins, such as albumin or fibrinogen, generally using glutaraldehyde to cross-link the proteins. 25

26. Setup for producing FMB by oil emulsion method 26

27. Natural Polymers Blends of collagen and glycosaminoglycans (GAG) have been utilized extensively for dermal regeneration. Chondroitin sulfate has been added to collagen type I for dermal regeneration templates and aggrecan (chondroitin sulfate/ dermatan sulfate/keratin sulfate) to collagen type II for articular cartilage tissue engineering 27

28. Hyaluronan Composed of repeated disaccharide units of D- glucuronic acid and N- acetylglucosamine The unique properties of HA are manifested in its mechanical function in the synovial fluid, the vitreous humor of the eye, and the ability of connective tissue to resist compressive forces, as in articular cartilage . Plays a fundamental role during embryonic development and in wound healing 28

29. Hyaluronan scaffold for central neural tissue engineering 29

30. Collagen In the form of collagen sponge Porosity, biodegradability, and biocompatibility Can be modified using growth factors or other manipulations to promote chondrocyte growth and cartilage matrix formation Scaffolds made from a single collagen type or composites of two or more types Disadvantages Poor dimensional stability. Variability in drug release kinetics. Poor mechanical strength. 30

31. Chitosan It consists of β -1-4 linked 2 amino-2-deoxy gluco – pyranose moieties. Commercially manufactured by N- deacetylation of Chitin which is obtained from Mollusc shells. It is soluble only in acidic pH i.e. when amino group is protonated. Thereby it readily adheres to bio membranes. It is degraded mainly by Glycosidases & lysozymes. 31

32. Chitosan Scaffold Fabrication of bulk porous chitosan scaffolds Freezing of a chitosan-acetic acid solution Subsequent lyophilization Scaffold microstructure will depend on the shape of the mold used for freezing and on the freezer temperature. 32

33. Chitosan and Collagen-chitosan scaffold 33

34. Hydrogels as scaffold Cells are suspended within or adhered to the 3D hydrogel framework during or after formulation as scaffolds RGD (arginine–glycine–aspartic acid) adhesion peptide sequence. Inclusion of these RGD domains in hydrogels has shown improved cellular migration, proliferation, growth, and organization in tissue regeneration applications. Cells have been shown to favorably bind to the RGD-modified hydrogel scaffolds. These cells include endothelial cells (ECs), fibroblasts, smooth muscle cells (SMCs), chondrocytes and osteoblasts. 34

35. Alginate as Scaffold 35

36. Pure Alginate as Scaffold 36

37. Bioactive TGF- β1/ HA alginate-based scaffolds for osteochondral tissue repair 37

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