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Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.

The effects of tether length on cell adhesion to poly(methyl methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean separation between integrin-bound peptides of 15.6 +/- 1.4 nm for combs with long (22-mer) tethers, compared with 17.5 +/- 1.3 nm for short (10-mer) tethers, on films of comparable peptide density (approximately 2500 peptides/microm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addition, adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equivalent peptide density.

Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, N-carboxyanhydride polypeptide, poly(γ-propargyl-l-glutamate) (PPLG) macromers were generated by fully grafting the "clickable" side chains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by cross-linking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of cross-link density. Further, PPLG-containing gels, which retain an α-helical chain conformation, were more effective than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favorable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.

Part 1 of these studies described poly(methyl methacrylate-r-polyoxyethylene methacrylate) P(MMA-r-POEM) comb polymers that present Arg-Gly-Asp (RGD) peptides at a surface in nanoscale clusters on a protein-resistant background for control of cell adhesion. Here in part 2, we examine surface segregation of these peptide-modified and unmodified comb polymers blended with polylactide (PLA) as a self-assembly approach suitable for surface modification of porous tissue engineering scaffolds. Multiple thermodynamic driving forces for surface enrichment of the comb polymer are exploited by annealing PLA/P(MMA-r-POEM) blends above the glass transition of the blend components but below the melting point of PLA, while in contact with water. Predictions of the interfacial composition profiles of annealed blends were made using a self-consistent field (SCF) lattice model. The calculations predict strong enrichment of the comb in the top approximately 50 A of blends, and organization of comb molecules in quasi-2D conformations at the interface, similar to the apparent structure of pure comb surfaces in contact with water described in part 1. Experimentally, PLA/comb blend surfaces were characterized by contact angle measurements, XPS, quantification of ligand-cluster surface density and stability by AFM and fluorescent nanosphere labeling, and cell attachment assays. These data were consistent with SCF predictions, showing significant enrichment of the comb at water-annealed surfaces and RGD cluster densities consistent with 2D conformations for comb molecules in the surface layer. Bulk miscibility of the blends was verified by dynamic rheometry, small-angle neutron scattering, DSC and X-ray diffraction studies. Surface segregation of combs provided tunable cell adhesion on PLA through surface-localized nanoclusters of RGD atop a cell-resistant background.

Theoretical and experimental studies were conducted to elucidate the structure and properties of amphiphilic comb polymer thin films presenting nanoscale clusters of Arg-Gly-Asp (RGD) peptides for control of cell adhesion on biomaterials. Combs comprised of a poly(methyl methacrylate) backbone and short poly(ethylene oxide) side chains were synthesized, and peptides were tethered to the side chain ends to create nanoscale peptide clusters. In thin films, comb polymers containingor = 30 wt % six to nine unit PEO side chains completely resisted adhesion of a model fibroblast cell line in the presence of 7.5% serum over 24 h. These same polymers modified with RGD peptides elicited tunable cell adhesion when mixed with unmodified combs in varying proportion. A self-consistent field lattice model of the interface between comb polymer films and water predicts an organization of the top molecular layer of comb polymer with the backbone oriented parallel to the interface in quasi-two-dimensional confinement and hydrophilic side chains extended in a brushlike layer into solution. This picture of a quasi-2D configuration is consistent with the observed surface properties of comb films in water as well as measurements of the RGD cluster density on mixed comb surfaces using fluorescent nanosphere labeling of ligand clusters.