Peptide-Functionalized Hydrogels Modulate Integrin Expression and Stemness in Adult Human Epidermal Keratinocytes

Abstract

The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through β-integrin signaling. Wound-healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)-based hydrogel formulations with mechanical properties (elastic modulus, E = 20.9 ± 0.56 kPa) and bioactive peptide sequences that mimic the epidermal ECM. These hydrogels enable systematic investigation of the influence of cell-binding domains from fibronectin (RGDS), laminin (YIGSR), and collagen IV (HepIII) on keratinocyte stemness and β₁ integrin expression. Quantification of 14-day keratin protein expression shows four hydrogels improve stemness compared to standard techniques. Three hydrogels increase β₁ integrin expression, demonstrating a positive linear relationship between stemness and β₁ integrin expression. Multifactorial statistical analysis predicts an optimal peptide combination ([RGDS] = 0.67 mm, [YIGSR] = 0.13 mm, and [HepIII] = 0.02 mm) for maintaining stemness in vitro. Best-performing hydrogels exhibit no decrease in Ki-67-positive cells compared to standards (15% decrease, day 7 to 14; p < 0.05, Tukey Test). These data demonstrate that precisely designed hydrogel biomaterials direct integrin expression and promote proliferation, improving the regenerative capability of cultured keratinocytes for basic science and translational work.

Keywords: extracellular matrix; hydrogels; keratinocytes; peptides; poly(ethylene glycol).

Figure 1.

a) Hydrogels were synthesized by combining PEG-norbornene with dithiothreitol (DTT), short cysteine-terminated peptide sequences, and a photoinitiator in the presence of 365 nm light to produce peptide-functionalized cell culture platforms. b) Parallel-plate rheology results confirmed that hydrogel platform mechanical properties could be tuned to match those of human skin (E ≈ 15–25 kPa).^[16] Columns report mean ± SEM, n = 5. c) Dynamic rheological characterization during polymerization confirmed that the network was fully formed within 60 s of exposure to 365 nm light. Symbols represent mean ± SEM, n = 5).

Figure 2.

a) Representative fluorescent images of K₁₄ (red) and K₁ (green) immunostaining on the standard and one of the best-performing hydrogel surfaces. Scale bar, 50 μm. b) Keratinocyte stemness on hydrogel (gray) and standard (white) platforms representing a multifactorial screen measured by calculating the ratio of K₁₄ to K₁ corrected total cell fluorescence. Columns represent mean ± SEM, n = 3. c) Prediction profiles of stemness versus binding motif concentrations showed trends that can maximize stemness. Shaded region represents 95% confidence interval of multifactorial analysis. d) Main and interaction effects of binding motif concentrations in hydrogel platforms. Bars represent parameter estimates.

Figure 3.

a) Representative fluorescent images of the standard and one of the best-performing hydrogel surfaces for β₁ integrin expression (green) show increased levels of β₁ expression on hydrogels versus the standard. Scale bar, 50 μm. b) β₁ integrin expression as measured by corrected total cell fluorescence at day 2 on hydrogel platforms (dark gray) and standards (light gray). Columns represent mean ± SEM, n = 4. c) Keratinocyte stemness (K₁₄:K₁) plotted versus β₁ integrin expression. Dark gray dots represent hydrogel platform mean values and white dots represent standard platform mean values. Fit line is the result of a linear regression and indicates a positive correlation.

Figure 4.

a) Representative fluorescent microscopy images comparing proliferation (Ki-67, green) between one of the best-performing hydrogels and the standard platform on day 14. Scale bar, 1 mm. b) Quantification of Ki-67-positive cells demonstrates no significant difference in proliferation on the hydrogel over 14 days, while a significant decrease was observed on the standard between days 7 and 14 (p < 0.05, Tukey Test). Columns represent mean ± SEM, n = 4.