Local delivery of FTY720 and NSCs on electrospun PLGA scaffolds improves functional recovery after spinal cord injury

Abstract

Spinal cord injury (SCI) is a common issue in the clinic that causes severe motor and sensory dysfunction below the lesion level. FTY720, also known as fingolimod, has recently been reported to exert a positive effect on the recovery from a spinal cord injury. Through local delivery to the lesion site, FTY720 effectively integrates with biomaterials, and the systemic adverse effects are alleviated. However, the effects of the proper mass ratio of FTY720 in biomaterials on neural stem cell (NSC) proliferation and differentiation, as well as functional recovery after SCI, have not been thoroughly investigated. In our study, we fabricated electrospun poly (lactide-co-glycolide) (PLGA)/FTY720 scaffolds at different mass ratios (0.1%, 1%, and 10%) and characterized these scaffolds. The effects of electrospun PLGA/FTY720 scaffolds on NSC proliferation and differentiation were measured. Then, a rat model of spinal transection was established to investigate the effects of PLGA/FTY720 scaffolds loaded with NSCs. Notably, 1% PLGA/FTY720 scaffolds exerted the best effects on the proliferation and differentiation of NSCs and 10% PLGA/FTY720 was cytotoxic to NSCs. Based on the Basso, Beattie, and Bresnahan (BBB) score, HE staining and immunofluorescence staining, the PLGA/FTY720 scaffold loaded with NSCs effectively promoted the recovery of spinal cord function. Thus, FTY720 properly integrated with electrospun PLGA scaffolds, and electrospun PLGA/FTY720 scaffolds loaded with NSCs may have potential applications for SCI as a nerve implant.

Fig. 1. Scanning electron microscopy images of PLGA (A), PLGA/FTY720 (0.1%) (B), PLGA/FTY720 (1%) (C) and PLGA/FTY720 (10%) (D). The average fibre diameter (E). (*P < 0.05, n = 3).

Fig. 2. Water contact angles of PLGA, PLGA/FTY720 (0.1%), PLGA/FTY720 (1%) and PLGA/FTY720 (10%). (*P < 0.05, n = 3).

Fig. 3. Proliferation of NSCs cultured with PLGA, PLGA/FTY720 (0.1%), PLGA/FTY720 (1%) and PLGA/FTY720 (10%) for 1, 3, and 7 days in vitro. (*P < 0.05, n = 3).

Fig. 4. Differentiation of NSCs. Immunofluorescence staining of differentiated neurons, astrocytes and oligodendrocytes in different groups. Immunostaining was conducted using antibodies against Oligo (green) to label oligodendrocytes, Tuj-1 (green) to label neurons, and GFAP (green) to label astrocytes, as well as DAPI (blue) to label nuclei. The scale bar represents 200 µm.

Fig. 5. qPCR analysis of GFAP (A), Tuj-1 (B) and Oligo (C) expression after NSCs were cultured for 7 days with the different scaffolds. (*P < 0.05, n = 3).

Fig. 6. BBB scores of SCI model rats. (*P < 0.05, n = 5).

Fig. 7. HE staining of spinal cord tissues at 6 weeks after the operation. Control group (A), PLGA group (B), PLGA/FTY720 group (C) and PLGA-FTY720/NSC group (D). The scale bar represents 200 µm. (E) The average cavity areas in spinal cord tissues from different groups. (*P < 0.05, n = 3).

Fig. 8. Immunofluorescence staining of neurons in the injury site after SCI. Images of spinal cord tissues from the control group (A), PLGA group (B), PLGA/FTY720 group (C) and PLGA-FTY720/NSC group (D) are shown. (E) Mean number of NeuN-positive cells per visual field.