Fabrication Of Polysaccharide-based In-situ Formable Hydrogels Via Michael Addition

In this paper, naturally derived polysaccharides(e.g., chitosan, hyaluronan, etc.) were chemical modified to obtain desired functional groups, which could provide in situ cross-linkable property via Michael addition reaction. The injectable, biocompatible composite hydrogels were prepared by self-crosslinking of functionalizaed polysaccharide derivatives, without using any extraneous chemical crosslinking agents, which have great potential to work as ideal in-situ forming tissue engineering scaffolds.CS-MA and CS-ITA were obtained by the introduction of maleoyl and itaconyl groups into chitosan N-terminal of the glucosamine units, respectively. 1H NMR, ATR-FTIR and UV-Vis results indicated the successful modification of chitosan with vinyl carboxylic acid groups. The vinyl carboxylic acid amount of obtained with optimal conditions CS-MA determined from UV-Vis method was 1860.68±42.81 μmol/g, while CS-ITA was 1068.94±26.49 μmol/g. The substitution degree of CS-ITA was lower than that of CS-MA, but the vinyl groups introduced from itaconic anhydride were more reactive than that from maleic anhydride.HA-SH and SA-SH were synthesized respectively via amide bond formation between the carboxylic acid groups and amine groups of L-cysteine in the presence of carbodiimide coupling agents. 1H NMR、UV-Vis results indicated the successful modification of hyaluronan and sodium alginate with thiol groups. The modification degree of obtained with optimal conditions HA with free thiol groups was 330.62±18.83 μmol/g(by standard Ellman’s assay), while SA was 97.35±10.19 μmol/g.The in-situ formable polysaccharide hydrogels of CS-MA/HA-SH, CS-ITA/HA-SH, and CS-MA/SA-SH was facile prepared by Michael addition reaction of the vinyl groups and thiolated anions, without using any extraneous chemical crosslinking agents. The hydrogels were characterized by oscillatory rheology, SEM, swelling ratio, etc. Results showed that the hydrogels formed in-situ within 15 min and their physical and chemical properties were dependent on the molar ratio of free thiol/vinyl groups. With increasing of vinyl ratios, storage modulus in rheological analysis increased, while porosity and swelling ratio decreased.The dual in-situ formable polysaccharide hydrogels of CS-MA/HA-SH and CS-ITA/HA-SH were facile prepared by the introduction of chitosan/β-glycerophosphate based physical crosslinked network into the initial Michael addition chemical cross-linkable gelling system. Results showed that their physical and chemical properties were also dependent on the molar ratio of free thiol/vinyl groups, which was closely consistent with the results of chemical cross-linking hydrogels. The physical crosslinked network introduced resulted in higher crosslinking density, and significantly lead to mechanical properties enhancement. Furthermore, the hydrogels(nthiol/nvinyl=4:4) reported presented obvious antibacterial activities against Staphylococcus aureus(S. aureus), which will be benefit for biomedical applications.The dual in-situ formable polysaccharide hydrogels of CS-MA/SA-SH were facile prepared by the introduction of SA-SH/Ca2+ based ionic crosslinked network into the initial Michael addition chemical cross-linkable gelling system, which could strengthen the network structure and provide self-healing ability. The rheological recovery, compression measurement, macroscopic and microscopic observation results suggested that the self-healing ability of the chemical cross-linking hydrogels might be attributed to the formation of disulfide bonds via the oxidation of unreacted free thiol groups or dynamic disulfide bonds, with healing efficiency about 79.3% and a long healing time. Ionic crosslinked network introduced could result in fast healing within 30 s with healing efficiency about 79.3%, which could not only heal with hydrogels themselves, but also with lyophilizated scaffolds. Furthermore, the tunable shape property of hydrogels could be obtained via this dual cross-linkable characteristic, which provided advantage to optimize the shape of hydrogels for more biomedical applications.