Thermosensitive quaternized chitosan hydrogel scaffolds promote neural differentiation in bone marrow mesenchymal stem cells and functional recovery in a rat spinal cord injury model

摘要

A thermosensitive quaternary ammonium chloride chitosan/beta-glycerophosphate (HACC/beta-GP) hydrogel scaffold combined with bone marrow mesenchymal stem cells (BMSCs) transfected with an adenovirus containing the glial cell-derived neurotrophic factor (GDNF) gene (Ad-rGDNF) was applied to spinal cord injury (SCI) repair. The BMSCs from rats were transfected with Ad-rGDNF, resulting in the expression of GDNF mRNA in the BMSCs increasing and their spontaneous differentiation into neural-like cells expressing neural markers such as NF-200 and GFAP. After incubation with HACC/beta-GP hydrogel scaffolds for 2 weeks, neuronal differentiation of the BMSCs was confirmed using immunofluorescence (IF), and the expression of GDNF by the BMSCs was detected by Western blot at different time points. MTT assay and scanning electron microscopy confirmed that the HACC scaffold provides a non-cytotoxic microenvironment that supports cell adhesion and growth. Rats with SCI were treated with BMSCs, BMSCs carried by the HACC/beta-GP hydrogel (HACC/BMSCs), Ad-rGDNF-BMSCs, or Ad-rGDNF-BMSCs carried by the hydrogel (HACC/GDNF-BMSCs). Animals were sacrificed at 2, 4, and 6 weeks of treatment. IF staining and Western blot were performed to detect the expression of NeuN, NF-200, GFAP, CS56, and Bax in the lesion sites of the injured spinal cord. Upon treatment with HACC/BMSCs, NF200 and GFAP were upregulated but CS56 and Bax were downregulated in the SCI lesion site. Furthermore, transplantation of HACC/GDNF-BMSCs into an SCI rat model significantly improved BBB scores and regeneration of the spinal cord. Thus, HACC/beta-GP hydrogel scaffolds show promise for functional recovery in spinal cord injury patients.