Biocompatible Materials: Cells Interacting with Materials 02_cells and materials_rottmar_2024 PDF

376-1714-00L Biocompatible Materials Cells interacting with and remodeling their surrounding 02.10.2024 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 The human body –...

376-1714-00L Biocompatible Materials Cells interacting with and remodeling their surrounding 02.10.2024 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 The human body – a functional piece of art “we live“ heart failure bone fractures atherosclerosis cartilage degeneration 2 Biomedical implants – interactions across scales Implants have become a standard approach in medicine dental implants to restore the biological function of diseased tissue. stents Blood contacting devices: Implants: heart valves high thrombogenic potential à require systemic anti-coagulation risk of risk of (Apostu et al,. 2018) (Thompson et al., 2021) hip replacements bleeding thrombosis Aseptic bone loss due to loosening bacterial infection (mismatch) à requires revision surgery è success depends on how cells & tissues interact with materials 3 Teaching goals You understand and can describe: - interaction of tissues/ cells with biomaterials - cell functions regulated by cell architecture - how cells are embedded in a tissue - the role of ECM - interaction point between cells and their surrounding - integrins: structure and function - examples for engineered substrates which challenge biology 4 Cells and tissues – the building blocks of the human body Cell: base unit of tissues Tissues: functional aggregates of cells (and matrix) Main tissue types: 1. nervous tissue 2. epithelial tissue 3. muscle tissue 4. connective tissue (bone, cartilage, tendon...) - blood and lymph TEM of a lymphoycte 5 Different tissues are made up of highly specialized cell types Much like society 6 Example: connective tissue Loose Types: - loose (e.g. adipose) - dense (e.g. tendon, regular; dermis, irregular) - special (e.g. blood, only plasma and cells) Cells: - fibroblasts, which synthesize and secrete Dense the extracellular matrix (ECM) - osteocytes, chondrocytes, tenocytes... ECM: - glycosaminoglycans, - proteoglycans, (fibrous) - proteins (e.g. collagen) most connective tissues contain fat cells (adipocytes) and are well supplied with blood vessels (exception: e.g. cartilage) 7 The optimal materials surface Material Dental implant Pacemaker mimic the natural ECM ! Hip implant etc. 8 Mimicking the natural ECM native ECM material in contact with cells/tissue - adequate mechanical properties - biodegradable/bioresorbable - enzymatic remodelling - suitable mechanical properties - molecular interactions with cells - adequate bio-/ physico-/ chemical properties - deposition and release of growth factors to direct cell-material interaction - non-immunogenic - shape and structure of the tissue to be replaced - ease in production - biocompatibility? material characteristics and biological stimuli provide 2D/3D-guidance for cells resemble natural cell/ tissue niche provide long-term functionality 9 9 Cell behavior is influenced by a broad range of external cues Biomaterials present a combination of cues to cells/tissues à Synergistic effects can lead to drastically different outcomes Gaharwar et al. Nat Rev Mater, 2020, 5, 686–705 Zhang et al. Adv. Sci. 2021, 2100446 10 Example: surface topography, cell shape & differentiation § Most tissues show high degree of anisotropy § Cell shape influences lineage commitment (cells align with the direction of matrix fibers of MSCs in tissues) § On flat surfaces, cells are randomly oriented à Cell shape and alignment can be induced by anisotropic biomaterial surfaces Teixeira, A.I. J Cell Sci 2003 116: 1881-1892 McBeath, R. Dev Cell. (2004) 6(4):483-95 Kilian, K.A. Proc Natl Acad Sci U S A. (2010) 107(11):4872-7 11 Scale of cell-material and tissue-material interactions Material Protein Cell Tissue nm µm mm - cm Events at the interface Tissue Remodeling Tissue Formation Cell-Cell Interactions Cell Response Cell Adhesion Protein Adsorption seconds to minutes days to weeks months to years 12 Cell-material and material-tissue interaction Proteins and cells at surfaces.….of implants/ medical devices Tagaya, Polymer Journal 47, 599-608 (2015) 13 13 Plasma proteins – the major source for surface adsorbed proteins > 2000 soluble serum proteins à all compete for the surface! Plasma Molecular Protein type concentration weight cells never “see” bare surface (mg/ml) (daltons) always encounter adsorbed proteins Prealbumin 10 - 40 54,900 low M.W. Albumin 35 - 45 highest 66,500 abundance IgG 6 - 17 155,000 Fibrinogen* 2.0 - 4.0 relevant 340,000 for cells For attachment-dependent cells, Fibronectin* 0.26-0.38 250,000 adhesion is essential for: mass transfer flux favors high albumin loading on cell migration cell survival all surfaces cell shape cell matrix assembly adhesion proteins have integrin binding sites; cell differentiation gene expression albumin non-adhesive cell proliferation mechanosensing 14 Proteins at surfaces…of implants/ medical devices desorption adsorption denaturation positive charge negative charge other residues hydrophobic / hydrophilic At low surface energy (hydrophobic) surfaces, proteins adsorb strongly and irreversibly. Protein becomes deformed = denatured Q1 – what happens to proteins on hydrophilic/hydrophobic surfaces? At high surface energy (hydrophilic) surfaces, proteins adsorb weakly and reversibly. Protein remains in its proper 3D-fold = functional active 15 Interaction of surfaces with blood (-proteins) Protein adsorption and blood coagulation is affected by surface properties Ti SLA-Hphob Ti SLActive-HphilNS Ti SLA-HphobNS Ti SLA-Hphil TiZr SLA-Hphob TiZr SLActive-HphilNS Surface nanostructures enhance protein adsorption Hydrophilicity and surface nanostructures synergistically promote protein adsorption and blood coagulation Kopf B. et al. J Biomed Mater Res A 2015 16 Protein adsorption regulates blood-material interaction Protein conformation is surface dependent! (Example: Pd-metallic glass vs crystalline Ti64) Accessibility of fibrinogen γ-chain is higher on Pd compared to Ti64. Platelet adhesion, but not their activation is promoted on Pd surfaces! Cihova M. et al. Adv. Funct. Mater 2022 17 Protein adsorption regulates blood-material interaction Protein conformation is surface dependent! (Example: Pd-metallic glass vs crystalline Ti64) Bone progenitor cells on Pd Pd77.5 Si16.5 Cu6 Pd43 Cu27 Ni10 P20 Promising for blood- 24min contacting Promising devices for implants Cihova M. et al. Adv. Funct. Mater 2022 Lackington W. et al. Adv. Funct. Mater 2023 18 Blood-material interaction differently affects cell types Blood incubation differently influences cell spreading, Lackington W. et al. Mater Today Bio 2022 especially negatively for HGKs on rough surfaces 19 Attachment-dependent cells are adhering to surfaces under tension How is this tension produced?? 20 Tensegrity concept for cell cytoskeleton D. Ingber (Harvard) theory (controversial) Tensegrity (Buckminster Fuller): a system stabilizes itself mechanically by balancing local compression with continuous tension D. Ingber suggested that the cytoskeleton is made up this way – allowing it to rapidly change shape, but requiring cell-surface tension to create tension and compression ** cell attachment and spreading as a necessary cell behavior for functionà tension Ingber, J Bodyw Mov Ther. 2008, 12(3): 198–200. 21 Tensegrity in cells? The smallest collagen fibers in the ECM attach to proteoglycans and to fibronectin. Proteoglycans (long sugar molecules combined with long protein molecules and many sulfated sugars as side chains). à rigid and resist compression and the sulfated sugars allow the ECM to change the turgor. Integrin molecules (transmembrane proteins) connect the ECM with the cytoplasm of the cell. Focal adhesion complex, linked to actin filaments via α-actin, transmits forces throughout the cell to the nucleus and to other signaling organelles. è biotensegrity of whole body to almost instantly transmit information from the outside world to the inside of a cell and visa versa. Ravin T. AAMM. 2011 22 Tensegrity in cells? Boghady et al. APL Bioeng. 5, 041501 (2021) 23 Tensegrity in cells? Cell migration Cell division Fig. 16-21 Mol Biol of the Cell, 6th Edition Fig. 16-2 Mol Biol of the Cell, 6th Edition Fig. 17-49 Mol Biol of the Cell, 4th Edition § tension is mediated through integrins (transmembrane adhesion receptors) that link the ECM on the outside of the cell with the cell's actin cytoskeleton § cell tension causes cells to round up when integrin-directed adhesions are perturbed (as with trypsin enzyme) 24 How to measure cellular tensional forces? Molecular scale Cellular scale Tissue scale è Tension = signal è Tension = (red) signal Boghady et al. APL Bioeng. 5, 041501 (2021) 25 Interactions of cells with ECM Integrins cell membrane Integrin activation (example: platelets) Fig. 19-58 Mol Biol of the Cell, 6th Ed 26 26 Integrins 18 alpha subunits 8 beta subunits à integrins: 24 cell membrane Morphictx.com Tab. 19-3 Mol Biol of the Cell, 6th Ed 27 Cell-matrix interactions Interactions between a cell and its environment can result in altered cell behavior (e.g. spreading, proliferation) = “outside-in” signaling Conversely, a cell may secrete molecules or rearrange contacts to alter the ECM = “inside-out” Shattil et al. Nat Rev Mol Cell Biol 2010 28 Cell-adhesive matrix proteins secreted bound to surfaces soluble adsorbed/processed recognized by cells collagen structural proteoglycans laminin fibronectin vitronectin osteopontin tenascin matricellular thrombospondin = proteins are rapidly turned over and have osteonectin regulatory roles 29 Structural environment of cells: the extracellular matrix (ECM) § ECM is composed of water, proteins and polysaccharides à each tissue has a highly heterogenous ECM with unique composition and topology Krantz et al (2010) J Cell Sci 123, 4195-4200 Proteoglycans Fibrous proteins & glycoproteins aggrecan aggrecan aggregate collagen fibronectin 30 30 30 Structural environment of cells: the ECM is highly dynamic Krantz et al (2010) J Cell Sci 123, 4195-4200 31 The ECM is very complex - What to mimic? Fig. 19-53 Mol Biol of the Cell, 6th Ed 32 Known cell adhesion motifs on ECM molecules glycoprotein binding sites heparin-binding sites on ECM And combinations: heparin binds fibronectin binds collagen binds heparin… Masters et al. 2004, Adv Chem Eng, 29:7-46 33 Integrins partner with growth factor receptors and other membrane proteins Receptors for insulin, platelet- derived growth factor (PDGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) are optimally activated by their ligands only under cell attachment conditions 34 Cells interacting with ECM Focal adhesions FA Osteoblasts attaching and spreading on PMMA (stained for vinculin) Focal complexes Focal adhesions (super)mature adhesions 35 Biggs et al., 2009 Biomaterials 30(28), 5094–5103 Cells interacting with ECM – focal adhesions Stress fibers terminating in a focal contact (arrow) - TEM image Actin + Vinculin Integrin clustering induced by binding to ECM leads to formation of stress fibers 36 Cells interacting with ECM – focal adhesions ECM trans-membrane integrins focal adhesion complex 37 Cells interacting with ECM – the «adhesome» Zaidel-Bar et al. (2007) Nat Cell Biol 9, 858–867 38 38  Focal Adhesions essential for numerous cell functions THP-1 cells cells in suspension: integrins uniformly distributed in membrane no focal adhesions adherent cells: focal contacts with integrin clustering à formation of stress fibers à Cell proliferation Cell motility Cell survival Differentiation 39 Wehrle-Haller (2012) Curr Opin Cell Biol 24,1:116-124 Cell spreading and cell survival (I) Effect of cell spreading on apoptosis. (A) Phase contrast-fluorescence micrographs of capillary endothelial cells cultured in suspension in the absence or presence of different-sized microbeads or attached to a planar culture dish coated with FN for 24 hours. (B) Apoptosis in cells attached to different-sized beads, in suspension, or attached to a dish. Chen et al. (1997) Science 276 40 Cell spreading and cell survival (II) (A) Differential interference-contrast micrographs of cells plated on substrates micropatterned with differently sized islands coated with FN on an otherwise non-fouling background. (B) Apoptotic index and DNA synthesis of cells attached to different-sized adhesive islands coated with a constant density of FN for 24 hours. Chen et al. (1997) Science 276 41 Quiz - Cell spreading and proliferation on Fn micropatterns 30 microns 5 microns 3 microns The same amount of Fn in each pattern. Q2 - What happens to cell spreading and proliferation? Chen et al. (1997) Science 276 42 Influence of inter-ligand spacing on cell adhesion and spreading biofunctionalization of Au particles with c(-RGDfK-)-thiol ligands M-PEG-Si(OMet)3 = self-assembled monolayer to prevent cell adhesion Huang et al., (2009) Nano Lett. 9(3): 1111–1116 43 Influence of inter-ligand spacing on cell adhesion and spreading PS homopolymers = AFM images of corresponding ordered (a,b,c,d) and disordered (e,f,g,h) ordering-interference reagent gold nanopatterns at different interparticle-distances Huang et al., (2009) Nano Lett. 9(3): 1111–1116 44 Influence of inter-ligand spacing on cell adhesion and spreading Osteoblast adhesion and spreading is reduced with increasing ligand spacing ordered Cell adhesion and spreading is better supported on disordered than on ordered nanopatterns disordered Huang et al., (2009) Nano Lett. 9(3): 1111–1116 45 Influence of inter-ligand spacing on cell adhesion and spreading Actin (cytoskeleton) Vinculin (focal adhesions) Formation of actin (stress) fibers is reduced with increasing ligand spacing Formation of focal adhesions and actin (stress) fibers is better supported on disordered than on ordered nanopatterns Huang et al., (2009) Nano Lett. 9(3): 1111–1116 46 Influence of inter-ligand spacing on cell adhesion and spreading Q3 – Why is cell spreading and proliferation enhanced on disordered patterns?? Presumption: integrins that potentially bind RGD-ligands over the nanopattern can be classified as clustering integrins; non-clustering integrins result from interdistances above a critical value. Even at a global average inter-ligand spacing of >70 nm, a disordered nanopattern still displayed some clustering integrins, which was not the case for ordered patterns with inter- ligand spacings of >70 nm. Huang et al., (2009) Nano Lett. 9(3): 1111–1116 47 Influence of inter-ligand spacing & substrate stiffness Due to actomyosin-based mechanotransduction and YAP/TAZ activation On soft substrates: altered loading of individual integrin-ligand bonds to activate focal ahesion formation Zhang et al., (2021) Biomater. 268:120543 48 Cells adapt their structure to suit the substrate they are on § cells adhering to firm, highly adhesive substrata, generate considerable tension and exhibit highly organized actin bundles and adhesion complexes. § on more soft, pliable substrates (e.g. hydrogels), cells exhibit less organized actin and smaller, weaker focal adhesions. …behaving as a fluid on soft substrates and as an elastic solid on stiff substrates through a large scale remodelling of the cytoskeleton. Gupta et al., 2015 Nat Comm, doi:10.1038/ncomms8525 49 Cells adapt their structure to suit the substrate they are on Lo, C.M. Biophys J. (2000) 79(1): 144–152. § Cells migrate towards higher stiffness (“durotaxis”) § Cell migration is slow on very soft and very hard substrates § Cell migration is fastest on intermediate stiffness 50 Role of substrate stiffness and ECM component § Substrate stiffness (y axis) and adhesive ligand type (x axis) combine to regulate MSC morphology. § Human MSCs spread more with increasing stiffness, but cells on laminin- coated hydrogels are smaller than those on other ECM protein coatings. Scale bar: 50µm MSC = mesenchymal stromal cell Caliari and Burdick (2016) Nat. Met. 51 Cells adapt their phenotype according to their substrate Engler, A.J. J Cell Biol. (2004) 166(6):877-87 Engler, A.J. Cell (2006) 126(4):677-89 è cell differentiation can be influenced by tuning the substrate stiffness 52 Macroscopic structure influences cell/tissue responses Macroscopic porosity determines § Mechanical properties and fracture resistance § Diffusion of nutrients and growth factors § Q4 – what role plays Cell migration into the material macroporosity for a biomaterial? 53 Going from “repair” to “regeneration” Today (repair) Tomorrow (regeneration) § dental implants § coronary bypass that heals itself (ceramic, metals..) § cartilage in joints that does not § hip implants disappear (TiO2, metal alloys) § archilles tendon that reconnects § arteries/ veins in lower extremities (PET, ePTFE, Dacron..) Tissue engineering 54 Designer matrices à cell-instructive (soft) environments/materials Controlling cell behavior: polymer density/ stiffness, peptide, degradability etc Example: Influence of matrix stiffness on intestinal stem cells (ISC) organoid differentiation and morphogenesis Gjorevski at al., (2016) Nature 55 Designer matrices à cell-instructive (soft) environments/materials ISC expansion requires higher matrix stiffness, differentiation and morphogenesis a softer matrix & laminin-based adhesion Gjorevski at al., (2016) Nature 56 Designer matrices à cell-instructive (soft) environments/materials § Controllable mechanical properties § PVA-gels support (slow) cell spreading & proliferation § MMP-dependent § Light-guided 3D cell invasion degradation 57 Qin et al, Adv Mater, 2018, 1705564 Designer matrices à cell-instructive (soft) environments/materials 58 Qazi et al, Biomater Biosys, 2021 Puiggali-Jou et al, Acta Biomater, 2023  Summary: cell-material biointerface - molecular surface recognition § cells never see a bare materials surface, but a layer of adsorbed proteins à crucial for subsequent events (healing, material integration/non-integration, biocompatibility…) 59 Questions? 60