Sequential crosslinking to control
cellular spreading in 3-dimensional
hydrogels
S. Khetan, J. Katz, and J. Burdick
University of Pennsylvania
Soft Matter 2009
Background
• Previous artificial cell scaffolds were
biologically inert
• Now clear that effective scaffolds must
mimic dynamic interplay between cells and
the natural ECM
• In vivo, cell shape/spreading influences
cell functions and can influence stem cell
differentiation
Previous Work
Figure 6. Expression of nestin (A), GFAP (B) and neurofilament (C) in undifferentiated and differentiated BMSC
Expression of nestin (A), GFAP (B) and neurofilament (C) in undifferentiated and differentiated BMSC.
Untreated cells were labeled for nestin (A1), whose expression decreased with treatment 1 (A2,3) but persisted
after treatment 2 (A4). GFAP immunoreactivity was negative in untreated cells (B1), but after 5 (B2) and 24
hours (B3) of treatment 1 some cells were GFAP positive. Treatment 2 determined strong GFAP
immunostaining after 7 days (B4). Immunoreactivity for neurofilaments, totally absent in untreated BMSC (C1),
was present in some cell bodies and processes after treatment 1 (C2,3; higher magnification in C3 insert) and
treatment 2 (C4). Cells were also stained with Hoechst dye 33258. Bar: 40 μm. Insert bar: 10 μm.
•Scintu et al. induced bone marrow stem cells into
neuronal differentiation through growth factor treatment,
noting phenotype change in cell morphology
•Curran et al. saw differences in morphology of hMSC
based on glass surface chemistry that led to differences in
differentiation
Motivation
• Despite advances in tailoring the
chemistry, matrix stiffness, MMP
degradation, and bioactive factor
incorporation of cell scaffolds to control
cell spreading and differentiation, more
work needs to be done to achieve spatial
control in cytocompatible manner
• This paper employs sequential 2-step
crosslinking to achieve bioactive hydrogels
with light exposure derived spatial control
of cell spreading
Materials
• Acrylated Hyaluronic acid (AHA):
– HA is chief GAG component of ECM
– primary structural component in these
hydrogels
– Biocompatible, hydrophilic, interactions
with cells via surface receptors
• Adhesive Peptide:
– Promotes cell adhesion and spreading
but terminates acrylate crosslinking sites
on AHA
• MMP-cleavable dithiol crosslinker
– Crosslinks the AHA to form a network
– Provides cell-mediated degradation to the
scaffold
HA
Acrylated
HA
Scaffold Synthesis Scheme
• Step 1 (“permissive”): A fraction of acrylate
groups react via addition mechanism with
thiols on cysteine residues on peptides
• Step 2 (“inhibitory”): Remaining unreacted
acrylates a photopolymerized with UV-light
exposure
Crosslinking Protocol
• Dissolve AHA in triethanolamine-buffere
saline containing Irgacure2959 (initiator)
• Cell adhesive peptide added to AHA
solution corresponding to 5% acrylate
groups. React at 37°C, 1 hour.
• Resuspend cells in this solution
• Add MMP-cleavable peptide at desired
concentration and react at room T, 15 min
• Then expose gels to 365 nm UV light, 4 min
Acellular Gel Characterization
• Modulus: Photo alone>75%>50%
• Swelling: 50%>75%>Photo alone
• Photopolymerizing after a pre-addition has similar effects
 Acrylate photopolymerization leads to denser network
than dithiol addition with MMP-cleavable peptides
Degradation of Gels
• Comparing degradation of
gels from 100% addition
reaction or 100%
photopolymerization
• Exogenously added MMP-1
degrades Addition gels in 7
days
• Only see minor degradation
(ester linkage hydrolysis?) in
photopolymerized gels.
• Modulus and degradation
results suggest potential
tunability of this sequential
crosslinking system
Cell Spreading Studies in Bulk Polymerized Gels
• Only gels with RGD
adhesion and MMP
cleavable peptide
adhesion feature
spreading hMSC
• All controls:
-RGD,
-MMP (dithiothreitol)
photo alone
do not show cell spreading
DTT subs for
MMP cleavable peptide
Cell Spreading in Sequentially Crosslinked AHA Hydrogels
• See max spreading for
cells in gels with only
“permissive” crosslinking
• Cells in gels with both
permissive and inhibitory
crosslinking showed more
rounded morphology
– Claim to see greater
aspect ratios for cells in
75% addition vs. 50%
addition due to greater
amount of inhibitory
crosslinking in 50% gels.
• Live/Dead staining
suggests similarly high cell
viability (88-94%) for all
hydrogel compositions
Spatially controlled encapsulated cell spreading
• Synthesize 50% addition
gels
• Apply photomask to ½ the
gel during photocrosslinking
• See drastic differences in
cell spreading between two
sides
Conclusions
• Cell spreading cues can lend control over cell
signaling and potentially differentiation
• Spatial Control over this differentiation and
morphology would be beneficial for anisotropic
tissue engineering applications (vasculature,
nervous tissue)
• Sequential crosslinking approach featuring
selectively exposure to UV provides this spatial
control capability.
• Could also tune AHA wt%, peptide
concentrations, cell adhesion moieties in a
flexible cell scaffolding paradigm.
Step 1
• Use modified Huisgen cycloaddition to link azide and alkyne without
toxic Cu catalyst (ring strain + fluorine withdrawing = reactive)
• Four-armed PEG and bifunctional peptide yield 3D network
Gel formation kinetics + Cell Viability
• Viscoelastic solid forms after 5 minutes when G’ and G” cross
•NMR suggests full network formation completes after ~1 hour
•Cell Live/Dead Staining shows high cell viability
Step 2: Photopolymerization
• Radical-mediated reaction between cysteine residues on peptides
and vinyl groups on peptide in the network backbone
• Can vary UV exposure to control amount of conjugation in step 2
• UV exposure is cytocompatible as shown in Live/Dead stain
Biochemical Patterning with Photolithography
• Can spatially control the incorporation of different fluorescently
labeled peptides in the same gel. (Requires long time to sequentially
incubate peptides, pattern, wash out, and repeat several times)
• Can also use confocal laser to created more complex 3D structures
Patterning cell-cleavable peptide
• Can conjugate via the thiol-ene reaction a di-fluorescein collagenasesensitive peptide (DiFAM)
• Fluorscent markers undergo intramolecular self-quenching unless
the peptide is cleaved to separate the fluorescein
• See strong fluorescence where cells induced collagenase cleavage
Patterning RGD peptide to localize cell adhesion
• Via thiol-ene reaction, pendantly link RGD containing peptide to
induce cell adhesion
• Control cell-adhesion by localizing RGD conjugation with a
photomask
Conclusion:
Use novel bio-orthogonal click chemistries to develop flexible, tunable
3D patternable system to observe and influence cell behavior