Biocompatible Materials PDF

376-1714-00L Biocompatible Materials Physical properties of biomaterials HS2024: 09.10.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces/ D-HEST Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication October 09, 2024...

376-1714-00L Biocompatible Materials Physical properties of biomaterials HS2024: 09.10.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces/ D-HEST Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication October 09, 2024 Katharina Maniura 1 Physical properties of biological tissues biological tissues show specific variations in physical properties on different scales: stiffness elasticity roughness porosity geometry (optical) (electromagnetic) bone porosity and roughness on nanometer to millimeter scale cells in fibrous extracellular matrix October 09, 2024 Katharina Maniura 2 Why are physical properties of biomaterial relevant? ▪ Biocompatible materials have to match the physical characteristics of their host tissue ▪ We need to understand how cells respond to different physical properties in order to control biocompatibility ▪ The physical properties of biomaterials have to be tuned in a controlled and reproducible way Biological response to physical properties is independent of the specific material and therefore needs to be studied separately. October 09, 2024 Katharina Maniura 3 Responses to physical properties ▪ cellular responses (minutes to days): ▪ adhesion ▪ spreading ▪ orientation ▪ migration ▪ proliferation ▪ force generation ▪ differentiation ▪ death ▪ generally: length scales from nm to mm, time scales from seconds to years October 09, 2024 Katharina Maniura 4 Geometry on different length scales determines cellular response by controlling cell adhesion and shape October 09, 2024 Katharina Maniura 5 From last week…. October 09, 2024 Katharina Maniura 6 Geometry on different length scales determines cellular response by controlling cell adhesion and shape Vogel V, Sheetz MP. Nat Rev Mol Cell Biol 2006;7(4):265-275. October 09, 2024 Katharina Maniura 7 Teaching objectives ▪ You understand why nano-roughness has an influence on protein adsorption and cell adhesion ▪ You can explain contact guidance and describe approaches to guide cell alignment on biomaterials ▪ You know how substrate stiffness influences cell response and how it can be tuned and measured ▪ You can explain why porosity and macroscopic geometry are important properties of biomaterials BD Ratner et al., “Biomaterials Science”, 3rd edition, Elsevier 2013 P Ducheyne et al., “Comprehensive Biomaterials”, Elsevier 2011 October 09, 2024 Katharina Maniura 8 1. Roughness, Patterning, Anisotropy ▪ Roughness, protein adsorption and adhesion ▪ Cell alignment and contact guidance 2. Mechanical properties ▪ Cell response to different stiffness ▪ How to tune and measure stiffness ▪ Other mechanical properties 3. Geometry, Porosity, Curvature ▪ The role of porosity for biocompatibility ▪ How to control porosity and curvature ▪ Substrate geometry and tissue patterning October 09, 2024 Katharina Maniura 9 1. Roughness, Patterning, Anisotropy ▪ Roughness, protein adsorption and adhesion ▪ Cell alignment and contact guidance 2. Mechanical properties ▪ Cell response to different stiffness ▪ How to tune and measure stiffness ▪ Other mechanical properties 3. Geometry, Porosity, Curvature ▪ The role of porosity for biocompatibility ▪ How to control porosity and curvature ▪ Substrate geometry and tissue patterning October 09, 2024 Katharina Maniura 10 Roughness increases protein adsorption ▪ Most material processing techniques (e.g. machining) create uncontrolled surface roughness and texture ▪ Higher roughness leads to more exposed surface area for proteins to interact with ▪ The effect of roughness on protein adsorption is stronger for larger proteins (e.g. fibrinogen) compared to smaller proteins (e.g. albumin) ▪ This is probably due to additional effects due to enhanced denaturation (unfolding) of large proteins on rough surfaces October 09, 2024 Katharina Maniura 11 Roughness and blood activation ▪ Enhanced protein adsorption also leads to enhanced blood clot formation and platelet activation on rough compared to smooth surfaces ▪ The effect of roughness can be modulated by additional alkali treatment (or other means) to render surfaces hydrophilic Milleret V, Tugulu S, Schlottig F, Hall H. Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation. Eur Cell Mater. 2011;21:430-44. October 09, 2024 Katharina Maniura 12 How do cells respond to nanoscale roughness? Matrix Fibers Filopodia Cell 40 nm pits October 09, 2024 Katharina Maniura 13 How do cells respond to nanoscale roughness? Filopodia Lamellipodia Both actin based cell protrusions Highly organized, tightly Thin sheet-like branched cross-linked long network of actin bundles of unidirectional filaments and parallel actin filaments www.mechanobio.info During cell migration, and in the absence of filopodia, lamellipodia detect the stiffness of the surrounding ECM in a process called rigidity sensing. Several models have been proposed that describe this process October 09, 2024 Katharina Maniura 14 How do cells respond to nanoscale roughness? Filopodia adhesion mechanism 2D vs. 3D The role of filopodia in the recognition of nanotopographies Albuschies, J. & Vogel, V.; Scientific Reports, 2013 October 09, 2024 Katharina Maniura 15 Nanoporosity and cell adhesion Q1: What cell response do you expect on the different substrates? October 09, 2024 Katharina Maniura 16 Nanoporosity determines cell adhesion spacing October 09, 2024 Katharina Maniura 17 Influence of adhesion spacing on cell differentiation ▪ Same size of pits (100nm), but different spacing; created by EBL DSQ50 DEX Filopodia Osteocalcin formation = marker for bone ALP Irregular spacing (DEX) induces more osteogenic differentiation of mesenchymal stem cells than regular (DSQ50) spacing Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, Wilkinson, CDW, Oreffo, ROC. Nat Mater. 2007;6(12):997–1003. October 09, 2024 Katharina Maniura 18  Controlled spacing of adhesive molecules ▪ No roughness, only spacing of adhesive sites ▪ 8nm Au dots functionalized with RGD 28nm 58nm 73nm Arnold M, Cavalcanti-Adam EA, Glass R, Blümmel J, Eck W, Kantlehner M, Kessler, H, Spatz, JP. Activation of integrin function by nanopatterned adhesive interfaces. Chemphyschem. 2004;5(3):383–8. October 09, 2024 Katharina Maniura 19 Summary on roughness and nanoporosity ▪ Nanoscale roughness increases protein adsorption ▪ Large proteins are more sensitive to roughness ▪ Cells “see” roughness through the spacing of adhesion sites ▪ Spacing of 50-70 nm supports clustering of adhesions ▪ Proliferation and differentiation depend on adhesion spacing Response to biomaterial roughness is determined by protein adsorption and cell adhesion. October 09, 2024 Katharina Maniura 20 1. Roughness, Patterning, Anisotropy ▪ Roughness, protein adsorption and adhesion ▪ Cell alignment and contact guidance 2. Mechanical properties ▪ Cell response to different stiffness ▪ How to tune and measure stiffness ▪ Other mechanical properties 3. Geometry, Porosity, Curvature ▪ The role of porosity for biocompatibility ▪ How to control porosity and curvature ▪ Substrate geometry and tissue patterning October 09, 2024 Katharina Maniura 21 Contact guidance of cells by alignment of ECM October 09, 2024 Katharina Maniura 22 Contact guidance – general observations ▪ Cells align with the direction of matrix fibers in tissues ▪ Most tissues show a high degree of anisotropy ▪ On flat, adhesive surfaces, cells are randomly oriented ▪ Alignment can be induced by anisotropic biomaterial surfaces October 09, 2024 Katharina Maniura 23 How to fabricate anisotropic biomaterial surfaces ▪ 2D: Micropatterning of stripes of adhesive proteins on a flat and otherwise passivated surface Collective migration of epithelial cells on patterns created by microcontact printing 250µm wide / 120µm spacing 10µm wide / 120µm spacing Vedula SRK, Hirata H, Nai MH, Brugués A, Toyama Y, Trepat X, Lim CT, Ladoux B. Epithelial bridges maintain tissue integrity during collective cell migration. Nat Mater. 2014;13(1):87–96. October 09, 2024 Katharina Maniura 24 How to fabricate anisotropic biomaterial surfaces ▪ 2.5D: Etching or molding of ridges and channels of different width and depth on an adhesive surface ▪ 3D: Anisotropic fiber matrix mimicking the real ECM, generated e.g. by electrospinning or 3D printing isotropic aligned Primary heart cells produce less ANP (harmful neurohormone) when growing on aligned fibers October 09, 2024 Katharina Maniura 25 Biofabrication and anisotropy Hydrogel-Based Fiber Biofabrication Techniques for Skeletal Muscle Tissue Engineering Marina Volpi, Alessia Paradiso, Marco Costantini, and Wojciech Świȩszkowski ACS Biomaterials Science & Engineering 2022 8 (2), 379-405 DOI: 10.1021/acsbiomaterials.1c01145 October 09, 2024 Katharina Maniura 26 Feedback of cell and matrix alignment ▪ Cells align with the extracellular matrix ▪ Cells also align matrix fibers by pulling on them In tissues, this can lead to emergent long-range structures W. Matthew Petroll, UT Southwestern October 09, 2024 Katharina Maniura 27 1. Roughness, Patterning, Anisotropy ▪ Roughness, protein adsorption and adhesion ▪ Cell alignment and contact guidance 2. Mechanical properties ▪ Cell response to different stiffness ▪ How to tune and measure stiffness ▪ Other mechanical properties 3. Geometry, Porosity, Curvature ▪ The role of porosity for biocompatibility ▪ How to control porosity and curvature ▪ Substrate geometry and tissue patterning October 09, 2024 Katharina Maniura 28 Stiffness and cell adhesion – common observations ▪ on very soft, linear elastic substrates, cells do not spread ▪ cell spreading increases with increasing substrate stiffness ▪ the same cell type may look very different depending on the stiffness of the substrate, here for human MSCs: Collagen I Collagen IV October 09, 2024 Katharina Maniura 29 Stiffness and cell migration Q2: How do cells respond to a stiffness gradient? October 09, 2024 Katharina Maniura 30 Measuring elastic properties Nanoindentation of caries lesions ▪ bulk stiffness rheometer tensile test ▪ local stiffness on surface Atomic Force Microscopy AFM indentation test ▪ local stiffness in bulk micro rheology (e.g. optical, magnetic) ultrasound elastography 1. He L-H, Swain MV. Microindentation. Chapter 3.306 MRI Comprehensive Biomaterials. Elsevier; 2011 October 09, 2024 Katharina Maniura 31 Substrate stiffness and stem cell differentiation ▪ linear elastic polyacrylamide (PA) hydrogels functionalized with type 1 collagen ▪ stiffness variation by changing ratio of acrylamide / bisacrylamide ▪ on soft PA substrates, differentiation tends towards soft tissue types (brain) ▪ on hard PA substrates, differentiation towards hard tissues (bone) is enhanced Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677 –89. October 09, 2024 Katharina Maniura 32 Mechanical properties of native ECM components Storm C, Pastore JJ, MacKintosh FC, Lubensky TC, Janmey PA. Nonlinear elasticity in biological gels. Nature. 2005;435(7039):191-194. October 09, 2024 Katharina Maniura 33 Measurements of viscoelasticity Chaudhuri O. Viscoelastic hydrogels for 3D cell culture. Biomat Sci. 2017;22(19):287. October 09, 2024 Katharina Maniura 34 Substrate stress relaxation regulates cell behaviour cell spreading stem cell differentation Chaudhuri O, Gu L, …, Mooney DJ. Nat Commun 2015;6:6365. Chaudhuri O, Gu L, …, Mooney DJ. Nat Mater 2015;15(3):326-334. October 09, 2024 Katharina Maniura 35  How do cells “feel” stiffness? ▪ cells adhere and apply cytoskeletal force (stress) to the substrate, thereby deforming their microenvironment (strain) ▪ cellular functions depend on cell shape, cytoskeletal organization and force-induced biochemical signals Vogel V, Sheetz MP. Nat Rev Mol Cell Biol 2006;7(4):265-275. October 09, 2024 Katharina Maniura 36 How do cells “feel” stiffness? Chaudhuri O, Gu L, …, Mooney DJ. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat Mater 2015;15(3):326-334. October 09, 2024 Katharina Maniura 37 Stiffness of biologically relevant substrates is scale-dependent ▪ the stiffness of biologically relevant substrates is only defined on a certain scale (hierarchy, anisotropy, texture) ▪ often, only bulk stiffness is reported, and linear elasticity assumed ▪ the stiffness seen by a cell can be very different Example: cells pulling on fibers in 3D matrix experience higher stiffness than measured e.g. by a bulk rheometer W. Matthew Petroll, UT Southwestern October 09, 2024 Katharina Maniura 38 How can we control material/substrate stiffness? ▪ many bulk materials (metal, ceramics) have a defined stiffness that can only be varied within a very limited range ▪ the same material can have different elasticity depending on the internal structure (e.g. porosity) ▪ polymers or polymer coatings offer more possibilities by tuning monomer length and crosslink density ▪ generally, bulk stiffness of artificial as well as natural polymers / biomaterials (e.g. collagen fibers) is orders of magnitude higher than that of most tissues Q3: How can we make soft biomaterials from very stiff materials? October 09, 2024 Katharina Maniura 39 Crosslinking density of hydrogels controls the substrate stiffness and stem cell fate adipogenic osteogenic Trappmann B, Gautrot JE, Connelly JT, Strange DGT, Li Y, Oyen ML, Cohen Stuart MA, Boehm H, Li B, Vogel V, Spatz JP, Watt FM, Huck WTS. Extracellular-matrix tethering regulates stem-cell fate. Nat Mater. 2012;11(7):642–9. October 09, 2024 Katharina Maniura 40 Summary mechanical properties of biomaterials Paluch EK, Nelson CM, Biais N, Fabry B, Moeller J, Pruitt BL, Wollnik C, Kudryasheva G, Rehfeldt F, Federle W. BMC Biol 2015;13(1):47. October 09, 2024 Katharina Maniura 41 1. Roughness, Patterning, Anisotropy ▪ Roughness, protein adsorption and adhesion ▪ Cell alignment and contact guidance 2. Mechanical properties ▪ Cell response to different stiffness ▪ How to tune and measure stiffness ▪ Other mechanical properties 3. Geometry, Porosity, Curvature ▪ The role of porosity for biocompatibility ▪ How to control porosity and curvature ▪ Substrate geometry and tissue patterning October 09, 2024 Katharina Maniura 42 Natural and artificial macroporosity Coralline Human cancellous CVP-deposited hydroxyapatite bone tantalum on carbon skeleton Macroscopic porosity determines: ▪ mechanical properties and fracture resistance ▪ diffusion of nutrients and growth factors ▪ cell migration into the material October 09, 2024 Katharina Maniura 43 Fabrication of porous materials ▪ Most materials can be made porous, for example ceramics, metals, as well as natural and synthetic polymers ▪ Fabrication methods include ▪ Plasma spraying ▪ Sintering ▪ Combustion synthesis ▪ Vapor deposition ▪ Freeze drying ▪ Gas foaming ▪ Electrospinning ▪ Control of crosslinking October 09, 2024 Katharina Maniura 44 Defined pore size: inverse colloid crystal (ICC) In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry PS beads Silica gel BM stroma cells Comparative analysis for CD34+ HSC proliferation in ICC and TCPS plates 110 µm PS beads Cells isolated from BM – bone marrow; 15-25 µm interconnected pores after annealing CB – cord blood or PB – peripheral blood Nichols JE, Cortiella J, Lee J, Niles JA, Cuddihy M, Wang S, et al. In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry. Biomaterials. 2009;30(6):1071 –9. October 09, 2024 Katharina Maniura 45 Macroscopic geometry in 2D: adhesive islands ▪ Emergent patterns of proliferation and differentiation on islands of adhesive protein due to distribution of mechanical stress Epithelial cells Proliferation FEM simulations of principal mechanical stress Red = Adipogenic; Blue = Osteogenic Nelson CM, Jean RP, Tan JL, Liu WF, Sniadecki NJ, Spector AA, Ruiz SA, Chen CS. Emergence of Patterned Stem Cell Chen CS. Emergent patterns of growth controlled by multicellular Differentiation Within Multicellular Structures. Stem Cells. John form and mechanics. PNAS. 2005;102(33):11594–9. Wiley & Sons, Ltd; 2008;26(11):2921–7. October 09, 2024 Katharina Maniura 46 3D shape and epithelial branching ▪ 3D geometry determines where tissue branches form ▪ Physical properties of ECM co-regulate morphogenesis Frequency map of tubule formation: Confocal image of tubules stained for actin (green) and nuclei (blue) before and after induction of branching. Induction via addition of EFG (epithelial growth factor) Branching position is determined by tubule geometry and is consistent with the concentration profile of secreted diffusible inhibitor(s). Frequency maps 24 hours after induction of branching for (A) curved tubules, (B) bifurcated tubules, and immunofluorescence Nelson CM, Vanduijn MM, Inman JL, Fletcher DA, Bissell MJ. Tissue staining of actin (red) and nuclei (green) of (C) fractal trees. geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science. 2006;314(5797):298–300. October 09, 2024 Katharina Maniura 47 Summary: physical properties on different scales by John W.C. Dunlop Nanometer Micrometer Millimeter Protein adsorption Cell adhesion Cell and ECM orientation Receptor clustering Cell shape and differentiation Tissue patterning Roughness Topology Anisotropy Stiffness Porosity Curvature October 09, 2024 Katharina Maniura 48 Mechanobiology – exciting field of research DOI: 10.1007/s00018-018-2830-z October 09, 2024 Katharina Maniura 49  Questions? October 09, 2024 Katharina Maniura