Download physical hydrogel

Strategies and Molecular Design Criteria
for 3D Printable Hydrogels [ Chem. Rev. 116, pp 1496-1539 (2016)]
1. Introduction
- Additive manufacturing (AM), rapid prototyping, solid free-form fabrication, 3D printing
- Object production via layer-by-layer fashion with CAD or CAM (comp.-aided manufacturing)
- no mold, high degree of design freedom, direct production of structures
with high precision and low-cost printers
- Biofabrication: AM for tissue engineering (cells, prefabricated scaffolds, growth factors)
cell-loaded constructs with living and nonliving materials
- “Bioinks”: cell loadable and printable materials
- Hybrogels: biomedical applications
i) chemical hydrogel (polymeric); ii) physical hydrogel (supramolecular) as bioink
- Injectable hydrogels [(cf.) printable hydrogels]
shear thinning properties with viscosity decrease with increased shear stress
stable cell-loaded bioink
nozzle diameters for high resolution structures
rapid gelation
- self-supporting, no post-processing , lack of geometrical constraints, rapid gelation
- new bioink development: sterile and endotoxin free
2. Fabrication Systems
- heat for thermoplastic materials
- laser for two-photon polymerization (2PP) and stereolithography (SLA)
top-down vs. bottom-up systems
- choice of fabrication system: materials, size, architecture, resolution
- printing of hydrogels under cell-friendly conditions
2.1 Laser-induced forward transfer (LIFT)
- three components: pulsed laser, donor slide (ribbon), receiving substrate
- donor slide = laser transparent support layer + laser absorption layer + deposition material
- light absorbing matrix: hydrogel, cell medium, glycerol, ECM
Figure 1. Laser-induced forward transfer (LIFT). (A) Schematic drawing of the process. (B) Close-up view of
the jet generated by the incident laser pulse. (C) Cell pattern printed with LIFT.
2-2 Inkjet printing
- continuous inkjet (CIJ) process and drop-on-demand (DOD) printing
- i) ink ejection; ii) drop formation during flight; iii) impact and interaction of drops after
collection on the substrate
Figure 2. Inkjet printing. (A) Schematic drawing
of the process. (B) Example for propelled
material after ejection at different time points
using a drop-on-demand printer. (C) Surface
profile of a linear pattern printed with inkjetprinted poly(lactic acid-coglycolic acid) (PLGA). (D,
E) Different patterns of PLGA printed with a
piezoelectric inkjet.
2-3 Robotic dispensing
- pneumatic or mechanical (piston and screw) dispense
- continuous strands, nozzle movement relative to the building plate, LbL assembly
- thermopolymer extrusion, high-viscosity materials
Figure 3. Robotic dispensing. (A) Schematic
drawing of robotic dispensing showing the
different mechanisms of ejection. (B) Magnified
view showing material dispensing from the
needle and collection onto a substrate. (C)
Stereomicroscopic image of a printed Pluronic
F127 construct.
2-4 Comparison of the fabrication methods
- i) material and structural; ii) processing; iii) economical
- material filament vs. droplet ---- structural integrity
- resolution; amount of material; fabrication and preparation time; material cost
3. Rheological Considerations
- bioink design and development
- molecular properties of inks (molecular architecture, molecular interaction, ink formation,
ion strength, colloidal components, reactive processes, etc.)
- nozzle-based dispense
3-1 Rheology of non-Newtonian liquids
- Newtonian fluids (shear-rate-independent viscosity)
and non-Newtonian fluids (shear-rate-dependent viscosity)
- shear thinning, thixotropic (time-dependent)
- shear thickening, rheopectic (time-dependent)
3-2 Important aspects of the printing process
- ideal ink for hydrogel printing
(i) physical gel formation with shear thinning and
no thixotropic behavior
(ii) rapid regelation for shape fidelity
at high resolution
(iii) little extrudate swell (dye swell) -- entropy
(resolution)
Figure 4. Viscosity against shear rate for
Newtonian and different non-Newtonian fluids.
3-3 Underlying molecular concepts: colloidal solutions
- non-Newtonian fluids: interparticle and particle-liquid interactions
- shear increase --- withstand --- reversible interactions --- permanent disintegration of
aggregates and non-restored particle distribution --- velocity gradient
- colloidal gel-based inks: analyzing and controlling interparticle forces
to tailor the viscoelastic properties of the system
3-4 Underlying molecular concepts: polymer solutions
- entanglement of polymers --- non-Newtonian property (polymer-polymer, polymer-liquid)
- the zero shear viscosity (high) and the onset of shear thinning (low value)
Figure 5. Viscosity against shear rate for high and low
polymer concentrations (c) of a homopolymer solution in a
good solvent.
4. Chemical Cross-linking
- Physical hydrogels: non-covalent interactions --- dynamic gel with self-healing properties
low mechanical strength and low stability
- Chemical hydrogels: covalent network --- less dynamic but stable
3D printing --- discontinuous printing with stop-go phases (not suitable)
two component systems (isophorone diisocyanate for X-linking of
PEG and glycerin) ---- not cytocompatible
- in situ gelation via chemical cross-linking (still numerous X-linking hydrogels)
4-1 Post- and prefabrication cross-linking
- photopolymerization (acrylates) for postfabrication stabilization of the printed constructs
- photo-initiated thermal X-linking as pre- and post-; chemical X-linking as pre- different intermolecular stabilization (ionic interaction followed by photopolymerization)
4-2 Application of dynamic covalent bonds for printing
- chemical bonds: stable becomes labile under some conditions
switching of disulfides or thioester exchanges
- dynamic imine chemistry (reversible crosslinking network)
i) oxidized hyaluronic acid (aldehyde) + chitosan (amine)
ii) partially oxidized alginate + gelatin --- cytocompatibility
- combination of reversible chemical X-linking with shear thinning and self-healing property
5. Molecular Physical Gels
- physical hydrogels with dynamic and reversible nature (alginate, etc.) as 3D printable gels
Figure 6. Basic physical molecular interactions exploitable for physical gel formation.
- supramolecular polymer network
i) noncovalently bound monomer based polymer
ii) covalently bound monomer-based polymer with noncovalent chain interconnection
- macrocycles for functional supramolecular gels
crown ethers, cyclodextrins, spiroborate cyclophanes
temp/pH change; pressure-responsive, host-guest interaction
- host-guest interaction (sol-gel transition with light and redox reaction)
nanosheets, nanotubes, vesicles, micelles
- p-p interaction (nanofibers, columns, helices)
- dynamic nature of functional supramolecular polymers
metal coordination bonds, multiple hydrogen bonds, host-guest, donor-acceptor
ex) self-healing supramolecular gels
Figure 7. Concept of self-healing materials relying on the reversibility of physical interactions.
- injectable hydrogels: shear thinning hydrogels
reversible linkages in adaptable hydrogel networks for cell encapsulation
- Hydrogel formation strategies: supramolecular polymer, supramolecular (low mol wt
gelators), macromolecular, and colloidal (solid particles)
Figure 8. Supramolecular, macromolecular, and colloidal strategies for hydrogel formation.
5-1 Supramolecular approaches
(1) Ionic interactions and coordination bonds
- chain-end-dithiolated PEG with Au+ as zigzag X-linker
at pH 11, thiols with strong nucleophilicity --- thiolate/Au-S exchange --- dynamic gel
at pH 3.1, weak nucleophilicity --- less thiolate/Au-S exchange --- stable gel
- G’ (storage modulus) and G’’ (loss modulus) used to evaluate microstructure
G’ > G’’ when applied force is smaller than molecular or interparticle forces
--- material’s capability to store some energy ---- materials return (elastic)
G’’> G’ with large applied force ---- collapsed microstructure
--- materials flow (viscous)
G’ = G’’ the applied mechanical force overtakes molecular or interparticles forces
--- materials yield (or flow)
- ionic interactions: (di/tri)carboxylic acids (citric acid) and
(di/tri)alkylamines (tetramethyl-1,3-propanediamine) with proton transfer
G’’ > G’ after the gel-sol transition temp (20 to 60oC)
- Shear thinning gels in acetonitrile
2,6-bis(1’-methylbenzimidazolyl)-4-oxypyridine for Zn2+ or La3+
sol transition with acid or heat
(2) Hydrogen bonds
- ureidopyrimidinone (Upy)/urea-end functionalized
Poly-(caprolactone) + Upy/urea-end-func peptides
quadruple H-bonding (Ka = 107 M-1 in CHCl3)
stable and flexible, melt at 80oC
--- fibers via electrospinning, films, scaffolds
- thermoreversible (gel-sol transition)
- shear thinning property (G’’ > G’)
- PEG with Upy and urea on both chain ends
at pH 8.5, Upy deprotonation, weak H-bonds
--- becomes solution
shear thinning property
(3) Host-guest and aromatic donor-acceptor interactions
- crown ethers + protonated alkylammonium group (secondary amines)
building blocks for supramolecular polymeric gels (hydrogen bonds)
+ [PdCl2(PhCN)2] as X-linker
sol by pH, temp, cations
shear thinning and self-healing properties
- pillararene-based supramolecular polymer gels
reversible gel-sol transition via temp. change
self-healing property
- amphiphilic calix[4]arenes
- copolymer with p-electron-deficient naphthalenediimide units and
p-electron-rich pyrenyl-end-capped polyamides
p-p stacking for self-healing materials
donor-acceptor interactions
(4) Low molecular weight gelator
- Fmoc-containing peptides for p-p stacking and thermoreversible properties
Fmoc-Cys(Acm)-His-Cys-OH + Fmoc-Cys-His-Cys-OH (gel with shear-thinning)
Fmoc- and methyl ester-containing dipeptides
protease --- methyl ester hydrolysis --- p-p stacking --- hydrogel --- Tm
- cyclic dipeptide derivatives
cyclo(Phe-Lys) and cyclo(Tyr-Lys) possessing N-acetylated D-(+)-gluconic acid
- aromatic groups
4-(octanoylamino)benzoic acid and 4-alkoxyanilines
naphthalene-based gelators
quinquethiophene-oligopeptide
- 3D printing of cell-loaded spider silk protein hydrogels (b-sheet formation)
5-2 Macromolecular Gels
(1) Naturally occurring biopolymers used for hydrogel formation
- biopolymers: bioactivity, biodegradability, injectable hydrogel for biofabrication
(1-1) Polysaccharides
- hydrogels via bonding (agarose) or intermolecular electrostatic interactions (alginate)
- alginate: homopolymeric blocks of 1,4-linked b-D-mannuronate and a-L-guluronate
biocompatible --- biofabrication with cells
supporting cell survival and differentiation
gelation at pH < 3 --- acidic gels via intermolecular H-bonds
gelation with Ca2+ --- intermolecular electrostatic interactions
bioink: low mechanical stability (1% for high cell viability)
- agarose: (1-3) b-D galactopyranose-(1--> 4)-3,6-anhydro-a-L-galactopyranose
+ ionized sulfate groups
thermal gelation & tunable elastic moduli of physical gel
double-network hydrogels (polyacrylamide or polystearyl methacrylate)
--- self-healing property
bioink: agarose into 3% gelatin medium bath
- bacterial extracellular polysaccharides (EPSs)
xanthan gum, gellan gum, dextran, bacterial alginate, bacterial cellulose
(1-2) Polypeptides/proteins
- collagen: mechanical support to control cell adhesion, cell migration, tissue repair
easy processing and modifications, abundance, non-antigenicity, biodegradability
단점) batch-to-batch variations, loss of shape and consistency (shrinkage)
poor mechanical property, hard to sterilize
- gelatin: partially denatured collagen
RGD-motifs, biodegradability, biocompatibility, water solubility
단점) not stable, must be X-linked
- fibrin: thrombin-initiated conversion of fibrinogen into fibrin network
biocompatible, cell adhesive, biodegradable (enzyme degradation)
poor mechanical property, fast disintegration
- silks from spiders or insects
crystalline domains periodically interrupted by helical or amorphous regions
(highly repetitive)
no toxicity, slow degradation, no immunogenicity, extraordinary mechanical prop.
단점) frequent clogging due to shear-induced b-sheet crystallization
- natural proteins
inconsistent or unwanted biological responses
disease transmission and immunogenic responses
(2) Synthetic peptides and proteins
- polypeptoids + single DNA --- shear-thinning hydrogel with complementary DNA
--- forming nanofibrils and hydrogels
- peptide/protein-based hydrogels
a-helix, b-sheet, b-hairpin, and coiled-coil --- shear thinning and rehealing
- biomimetic peptide-heparin or receptor-ligand interactions
four-arm PEG with positively charged basic peptides in the presence of heparin
PEG-biotin with tetrametic avidin
Figure 10. Stepwise formation from
amino acids to hydrogel networks.
(3) Host-guest interaction
- storage modulus, loss modulus, viscosity, recovery, host/guest ratios
- poly(ethyl acrylate)-containing protonated dibenzylammonium moieties X-linked
with dibenzo-24-crown-8 bis(crown ether)
* pH and temp-responsive gel-sol transition
* self-healing property / full recovery after the strain removal
- hyaluronic acid-based hydrogels
adamantine and b-cyclodextrin (CD)
as host-guest interaction
+ methacrylate (UV-induced X-linking)
- CD and linear PEG (rotaxanes)
viscoelastic gel / pressure responsive
CD sliding over PEG chain
tunable shear thinning properties
- supramolecular host-guest materials containing
CD, cucurbit[n]urils, calix[n]arenes
Ex) poly(vinyl alcohol)-containing viologen
+ hydroxyethyl cellulose with naphthalene
---- host-guest complexes with cucurbit[8]uril
---- high water content, shear-thinning, recovery
Figure 11. Supramolecular hydrogel with
viologen/hydroxyethyl-functionalized
(Viologen: bipyridinium derivatives)
cellulose and naphthalene-functionalized
poly(vinyl alcohol) forming host−guest
complexes with cucurbit[8]uril.
(4) Ionic interactions and coordination chemistry
- negative hyaluronic acid + positive four-arm star
PEG-block-poly(2-aminoethyl methacrylate)
* (PEG113-b-PAEM12)4 + hyaluronic acid
at 1:1 charge ratio in water
- mechanoresponsive polyelectrolyte brushes
cationic poly[2-(methacryloyloxy)ethyl]trimethyl
ammonium chloride + 5(6) carboxyfluorescein
--- pressure-dep fluorescence quenching
with AFM
- gels X-linked via coordination chemistry
Figure 12. Hyaluronic acid- and PEG-based
with palladium- and amine-based ligands
hydrogels.
Ex) poly(4-vinylpyridine) X-linked with bis-Pd(II) compound
in DMSO or DMF (dimethylformamide)
- metallopolymer film
poly(butyl acrylate) containing 2,6-bis(1’-methylbenzimidazolyl) pyridine
for complexation of Cu2+, Zn2+, Co2+
(5) Hydrogen bonds
- poly(n-butyl acrylate) containgin acrylamidopyridine, acrylic acid, carboxyethyl acrylate,
ureidopyrimidinone acrylate in different ratios
- high glass transition temp, high G’, high zero shear viscosity
- NIPAAm + dopamine methacrylate forms gel in DMSO with NaOH
dopamine deprotonation with base --- gelation
- NIPAAm X-linked via H-bonds with diaminotriazine or cyanuric acid groups at the side chain
--- addition of complementary bismaleimide or Hamilton wedge
--- gelation in methanol/chloroform (1:1)
5.3 Colloidal Systems
- solid particles with polymers or small organic chemcials --- supramolecular hydrogels
- silica NP, laponite, CNT, GO sheets, titania sheets, AuNP, AgNP
- shear thinning, temp/pH-induced reversible sol-gel transitions, self-healing properties
(1) Silica NPs and laponite-based hydrogels
- hydroxide-rich surface of silica NPs
covalent modification
Ex) b-CD on SNPs + mono-end-func. PEG
--- viscoelastic hydrogel with a-CD addition
- laponite (clay nanosheet, CNS)
Na0.66[Mg5.34Li0.66Si8O20(OH)4]
- oxyanion of CNS + cationic compound
Ex) DMA/NIPAAm + guanidinium-pendant
methacrylate + CNS
G’’ > G’ at 10% strain
- large storage modulus (G’) with high CNS content
(2) Carbon-nanotube and graphene-sheet rich compounds
- SWCNT and bile salt sodium deoxycholate (NaDC)
hydrophobic steroid group with CNT surface + carboxyl group with water
- MWCNT (0.015% to 0.5%) + N-ethylamine-functionalized poly(ethylene imine)
pH, temp, NIR light / self-healing property
- Pluronic polymer: hydrophobic poly(propylene oxide) for CNT
and hydrophilic poly(ethylene oxide) for a-CD ---- gelation upon a-CD addition
- pyrene-based compounds with CNT via p-p interaction
b-CD with pyrene + SWCNT --- poly(acrylic acid) containing 2% dodecyl groups
solution with competitive guest (sodium adamantine carboxylate)
or competitive host (a-CD)
- PNIPAAm with pyrene (for CNT) and MPEG (for a-CD interaction)
- high gel-sol transition temperature and increased G’ with CNTs
- reduced graphene oxide (RGO) + short peptides (Fmoc-Tyr/Phe) at only 0.5% and 0.55%
thermoresponsiveness for gel-sol transition
- GO with carboxyl and hydroxyl groups + protonated amine or other positive functionalities
such as amino acids (Arg, Trp, His) and polyamines (spermine, spermidine,
tris(aminoethyl)amine)
* good crosslinking ability, high storage modulus
* pH dependence
- Ca2+-mediated X-linking between poly(acryloyl-6-amino-capronic acid) and GO (carboxyl)
porous hydrogel with high stretchability, self-healing at pH<3, solidified at pH>7
- thermoresponsive polymers as X-linkers for GO
b-CD modified GO + azo-PDMA-b-PNIPAAm
gelation via host-guest between b-CD and azophenyl group
- GO orientation control with magnetic field located within PDMA (+MBA)
(3) Metal-based gels
- titania nanosheets (via external magnetic field) + PDMA (+MBA as X-linker) with UV exp.
- gold with monothiolated b-CD + Pluronic
gelation via PEO and b-CD interaction / solution with 1-adamantanamine HCl
- AuNPs and AuNRs with monothiolated mPEG
gelation with a-CD: thermo- (60-70oC)and mechanoresponsivity, high G’
shear-thinning property, low molecular weight gelators (LMWGs) 0.45% to 3%
6. Biotechnological Approaches toward Bioinks
- develp and design new biopolymers with complexity and novel functionality
- natural or artificial proteins: mechanics, degradation, porosity, cell interaction, and
cytocompatibility
- genetically engineered recombinant proteins
RGD or IKVAV containing proteins
hyaluronic acid with anchor peptides (docking polypeptides)
or methacrylate (photopolymerization)
6-1 Designing biopolymers: recombinantly produced proteins
(1) Methods for recombinant protein production
- E. coli (G-)
- Salmonella typhimurium (G-): protein secretion
- Bacillus subtilis (G+): protein secretion in large quantity
- Yeast Saccharomyces cerevisiae and Pichia pastoris (high cell density culture)
glycosylation
- transgenic plants: Arabidopsis thaliana and Nicotiana tabacum
large scale and at lower costs / complicated genetic manipulation
- insect cells: high expression efficiency, low feeding costs, protein secretion,
simple purification, post-translational modifications / complex cloning, long time
- transgenic animals: body fluids such as milks or urine
high protein yield / time consuming, complex procedure, difficult purification
(2) Silk-like proteins
- SLPs: number of repeats, types of motifs, spacing between the motifs
- b-sheet rich spider silk (in comparison with silk from Bombyx mori)
shear thinning, 16 layers on top of each other, cell-loaded spider silk constructs
(3) Recombinant collagen/collagen-like proteins
- biologically safe (no infectious contamination) [cf. native collagen]
- quite complex expression
prolyl 4-hydroxylase required
only in mammalian cells / multigene expression tech in E. coli or yeast
- difficulty on higher order structure and incorporation of biologically relevant motifs
(4) Elastin-like polypeptides (ELPs)
- VPGXG repeats as elastin-inspired sequence
temperature, ionic strength, redox state, pH
physically X-linked network at high temp.
excellent mechanical properties, minimal cytotoxicity
chemical X-linking with transglutaminase
(5) Resilin-like polypeptides (RLPs)
- resilin found in insect cuticle, role in insect flight or jump
- 12 repeats of the resilin consensus sequence: cell-binding, MMP-sensitive, polysaccharidesequestration domains in hydrogels
- randomly coiled, isotropic three-dimensional network: ideal rubber with near-perfect
reversible long-range elasticity
- RLP hydrogel with tris[(hydroxymethyl)phosphine] for mesenchymal stem cells
(6) Hybrid proteins
- SELP: silk-repeats + ELP sequences
silk for mechanical properties/ELP for high solubility and environmental sensitivity
two step self-assembly: (1) crystallization of silk-like domain, (2) hydrophobic
interaction between elastin blocks
temperature-sensitive behavior
- C2SH48C2: 48 silk-like octapeptides + two 99 amino acid collagen-like sequences
nanofibrillar hydrogels: long-term stability, self-healing, mechanical property
- RECs: resilin-, elastin-, and collagen-like engineered polypeptides
high resilience and elasticity
self-assembly property
- ELP-PEG hydrogel system
tenability of ELP, flexibility, tunable stiffness, RGD ligand density
6-2 Designable biopolymers: polynucleotides
- DNA: secondary structures and stimulus-sensitive features
biocompatible, biodegradable, inexpensive, easy molding
hydrogel under physiological condition, cell encapsulation
- CpG-DNA: immunostimulatory to induce helper T-cell cytokines
- ligase-free, injectable, self-gelling, biodegradable DNA hydrogels with oligonucleotides
- pH-switchable DNA hydrogel with shape-memory effects
- DNA hydrogel deliverying tumor antigens for induction of antigen-specific immune
responses
7. Conclusions and Outlook
- biofabrication: hierarchical structures through the simultaneous printing of cells and
supporting materials
- 3D hydrogel printing: (1) LIFT, (2) Inkjet printing, (3) robotic dispensing
- rheological behavior of non-Newtonian liquids
shear-thinning property (not being thixotropic)
no extrudate swell
viscosity without affecting cell’s viability
cell tolerable shear forces for printing
rapid gelation for good shape fidelity
- physical vs. chemical hydrogels
- combination of both types of gel
physical component for rheological behavior
chemical X-linking for post-fabrication and stabilization
- bioink development instead of optimizing formulations
multiple conjugation with polymers for multivalently interacting building blocks
supramolecular polymers
stimulus-switchable chemical reactions
thermosensitive physically cross-linked hydrogels
shear-stress-sensitive chemical bonds
bioinspired designer structures (recombinant proteins with multiple functionalities)