Hydrogels Based on Dual Curable Chitosan-graft-Polyethylene Glycol-graft-Methacrylate: Application to Layer-by-Layer Cell Encapsulation

Abstract

Ultraviolet (UV) photo-cross-linkable hydrogels have been commonly used for three-dimensional (3D) encapsulation of cells. Previous UV cross-linkable hydrogels have employed one-shot hardening of mixtures of hydrogels and cells. Here we propose an alternative method of making hydrogel-encapsulated cell constructs through layer by layer (LBL) buildup of alternating layers of cells and hydrogel. The LBL method potentially permits better spatial control of different cell types and control of cell orientation. Each hydrogel layer must be hardened before deposition of the next layer of cells. A UV-curable gel precursor that can also be gelled at physiological temperature is desirable to avoid repeated UV exposure of cells after deposition of each successive hydrogel layer. We designed, synthesized, and applied such a precursor, dual-curable-both thermoresponsive and UV-curable-chitosan-graft-polyethylene glycol-graft-methacrylate (CEGx-MA) copolymer (x is the PEG molecular weight in Daltons). We found that CEG350-MA copolymer solutions (5 wt % polymer) formed physical gels at approximately 37 degrees C and could be further photopolymerized to form thermally stable dual-cured hydrogels. This material was applied to the creation of a two-layer LBL smooth muscle cell (SMC)/hydrogel construct using temperature elevation to approximately 37 degrees C to gel each hydrogel layer. The physically gelled two-layered hydrogel/cell construct was finally exposed to a single UV shot to improve its mechanical properties and render it thermally stable. CEG350-MA solution and gel are nontoxic to SMCs. Cells remained mostly viable when they were encapsulated inside both physically gelled and dual-cured CEG350-MA and suffered little damage from the single brief UV exposure. The combination of LBL tissue engineering with a dual curable hydrogel precursor such as CEG350-MA permits the buildup of viable thick and complex tissues in a stable, biocompatible, and biodegradable matrix.