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Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels S. Khetan, J. Katz, and J. Burdick University of Pennsylvania Soft Matter.

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Presentation on theme: "Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels S. Khetan, J. Katz, and J. Burdick University of Pennsylvania Soft Matter."— Presentation transcript:

1 Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels S. Khetan, J. Katz, and J. Burdick University of Pennsylvania Soft Matter 2009

2 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

3 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 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

4 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

5 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

6 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

7 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

8 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

9 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

10 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

11 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

12 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

13 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.

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15 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 Step 1

16 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

17 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

18 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

19 Can conjugate via the thiol-ene reaction a di-fluorescein collagenase- sensitive 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 cell-cleavable peptide

20 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

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