1. SCAFFOLDS FOR TISSUE ENGINEERING ENGINEERING Tissue Engineering & Drug Delivery BBI 4203 LECTURE 7 Much of this lecture was taken from Nur Farahiyah Binti Mohammad Email : farahiyah@unimap.edu.my

2. Introduction Scaffold: serves as temporary or permanent artifical Extracelular Matrices (ECM) to accommodate cells and support 3D tissue regenerations What is ECM? blend of macromolecules (protein) around cells—as space filler.

3. An ideal scaffold for TE should…. Act as template for tissue growth in 3D Have an interconnected macroporous network for vascularisation, tissue ingrowth and nutrient delivery Bond to the host tissue without the formation of scar tissue Resorb at the same rate as the tissue is repaired Influence the genes in the cells of the tissue to enable efficient cell differentiation and proliferation Be easily and cheaply produced to ISO/FDA/CE standards (must be easily sterilised) Produce a construct with mechanical properties similar to the host tissue

4. Classification of potential scaffold materials Bioinert: non toxic response from the body on implantation. Usually results in fibrous encapsulation (scar tissue formation). Bioresorbable: undergoes degradation in the body. Dissolution products are harmless and can be secreted naturally. Bioactive: Produces a biological response from the body that results in a bond between the material and the host tissue.

5. Processing polymers: porous scaffolds phase separation – Low pore diameter, difficult to control pore size fibre bonding – Lack of mechanical strength of bonds porogen leaching/salt leaching – Closed pores freeze drying high-pressure CO2 rapid prototyping/ solid freeform fabrication

6. Optimal Pore Sizes for Cell Proliferation & Tissue Growth

7. Thermally Induced Phase Separation (TIPS) Developed 1970s-1980s Used for production of microporous membranes Solid liquid separation of polymer solution induced by cooling: – Solvent crystallisation – Polymer precipitation

8. Thermally Induced Phase Separation (TIPS)

9. TIPS Scaffold Morphologies

10. Thermally Induced Phase Separation (TIPS)

11. Nam YS and Park TG, 1999 Phase separation examples

12. Spraying the polymer solution (alternative) Fiber bonding

13. Fibre Bonding Technique

15. Solvent Casting and Particulate Leaching Technique (SCPL)

16. SCPL / Porogen leaching method

17. Solvent Casting and Particulate Leaching Technique (SCPL)

18. Supercritical CO2 Scaffold Production

19. Rapid prototyping

20. Rapid prototyping/solid freeform fabrication

21. Leong KF at al, 2003 Limited resolution degradation can destroy repeatable structure Rapid prototyping - examples

22. Advantages of rapid prototyping Pore network defined by CAD file Pore network can be tailored to the CT scan of a patient ’ sdefect A pore size gradient can be obtained

23. Disadvantages of rapid prototyping Mechanical properties poor? Not all materials can be used in the techniques yet. Expensive equipment.

24. Or around a mandrel to get alignment or tubular scaffolds Electrospinning

25. Non-aligned Aligned Low porosity is usually a problem for cell infiltration 10  m Co-spinning of cells and polymer fibers (Wagner at al. ) Electrospinning (examples)

26. Freeze-drying of porous collagen 1. addition of 3.8% acetic acid to the basic collagen suspension (1.8 wt% bovine collagen type I) 2. the collagen suspension is frozen under uniform conditions with a temperature gradient of 50C/cm and an ice front velocity of 30mm/s. 3. these parameters lead to a homogeneous plate-like ice crystal morphology with the smallest distance between the ice crystal layers. 4. vacuum-drying to remove the ice crystals by sublimation 5. collagen cross-linking (u.v.) 6. sterilisation by ethylene oxide or gamma irradiation

27. Emulsion between polymer solution and water is frozen in liquid nitrogen Scaffold is then freeze-dried at – 55 o C Highly porous scaffold (95%) but relatively small pores 15-35  m Faster drying yields smaller pores Similar methods used for formation of collagen and alginate scaffolds Freeze drying combined with salt leaching increases pore size Emulsification/freeze drying

28. Different tissue types Examples: skin, bone, nerves,blood vessel, cartilage, tendon, ligament, muscles

29. SCAFFOLD MATERIALS: Polymer Two categories: A) Materials for porous solid-state scaffolds and B) Materials for hydrogel scaffolds  The chosen of scaffolding materials depends on the environment of original ECM due to specific application for scaffold. Ex:CartilageECM=Hydrated,Bone ECM=Dense

31. Materials for porous solid- state scaffolds Application: Bone tissue engineering Material properties: Solid and stable porous structures. Not dissolve or melt under in vitro tissue culture condition or when implanted in-vivo Degrade through hydrolysis of the ester bonds Materials for hydrogel Scaffolds Application: Blood vessel, skin, cartilage, ligaments, and tendons Material properties: Ability to fill irregularly shaped tissue defects. the allowance of minimally invasive procedures such as arthroscopic surgeries the ease of incorporation of cells and bioactive agents

32. Popular Scaffolds MaterialsProperties Polyglycolic acid (PGA)–Most widely used scaffolding polymers –PGA is hydrophilic nature so that it degrades rapidly in aqueous solutions or in vivo, and loses mechanical integrity between two and four weeks. –processed into non-woven fibrous fabrics Polylactic acid (PLA)–The extra methyl group in the PLA repeating unit (compared with PGA) makes it more hydrophobic, reduces the molecular affinity to water, and leads to a slower hydrolysis rate. –It takes many months or even years for a PLA scaffold or implant to lose mechanical integrity in vitro or in vivo Collagen–a major natural extracellular matrix component –fabricated scaffolding materials

33. MATERIAL PROPERTIES VS IDEAL PROPERTIES Most of the polymer properties meets the basic requirements of an ideal ECM properties: a) Porosity  Ideal properties: High porosity, high surface area and proper pore size  Material properties: polymer is chose because it is easy to scale up (pores size, shape) b) Degradation rate  Ideal properties: proper degradation rate  Material properties: polymer is a biodegradable material. Polymer can control degradation rate and tissue quantity and quality cells seeded is

34. c) Mechanical properties APPLICATIONPERCENTAGE COLLAGEN PERCENTAGE ELASTIN ELASTIC MODULUS Bone30%0%20GPA Cartilage15%0%30Gpa Tendon20%3%1GPa Skin10%3%1GPa Elastic Modulus of Polymers: 1Mpa-3000 GPa

35. d) Biocompatibility  Ideal properties: biocompatible, non- toxic to the cells (i.e. biocompatible) and non-carcinogenic  Material properties: some of the polymer is not biocompatible especially synthetic polymer. Therefore with controlled degradation rate, we can increase the biocompatibility between polymer and host

36. Scaffold Materials: Ceramics 1. Hydroxyapatite Ca10(PO4)6(OH)2 Mineral component of bone and corral “corraline” HA Synthetic HA

37. Scaffold Materials: Ceramics 2. Bioactive glasses

40. SCAFFOLD MATERIALS: Composites Aim is to combine the stiffness of a ceramic (+ bioactivity?) with the toughness (+ resorbability?) of a polymer to tailor the properties of a scaffold to that of the host tissue.

41. Collagen-HA composites of bone

42. Precipitated HA and Chitosan

43. Composites: HAPEX®

44. Summary There are many criteria for an ideal scaffold It is important to mimic the structure of the tissue as closely as possible when designing a tissue engineering scaffold It is important to select materials specific to the application An ideal scaffold material should be tailorable to the exact needs of individual patients Cells will be affected by material composition, curvature, surface chemistry and surface roughness Culture conditions must also be optimised