脊髓损伤
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Figure 1｜Characterization of the collagen scaffold with axially-aligned luminal conduits.
The collagen scaffold with axially-aligned luminal conduits was fabricated as a cylindrical scaffold of 3-mm diameter and 3-mm thickness, with axially-aligned luminal conduits for directional axonal growth and promoting connections between distal and proximal axons (Figure 1A and B). SEM images showed this collagen scaffold had uniform sponge-like pores, suggesting that it should provide enough space for the adhesion and growth of seeded cells (Figure 1C and D). Figure 1E shows a sketch of the mold for preparing the collagen scaffold with axially-aligned luminal conduits.
The mechanical properties of the scaffold are shown in Figure 1F and G. The compressive strength of the scaffold was 107.73–233 kPa (Figure 1F). After 10 compression cycles, the collagen scaffold could partially maintain its structure (Figure 1G). Thus, the scaffold should have sufficient mechanical strength for transplantation.
Figure 2｜Biocompatibility of the collagen scaffold with axially-aligned luminal conduits.
Calcein-AM/PI double staining showed that NSCs had good adhesion and growth on the collagen scaffolds (Figure 2A), which was confirmed by SEM analysis (Figure 2B). The data indicate that the NSCs on the collagen scaffolds had a similar viability at 3 days, but a markedly higher viability at 5 days, compared with NSCs on cell slides (Figure 2C). The growth and differentiation of NSCs in the conduit structure of the scaffold is shown in Figure 2D. The cells grew linearly along the conduit structure, which helps to connect the damaged nerves and promote recovery following SCI. The differentiation of NSCs on the collagen scaffolds was investigated using immunostaining for Tuj-1, a neuronal marker, and GFAP, an astrocyte marker (Figure 2E). The collagen scaffolds supported the differentiation of NSCs and the neuronal and astrocytic differentiation of NSCs, similar to NSCs in normal two-dimensional culture (Figure 2F and G). These results indicate that our collagen scaffold has very good biocompatibility and is suitable for adhesion, growth and differentiation of NSCs in vitro.
Figure 3｜Effect of transplantation of collagen scaffolds loaded with NSCs on motor functional recovery in SCI model rats.
NSCs were loaded into axially-aligned luminal conduits of collagen scaffolds and transplanted into the defect of the completely-transected T8 spinal cord (Figure 3A and B). The recovery of locomotor function is the most important indicator and goal of SCI therapy (Li et al., 2017). The BBB score of all rats was 21 before operation. The hindlimbs of rats were completely paralyzed immediately after SCI, with a BBB score of 0. The rats in the SCI group had very limited locomotor recovery of the hindlimbs, with BBB scores of 2–3 at 8 weeks post-surgery (Figure 3C). Collagen scaffold transplant only increased slightly the BBB score, about 4–5 points, at 8 weeks, without a significant difference from the SCI group. In contrast, rats with NSCs/collagen scaffold transplantation had consistent recovery in locomotor function during the observation period, with a significantly higher BBB score (about 7–8 points at 8 weeks) compared with the SCI and collagen scaffold alone groups (P < 0.05; Figure 3C). These results indicate that the collagen scaffold loaded with NSCs in axially-aligned luminal conduits markedly promotes the recovery of locomotor function in paraplegic rats after complete-transection SCI.
Figure 4｜Effect of transplantation of collagen scaffold loaded with NSCs on the histopathology of the spinal cord in the SCI rat model.
Functional recovery is attributed to changes in tissue structure and pathophysiology (Fan et al., 2018). Hematoxylin and eosin staining was used to observe structural changes in the injured spinal cord at the indicated time points (Figure 4A). In the SCI group, the tissue at the site of injury showed a loose and disordered structure, accompanied with cavities of uneven size. In contrast, the collagen scaffold and NSCs/collagen scaffold groups both had compact tissues at the lesion site, and no obvious cavity was observed. The structure of the lesion/scaffold site can be seen clearly in high-power images (Figure 4B). The degradation of collagen scaffold transplants in SCI repair is a major concern (Sun et al., 2019), thus Masson staining was performed to investigate the collagen deposits at the site of injury after transplantation at different time points (Figure 4C). At 1 week, Masson staining was very intense both in the collagen scaffold and NSCs/collagen scaffold groups, indicating collagen was mostly undegraded at the lesion site. At 3 weeks, the intensity of the blue faded, and the blue-stained area markedly decreased in the collagen scaffold and NSCs/collagen scaffold groups, suggesting the transplanted collagen scaffolds had dramatically degraded in the injured spinal cord. Notably, at 5 and 8 weeks, Masson staining was very weak in the collagen scaffold and NSCs/collagen scaffold groups. These results indicate that our collagen scaffold had a good degradation rate in vivo and was almost completely degraded within 5 weeks after transplantation.
Figure 5｜NSCs/collagen scaffold treatment promotes nerve regeneration in SCI model rats.
Recent studies suggest that newly differentiated neurons derived from endogenous or exogenous NSCs formed a relay to transmit the activity of surviving damaged host axons above and below the injury levels (Lu et al., 2012; Lai et al., 2016). Immunofluorescence staining of longitudinal sections showed that the collagen scaffold group had more Tuj-1-positive cells in the injured spinal cord than the SCI group (P < 0.01), while the NSCs/collagen scaffold group had much more Tuj-1-positive cells than the collagen scaffold and SCI groups (P < 0.001; Figure 5A and B). In contrast, hypertrophic GFAP-positive astrocytes were observed in the SCI and collagen scaffold groups, while the NSCs/collagen scaffold group had a notable decrease in reactive astrogliosis (Figure 5A and C).
It is crucial for regenerating axons to transmit neural signals across the injured spinal cord segment. 5-HT nerve fibers participate in the activity of the spinal cord neural network involved in vertebrate movement after SCI (Liu et al., 2019). Here, immunostaining for NF and 5-HT was used to assess axon regeneration (Figure 5D–F). NF immunoreactivity was distributed throughout the lesion site in the three groups at 8 weeks after surgery. Quantitative analysis showed that the collagen scaffold group had more NF-positive axons than the control group (P < 0.01; Figure 5E). The NSCs/collagen scaffold group had more NF-positive axons compared with the collagen scaffold and SCI groups (Figure 5E). Similar to NF staining, the NSCs/collagen scaffold group had more 5-HT-positive axons than the collagen scaffold and SCI groups (P < 0.001). In addition, 5-TH staining was heavier in the collagen scaffold group compared with the SCI group (P < 0.01; Figure 5F).
Figure 6｜NSCs/collagen scaffold treatment decreases the inflammatory response in the rat SCI model.
The normal rectal temperature was 38.57 ± 0.49°C in rats, but was markedly lower in all rats after surgery. Consecutive rectal temperature monitoring showed that there was no statistical difference among the three groups at the indicated time points (Figure 6A). The serum CRP concentration was 758.71 ± 174.5 ng/mL before surgery, and was markedly increased to 1,349.56 ± 451.41 ng/mL at day 1 post-surgery, and then quickly returned to normal levels in the SCI group. The scaffold and NSCs/scaffold transplant groups did not show changes in CRP levels at the indicated times, other than day 5 post-surgery, and the NSCs/collagen scaffold group had a higher CRP level than the collagen scaffold group (P < 0.05; Figure 6B). To investigate macrophage infiltration at the lesion site, immunostaining for CD68 and CD206 was performed at 1, 4 and 8 weeks after surgery. Evident positive staining for CD68 and CD206 was observed among the three groups at all time points (Figure 6C–E). There were more CD68-positive macrophages in the NSCs/collagen scaffold group, compared with the SCI group at 1 week post-surgery. We noticed that the CD68-positive cells in the collagen scaffold and NSCs/collagen scaffold groups had an appearance distinct from those in the SCI group, which exhibited a round morphology. CD206 is a marker of M2 macrophages, which are considered to play an anti-inflammatory role in SCI repair (Chedly et al., 2017). CD206 staining showed that the scaffold and NSCs/scaffold transplants increased M2 macrophage infiltration into the lesion site at 8 weeks after SCI (Figure 6C–E). These results clearly indicate that our collagen scaffold has low immunogenicity and does not elicit an obvious adverse inflammatory response following SCI.