Bioinks and Crosslinking: Fundamentals and Applications PDF

Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Bioinks and crosslinking Bioprinting: fundamentals and applications Prof. Dr. Petra Mela Garching, 16.05.2024...

Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Bioinks and crosslinking Bioprinting: fundamentals and applications Prof. Dr. Petra Mela Garching, 16.05.2024 1 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Recap. last week: extrusion-based bioprinting Deposition Vat polymerization (VP) Specialized modalities Magnetic bioprinting Microfluidic bioprinting Acoustic bioprinting Aspiration-assisted bioprinting Organoid bioprinting Laser-induced forward transfer … 2 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Recap. last week: extrusion-based bioprinting mechanical extrusion Malda et al (2013). Advanced materials, 25(36), 5011-5028. Schwab et al (2020). Chemical Reviews, 120(19), 11028-11055. 3  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Bioinks Bioinks can, but do not have to, contain biomolecules such as growth factors, DNA, miRNA, cytokines, exosomes Bedell et al. Chemical Reviews (2020). 120, 19, 10744–10792 4 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material-based bioinks Thiele et al. 2014 These are mainly hydrogel precursors and hydrogels (gel-phase). Hydrogel: Water-swollen (insoluble) polymer network Viscoelastic behavior High water content, may contain more than 90% of water Of natural or synthetic origin Needed: cytocompatible and biodegradable → cell proliferation and migration, remodelling Hydrogel+cells True 3D (ideal) environment for cell → cell instructive environment Suitable for suspension of cells and biologically active materials Mechanically weak Native tissue (cells+extracellular matrix+extracellular fluid) 5 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties for bioprinting Cell compatibility (cell adhesion, stiffness) Biological tissue mimicry Porosity: diffusion properties for nutrient supply Viscosity (e.g. for uniform cell encapsulation and printability) Gelation kinetics (structural fidelty) Printability 6  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – cell adhesion Adhesion of mesenchymal stem cells (MSCs) on ink surface after 3 days in culture. The ink consisted of GG-MA with 0%, 25% and 30% (w/w) of Hap particles. Evalualtion via phalloidin- rhodamine/Hoechst staining. Scale bar is 500 μm for the low magnification and 50 μm for the high magnification images. GG-MA: gellan gum methacrylate Hoechst: stains DNA Phalloidin-rhodamine staining: stains actin filaments Müller et al. Materials Science and Engineering: C 108 (2020): 110510. Chimene et al. Annals of biomedical engineering 44.6 (2016): 2090-2102. 7  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – stiffness Dense polymer network limits cell migration, growth, and differentiation. Cells thrive best in soft hydrogels. cell polymer network Main contributing factors ▪ molecular weight of polymer ▪ polymer concentration ▪ crosslinking density Malda et al. Advanced materials 25.36 (2013): 5011-5028. Schwab et al. Chemical Reviews 120.19 (2020): 11028-11055. Chimene et al. Annals of biomedical engineering 44.6 (2016): 2090-2102. 8  Interpretation Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Live/Dead Staining (A): High proportion of green-stained cells would indicate good viability of hBMSCs in the BldECM, while red-stained cells indicate cell death. Material properties – stiffness F-actin Staining (B): Well-organized actin filaments suggest healthy cytoskeletal development and cell spreading, important for tissue formation. Proliferation Assays (C): Higher proliferation rates (indicated by CCK-8 and DNA tests) in the BldECM group compared to collagen would suggest that BldECM provides a more conducive environment for hBMSCs growth and activity. human bone In vitro biofunctionality assessment of the marrow stem hBMSCs-laden BldECM bioink. (A) Live/deadcells blood-deriv staining of hBMSCs in the BldECM for 10 days ed extracellula (scale bar = 200 μm); live cells (green), dead cells r matrix (red). (B) F-actin staining of the BldECM constructs on day 10 (scale bar = 100 μm); DAPI (blue, cell nuclei), F-actin (green, actin filaments). (C) Comparison of cell proliferation in collagen and BldECM groups for 10 days: (i) CCK-8 assay test and (ii) DNA quantification test (* p < 0.05 and ** p < 0.01). Chae et al. (2022): Micromachines (Basel). 2022 Feb; 13(2): 277. DOI:10.3390/mi13020277 9  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – stiffness / biological tissue mimicry Butcher et al. (2009): Nature Reviews Cancer, 9, pages 108–122. DOI:10.1038/nrc2544 10  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – biological tissue mimicry Biological Tissue Mimicry: The ability of the bioink and printed constructs to replicate the physical, mechanical, and biochemical properties of natural Example: stress relaxation tissues. Stress Relaxation Occurrence: Immediately after printing, the scaffold may initially exhibit high stress to maintain its shape due to the viscoelastic properties of alginate. Over time, however, the stress in the printed structure gradually decreases as the alginate chains reorganize and adjust to the applied strain. Importance: Understanding stress relaxation is crucial for several reasons in bioprinting: Structural Integrity: It ensures that the printed structure retains its shape and mechanical stability after printing, which is essential for supporting cell attachment, proliferation, and differentiation. Cell Viability: Excessive stress or deformation can adversely affect cell viability and function within the printed construct. Stress relaxation allows the scaffold to settle into a stable state, reducing mechanical strain on encapsulated cells. Long-term Functionality: By managing stress relaxation, bioprinted tissues can better mimic the mechanical properties of native tissues, promoting their integration and functionality post-implantation or in vitro culture. Chaudhuri et al. Nature 584.7822 (2020): 535-546. Chimene et al. Annals of biomedical engineering 44.6 (2016): 2090-2102. 11 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering when dealing with hydrogel-based bioinks, the "fabrication window" refers to a critical range of parameters within which the bioink can be successfully printed into a desired structure with Caveat: fabrication window! optimal fidelity and mechanical integrity. 12  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – porosity (diffusion) Duffision properties for nutrient supply O2 O2 O2 O2 well O2 O2 O2 Oxygen is required for medium nutrients, cellular respiration O2 biomaterial CO2, waste cell products 13  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties – viscosity Confocal images of 3T3 cells encapsulated in various bioinks for 1 h. For uniform cell encapsulation cell sedimentation bioink #2 bioink bioink #1 PEGDA: Poly(ethylene glycol) diacrylate XG: Xanthan gum Alg: Alginate GelMa: Gelatin methacryloyl RAPID: Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink Dubbin et al. Biofabrication 9.4 (2017): 044102. P: Peptide Chimene et al. Annals of biomedical engineering 44.6 (2016): 2090-2102. 14  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties– gelation kinetics For structural fidelty structural fidelity „bad“ „good“ Dong et al. RSC advances 9.31 (2019): 17737-17744. Chimene et al. Annals of biomedical engineering 44.6 (2016): 2090-2102. 15  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material properties– printability Depends on bioprinting modality Adapted from: Schwab et al. Chemical Reviews 12 (2020): 11028-11055 16 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Printability Several methodologies, both quantitative and qualitative, have been proposed to assess printability. Common metrics such as print resolution and shape fidelity are highly dependent not only on the (bio)ink but also on printing process conditions such as flow rate, pressure, nozzle size, path design, temperature, etc Rheological properties are quantitative metrics for evaluating (bio)ink independently from the process Generally, for extrusion-based bioprinting, printability refers to the ability of the material: − to be extruded through a syringe − to form consistent filaments during deposition − to maintain shape following printing Exercise on printability assessment (Kilian Mueller) 17 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Hydrogel: material classes Classification by origin: Natural hydrogels Synthetic hydrogels Semi-synthetic hydrogels Lim et al (2018). In Functional hydrogels as biomaterials (pp. 1-29). Springer, Berlin, Heidelberg. 18  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Natural hydrogels Materials composed of naturally derived biopolymers: ▪ Protein-based (e.g., collagen, fibrin, gelatin, silk) ▪ Polysaccharide-based (e.g., alginate, agarose, chitosan, hyaluronic acid, cellulose) ▪ Decellularized tissue-based (extracellular matrix, e.g. Matrigel) Due to natural origin, they provide important biological molecules (e.g., cell adhesive proteins, growth factors) Cells benefit from the abundance of chemical signals present in these hydrogels, resulting in high viability and proliferation rates Provide basic control of material properties (e.g., mechanical stiffness) through chemical and physical modifications Natural biopolymer materials may suffer from batch-to-batch variability, resulting in reproducibility issue during experimentation: ▪ Lack consistent properties ▪ Contain impurities Lim et al (2018). In Functional hydrogels as biomaterials (pp. 1-29). Springer, Berlin, Heidelberg. 19 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Recap: natural polymers Polymers found in nature in animals and plants: Proteins (built from aminoacids) e.g. collagen, elastin, albumin, silk, gelatin Nucleic acids (built from nucleotides) e.g. DNA, RNA Polysaccharides (built from sugars) e.g. starch, cellulose, chitosan Natural rubber (built from isoprene monomers) 20 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Decellularized hydrogels Decellularization aims to remove cells from tissue while protecting the composition and integrity of the native tissue properties. The elimination of cells removes potential antigens which can induce an inflammatory and immune response. Decellularized materials provide a rich reservoir of https://www.youtube.com/watch?v=kFfIlCkrQGg&t=3s various molecules, such as cell adhesive proteins and growth factors found in the native tissue. Methods such as chemical, enzymatic, and physical approaches and the combination of them have been investigated. Decellularization is a critical process in tissue engineering aimed at creating scaffolds that are both biocompatible and functional. By removing cellular elements and retaining the native ECM's structural and biochemical properties, decellularized tissues provide an ideal environment for cell infiltration and tissue regeneration. Various methods, often used in combination, ensure effective decellularization while maintaining the integrity of Macroscopic observation of heart decellularization. the ECM. Kabirian et Mozafari (2020). Methods, 171, 108-118. More information: https://www.jove.com/de/v/50825/nonhuman-primate-lung-decellularization-recellularization-using 21  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Decellularized hydrogels Example Decellularized tendon Processing native tendon tissue to a bioink. Native fresh tissues A) were snap-frozen and minced into thin slices after removing the synovial sheath, and B) were decellularized by optimized combination of physical, enzymatic, and chemical methods. Decellularized tissue samples C) were thoroughly phosphate-buffer washed in PBS supplemented with antibiotic, and ed saline D) freeze-dried and ground to form uniform fine powders. E) dtECM powders were partially digested by pepsin to obtain the pre-gel solutions. F) Stable hydrogels were formed upon neutralization of dtECM decellularized tissue ECM solution and brief incubation at 37 °C. G) Cell-laden dtECM bioinks were bioprinted by SU3D bioprinter through implementation of the aspiration–extrusion bioprinting Toprakhisar et al (2018). Macromolecular bioscience, 18(10), 1800024. 22  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Bladder model from decellularized bladder bioink In vitro model of the urinary bladder that can simulate 3D biomimetic tissue structures and dynamic microenvironments to replicate the smooth muscle functions of an actual human urinary bladder medium. 3D bioprinted constructs were dynamically conditioned 10 days after printing. Chae et al. (2022): Micromachines (Basel). 2022 Feb; 13(2): 277. DOI:10.3390/mi13020277 23  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Synthetic hydrogels Materials based entirely on synthetic polymers. They are: ▪ Reproducible ▪ Well-defined ▪ Tunable Synthetic materials permit almost limitless possibilities to modify material properties, allowing for the precise tuning of physical and biochemical properties e.g. to mimic native tissue However they lack biological recognition sites for cell attachment, migration and proliferation Examples: ▪ Poly(ethylene glycol) (PEG) (up to 900 kPa) ▪ Pluronics ▪ Poly(vinyl alcohol) (PVA) hydrogels (up to 20 MPa) ▪ Poly(N-isopropyl acrylamide) (PNIPAAm) Lim et al (2018). In Functional hydrogels as biomaterials (pp. 1-29). Springer, Berlin, Heidelberg. 24 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Semi-synthetic or biosynthetic hydrogels Combination of natural and synthetic materials Combination of both advantages ▪ Tunability of physical and biochemical characteristics ▪ Biological recognition sites to promote cell attachment, migration and proliferation Mostly applied approaches: ▪ Mix natural with synthetic materials (blend) ▪ Functionalize natural materials to add chemical reaction groups (e.g., methacryloyl groups in GelMA) ▪ Incorporate biomolecules (e.g., polysaccharides, proteins, growth factors, etc) into synthetic hydrogel networks to add bifunctionality. Example: four-arm poly(ethylene glycol) PEG-peptide conjugates (equipped with cell-adhesive peptide and MMP cleavable sites) + RGD (peptide) → DOI: 10.1016/j.biomaterials.2010.02.044 25  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Semi-synthetic or biosynthetic hydrogels Synthetic Biological Incorporation mechanism Reference PEG RGDS peptide Thiol end group of RGDS was photopolymerized with acrylate groups of PEG Salinas et al (2007). through thiol-ene rection Tissue Eng 13(5):1025–1034 PEG Fibrinogen Fibrinogen was conjugated to acrylated PEG then Dikovsky et al photopolymerised into hydrogels (2006). Biomaterials 27(8):1496–1506 PEG Laminin Acrylated PEG was conjugated onto laminin. The PEGylated laminin was then Francisco et al crosslinked with PEG-dithiol through Michael-type addition (2013). Biomaterials 34(30):7381–7388 PEG Collagen type I Collagen was modified with free thiols then reacted with PEG- diacrylate via Singh et al (2013. Michael- type addition Biomaterials 34(37):9331–9340 PVA Heparin Methacrylated heparin was copolymerised with methacrylated PVA Young et al (2013). Biomicrofluidics 7(4):044109 Lim et al (2018). In Functional hydrogels as biomaterials (pp. 1-29). Springer, Berlin, Heidelberg. 26 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Material examples 27 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Most used hydrogels Combination of the 10 most used hydrogel materials for bioprinting. The materials are shown in the external ring (total of papers and name). The middle ring segments represent one-material hydrogels and hydrogels, marked with (*), that are a combination of this material with non-selected materials (one-material papers mixed with non-selected materials papers). Inner lines represent hydrogels with two of the selected materials, but in some case other non- selected materials can be included (number of papers next to each inner line). In total, 118 publications were screened for this study. Sánchez et al (2020). Frontiers in Bioengineering and Biotechnology, 8. 28 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Natural hydrogels: alginate Naturally occurring anionic polymer typically obtained from brown seaweed High biocompatibility Low toxicity Relatively low cost Good rheological properties: printable Mild gelation by addition of divalent cations such as calcium ions (Ca2+) 29 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Natural hydrogels: gelatin (or gelatine) Denatured collagen High biocompatibility Water solubility Abundance of biological recognition sites Temperature sensitivity Ease of chemical modification 30 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Natural hydrogels: gelatin Low structural fidelity after printing Mostly used in combination with other materials Function: provide biological recognition sites for cell attachment, migration and proliferation 31 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Synthetic hydrogels: poloxamers Excellent printability Poloxamers: block copolymers composed of central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) Trade names: Pluronic, Kolliphor, Lutrol Pluronic example: ▪ Mixing 40 % (w/v) Pluronic F-127 (poloxamer 407) at 4°C DI water for 15 min at 400 rpm ▪ Storage in fridge at approx. 4°C DOI: 10.3390/molecules26123610 32 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Synthetic hydrogels: poloxamers Example: Pluronics F-127 Characteristics: thermoresponsive material ▪ Printable at room temperature ▪ Fluid at approx. 4°C Application as a sacrificial material 34 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Hydrogels Water-swollen polymer network (hydrophilic precursor, insoluble network) The polymer network is held together by crosslinks, i.e. bonds between polymer chains. Crosslinking results in ‘stabilization’ through multiple connections of polymeric chains → network It can be obtained by physical or chemical binding or a combination of the two. The degree of crosslinking affects the physical properties of the hydrogel Crosslinking affects: polymer chains Viscosity Elasticity Solubility ≠ Swelling behaviour Diffusion properties (e.g. pore sizes, water type (primary and secondary bound water, and free water)) Biocompatibility (in the sense of cell-friendliness) bonds/crosslinks Adapted from https://www.lubrizol.com/Coatings/Blog/2019/06/What-is-Crosslinking 35  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking strategies LCST (Lower Critical Solution Temperature) and UCST (Upper Critical Solution Temperature) hydrogels are types of stimuli-responsive hydrogels that change their physical properties in response to temperature changes. 36 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Recap: polymer basics Polymerisation (polymer forming reaction): Small molecules (monomers) are linked together by chemical reaction (polymerisation) to form a long chain (polymer). CH2 CH2 CH2 H2C = CH2 + H2C = CH2 + H2C = CH2 CH2 CH2 CH2 Monomer: one single repeating unit Dimer: two monomers joined by chemical reaction Trimer, tetramer, pentamer, … Oligomer: short chain consisting of “a few” monomers Polymer: long chain (> 20) of monomers Homopolymer: one monomer type, all repeating units are the same Heteropolymer / copolymer: more than one species of monomer Macromolecule: any molecule containing a very large number of atoms: (synthetic) polymer chains, proteins, DNA Macromolecule: A large molecule with a very high molecular weight, which includes synthetic polymer chains, proteins, and DNA. 37 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Representation of a macromolecule Monomer Polymer (C2H4)n or: Ethylene Polyethylene (PE) GRAPHIC REPRESENTATIONS (CHEMICAL FORMULAE) OF MACROMOLECULES (IUPAC Recommendations 1994) https://doi.org/10.1351/pac199466122469 38 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Chain-growth versus step-growth Two main classes of polymerisation reaction mechanisms: Chain-growth polymerization: the polymer chains grow by adding one monomer unit during each reaction step. Step-growth polymerization: monomers react to form first dimers, then longer oligomers and eventually long chain polymers. Chains of any length can react with each other. chain-growth step-growth 39 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Chain-growth polymerisation – radical polymerisation a method widely used for synthesizing polymers H H X* + C=C H H free radical ethylene making them reactive and suitable for polymerization. Unsaturated molecules: double bonds between carbon atoms Reaction sequence: Initiation: radical formation (e.g. UV photoinitiator) Propagation: chain growth by adding one monomer at the time, the radical stays at the growing end of the chain. Radical Scavenging: The active radical can be neutralized by reacting with impurities or other substances (e.g., oxygen or water). Termination reactions: radical-radical combination, radical scavenging (water, impurities). Since a terminated chain cannot grow anymore, not all chains have the same length. No formation of reaction by-products. 40 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Step-growth polymerization Two or more reactive (functional) groups necessary per monomer Homopolymers, with two different functional groups on the monomer Copolymers with different functional groups on each monomer Monomers with more then two functional groups: branching, 3D network (e.g. epoxy resins) Each formed oligomer still has reactive groups at the ends: pairs of reactants of any lengths can combine (A reacts with B) 43 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Step-growth polymerization Two different reaction mechanisms Polyaddition Polycondensation 44 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Step-growth polymerization – polyaddition Two or more reactive groups required (e.g. -N=C=O; -OH; -C=O; -NH2). Each formed oligomer still has reactive groups at the ends: pairs of reactants of any lengths can combine. No formation of reaction by-products Typical polyaddition polymers: polyurethane (PU), epoxy resins, cyanoacrylates, polyoxymethylene (POM) urethane bond The properties of the polyurethane, ranging from soft, flexible to brittle and hard, depend (among others) on the size and properties of the R’ and R” groups in the monomers. Example: 45 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Step-growth polymerization – polycondensation During the polymerisation reaction, small molecules are formed as by-product (water, methanol, …). Two or more reactive groups required (e.g. -OH; -C=O; -COOH, -NH2) Equilibrium reaction: also the reverse reaction is possible (hydrolysis). Each formed oligomer still has reactive groups at the ends: pairs of reactants of any lengths can combine. Typical polycondensates: polyamides (PA, nylon), polyesters (PET), silicones, polypeptides (proteins) Example polyamide: The polymer properties depend (among others) on the R’ and R” groups. For polyamide 66 (nylon): R’ = (CH2)4 R” = (CH2)6 46 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Polycondensation – polyesters An ester is the chemical bond between an organic acid (carboxylic acid -COOH) and an alcohol (-OH). Typical polyesters: polyethylene terephthalate (PET), polylactic acid (PLA), polycarbonate (PC) Polyethylene terephthalate (PET): ester The occurrence of the reverse hydrolysis reaction (degradability) is depending on the properties of the monomers and the environment (e.g. acidity, temperature). 47 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Polycondensation – polypeptides A peptide is a chemical bond between an organic acid (carboxylic acid -COOH) and an amine (-NH2). Amino acids have both a carboxylic acid and an amine group. Proteins are built up from specific sequences of different amino acids and are therefore polypeptides. The reverse reaction, hydrolysis, occurs in the body during digestion of proteins. + 49 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Combining polymer properties Copolymerisation: Reaction between 2 or more monomers, forming chemical bonds within the resulting chain Blends: Mixtures of 2 or more base polymers No formation of chemical bonds Cohesion through physical forces 50 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Copolymerisation Polymerisation of different monomers with each other Statistical (random) copolymerisation Monomer A Alternating copolymerisation Monomer B Block copolymerisation Graft copolymerisation 51 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Covalent bonds Chemical atomic bond between atoms within polymer chains (intramolecular forces) Also called electron pair bonding - Each bond-line consists of two shared electrons Temperature-independent, atoms stay at fixed distance Example: PS Binding energies: 40 – 800 kJ/mol (e.g. C-C: ca. 350 kJ/mol) 52 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Intermolecular forces Physical atomic bonding between chemically unbound macromolecules Binding energies: 2-20 kJ/mol - 10-100x lower than covalent forces - Strength decreases with increasing temperature and distance 53 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Hydrogels Water-swollen polymer network (hydrophilic precursor, insoluble network) The polymer network is held together by crosslinks, i.e. bonds between polymer chains. Crosslinking results in ‘stabilization’ through multiple connections of polymeric chains → network It can be obtained by physical or chemical binding or a combination of the two. The degree of crosslinking affects the physical properties of the hydrogel Crosslinking affects: polymer chains Viscosity Elasticity Solubility ≠ Swelling behaviour Diffusion properties (e.g. pore sizes, water type (primary and secondary bound water, and free water)) Biocompatibility (in the sense of cell-friendliness) bonds/crosslinks Adapted from https://www.lubrizol.com/Coatings/Blog/2019/06/What-is-Crosslinking 54 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking Before printing: freely moving polymer chains → printable but will flow on the print bed Crosslinking is restricting the ability of movement of individual polymer chains → network formation After printing: crosslinked polymer chains → maintain shape Constructs are stable in watery environment (cell medium) Ribeiro et al (2017). Biofabrication, 10(1), 014102. 55 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking Chem. Soc. Rev. 2019, https://doi.org/10.1039/C7CS00718C 56 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking General approach crosslinking step crosslinking step Bottom-up crosslinking approach with layer-by-layer deposition of a material-based bioink to produce a cylindrical construct, including gelation between deposition of single layers Schwab et al (2020). Chemical Reviews, 120(19), 11028-11055. 57 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking – gelation Gelation results from crosslinking of the hydrogel precursors - It describes the sol-gel transition from a fluid to a viscoelastic solid. - The critical point where gel first appears is called the “gel point”. - In rheology this corresponds to the state where G’=G’’. storage modulus (G') equals the loss modulus (G''). gelation Redaelli et al (2017). In Bioresorbable polymers for biomedical applications (pp. 205-228). Woodhead Publishing. 58 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking Physical crosslinking consists of weak intermolecular interactions, such as: Ionic (electrostatic interaction): involves ions with opposite electric charges Hydrogen bond (electrostatic interaction): involves a hydrogen-containing group and a group that has an atom with a lone pair of electrons such as nitrogen (N), sulfur (S), fluorine (F), chlorine (Cl) and oxygen (O) Van der Waals forces Polymer chain entanglement: polymer chains ‘crossing’ each other and ‘hooking’ each other Host-guest interaction: involves two chemical entities (e.g. ligand and receptor) that can form stable complexes through unique structural relationships and non-covalent binding. Also referred to as molecular recognition, this type of interaction is widely found in biorecognition processes, such as enzyme–inhibitor and antigen–antibody interactions. DOI: 10.1002/adhm.202203148 59 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking Physical crosslinking is a method of creating hydrogels where polymer chains are held together by non-covalent interactions such as hydrogen bonds, ionic interactions, hydrophobic interactions, and crystallization. Strategies include: Crystallization: the polymer chains are pushed together by ice crystals in freezing and thawing cycles, forming crystalline Involves freezing and thawing cycles that lead to the formation of ice crystals. As the ice forms, it zones by e.g. hydrogen bonding and hydrophobic interactions pushes polymer chains closer together, promoting hydrogen bonding and hydrophobic interactions, resulting in crystalline zones. Thermal transition: involves molecular conformational changes with temperature with consequent aggregations → sol-gel transition Dual crosslinking: also in combination with chemical crosslinking Physical crosslinking is reversable Physical bond forces are dependent on temperature and distance Physical crosslinking generally results in a more cell-friendly environment than chemical crosslinking 60 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking – hydrogen bonds Gelatin Denaturated collagen Random coil Temperature sensitive → transition temperature (25-35°C) at which a conformational change occurs, from random coil to partially triple helix on cooling → gelation Gelation in water mainly due to hydrogen bonds between the chains in the triple helix https://doi.org/10.1016/j.foodres.2010.09.008 61 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking – hydrogen bonds Agarose Natural polysaccharide Temperature sensitive → transition temperature (~45 °C) at which a conformational change occurs, from random coil to double helix on cooling → double helix bundles → gelation Gelation mainly due to hydrogen bonds between the chains in the double helix (high T) cooling heating Adapted from: https://www.nature.com/articles/s41598-017-17486-9 and https://repository.library.northeastern.edu/files/neu:cj82sr887/fulltext.pdf 62 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking – ionic interaction Physical crosslink with molecules of opposite electric charges + Polyion complex Multivalent cation Ionotropic hydrogel Adapted from: Ghobril, C., & Grinstaff, M. W. (2015). Chemical Society Reviews, 44(7), 1820-1835. 63 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Physical crosslinking – ionic interactions Alginate Natural polyanionic polysaccharide Can be crosslinked by divalent cations, such as calcium (Ca2+), barium (Ba2+) and magnesium (Mg2+). Divalent cations bind the guluronate (G) blocks from adjacent alginate chains. Egg-box supramolecular structure Adapted from: Bruchet, M., & Melman, A. (2015). Carbohydrate polymers, 131, 57-64 and DOI: 10.3144/expresspolymlett.2017.26 64 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking by crystallization Poly vinyl alcohol (PVA) Crystallization: the polymer chains are pushed together by ice crystals in freezing and thawing cycles, forming crystalline zones by e.g. hydrogen bonding and hydrophobic interactions. 65 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Chemical crosslinking Chemical crosslinking consists of covalent bonds between polymer chains. This can be achieved with different methods such as: Free radical polymerization Enzyme catalyzed reaction Click chemistry: not a single specific reaction, but describes reactions with high specificity, high yield under mild conditions (favourable for cells), minimal and inoffensive byproducts, fast, in ‘one-pot’, does not necessarily need a catalyst (e.g. copper-free click chemistry) − Diels–Alder reaction − Schiff base formation − Oxime formation − Michael type-addition Involves the formation and breaking of covalent bonds in a reversible manner under specific conditions, allowing for dynamic and Dynamic covalent chemistry * adaptable materials. Chemical crosslinking is generating mechanically stronger, more stable hydrogels Normally irreversible, however latest development in * dynamic covalent chemistry for reversibility 66  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Chemical crosslinking – free radical polymerization It requires the presence of unsaturated groups Radical generation by thermal or photo-induced reaction Photopolymerization is one of the most common methods, it requires a photoinitiator (molecule that absorbs light and undergoes a photochemical reaction, which generates radical species) Example: GelMA (gelatin methacrylate) Methacrylate Gelatin chain Lee et al (2020). Chemical Reviews, 120(19), 10950-11027. 67  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photoinitiated radical polymerization Photoinitiators (PIs) Lithium-acyl phosphinate (LAP) Irgacure 2959 Riboflavin Ruthenium-based crosslinkers (i.e., Ru-aldehyde, [Ru(bpy)2(3- pyridinaldehyde)2]Cl2) And many more … Be careful: PIs are mostly toxic to cells PIs have specific light absorbance values depending on the wavelength Concentration is critical to achieve sufficient crosslinking and no toxicity Lee et al (2020). Chemical Reviews, 120(19), 10950-11027. Bagheri et Jin (2019). ACS Applied Polymer Materials, 1(4), 593-611. 70 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photoinitiated radical polymerization Example: GelMA Parameters: 0.05% (wt/vol) Irgacure 2959 by 15 min exposure to 365 nm light at an intensity of 2.6 mW/cm2 Lee et al (2020). Chemical Reviews, 120(19), 10950-11027. 71 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photoinitiated radical polymerization Photo-crosslinking parameters for Irgacure 2959. Lim et al (2020). Chemical reviews, 120(19), 10662-10694. 72  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photoinitiated radical polymerization Absorption spectra and molar attenuation coefficients of commonly used photoinitiators for light-based bioprinting. Lim et al (2020). Chemical reviews, 120(19), 10662-10694. 73  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photopolymerization in extrusion-based bioprinting A) pre-crosslinking; B) post-crosslinking; C) in-situ crosslinking. doi:epdf/10.1002/smll.202002931 74  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Photopolymerization in extrusion-based bioprinting For extrusion-based 3D bioprinting, it would be better to crosslink the bioinks at the nozzle outlet (in situ, C) or immediately after extrusion (B) rather than pre-crosslinking (A) doi:epdf/10.1002/smll.202002931 doi:10.1021/acs.chemrev.9b00812 75  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Chemical crosslinking Purely synthetic https://onlinelibrary.wiley.com/doi/10.1002/marc.202000295 DOI: 10.1016/j.biomaterials.2010.02.044 https://doi.org/10.1016/j.biomaterials.2016.12.015 77  Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Enzymatic crosslinking Enzymes are proteins that act as biological catalysts (biocatalysts). Enzymes can be employed as catalysts to promote the formation of covalent bonds between protein-based polymers. Enzymes are substrate-specific. Enzymatic crosslinking is attractive for bioprinting due to the mildness of the enzymatic reactions. enzyme + enzyme covalently bonded polymer chain Adapted from: Nezhad-Mokhtari et al (2019). International journal of biological macromolecules, 139, 760-772. 78 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Enzymatic crosslinking: example Gelatin crosslinked by transglutaminase enzyme Transglutaminase Transglutaminase Thi et al (2019). Journal of Industrial and Engineering Chemistry, 78, 34-52. 79 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Pluronics F-127 for Sacrificial bioprinting is a cutting-edge technique used to create intricate vascular networks within three-dimensional tissue constructs. One of the key materials used in sacrificial bioprinting this process is Pluronics F-127, a triblock copolymer Three-dimensional vascularized tissue fabrication. (A) Schematic illustration of the tissue manufacturing process. (i) Sacrificial/fugitive (vascular) ink, which contains pluronics and a bioink, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces rapid polymerization of fibrinogen into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly crosslinks the gelatin and fibrin. (iii) Upon cooling, the sacrificial ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. Kolesky et al. Proceedings of the national academy of sciences 113.12 (2016): 3179-3184. 80 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Pluronics F-127 for sacrificial bioprinting Print vasculature structure with a sacrificial material Pluronic F-127 is liquefied after printing and can be washed out Vascular structures are seeded with Kolesky et al. Proceedings of the national academy of sciences 113.12 (2016): 3179-3184. endothelial cells 81 Chair of Medical Materials and Implants | Department of Mechanical Engineering TUM School of Engineering and Design | Munich Institute of Biomedical Engineering Crosslinking Adding crosslinks between polymer chains affect the physical properties of the polymer depending on the degree of crosslinking Main parameters: Viscosity Elasticity Solubility Swelling behaviour Diffusion properties (e.g. pore sizes, water type (primary and secondary bound water, and free water)) Biocompatibility (in the sense of cell-friendliness) 82