Preparation and evaluation of thermo-reversible copolymer hydrogels containing chitosan and hyaluronic acid as injectable cell carriers

Poly(N-isopropylacrylamide) end-capped with a carboxyl group (PNIPAM-COOH) was grafted to chitosan for synthesizing thermo-reversible chitosan-g-poly(N-isopropylacrylamide) (CPN), which was further grafted with hyaluronic acid (HA) to form hyaluronic acid-g-CPN (HA-CPN). PNIPAM-COOH, CPN and HA-CPN formed injectable free-flowing aqueous solutions and exhibited reversible sol-to-gel phase transition (above 5% polymer concentration) at 30 °C. Chemical properties and temperature-dependent physical properties of the polymer hydrogels, such as rheological behavior, phase transition kinetics, and water content were characterized in detail. The mechanical stiffness of hydrogels increased with the presence of chitosan in the copolymer, but decreased after conjugation with HA. Chitosan and HA grafting also endowed higher water content and resistance to volume contraction during phase change of the copolymer solution. In vitro cell culture experiments with chondrocytes and meniscus cells in HA-CPN hydrogel showed beneficial effects on the cell phenotypic morphology, proliferation, and differentiation. Progressive tissue formation was demonstrated by monotonic increases in extracellular matrix contents and mechanical properties.