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.

Keratinocyte-Specific Peptide-Based Surfaces for Hemidesmosome Upregulation and Prevention of Bacterial Colonization

“…However, we have previously shown that GL13K does not interfere with early osseointegration in a rabbit model compared to noncoated controls . Responses, or lack thereof, to LamLG3 are likely integrin mediated, as HGFs and OKs have different integrin expression profiles. , An exact, mechanistic understanding of our observed cell selectivity phenomena is one branch of our current work which may enable further peptide-mediated control of keratinocytes . While past work has successfully engineered cell-specific surfaces, , to the best of our knowledge this is first report of a cell-specific, multifunctional peptide coating.…”

Pulmonary fibrosis distal airway epithelia are dynamically and structurally dysfunctional

“…Norbornene-functionalized poly(ethylene glycol) was synthesized following established protocols 73 . Briefly, PEG-hydroxyl (PEG-OH; MW 40 kg/mol, 8-arm, hexaglycerol core; JenKemUSA) was combined with a 5× molar ratio of pyridine (Fisher Scientific) and a 0.5× molar ratio of 4-(dimethylamino)pyridine (DMAP; Sigma-Aldrich) with respect to PEG hydroxyls in dichloromethane (DCM; Fisher Scientific).…”

“…Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) photoinitiator was synthesized utilizing established methods 73 . Briefly, equimolar amounts of dimethyl phenylphosphonite (Alfa Aesar) and 2,4,6-trimethylbenzoylphosphonite (Acros Organics) were added to a round-bottom flask and stirred overnight at room temperature under argon.…”

“…PEG-NB hydrogels were polymerized using established methods 73 . The hydrogel prepolymer solution was created by combining 5 wt% 8-arm, 40 kg/mol PEG-NB, dithiothreitol (DTT; Sigma-Aldrich), 0.05 wt% LAP, and 2 mM of CGRGDS peptide in sterile phosphate buffered saline (PBS, pH = 7.2; Life Technologies).…”

“…The elastic modulus (E) of swollen hydrogel samples was measured utilizing established methods 73 . Briefly, cylindrical hydrogel samples (volume = 50 uL) were polymerized between hydrophobic glass slides separated by a 0.5 mm silicone gasket, allowed to swell to equilibrium in PBS, and then cut to 8 mm diameter with a circular punch.…”