Dual mechanism β-amino acid polymers promoting cell adhesion

Abstract

Cell adhesion has tremendous impact on the function of culture platforms and implants. Cell-adhesive proteins and peptides have been extensively used for decades to promote cell adhesion, however, their application suffers from their easy enzymatic degradation, difficulty in large-scale preparation and expensiveness. To develop the next-generation cell-adhesive materials, we mimic the cell adhesion functions and mechanisms of RGD and KRSR peptides and design cell-adhesive cationic-hydrophobic amphiphilic β-amino acid polymers that are stable upon proteolysis and easily prepared in large scale at low cost. The optimal polymer strongly promotes cell adhesion, using preosteoblast cell as a model, by following dual mechanisms that are independent of sequence and chirality of the statistic copolymer. Our strategy opens avenues in designing the next-generation cell-adhesive materials and may guide future studies and applications.

Fig. 1. High-throughput screening of β-amino acid polymers for preosteoblast cell adhesion.

a Cell adhesion mechanisms of RGD, KRSR, and β-amino acid polymer-modified surfaces. b Subunits to compose β-amino acid polymers. All subunits are racemic and the resulting polymers are heterochiral. c The general synthesis of the 76 β-peptide polymers with a chain length of 20 amino acid residue. d An OEG8 antifouling layer was used to resist protein adsorption and non-specific cell adhesion to the substrate. e Thiol-terminated polymers were covalently attached to maleimide-functionalized glass surface for high-throughput screening of cell adhesion, using RGD (RGDSPC) and KRSR (KRSRGYC) peptide-modified surfaces for comparison. f High-throughput screening of cell adhesion evaluated by fluorescence scanning and quantification on live/dead stained cells, calcein AM (green) for live cells. g Heat map showing the relationship of preosteoblast adhesion vs. polymer composition. The fluorescence intensity was normalized with RGD-modified surface. h Micrograph of F-actin (green) stained preosteoblast cell adhesion to surfaces modified with DM_xCO_y (x + y = 100, x = 40, 50, 60, 70, 80, 90), RGD, KRSR, and OEG8, respectively. Scale bar: 500 μm. i XPS spectra of bare glass, NH₂-glass, OEG8, and DM₅₀CO₅₀-modified surfaces. j 3D AFM images of bare glass, RGD, and DM₅₀CO₅₀-modified surfaces. k Water contact angle of bare glass, OEG8, RGD, and DM₅₀CO₅₀-modified surfaces.

Fig. 2. Adhesion and proliferation of preosteoblast cells.

a–c Fluorescence confocal microscope images (green, actin; red, vinculin; blue, nucleus) of preosteoblast cells on RGD, KRSR, and DM₅₀CO₅₀-modified surfaces after cell seeding for 3 h (a) and 24 h (b), Scale bar: 50 μm. Lower magnification images (c) after cell seeding for 3 and 24 h, Scale bar: 100 μm. d Cell proliferation after 1, 3, and 5 days of culture. Quantification of cell density was done with Alamar Blue assay. e Bright field images of preosteoblast cells cultured on NH₂, RGD, KRSR, and DM₅₀CO₅₀-modified surfaces for 5 days. Scale bar: 100 μm. Data (in d) represent mean ± s.d. (n = 3). Statistical analysis: one-way ANOVA with Tukey post-test, ^#p < 0.05, ***p < 0.0001.

Fig. 3. RNA-seq analysis on DM₅₀CO₅₀-modified surface compared to RGD-modified surface.

a From 17,267 gene transcripts in analysis, 305 differentially expressed genes showed greater than 50% change in expression level after 2 days of culture. b Significant GO terms of associated biological processes, cellular components, and molecular functions from differentially regulated genes. c KEGG pathway enrichment. The y-axis label represents KEGG pathway, and the x-axis shows -lgP. The size of the dots represents the number of gene enrichment.

Fig. 4. Cells adhesion via both integrin-dependent and -independent mechanisms.

a Fluorescent micrographs of live/dead stained cells, b cell area analysis, and c cell density analysis, of cell adhesion to DM₅₀CO₅₀ and RGD-modified surfaces in serum-containing media for 3 h, with or without EDTA treatment. d Fluorescent micrographs, e cell area analysis, and f cell density analysis, of cell adhesion to DM₅₀CO₅₀ and RGD-modified surfaces in serum-free media for 3 h, with or without EDTA treatment. g Cell adhesion mechanisms of DM₅₀CO₅₀ and RGD-modified surfaces in different conditions. Data in (b, c, e, f) represent mean ± s.d. (n = 9). Statistical analysis: two-tailed t-test, ***p < 0.0001, ns = not significant.

Fig. 5. Effect of adsorbed serum proteins on preosteoblast cell adhesion to DM₅₀CO₅₀-modified surfaces.

a The amount of surface-adsorbed total protein after variable surfaces were incubated with a serum-containing cell culture medium for 2 h. b, c AFM images of DM₅₀CO₅₀ and RGD-modified surfaces after incubation with serum-containing cell culture medium (b) and pure fibronectin (c) for 2 h. d The amount of surface-adsorbed individual protein (fibronectin, vitronectin, collagen, laminin) on DM₅₀CO₅₀ and RGD-modified surfaces after cells were cultured on the surfaces for 2 days. Fluorescence micrographs of: e live/dead stained cells, f spreading area, and g density analysis, of cells 2 h after seeding on DM₅₀CO₅₀ and RGD-modified surfaces, which were first incubated with serum-containing medium and then treated with or without integrin αvβ3 before cell seeding. h Carton illustration of results in (e–g). Data in (a, d, f, g) represent mean ± s.d. (n = 3 for a, d and n = 9 for f, g). Statistical analysis: two-tailed t-test, **p < 0.001, ***p < 0.0001.

Fig. 6. Effect of membrane proteins and polysaccharides on preosteoblast cell adhesion.

Fluorescent micrographs of: a live/dead stained cells, b spreading area, and c density analysis, of cells adhered to RGD, KRSR, and DM₅₀CO₅₀-modified surfaces for 1 day in a serum-free medium. Cells were treated with plasmin, collagenase, heparinase, and hyaluronidase, respectively, before seeded on the surfaces. Untreated cells were used as the control. Data in (b, c) represent mean ± s.d. (n = 9). Statistical analysis: one-way ANOVA with Tukey post-test, *p < 0.01.

Fig. 7. Demonstration of cell adhesion impact of DM₅₀CO₅₀-incorporated hydrogel on in vivo bone regeneration using a critical-size rat calvarial defect model.

a Synthesis of bare PEG hydrogel and DM₅₀CO₅₀-modified PEG hydrogel. b Comparison before and after PEG hydrogel swelling. c Preosteoblast cells adhesion to bare PEG hydrogel and DM₅₀CO₅₀-modified PEG hydrogel after cell seeding for 2 days. Cells were subjected to live/dead staining. d Masson’s trichrome stain, e 3D micro-CT analysis, and f bone volume analysis, of the cranial bone samples from bare PEG hydrogels, DM₅₀CO₅₀-modified PEG hydrogels, GelMA, and PLA after 8 weeks of implantation. Data in (f) represent mean ± s.d. (n = 6). Statistical analysis: two-tailed t-test, *p < 0.05.