12.2 Bioinks and printable materials

Bioinks are the lifeblood of 3D bioprinting, combining cells and materials to create living tissues. They need to be printable, support cell growth, and mimic natural environments. Getting the right mix of properties is key to successful printing and tissue formation.

Different bioink types offer unique advantages. Natural polymers like alginate and collagen provide biocompatibility, while synthetic options like PEG offer tunability. Combining materials in composite bioinks can achieve the best of both worlds for specific applications.

Bioinks and their properties

Essential properties for successful 3D bioprinting

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• Encapsulate living cells and support their growth and function
• Possess biocompatibility to ensure cell survival and prevent immune responses
• Exhibit printability, allowing precise deposition and maintaining structural integrity post-printing
• Demonstrate mechanical stability to withstand the printing process and support tissue development
• Promote cell adhesion, proliferation, and differentiation by providing appropriate biochemical and physical cues

Crosslinking mechanisms and their effects

• Physical crosslinking methods (ionic interactions, hydrogen bonding, hydrophobic interactions) provide reversible gelation
• Chemical crosslinking (covalent bonds, photopolymerization, enzymatic reactions) offers permanent and stable structures
• Crosslinking mechanism affects bioink stability, influencing its ability to maintain shape and support cell viability
• Appropriate crosslinking kinetics are crucial for successful printing, avoiding nozzle clogging or poor shape fidelity

Microenvironment provided by bioinks

• Suitable porosity allows nutrient diffusion, waste removal, and cell migration
• Permeability enables exchange of oxygen, nutrients, and growth factors between cells and the surrounding environment
• Degradation rate should match the rate of tissue formation, providing space for cell growth and matrix deposition
• Bioinks should mimic the native extracellular matrix, providing tissue-specific biochemical and mechanical cues

Bioink types and classifications

Natural polymer-based bioinks

• Alginate, a polysaccharide derived from brown algae, forms hydrogels through ionic crosslinking with divalent cations (calcium chloride)
• Gelatin, a denatured form of collagen, offers inherent biocompatibility and can be modified with methacrylate groups for photopolymerization
• Collagen, the most abundant protein in the extracellular matrix, provides a native-like environment for cell growth and differentiation
• Fibrin, a protein involved in blood clotting, forms bioinks through enzymatic crosslinking with thrombin
• Hyaluronic acid, a glycosaminoglycan found in connective tissues, can be modified with functional groups for enhanced stability and cell adhesion
• Silk fibroin, a protein derived from silkworm cocoons, offers excellent mechanical properties and slow degradation rates

Synthetic polymer-based bioinks

• Polyethylene glycol (PEG), a hydrophilic polymer, can be functionalized with various bioactive molecules and crosslinked through photopolymerization
• Polycaprolactone (PCL), a biodegradable polyester, provides mechanical strength and slow degradation rates suitable for long-term tissue support
• Pluronic, a triblock copolymer with thermoreversible gelation properties, enables injectable and self-healing bioinks

Hydrogel-based and composite bioinks

• Natural hydrogels (agarose, chitosan, gellan gum) offer biocompatibility and can be blended with other polymers to improve printability
• Synthetic hydrogels (polyacrylamide, poly(N-isopropylacrylamide)) provide tunable mechanical and responsive properties
• Decellularized extracellular matrix (dECM) bioinks, derived from native tissues (heart, liver, bone), contain tissue-specific biochemical cues
• Composite bioinks combine multiple materials (natural and synthetic polymers, hydrogels and dECM) to achieve desired properties and biological functions

Rheological properties of bioinks

Key rheological properties for printability

• Viscosity, a measure of a bioink's resistance to flow, determines its ability to be extruded through a nozzle
• Shear-thinning behavior allows bioinks to flow under applied shear stress and recover viscosity post-printing, maintaining the printed structure
• Viscoelasticity, a combination of elastic and viscous properties, influences the bioink's ability to deform and recover after printing
• Yield stress, the minimum stress required to initiate flow, is critical for achieving high-resolution printing and preventing cell sedimentation

Crosslinking kinetics and printability

• Rapid crosslinking can lead to nozzle clogging and difficulty in extruding the bioink
• Slow crosslinking may result in poor shape fidelity and collapse of the printed structure
• Optimal crosslinking kinetics allow smooth extrusion and rapid gelation post-printing, maintaining the desired geometry

Techniques for assessing printability

• Filament formation tests evaluate the bioink's ability to form continuous filaments and maintain shape after extrusion
• Extrusion force measurements provide insights into the bioink's flow behavior and the required pressure for printing
• Post-printing shape fidelity analysis assesses the printed structure's ability to maintain its geometry over time
• Rheological characterization techniques (oscillatory shear tests, flow curves) quantify the bioink's viscoelastic properties and shear-thinning behavior

Challenges and strategies for bioink development

Balancing biocompatibility and mechanical properties

• Increasing mechanical strength often compromises cell viability and function due to the use of harsh crosslinking conditions or high polymer concentrations
• Strategies to improve biocompatibility include incorporating cell-adhesive ligands (RGD peptides), growth factors (VEGF, BMP-2), and tissue-specific ECM components (laminin, fibronectin)
• Mechanical properties can be enhanced by using high molecular weight polymers, increasing polymer concentration, or introducing interpenetrating networks

Stimuli-responsive and multimaterial bioinks

• Temperature-responsive bioinks (gelatin, pluronic) enable sol-gel transitions and provide injectable and self-healing properties
• pH-responsive bioinks (alginate, chitosan) allow for controlled gelation and release of bioactive molecules in response to pH changes
• Light-responsive bioinks (methacrylated gelatin, PEG-diacrylate) offer spatial and temporal control over crosslinking, enabling the creation of complex geometries
• Multimaterial bioprinting combines bioinks with complementary properties (mechanical strength, bioactivity) to create heterogeneous tissue constructs

Advanced bioink formulations

• Nanocomposite bioinks incorporate nanoparticles (hydroxyapatite, graphene oxide) to improve mechanical properties and bioactivity
• Supramolecular bioinks utilize non-covalent interactions (host-guest complexation, hydrogen bonding) for reversible and self-healing properties
• Conductive bioinks, containing conductive polymers (polypyrrole, polyaniline) or nanoparticles (gold, silver), enable electrical stimulation of cells for applications in cardiac and neural tissue engineering
• Gradient bioinks, with spatially varying compositions or properties, mimic the heterogeneity of native tissues and promote tissue-specific cell differentiation