Spinal cord injury (SCI) produces an inflammatory microenvironment characterized by damage-associated molecular patterns (DAMP) and immune cell activation, which exacerbates secondary injury and impairs neurological recovery. In addition, unbalanced polarization of macrophages occurred in the injured spinal cord. The activation levels of proinflammatory M1 phenotype macrophages continued to rise, while the activation levels of potentially restorative M2 phenotype macrophages decreased, resulting in prolonged inflammation duration and tissue remodeling failure. On the other hand, SCI inflammatory microenvironment is not conducive to the survival, migration and differentiation of neural stem cells, which seriously impedes the axon growth and nerve differentiation and regeneration of endogenous neural stem cells/progenitor cells after injury, and affects the reconstruction and functional recovery of neural networks in the injured area. Therefore, the reconstruction of SCI immunoinflammatory microenvironment is crucial to improve the effect of spinal cord regeneration and repair.
Figure 1: Schematic illustration of an immune-regulatory hydrogel scaffold promoting nerve regeneration after spinal cord injury
Left: photocrosslinked gelatin hydrogel modified with cationic polymer polyamide and anti-inflammatory cytokine IL-10. The scaffold promotes M2 macrophage polarization and neuronal differentiation in vitro. Right: Transplant a bifunctional immunomodulatory scaffold to the site of spinal cord injury in mice, where DAMPs are cleared, inflammation is reduced, and tissue remodeling and nerve regeneration are enhanced.
Figure 2: Characterization of hydrogel scaffolds
(A) SEM images of GL(gelatin), SCAV-GL (gelatin +PAAM-G3), Delv-GL(gelatin +IL-10) and Scav/Delv-GL(gelatin +PAAM-G3 +IL-10) scaffolds showed that all scaffolds had similar pore sizes ranging from 60 to 180μm. (B) Compression modulus of the support. (C) In vitro degradation rates of hydrogel scaffolds at days 7, 14, 21, and 28. (D) Percentage of total protein clearance in DAMP solution. (E) Levels of HMGB1 and (F) extracellular DNA after incubation with the SCAV-GL scaffold. (D-F) showed that the amount of cell-free DNA(cfDNA) and HMGB1 protein (anionic DAMP) and the amount of total protein in solution were reduced after treatment with scaffolds containing PAAM-G3. (G) After one day, Delv-GL and Scav/Delv-GL hydrogels released approximately 24.4% and 23.7% of the loaded IL-10, respectively, followed by a gradual release of IL-10, with approximately 52.4% of IL-10 releasing 21d in the SCAV/Delv-GL scaffold. The addition of PAMAM-G3 to the hydrogel had no significant effect on the sustained release behavior of IL-10.
Figure 3: Regulation of proinflammatory activity of macrophages and microglia by Scav/Delv-GL scaffolds
Quantitative expression of (A) interleukin (IL-1β), (B) TNF-α and (C) iNOS in M1-polarized RAW264.7 cells after DAMPs stimulation and treatment with PBS, GL, SCAV-GL, Delv-GL or Scav/Delv-GL scaffolds. (D)iNOS immunofluorescence staining, the number of iNOS positively stained cells decreased in the Scav /Delv-GL group. (E) gene expression levels of IL-1β, (F)TNF-α and (G)iNOS. (H) Immunofluorescence images of iNOS of M1-polarized BV2 cells treated with PBS, GL, SCAV-GL, Delv-GL, or Scav/Delv-GL scaffolds after DAMPs stimulation. The iNOS expression in the Scav/Delv-GL treatment group was significantly lower than that in all other groups. These results suggest that immunomodulatory scaffolds (SCAV-GL, Delv-GL, and Scav/Delv-GL) significantly inhibit DAMP-induced pro-inflammatory responses in macrophages and microglia.
Figure 4: The role of Scav/Delv-GL scaffolds in neuronal differentiation
Immunostaining image of tubulin (Tuj-1), a marker of neurospecific differentiation (neuron: Tuj-1, green; Nucleus: DAPI, blue.) . After adding DAMPs to the neurogenic differentiation medium, few Tuj-1 positive cells were observed, indicating the inhibitory effect of DAMPs on neurogenesis. Blank hydrogels (GL) did not mitigate the effects of DAMPs on NSCs. The number of Tuj-1 positive nerve cells in the Scav/Delv-GL and SCAV-GL groups was significantly higher, indicating greater neurogenic differentiation. The number of Tuj-1 positive cells exposed to DAMPs also increased in the Delv-GL group, but were less differentiated than in the Scav/Delv-GL and SCAV-GL treatment groups.
Figure 5: In the acute phase, implantation of Scav/Delv-GL scaffolds reduced the expression of pro-inflammatory cytokines and reduced the number of macrophages and microglia at the lesion site
(A) Heat map analysis of changes in levels of inflammatory cytokines in the spinal cord tissue of the control group, group GL, group SCAV-GL, group Delv-GL and group Scav/Delv-GL on the 7th day after SCI showed that the concentration of pro-inflammatory cytokines in group Scav/Delv-GL was significantly reduced. (B) Images of IL-1β and TNF-α expression in spinal cord tissue of SCI mice in each group, and (C-D) is quantitative analysis of each group in (B). (E-J) The distribution and immunophenotype of macrophages and microglia in the acute lesion area of SCI were observed by immunostaining, and the expressions of ED1(reactive microglia/phage marker), F4/80(macrophage marker) and Iba-1(microglia marker) were detected. The images showed that the number of positively stained macrophages and microglia in the Scav/ Delv-GL group was significantly lower than in the other four groups. The results showed that implantation of Scav/Delv-GL and SCAV-GL scaffolds at the site of spinal cord injury could reduce the secretion of pro-inflammatory cytokines in the acute phase.
Figure 6: Scav/Delv-GL scaffold implantation of inflammatory cell subtypes
(A-D) immunofluorescence staining image and quantitative analysis of iNOS and ARG-1 in control group, GL, SCAV-GL, Delv-GL and Scav/Delv-GL groups at 7 days after injury. iNOS(type M1) and ARG-1(type M2). The images showed that on the 7th day after injury, the number of iNOS positive cells in the injury site and surrounding tissue area in Scav/Delv-GL group and SCAV-GL group was significantly lower than that in the control group and GL group. Implantation of hydrogel scaffolds in the injured area, especially Delv-GL scaffolds, increased the number of ARG-1 positive cells, suggesting that Scav/Delv-GL scaffolds regulated M1 / M2 polarization of macrophages and microglia in the injured spinal cord during acute SCI phase.
Figure 7: Long-term inflammatory response at the lesion site
(A-C) Expression levels of IL-1β and TNF-α in control group, GL, SCAV-GL, Delv-GL, and Scav/Delv-GL groups and immunofluorescence staining of ED1, F4/80, and Iba-1 at 8 weeks after surgery. The results showed that the expression levels of IL-1β and TNF-α in SCAV-GL, Delv-GL and Scav/Delv-GL groups were lower than those in untreated control group and GL group. In addition, significantly fewer ED1, F4/80 and Iba-1 positive cells were found in the central region of damaged tissue in mice implanted with Scav/Delv-GL scaffolds than in the other four groups. The results suggest that immunomodulatory stents, specifically Scav/Delv-GL stents, attenuate the long-term pro-inflammatory response and thus help reduce the inflammatory microenvironment in the chronic phase after SCI. These results suggest that immunomodulatory stents, specifically Scav/Delv-GL stents, attenuate long-term pro-inflammatory responses and thus help to improve the inflammatory microenvironment in the chronic phase after SCI.
Figure 8: Nerve regeneration and functional recovery
(A) Tuj-1 positive axon fluorescence staining at the lesion site of each group of mice on the 7th day of SCI. The images showed that seven days after surgery, the number of Tuj-1 positive newly generated neurons was higher in the Scav/Delv-GL and Delv-GL groups than in the SCAV-GL, GL and untreated controls. (B-C) : MAP2 expression levels in Tuj-1 positive cells from the lesion center and in spinal cord tissue samples from control groups 8 weeks after surgery. (D-F) Motor evoked potentials (MEPs) of the hind limbs of each group responded to electrical stimulation of the brain 8 weeks after surgery. Mice treated with Scav/Delv-GL scaffolds were observed (mean latency 3.39 ± 0.06 ms; The mean MEP amplitude of mice treated was 0.14 ± 0.02 mV, compared with mice treated with Scav-GL (mean latency 4.28±0.84 ms; Mean amplitude 0.12 ± 0.05 mV) or Delv-GL(mean latency 4.17 ± 0.79 ms; Average amplitude: 0.11 ± 0.04 mV) larger. (G) Hind limb field walking score (BMS) was scored for different groups of mice. These results suggest that both DAMP clearance and IL-10 sustained release are important for long-term motor function recovery in SCI models.
【 Summary 】
In spinal cord injury, the lesion site and its surrounding immune microenvironment play an important role in nerve regeneration and functional recovery. This paper develops a bifocal immunomodulatory scaffold that clears DAMP and slowly releases IL-10, which reduces pro-inflammatory cells while enhancing anti-inflammatory cell responses, accelerates nerve regeneration, and improves the long-term restorative function of exercise in vivo.
