Abstract:
The goal of our study was to compare the biological
responses of cells cultured on polyethylene glycol (PEG)
hydrogels functionalized with varying concentrations of the
widely used adhesion peptide, RGD, and the cell-binding domain
of fibronectin, III9-10. We used Michael addition chemistry
to covalently link cysteines in GRGDSPC and
glutathione S-transferase (GST) tagged III9-10 (GST-III9-10), to
the acrylate groups in PEG diacrylate (PEGDA). Conjugation
of GST-III9-10 to PEGDA occurred through cysteine residues
in GST. Ellman’s reagent and immunoblotting studies demonstrated
an efficiency of 90% or more for PEG conjugation
of 1 lM GST-III9-10 or GRDGSPC in 10% (wt/vol) PEGDA at
37
C for 1 h. Circular dichroism and limited proteolysis demonstrated
that conjugating PEGDA to GST-III9-10 did not significantly
perturb its native secondary structure. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis characterization
of the wash solution of PEG hydrogels after photo-polymerization demonstrated that >95% of the 1 lM GSTIII9-10
was incorporated into the PEG hydrogels after crosslinking.
PEG hydrogels derivatized with 1 lM GST-III9-10 had
significantly higher cell adhesion and spreading than PEG
hydrogels with 1 lM GRGDSPC. A comparable adhesion
response between GRGDSPC and GST-III9-10 was obtained
when the former was at millimolar and the latter at micromolar
concentration. The amount and type of conjugate in
the PEG hydrogel derivative was statistically more significant
than hydrogel rigidity in stimulating the biological
responses observed. This report presents new evidence of
the robustness of III9-10 in mediating cell adhesion and
spreading on PEG hydrogels.
