Tissue Engineering And Regenerative Medicine PDF

TISSUE ENGINEERING AND REGENERATIVE MEDICINE Topic 2: Tissue Engineering Principles The Learning Outcomes At the end of the lecture, you should be able to: 1. Describe various approaches and strategies to develop tissue replacements. 2. Explain the basic considera...

TISSUE ENGINEERING AND REGENERATIVE MEDICINE Topic 2: Tissue Engineering Principles The Learning Outcomes At the end of the lecture, you should be able to: 1. Describe various approaches and strategies to develop tissue replacements. 2. Explain the basic considerations in design and materials selection for scaffold biomaterials. 3. Describe the properties of extracellular matrix (ECM) and its functions as natural biomaterials. 4. Describe the common fabrication methods for tissue engineering scaffolds. 5. Describe the mechanical, biochemical and biological properties of tissue development 2  Cellular Implantation Tissue Tissue Engineering Implantation Approaches In Situ Regeneration Tissue Engineering Approaches (1) 1. Cellular Implantation ✓Implantation or infusion of freshly isolated or cultured cells directly into damaged tissue ✓Individual cells or small cellular aggregates combined with a degradable scaffold in vitro and then implanted damaged tissue  ADVANTAGES Avoid complication of surgery Replacement of only those cells that supply the needed function Permits manipulation of cells (1) Cellular before infusion Implantation DISADVANTAGES Potential failure of infused cells to maintain their function in the recipient Immunological rejection Tissue Engineering Approaches (1) o AUTOLOGOUS – from patient o ALLOGENEIC – from human donor who is not immunologically identical to patient o XENOGENEIC – from different species o Each category can be further delineated into whether: The cells are adult or embryonic stem cells Stages of maturation Tissue Engineering Approaches (2) 2. Tissue Implantation ✓Complete 3D tissue is grown in vitro using cells and a scaffold, and then implanted once it has reached maturity  GENERAL REQUIREMENTS Cultured cells have to be coaxed to grow on bioactive degradable scaffolds that provide physical and chemical cues to guide their differentiation and assembly into (2) Tissue 3D tissues. Implantation SCAFFOLDS & BIOMATERIALS Decellularization / Bioprinting Tissue Engineering Approaches (3) 3. In Situ Regeneration ✓Scaffold implanted directly into the injured tissue stimulates the body’s own cells to promote local tissue repair  Signal molecules – growth factors Purification and large-scale production Development of methods of delivery (3) In Situ Regeneration  What are the things to consider to engineer a tissue? Basic Considerations What are the factors affecting the success of an engineered tissue? 12  Biomaterials and Scaffolds 1. What is Biomaterials? 2. What is ECM? 3. Criteria for Scaffold biomaterials 4. Scaffold Fabrication and Techniques 13 Bio X Bioprinter - Cellink 14 1. What is Biomaterials? Material intended to interact with biological system to evaluate, treat, augment or replace any tissue, organ or function of the body Function: oProvide cells with a local environment that enhances and regulates their proliferation and differentiation for cell-based tissue regeneration Types of Biomaterials: o Synthetic o Natural 15  Synthetic Biomaterials 1 Single phase materials Ceramics (e.g. aluminum oxides, silica) Composites 2 A combination of single-phase Artificial polymers (e.g. PLLA, PLGA) biomaterials to obtain combination of desired 3 Applications characteristics Ceramic – Orthopedic and dental Advantage / Disadvantage 4 devices More controllable in Artificial polymers – catheters, terms of compositional vascular grafts, intraocular lenses and materials processing may not be recognized by cells due to the absence of biological signals  Natural Biomaterials 1 Examples Constituents of extracellular matrix Advantages 2 (ECM) of connective tissue Often identical to macromolecular substances in our body Readily recognized by cells Interactions between cells and 3 Applications biological ECM are catalysts to Collagen – helps in cartilage many critical functions in tissues and vascular regeneration and wound repair Fibrin – useful for cardiac tissue engineering with excellent cell seeding effects 4 Disadvantages? Properties of ECM – to serve as natural biomaterials Biochemical Signaling Scaffolding ECM molecules have specific Acts as scaffold for cells – to binding sites for cell receptors – attach, spread, migrate and influence cell behaviour communicate with neighboring cells 1 2 3 4 Composition Dynamic and Remodeling Complex network of fibrous Not static but constant proteins and proteoglycans remodeling. Cells within the that provides structural and tissue can synthesize, mechanical supports to cells degrade, and modify ECM components – tissue can adapt and repair themselves 2. What is ECM? A polymeric network (highly organized) of macromolecules in which small molecules, ions and water are trapped Functions: o To organize and support cells in space o To provide cells with environmental signals to direct site-specific cellular regulation o To separate one tissue space from another o To interact with cells Major types of macromolecules: 1. Fibrous proteins 2. Proteoglycans 19 2. What is ECM? 1. Fibrous proteins Collagen Elastin Fibronectin Laminin 2. Proteoglycans (Hydrophilic Heteropolysaccharides) Protein core attached to Glycosaminoglcan (GAG, polysaccharide chain) o Hyaluronan o Dermatan sulphate o Heparan sulphate o Keratan sulphate o Chondroitin sulphate 20  Fibrous Proteins 1. Collagen ONE – main component of bone 2. Collagen TWO – main component of cartilage 3. Collagen THREE – main component of recticular fibers Collagens 4. Collagen FOUR – forms the basement membrane / basal lamina Major component of skin and bone that constitutes ~ 25% of total protein mass in mammals > 90% of collagens in body are Collagen I, II, III and IV  Fibrous Proteins Also an important load-bearing tissue in the bodies of mammals and used in places where mechanical energy is required to be stored Serves an important function in arteries and is particularly abundant in large elastic blood vessels i.e. aorta Elastin Lungs, bladder and ligaments are other important tissues with more elastin A protein in connective tissue that is elastic and allows many tissues in the body to resume their shape after stretching or contracting  Fibrous Proteins Laminin The major non-collagenous component of Fibronectin the basal lamina Adhesive glycoprotein that serves as bridge between cell They are a family of glycoproteins that surface receptor and ECM has a lot of flexibility in connecting to various kinds of molecules Guides in cellular movement Promotes cell attachment  Glycoprotein vs. Proteoglycan Glycoprotein: ▪ 1 – 60% carbohydrate by weight ▪ Numerous, short, branched oligosaccharides Proteoglycans ▪ Up to 95% carbohydrate by weight ▪ Mostly long, unbranched GAG chains (typically 80 sugars) ▪ Can be enormous in size protein/carb ratio The major difference between them is the _______________ 24  Proteoglycans 25 Family of the molecules with protein GAG Chains core attached to one of more GAG side Unbranched polysaccharide chains chains Heterogeneity – Variants in protein Highly negative charge, core and type and size of GAG side strongly hydrophilic chains Occupy a large amount of Function mediated by protein core and space and form hydrated gels GAG chains Proteoglycans Regulates angiogenesis Orchestrates matrix assembly; Triggers cell proliferation Proteoglycans ❖ One of the most important proteoglycans Aggregates of proteoglycans are formed when multiple proteoglycans bind to hyaluronic acid, a non-sulfated GAG molecule.  Glycosaminoglycans (GAGs) Dermatan Sulfate Most biologically active GAG – serves as biological response modifier by Hyaluronan binding to wide range of molecules The simplest GAG In skin, blood vessels, heart – Concentrated in synovial fluid in the Coagulation and wound healing eyes and body joints Potential anti-cancer drug – interacts Has the property of biological with many growth factors / cytokines lubricant, reducing friction during involved in cancer formation & movement progression  Glycosaminoglycans (GAGs) Heparan Sulfate Keratan Sulfate Found on cell surfaces, lung, arteries Found cornea, cartilage and bone Binds to a variety of protein ligands – Highly hydrated molecules, can act as regulates wide range of biological a cushion between joints, to absorb activities, including developmental mechanical shock processes, angiogenesis Enhance formation of their In cornea, KS proteoglycans maintain receptor-signaling complexes even spacing of type I collagen fibrils, allow the light without scattering – essential role in ECM organizing  Glycosaminoglycans (GAGs) Articular Cartilage Chondroitin Sulfate Maintaining structural integrity Interweaving of of tissue Proteoglycans and Collagen Fibrils Concentrated in cartilage, skin, arteries Slightly different from meniscus Has the property of biological lubricant, reducing friction during movement Tissue Water Collagen Proteoglycans Articular Cartilage 68 – 85% 10 – 20% 5 – 10% Meniscus 60 – 70% 15 – 25% 1 – 2%  3. Criteria for Scaffold Biomaterials 1. Biocompatibility 2. Cell Adhesion 3. Biodegradability 4. Bioactivity 5. Reproducibility 31 I. Biocompatibility o To assist in the molecular and mechanical signalling systems and to optimise tissue regeneration, without causing any damage o Support cell growth and proliferation II. Cell Adhesion o Interaction of the surface promotes traction for the migration of the cells 32 III. Biodegradability o To leave a totally natural tissue replacement following degradation of the polymer o Degradation rate must be compatible with the cell growth rate to maintain the mechanical integrity o Scaffolds are not permanent implants 33 IV. Bioactivity o The ability of a scaffold biomaterial to produce an effect on living tissue o From inert biomaterial > biologically active biomaterial: Not blocking regeneration Provides biological cues that initiate and guide regeneration V. Reproducibility o Material should be easily and reliably reproduce into a variety of shapes and structures that retained their shape when implanted 34  Scaffold Biomaterials – Natural vs Synthetic Natural Synthetic Biocompatibility Mostly Better Mostly Poorer Cell Adhesion Mostly Better Mostly Poorer Biodegradability Generally Yes Generally No Bioactivity Mostly Higher Mostly Lower Reproducibility Maybe Yes Primary role of a scaffold? 35 to provide a temporary substrate to which transplanted cells can adhere  Scaffold Biomaterials – Other considerations Immunogenicity Blood Cost of Carcinogenicity Compatibility Manufacturing Provokes immune Interacts with Prohibitively costly Will it cause responses? blood to create? tumor formation? components? 36 Scaffold Design – Architectural / Mechanical Considerations Strong enough to resist physical Strength & forces within site of implantation Flexibility and prevent pores from collapsing Match to those of the tissues at Elasticity the site of implantation to aid in the vascularization processes High porosity to provide large void volume into which Porosity transplanted cells may be 37 seeded; will affect diffusion of nutrients, gas and waste products 4. Scaffold Fabrication and Techniques I. Electrospinning o Utilizes the electrostatic force for the production of polymeric fiber ranging from nanoscale to microscale o Diameter of fiber: 400 – 1100 nm o Porosity: 80 – 95% Applications: o Skin o Cartilage o Vascular o Nerve 38 4. Scaffold Fabrication and Techniques II. Freeze-drying o Drying process for converting solutions of labile materials into solids of sufficient stability Solution is frozen at low temperature (-70°C to -80°C) Frozen sample is located at the chamber with low pressure (partial vacuum) Unfrozen water in material is removed by desorption o Diameter of fiber: 50 – 500 nm o Porosity: 30 – 80% Applications: o Tendon o Bone o Skin 39 4. Scaffold Fabrication and Techniques III. Self-assembly o An autonomous organization of components into patterns or structures without human intervention o Nature example: phospholipids o Diameter of fiber: 5 – 300 nm o Porosity: 80 – 90% Applications: o Cartilage 40 3. Scaffold Fabrication and Techniques IV. Decellularisation o Removing cellular components (especially DNA and RNA) from natural tissues, leaving behind the ECM – retaining the tissues’ native architecture and bioactive molecules V. 3D Bioprinting 41 Summary Properties of ECM What is ECM? Compositions Fabrication methods Electrospinning Freeze-drying 1 2 3 4 Self-assembly Decellularisation TE Approaches Basic Considerations Cellular implantation Tissue implantation Biomaterial selection In situ regeneration Design  Right cells + Right scaffold + Right biomaterial Tissue Development Mechanical Properties Biochemical Properties Biological Properties Include tensile strength, Involve ECM with proteins Encompass cell differentiation, elasticity, compressibility, & GAGs guiding cell cell proliferation, migration, viscoelasticity, tissue behaviour, growth factors apoptosis, tissue morphogenesis stiffness etc. and cell-cell signaling and maturation etc. regulate cell processes etc.