Chitosan (Ch) is a deacylated form of chitin, which is widely distributed in nature. As is well known, positively charged chitosan-based matrices can support cell adhesion and proliferation. However, chitosan has rather low mechanical strength and degradation rate. To overcome these limitations, various copolymers of chitosan obtained using Ch amino groups could be proposed. Thus, chitosan-based composite matrices can be obtained by copolymerization of Ch with some other monomers/oligomers without additional cross-linking agents, which allows to reduce matrix cytotoxicity. Polylactic acid (PLA) approved by FDA for clinical use, is a promising synthetic polymer for grafting chitosan. Polylactic acid meets the requirements for biomaterials due to its biocompatibility and ease of matrix formation with the desired morphology. However, rather low negative charge of the PLA surface limits cell adhesion. The combination of the properties of Ch and PLA may be of great interest: 1) to adjust the hydrophilic-hydrophobic balance of chitosan-based matrices and their mechanical properties; and 2) to enhance cell adhesion to PLA-based matrices. Mesenchymal stromal stem cells (MSC) are known to possess tri-lineage differentiation ability, including osteogenesis. By varying the properties of the matrix, one can affect this process. For example, by adopting the matrix properties it is possible to enhance the osteoinductive potential of MSC.
The aim of this study was to obtain polymer matrices in the form of films (2D) and macroporous hydrogels (3D) based on chitosan, as well as its graft copolymers with oligolactides: to study their structure and some physicochemical properties; and to evaluate the growth and differentiation of MSC on/in them in an in vitro model. For the formation of 2D films and 3D hydrogels, we used copolymers of Ch, (MW 80 kDa, DA 0.1), with oligo (L, L- and / L, D-lactides) with MW 5 kDa, (Chit-L, L and Chit-L, D, respectively). The copolymers were obtained by the method of solid-phase synthesis¹. The structure of the hydrogels studied by confocal laser microscopy represented a system of interconnected macropores with an average size of 120–140 μm. The obtained matrixes were tested by examining the toxicity of the obtained extracts using the MTT-test after previous matrix incubation with culture medium for 24 h. The surface of the copolymers matrixes was found to ensure rather good adhesion of MSC isolated from human adipose tissue (confocal microscopy), while cell growth and proliferation at long-term cultivation for 7 days in the matrices was studied by MTT-test. Chit-L,D matrices (hydrogels) were shown to improve the proliferation of MSC, while copolymer films contributed to the cell differentiation in the osteo direction. MSC differentiation was assessed by estimating alkaline phosphatase activity (light microscopy) and by qRT- PCR which confirmed the expression of ALPL, RUNX2, SPP1 gene markers of osteogenesis on day 7 and 14 after induction.
Thus, hydrogels based on copolymers of chitosan with oligolactides are promising biomaterials for regenerative medicine.