Preparation and In Vitro Evaluation of Chitosan-g-Oligolactide Based Films and Macroporous Hydrogels for Tissue Engineering

In the current study, novel matrices based on chitosan-g-oligo (L,L-/L,D-lactide) copolymers were fabricated. In particular, 2D films were prepared by solvent casting, while 3D macroporous hydrogels were obtained by lyophilization of copolymer solutions. Copolymers of chitosan (Chit) with semi-crystalline oligo (L,L-lactide) (Chit-LL) or amorphous oligo (L,D-lactide) (Chit-LD) were obtained by solid-state mechanochemical synthesis. The structure of the hydrogels was found to be a system of interconnected macropores with an average size of 150 mu m. In vitro degradation of these copolymer-based matrices was shown to increase in the case of the Chit-LL-based hydrogel by 34% and decrease for the Chit-LD-based hydrogel by 23% compared to the parameter of the Chit sample. Localization and distribution of mouse fibroblast L929 cells and adipose tissue-derived mesenchymal stromal cells (MSCs) within the hydrogels was studied by confocal laser scanning microscopy (CLSM). Moreover, cellular response, namely cell adhesion, spreading, growth, proliferation, as well as cell differentiation in vitro were also evaluated in the hydrogels for 10-14 days. Both the Chit-LL and Chit-LD matrices were shown to support cell growth and proliferation, while they had improved swelling compared to the Chit matrix. Osteogenic MSCs differentiation on the copolymer-based films was studied by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Maximal expression levels of osteogenesis markers (alkaline phosphatase (ALPL), bone transcription factor (Runx2), and osteopontin (SPP1) were revealed for the Chit-LD films. Thus, osteodifferentiation was demonstrated to depend on the film composition. Both Chit-LL and Chit-LD copolymer-based matrices are promising for tissue engineering.