Work

A technique to create hydrogels with tethered concentration gradients of molecules in vitro

Year: 2025

Type: article

Abstract: mimics of aspects of biological environments, such as the environment around a hydrogel implanted in the body for cell delivery. We use a well-defined synthetic scaffold with a poly(ethylene glycol) (PEG)-norbornene backbone cross-linked with a matrix metalloproteinase (MMP)-degradable peptide, a standard material for cell encapsulation. The method to tether molecular concentration gradients is validated using a fluorescent PEG-thiol (FITC-PEG-SH), an ideal polymer. We first create a calibration curve by measuring the fluorescence intensity of hydrogels with known uniform concentrations of the tethered fluorescent molecule. The calibration curve is used to calculate spatial concentration from measured fluorescence intensity in hydrogels with polymer or protein concentration gradients. FITC-PEG-SH is diffused through our hydrogel in a flow chamber for 6, 24 and 48 hours. We make consistent gradients and quantify the concentration of the fluorescent molecule every 25 µm along the material. Next, we make tethered concentration gradients of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine found in the wound environment, after 24 hours of diffusion. These gradients are consistent when normalized by the concentration at the edge of the hydrogel, which varies due to pore clogging. From both molecular concentration gradients, we calculate an effective diffusion coefficient that is the same order of magnitude as the value calculated using the multiscale diffusion model. Significant advances made with this technique include limited confinement of the material, which enables isotropic swelling and facile nutrient diffusion, the ability to image through the device and the same hydrogel rheological properties across samples. This technique can be used in future work to characterize cell-laden hydrogels which present the same physical cues to cells and tethered concentration gradients of chemical cues using microrheology.

Source: Soft Matter

DOI: 10.1039/d5sm01194a

Authors: Thomas C. O’Shea, Ahmad Salem, Kelly M. Schultz

Institutions: Purdue University West Lafayette, Lehigh University

Language: English

Topic: Hydrogels: synthesis, properties, applications

Subfield: Molecular Medicine

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Funders: National Institutes of Health, National Institute of General Medical Sciences

Awards: G4265174288, G4617297381