Biocompatible Materials: Past Paper PDF 05 Biological Materials I

376-1714-00L Biocompatible Materials Materials of biological origin I 16.10.2024 Dr. Markus Rottmar, Empa, Lab for Biointerfaces Prof. Dr. Katharina Maniura, Empa, Lab for Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, Tissue Engineering and Biofabrication Classes of biomaterials...

376-1714-00L Biocompatible Materials Materials of biological origin I 16.10.2024 Dr. Markus Rottmar, Empa, Lab for Biointerfaces Prof. Dr. Katharina Maniura, Empa, Lab for Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, Tissue Engineering and Biofabrication Classes of biomaterials metals ceramics synthetic Biomaterials polymers natural macromolecules composites 2 Evolution of biomaterials Ramakrishna, S. et al. Biomaterials: A nano approach (CRC press 2010) 3 Motivation § synthetic biomaterials do not present the form and function presented by the human body (chemically, spatially, physically, temporally) § can we learn to adopt biomaterials by understanding how nature’s form produces function?= “Biomimicry” à biomimetic materials Gecko-feet inspired materials Hawkes et al. (2015) J. R. Soc. Interface 12: 20140675. 4 Biomimetic biomaterials – example bone TE Park, J.Y., et al (2018). https://doi.org/10.1007/978-981-13-0445-3_7 5 Biomimicry – a range of characteristics Native ECM Desired material characteristics § Adequate mechanical § Biocompatible properties § Biodegradable/bioresorbable § Enzymatic remodelling § Suitable mechanical properties § Binding and release of § Adequate physico-chemical growth factors properties to direct cell-material § Biocompatible interactions § Non-immunogenic § Shape and structure of the tissue to be replaced § Ease of production § Inexpensive natural materials can provide some/many/most of the required material characteristics and biological stimuli 6 Especially promising for tissue engineering & regenerative medicine (oftentimes) materials of biological origin Cells + Scaffolds + Signals Scaffolds Cells + Signals § Why are biological materials advantageous? A cell is the most Q1 sophisticated Why are controlled biological release system!materials - advantageous? right factors, right place, right time, right order, right dose à suitable scaffolds required 7 Materials of biological origin § enable the design of biomaterials that function at the molecular rather than at the macromolecular scale § low toxicity or foreign body reaction § degradation by naturally occurring enzymes, biomaterial disappears after some time (à design of lifetime of the implant) § collagens and polysaccharide materials are low immunogenic since they are highly conserved between species § some biopolymers are very immunogenic (host – donor issue) § isolation, modification and processing can be very challenging § Examples: - silk-based materials - bacterial cellulose - chitosan - fibrin - hyaluronic acid - de-cellularized matrix/tissue - collagen 8 Content 1. Fibrin § structure and function § fibrin as biomaterial 2. Native Extracellular Matrix § overview and function § constituents - glycosaminoglycans (GAG) - collagens - fibronectin - laminin § decellularization § applications 9 Teaching objectives § You know the structure and composition of fibrin and fibrin clots § You can describe typical clinical applications of fibrin § You can describe the major constituents and the corresponding function of native extracellular matrix (ECM) § You have a basic understanding of the ultrastructure of bone § You know what kinds of tissue transplant strategies exist § You can compare benefits and challenges of decellularization 10 § You know which decellularization methods exist Markus Rottmar Fibrin - overview Enzymatically processed, fibrillary assembled derivative of fibrinogen with dual function § Hemostasis: crucial component of the blood coagulation cascade to rapidly control bleeding at injury site § Provisional, remodelable matrix: Serves as initial scaffold for tissue repair by providing matrix for cell adhesion, migration and proliferation whole blood thrombin + fibrinogen Luyendyk, J.P. Blood. (2019) 133(6):511-520 Weisel, J.W. Adv Protein Chem. (2005) 70:247-99 11 Fibrin – overview of structure and polymerization steps Replicating the coagulation cascade in a controlled manner makes it possible to form materials (in situ)! Weisel, J.W. Adv Protein Chem. (2005) 70:247-99 12 Fibrin polymerization § Thrombin cleaves fibrinopeptides from the Aα chains of fibrinogen § Fibrin monomers align in staggered overlapping end-to-middle arrangement (D:E) to form double- stranded twisted protofibrils § Thrombin cleaves fibrinopeptides from the Bβ chains of fibrinogen § Protofibrils self-assemble laterally into branched fiber bundles § Factor XIIIa crosslinks the fibrils to form fibrin matrix Roberts IV. Macromol Biosci. (2019) 20, 1900283 13 Fibrin mechanical properties Mech. prop. of in vivo fibrin clots not well characterized § Important for hemostasis, rupture, embolization § Viscoelastic (or even poro-visco-elastic) § Determined by polymerization conditions: Ghezelbash F. J Mech. Behav. Biomed. Mater (2022) 128, 105101 § Lateral aggregation favored: thick fibers, few branch points § Q2 Lateral aggregation inhibited: thin – how fibers, manywill thepoints branch fibers look like? § Porosity and overall clot structure can be tuned § e.g. concentration of fiber diameter stiffness fibrinogen 14 Tuning of fibrin clot architecture thrombin concentration 0.25 IU/ml 25 IU/ml § Thinner fibers and decreased overall porosity with increasing thrombin concentration salt concentration 0.07M 0.13M 0.20M § Thinner fibers and decreased overall porosity with increasing medium salt concentration Seelich et al. Encyc Biomat Biomed Eng (2004), 1, 603-610 15 Fibrinolysis (dissolution of clot) § Plasminogen binds and is activated to plasmin, which then cleaves fibrin at specific sites, proceeding by transversely cutting fibrils and fibers § Balance between stability and degradation is important § Measure degradation: § ELISA to detect soluble, crosslinked D dimers § Absorbance of clot supernatant at 280 nm, changes in turbidity § Thromboelastography Roberts IV. Macromol Biosci. (2019) 20, 1900283 16 Fibrin as tissue scaffold / biomaterial § Historical perspective Commercialized: Tisseel®, Tissucol ®, 1944: as tissue adhesive for skin grafts Artiss ®, Beriplast P ® 1940: First reported use as tissue adhesive (peripheral nerve anostomosis) 1909: First clinical application of fibrin 1972:“blood glue“concept reintroduced (degradable hemostatic agent) (cryoprecipitate with higher concentration fibrinogen, Factor XIII, fibronectin etc.) 1900 1925 1950 1975 2000 Q3 Use of fibrin as patches for (poor adhesive sealant for hemostasis, growing hemostasis (e.g. in WW1) strength and poor platform for various applications durability) in tissue engineering and Why was it ~30 regenerative medicine years neglected Sierra, D.H. J. Biomater. Appl. (1993) 7, 309–352. 17 Fibrin as biomaterial § Production (fibrinogen, thrombin): isolation from blood plasma, recombinant production § Fibrin clot provides structural basis for tissue repair and serves as scaffold for cell adhesion, proliferation, migration § Fibrin has binding sites for integrins, CD44, VE-Cadherin, growth factors; other proteins (e.g. Fibronectin) bind by crosslinking via Factor XIII § Advantages: § “Biocompatible” and biodegradable § Uniform distribution of biological molecules and cells possible § Lifetime can be prolonged by addition of artificial polymers such as PEG or with protease inhibitors § Mechanical properties can easily be modified (e.g. fibrinogen/thrombin ratio, crosslinking, salts, pH…) 18 Fibrin mediates forces during tissue self-assembly § Cell-mediated compaction of fibrin or fibrin- collagen gels guided by geometric environment (pins or posts) for muscle, tendon and ligament engineering Hecker et al, Biotech Appl Biochem 2008 skeletal myoblasts cardiomyocytes Bian et al., Nat Protoc. 2009; 4(10): 1522–1534. 19 Application of fibrin as delivery vehicle § Cell delivery: e.g. for treating ulcers and chronic wounds (keratinocytes, fibroblasts) Differentiation of cells can be controlled by fibrin clot properties (e.g. mechanics) Example chondrocytes: chondrogenic differentiation, GAG retention, collagen-II synthesis improved in fibrin compared to e.g. PGA § Delivery of drugs and growth factors: binding and release can be controlled by clot properties Breen, A.; O’Brien, T.; Pandit, A. Tissue Eng. Part B 2009, 15(2), 201–214. 20 Clinical practice: fibrin glue § Fibrin glue approved for hemostasis, as adhesives and sealants, but also used off-label § Two component solution (fibrinogen and thrombin) applied via double syringe system § Composition depends on manufacturer: 21 Two-barrel syringe applicator Ahmed, T., Dare, E., Hincke, M., Fibrin: a versatile scaffold for tissue engineering applications. Tissue Eng Part B Rev (2008) vol. 14(2), 199-215 22 Fibrin as biomaterial - challenges § Degradation rate of fibrin gels § Variety of cells produce plasmin (counteract through addition of e.g. tranexamic acid) and matrix metalloproteinases (MMPs; counteract through addition of e.g. aprotinin) § Contraction of the hydrogel (especially cell-mediated) Q4polymers or as coatings à Combination with other synthetic/natural What § Limited challenges mechanical are associated with fibrin as biomaterial? properties § 0.94 to 6.49 kPa for fibrinogen concentrations of 0.5–3.0 mg/mL (27.5 kPa with 30mg/mL, but with impaired cell spreading, migration and survival) à Combination with other synthetic/natural polymers (but can alter cell response) § Batch-to-batch variability Sanz-Horta R. J Tiss Eng (2023) 14 23 Fibrin-based biomaterials - steering mechanical properties Vorwald C.E. et al, Acta Biomater (2020) 108, 142-152. 24 Content 1. Fibrin § structure and function § fibrin as biomaterial 2. Native Extracellular Matrix § overview and function § constituents - glycosaminoglycans (GAG) - collagens - fibronectin - laminin § decellularization § applications 25 Extracellular Matrix (ECM) § Tissues are not made up cells only: “intracellular space” is filled with ECM § The ECM is an intricate network of proteins and polysaccharides § ECM is locally secreted and assembled by tissue cells § Structure and composition are highly tissue- specific § Separation into structural and functional 26 molecules not possible Kubow, Klotzsch, Smith, Gourdon, Little, Vogel; Integrative Biology, 2009, (1(11-12)), 635-648 Markus Rottmar Functions of the ECM 1. Mechanical support (stability and elasticity of tissues) 2. Selective barrier between tissue types Mendibil et.al, Int. J. Mol. Sci. 2020, 21, 5447 (e.g. impermeable to cells) 3. Cell guidance: shape, proliferation (structural and biochemical features) 4. “Information” storage (e.g. growth factors or cytokines; ECM acts as reservoir controlled release and activation during remodeling of ECM by cells, e.g. during wound healing or development) Dynamic equilibrium / crosstalk between cells and matrix: - cells control the composition and structure of the ECM - ECM influences the phenotype and behavior of cells and tissues 27 ECM constituents Main classes of extracellular matrix molecules: 1. Polysaccharide chains: Glycosaminoglycans (GAGs) - usually covalently linked to proteins: Proteoglycans - form highly hydrated, gel-like substance in which fibrous proteins are embedded - resist compressive forces, highly permeable 2. Fibrous proteins: structural and adhesive functions - Collagen - Fibronectin 28 - Laminin - others (incl. Elastin, Tenascin) 3. Integrated molecules - growth factors, cytokines 28 Markus Rottmar Dimensions of ECM components Glycoproteins Proteoglycans (Protein >>> Carbohydrate) (Carbohydrate >>> Protein) 29 mono- or oligosaccharide chains, heteropolysaccharide chains, long short and often branched, may or Q5 and linear, negatively charged may not be negatively charged Whats the difference between GAGs glycoproteins and proteoglycans proteins cell surface connective tissues Fig. 19-31/33 Mol Biol of the Cell, 6th Ed cell-cell recognition, signaling structural support to ECM 29 Sourcing of ECM proteins § Isolation from tissues, from 2D/3D cell culture (e.g. fibroblasts) or via recombinant production (e.g with E.coli, yeast, CHO-cells) https://www.technologynetworks.com/ 30 30 Sourcing of ECM proteins § Isolation from tissues, from 2D/3D cell culture (e.g. fibroblasts) or via recombinant production (e.g with E.coli, yeast, CHO-cells) https://www.technologynetworks.com/ 31 31 Glycosaminoglycans (GAGs) § unbranched polysaccharides composed of repeating disaccharide units out of: 1) amino sugar (N-acetylglucosamine or N-acetylgalactosamine) 2) uronic acid (glucuronic / iduronic acid) Repeating disaccharide sequence of a heparan sulfate glycosaminoglycan (GAG) chain. These chains can consist of as many as 200 disaccharide units, but are typically less than half that size. § highly negatively charged; attract positively charges ions (Na+, Ca2+) § form native hydrogels where other ECM components can be embedded and permit diffusion of nutrients, metabolites, growth factors and hormones § approx.10 % by weight of the ECM but fill most of the extracellular space as porous hydrated gel -> structural role 32 32 Glycosaminoglycans (GAGs) § 4 groups of GAG chains can be distinguished according to their: - sugar chains - type of linkage - number and position of sulfate groups 1. Hyaluronan (hyaluronic acid); not sulfated 2. Chondroitin sulfate and dermatan sulfate 3. Heparan sulfate 4. Keratan sulfate § Functions of GAG chains: - binding of growth factors (FGF, EGF, VEGF) -> chemical signaling - clustering of receptors on the cell surface - hydrogel resists compressive forces on the matrix -> mechanical signaling 33 33 Hyaluronic acid § linear polysaccharide composed of repeating disaccharide units of N-acetly-glucosamine and D-glucuronic acid, linked by alternating b-1,3 and b-1,4 glycosidic linkages § the highest concentration is found in soft connective tissue à major component of the ECM § HA is highly abundant in hyaline cartilage, in synovial joint fluid, and in the skin tissue - both dermis and epidermis K.Wolf et al. ACS Biomater. Sci. Eng. 2019, 5, 8, 3753-3765 34 Hyaluronic acid-based biomaterials – cell material interactions K.Wolf et al. ACS Biomater. Sci. Eng. 2019, 5, 8, 3753-3765 35 Hyaluronic acid – biomedical applications Main forms § Hydrogels § Scaffolds § Nanoparticles § Composites § … M. Hemshekhar et al. Int J Biol Macromolecules 86 (2016) 917–928 36 Proteoglycans – polysaccharides in the ECM § Proteoglycans are GAG chains linked to a core protein Biomedical applications § Special link tetrasaccharide is attached to serine residues of core protein that serves as primer for GAG growth Rnjak-Kovacina, J., et al. Adv. Healthcare Mater. 2018, 7, 1701042 Fig. 19-35/37 Mol Biol of the Cell, 6th Ed 37 37 Proteoglycans – biomedical applications § Example: multilayered PEG-gels modified with CS+MMP-peptide, CS, or HA (for osteochondral defects) Rnjak-Kovacina, J., et al. Adv. Healthcare Mater. 2018, 7, 1701042 38 Collagens § up to 90% of dry weight of the ECM § More than 20 distinct types (fibrous, non-fibrous, filamentous and fibril-associated). Most common: - Type I: skin, tendon, vasculature, organs, bone (most abundant collagen) - Type II: cartilage (main collagen of cartilage) - Type III: reticulate (main component of reticular fibers), commonly found along type I - Type IV: basal lamina (epithelium-secreted layer of the basement membrane) - Type V: cell surfaces, hair, and placenta § Assembly requires ascorbic acid (Vitamin C) Fibroblast surrounded by collagen fibrils 39 Collagens (only FYI, not to memorize) Lin, K., et al., Adv. Funct. Mater. 2019 40 Collagen biosynthesis 41 41 Markus Rottmar Collagens arrangement/orientation in tissues § Fibrils have various diameters and and organized differently in each tissue: skin: - woven in pattern to resist isotropic tensile stress tendon: - organized in parallel bundles along the major axis of tension bone: - arranged in orderly plywood-like layer - parallel fibers within one layer oriented in 90°angle to fibers in adjacent layers 42 42 Collagen-based biomaterials § Excellent biocompatibility and biodegradability (nature-derived) § Collagen shows excellent cell attachment (via integrins), gelatin via adhesiveness towards e.g. fibronectin § Collagen and gelatin solutions can readily form gels. à culture cells in or on collagen gels § Excellent formability/processability 43 43 Collagen-based biomaterials § Collagen-based materials can be modified/functionalized to control material properties/gelation mechanisms J. Sapudom et al. Biomaterials 52 (2015) 367e375 44 50 µm à Coll I network density and stiffness increases with Coll I concentration 44 Markus Rottmar Collagen-based biomaterials § Collagen-based materials can be modified/functionalized to control material properties/gelation mechanisms 45 Q6 How do gels differ if made from atelocollagen? 45 Collagen-based biomaterials – TE applications 46 Lin, K., et al., Adv. Funct. Mater. 2019 46 Bone – a collagen-rich composite biomaterial § very dense specialized form of connective tissue § like reinforced concrete: bone matrix is a mixture of organic and inorganic components organic: cells and osteoid (~35% bone mass; ~50% volume) (collagen I fibers, proteoglycans and glycoproteins) inorganic: mineralized phase (hydroxyapatite crystals) (~65% bone mass; ~50% volume) § collagen I fibers tensile forces § hydroxyapatite compressive load 47 Bone tissue: collagen I fibers integrated into calcified matrix Distribution of mineral and non- collagenous proteins within and between collagen fibrils 48 Engineering collagen-hydroxyapatite scaffolds for bone TE § Collagen (CO) + rhBMP-2 Collagen-hydroxyapatite (CHA) + rhBMP-2 Hyaluronic acid hydrogel/gelatin + rhBMP-2 § Improved stability and drug release of CHA, leading to enhanced bone formation Lackington W. et al Mater Today Comm (2021) 29:102933 49 Fibronectin § Second most abundant matrix protein § large multidomain glycoprotein; covalently linked homodimer § found in all vertebrates beginning from early development § essential role for cell attachment and migration to and within ECM plasma fibronectin: - Soluble - circulates in the blood and other body fluids - helps to enhance blood clotting, wound healing and phagocytosis 50 fibronectin in the ECM: - Insoluble - assembles to filaments which are deposited on the cell surface or in the ECM 50 Fibronectin as biomaterial § Different strategies to functionalize materials with Fn: - Simple 2D coatings via covalent binding or adsorption of Fn in monolayers - Complex coated interfaces, where Fn is combined with other molecules to form bioengineered multilayered thin films and interfaces in 2.5D - Fn distributed in 3D hydrogels via physical dispersion and covalent cross-linking 51 Palomino-Durand,C et al. Appl. Sci. 2021, 11, 12111. Daum, Ret al.. Cells 2020, 9, 778. 51 Laminin § Glycoprotein (600-1000 kDa) § Composed of three long polypeptide chains § Consists of several functional domains mediating distinct functions - perlecan & nidogen binding - cell adhesion - self aggregation § Major component of basal lamina (with coll IV and perlecan) 52 Laminin as biomaterial § Different strategies to functionalize materials with laminin: - Simple 2D coatings via covalent binding or adsorption of laminin in monolayers - Complex coated interfaces, where laminin is combined with other molecules to form bioengineered multilayered thin films and interfaces in 2.5D - Fn distributed in 3D hydrogels via physical dispersion 53 and covalent cross-linking Hassan, G.; et al.. Processes 2021,9,45. 53 Using whole ECM instead of individual proteins in TE § Preserves tissue-specific structural and functional molecules in the same relative amounts as in native tissue § Preserves the native three-dimensional structure for guiding cellular organization Isograft - tissue graft that is harvested from a genetically identical donor (twin) Autograft Isograft Autograft - tissue graft that is harvested from and implanted in the same individual identical twins Q7 - disadvantage of creating a secondary morbid site upon harvest Allograft What graft types exist? Allograft non-identical - tissue graft that is harvested from one individual and transplanted in a genetically non-identical individual of the same species - potential risk of transmitting infectious diseases Xenograft Xenograft - tissue graft that is harvested from one individual and implanted in inter-species another individual of different species 54 Tissue decellularization Process of removing the allogeneic or xenogeneic cellular antigens from a tissue that would initiate an immune response while leaving behind an intact ECM comprising a mixture of structural and functional molecules. What to remove? - Nuclear material (nucleic acids) - Lipids (cell plasma membrane) - Proteins (cytoskeleton) Native liver tissue Decellularized liver tissue Srokowski, EM, Woodhouse, KA, Decellularized Scaffolds. Comprehensive Biomaterials (2011) section 2.221 55 Tissue decellularization Target Target Lipid-lipid Peptide interactions bonds Lipid- Phosphodi protein ester-bonds interactions Target Cell membranes J.Liu et al Exploration (2024) , https://doi.org/10.1002/EXP.20230078 56 Advantages and Disadvantages of Decellularization § Minimizes immunological response § Requires a tissue-specific process § Allows for the use of xenogeneic grafts § Cellular removal can be incomplete § Conserves mechanical integrity and § Efficiency depends on tissue source and architecture of the tissue architecture and is difficult to assess § Retains biochemical composition and § Manufacturing process may influence bioinductivity properties § Widely applicable, economical § Scaffold properties limited to tissue source properties § Long-term storage capability § Requires availability & infiltration by host cells § Potential for off-the-shelf product § Degradation can be faster than recellularization 57 Decellularized tissue derived biomaterials § Decellularized corneal ECM derived hydrogels Fernández-Pérez, J. et al. Sci Rep 9, 14933 (2019) 58 Example for de-cellularized tissue product Small intestinal submucosa (SIS) Srokowski, EM, Woodhouse, KA, Decellularized Scaffolds. Comprehensive Biomaterials (2011) section 2.221 59 Decellularization – tissues & whole organs Chen FM, Liu X. Prog Polym Sci. 2016 60 Whole organ decellularization § Decellularization by retrograde perfusion before 0.02% Trypsin 3% Triton-X100 4% DCA Acetic Acid/EtOH 61 Summary § Fibrin clots form by enzymatic cross-linking of fibrinogen, control hemostasis and serve as provisional cell matrix. Fibrin glue is widely applied in clinics. § ECM has structural as well as biochemical functions § ECM consists of GAGs, fibrous proteins (e.g. collagens, fibronectin), and bound soluble factors § Individual ECM components (GAGs, collagen, fibronectin, laminin) are widely used biomaterials as (components of) 3D matrices or for 2D surface functionalization § Decellularization is the de-facto “gold standard” to provide tissue-specific scaffolds for regenerative medicine § Physical, chemical and enzymatic strategies exist for decellularization § Decellularized matrix can be used as xenogenic tissue transplant 62 Markus Rottmar