Encoding Cell-Instructive Cues to PEG-Based Hydrogels via Triple Helical Peptide Assembly

Abstract

Effective synthetic tissue engineering scaffolds mimic the structure and composition of natural extracellular matrix (ECM) to promote optimal cellular adhesion, proliferation, and differentiation. Among many proteins of the ECM, collagen and fibronectin are known to play a key role in the scaffold's structural integrity as well as its ability to support cell adhesion. Here, we present photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs) that can be further conjugated to bioactive molecules via CMP-CMP triple helix association. Pre-formed PEGDA-CMP hydrogels can be encoded with varying concentration of cell-signaling CMP-RGD peptides similar to cell adhesive fibronectin decorating the collagen fibrous network by non-covalent binding. Furthermore, the triple helix mediated encoding allows facile generation of spatial gradients and patterns of cell-instructive cues across the cell scaffold that simulate distribution of insoluble factors in the natural ECM.

Fig. 1

A) Circular dichroism melting curves of PEGDA-CMP precursor solution and PEGDA-CMP gel photopolymerized at 4°C and 80°C. B) First derivatives of corresponding CD melting curves.

Fig. 2

Cumulative release profiles of CF-CMP-RGD from PEGDA-CMP gels and from PEGDA-only gels. Single-stranded CF-CMP-RGD were added to gels at varying concentration and left at 37°C overnight to allow triple helix-mediated binding. The gels were washed with PBS to remove unbound peptides and their CF absorbance was monitored over the following days at 37°C. Data reported as mean ± SD.

Fig. 3

Phase contrast micrographs of human dermal neonatal fibroblasts seeded onto PEGDA-CMP scaffolds modified with CMP-RGD (A), PEGDA-CMP modified with CMP (B), and PEGDA modified with CMP-RGD (C).

Fig. 4

WST-1 proliferation assay (absorbance at 440 nm) of fibroblasts seeded on PEGDA-CMP modified with CMP-RGD and control hydrogels: PEGDA-CMP modified with CMP and PEGDA hydrogel (without CMP) modified with CMP-RGD. CMP-RGD modified PEGDA-CMP scaffolds showed higher cell proliferation compared to control samples (Student’s t test, p < 0.005). Data reported as mean ± SD.

Fig. 5

A) Fluorescence micrographs and corresponding cell area histograms of fibroblasts stained with DAPI (stains nucleus in blue) and phalloidin (stains actin in green). Cells exhibited enhanced adhesion and spread morphology on PEGDA-CMP scaffolds modified with increasing concentrations of CMP-RGD. B) Cell area histograms for cells seeded on control scaffolds showing reduced cell adhesion. Data reported as mean ± SD.

Fig. 6

A) Image of PEGDA-CMP gel partially treated with CF-CMP-RGD and CF absorbance profile across the gel. B) Cell area histograms (fibroblasts after 24 h culture) corresponding to the three areas defined in A demonstrating modulation of cell morphology across PEGDA-CMP scaffolds with RGD gradient formed by triple helical CMP association. Data reported as mean ± SD.

Fig. 7

A) PEGDA-CMP gel modified with rhodamine (red) and CF (green) labeled CMP-RGD. Local absorbance measurements for rhodamine (564 nm) and CF (493 nm) labeled peptides show opposing gradients across the gel. B) PEGDA-CMP scaffolds modified into quadrant patterns with rhodamine and CF labeled peptides. Data reported as mean ± SEM (n ≥ 3).

Scheme 1

Design of photocrosslinkable Acrl-PEG-CMP peptide prepared by solid-phase peptide synthesis.