In vitro characterization of injectable collagen and collagen-genipin hydrogels for neural tissue engineering
Li, Jingwen 1990-
Nervous system injury leads to the permanent loss of sensory and motor functions. Injectable hydrogel containing therapeutic agents can be directly injected to the injured cavity as a promising approach for minimally-invasive treatment of nerve injury. However, such injectable hydrogels have not been well developed and documented in the literature. As inspired, this project aims to develop injectable collagen-based gels for nerve injury repair and to characterize in vitro for supporting neurite outgrowth of dorsal root ganglia (DRG) explants and dissociated neurons. To develop collagen-based gels, collagen at varying concentrations (e.g. 1.5, 2 and 2.5 mg/mL) were used to form gels under physiological conditions and genipin (0.25-5 mM) were applied as the chemical crosslinker. Characterization studies showed that collagen-based hydrogels could form porous and fibrillary gels within a time period of 40 s at 37 °C and genipin could significantly improve the mechanical property of gels and the resistance to degradation. To evaluate the cytotoxicity of the injectable hydrogels and compare the cell behaviour in two-dimensional (2D) and three-dimensional (3D) environments, rat primary Schwann cells (PRSCs) were seeded onto and encapsulated within the gels, and the cell viability was examined at Day 3 by the Live/Dead assay. The results showed that collagen gels provide superior support for PRSCs survival in both 2D and 3D cultures, for example, with a cell viability of 96 % and 95 %, respectively, for the collagen gel with a concentration of 1.5 mg/mL. Collagen chemically crosslinked by genipin at 0.25 and 0.5 mM exhibited a permissive but less favorable environment to PRSCs comparing with pure collagen. Genipin over 1 mM inhibited the PRSCs survival significantly in both 2D and 3D cultures. DRG explant and dissociated neuron cultures were examined as in vitro cell models to evaluate the therapeutic efficacy of collagen and collagen-genipin gels for nerve injury repair and the cellular response was also characterized and compared to each other. Preliminary 2D cultures were shown to greatly support neurite extension and 2.5 mg/mL collagen gel supported the most neurite extension and branching development. It was shown that genipin had a significant effect on the neurite density but not neurite length of DRG explants, whereas the dissociated neurons were more sensitive to genipin. Enrichment of culture medium with nerve growth factor (NGF) could significantly enhance the neurite length and density. PRSCs as the supportive cells were co-cultured with DRG explants/dissociated neurons in 3D hydrogels. Confocal microscopy showed that the neurites of DRG explants and dissociated neurons could extend freely within the physical collagen gels, and dissociated neurons exhibited pseudo-unipolar phenotype in 3D environment indicating true axonal extension. Moreover, genipin had a significantly inhibitory effect on dissociated neurons whereas the explants were more tolerant to genipin possibly due to the preserved cellular components and interactions. It was also shown that hydrogels infiltrated with PRSCs could enhance the neurite elongation and branches dramatically. Our research has determined the therapeutic potency of injectable collagen-based gels containing the PRSCs for nerve injury repair and gained new insights into the use of the injectable gel as a delivery substrate in neural tissue engineering.
DegreeMaster of Science (M.Sc.)
SupervisorCHEN, DANIEL; SCHREYER, DAVID
CommitteeHEDAYAT, ASSEM; ZHU, NING; MEDA, VENKATESH
Copyright DateAugust 2016
injectable hydrogel, collagen, genipin, nerve injury repair, tissue engineering