Thermosensitive heparin-poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury

Abstract

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs-based delivery system (called GFs-HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin-poloxamer (HP) hydrogel through self-assembly mode. This GFs-HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs-HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs-HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.

Keywords: basic fibroblast growth factor (bFGF); heparin-poloxamer (HP) hydrogel; nerve growth factor (NGF); neuroprotection; spinal cord injury.

FIGURE 1

Characterization of HP and GFs‐HP hydrogel. A, SEM images and the thermosensitive property of HP hydrogel. B, SEM images and the thermosensitive property of GFs‐HP hydrogels. The SEM images of both hydrogels were photographed at low magnification (left, scale bar: 200 µm) and high magnification (right, scale bar: 100 µm), respectively. Meanwhile, the temperature‐sensitive characteristic of both hydrogels was tested at 4 and 37°C condition

FIGURE 2

GFs‐HP improves the motor function recovery in SCI model rats. A, Footprint analyses of the sham, SCI, HP, GFs and GFs‐HP groups at 28 d post‐injury. B, BBB locomotion assessments of different groups at 1, 3, 7, 14, 21, 28 d post‐SCI. Values were expressed as the mean ± SEM, n = 10 per group. ^& P < .05, ^&& P < .01 vs the SCI group.^* P < .05, ^** P < .01 vs the SCI group. ^# P < .05, ^## P < .01 vs the GFs group

FIGURE 3

GFs‐HP decreases the damage of tissue structure and the loss of neurons at 28 d after SCI. A, Representative images from HE staining (40× and 200×) and Nissl staining (200×) on transverse sections in sham, SCI, HP, GFs and GFs‐HP groups at 28 dpi. Scale bar: 500 µm for low magnification images and 100 µm for high magnification ones. B, Counting analysis of the number of VMNs from (A) of HE staining. C, Quantitative analysis of the Nissl bodies numbers from Nissl staining. Values were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group. ^* P < .05, ^** P < .01 vs the SCI group. ^# P < .05, ^## P < .01 vs the GFs group

FIGURE 4

GFs‐HP hydrogel enhances neuronal survival at 28 d after SCI. A, Fluorescence images of transverse sections show the expressions of NeuN in the sham, SCI, HP, GFs and GFs‐HP groups. Scale bar = 100 µm. B, Quantifying the number of NeuN⁺‐positive cells from (A). Results were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group, ^* P < .05 vs the SCI group, ^# P < .05 vs the GFs group

FIGURE 5

GFs‐HP hydrogel reduces neuronal apoptosis at 28 d after SCI. A, Representative immunoblotting bands of Bcl‐2, BAX, cleaved caspase‐3 in the sham, SCI, HP, GFs and GFs‐HP groups. GAPDH was used as was used as the loading control and for band density normalization. B‐D, The optical density analysis of Bcl‐2, BAX, cleaved caspase‐3 proteins in five groups. Values were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group. ^* P < .05, ^** P < .01, ^*** P < .001 vs the SCI group. ^# P < .05 vs the GFs group

FIGURE 6

GFs‐HP hydrogel promotes axonal growth at 28 d after SCI. A, Representative fluorescence images for GAP43 of transverse sections from the injured spinal cord in each group. Scale bar = 50 µm. B, Protein expressions of GAP43 in each group via Western blotting detection. C, Densitometric analyses of GAP43 from (B). GAPDH was used for band density normalization. Values were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group. ^*** P < .001 vs the SCI group. ^### P < .001 vs the GFs group

FIGURE 7

GFs‐HP hydrogel promotes axonal regeneration and attenuates the reactive astrogliosis at 28 d after SCI. A, Immunohistochemical staining of NF‐200 and GFAP of longitudinal sections in the sham, SCI, HP, GFs and GFs‐HP groups at 28 d after SCI. Scale bar = 50 µm. B‐C, Quantitative analysis of GFAP and NF‐200‐positive area from A. D, Western blotting detected the protein levels of GFAP and NF‐200 in each group, GAPDH served as a protein loading control. E‐F, Quantification of GFAP and NF‐200 from D. Values were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group. ^* P < .05, ^*** P < .001 vs the SCI group. ^# P < .05, ^## P < .01 vs the GFs group

FIGURE 8

GFs‐HP hydrogel treatment through receptor‐medicated PI3K/AKT and MAPK/ERK activation. A, Immunoblotting for TrkA and FGFR1 in each group. B‐C, The optical density analysis of TrkA and FGFR1 from A. D, Immunoblotting for P‐AKT, AKT, P‐ERK and ERK in the spinal cord segment at the contusion epicentre. GAPDH was used for band density normalization. E‐F, Quantifying the ratio of p‐AKT/AKT, p‐ERK/ERK from (D). Values were expressed as the mean ± SEM, n = 4 per group. ^&&& P < .001 vs the SCI group. ^* P < .05, ^** P < .01, ^*** P < .001 vs the SCI group, ^# P < .05, ^## P < .01, ^### P < .001 vs the GFs group