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    Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord
  • Figure 1|Preparation and structural characterization of decellularized scaffolds.

    Both DON and DSN were processed from fresh porcine nervous tissue. The DON scaffold with many longitudinal channels and a three-dimensional topographic structure was prepared from fresh optic nerves decellularized using a chemical extraction method and a vacuum freeze-drying process (Figure 1A–D). Transverse and longitudinal scaffold sections were evaluated after hematoxylin and eosin staining. The DON scaffold showed uniformly distributed straight channels with uniform hole sizes (Figure 1E1 and F1). The DSN scaffolds had straight longitudinal channels, but the holes were not of uniform size (Figure 1E2 and F2). Compared with holes in transverse sections, the shorter channels in longitudinal sections showed no structural differences in the CS, and they were still randomly distributed, which can provide a larger cell-attachment surface (Figure 1E3 and F3).
    Next, we characterized the topographic structure of each scaffold using scanning electron microscopy (Figure 1G1–H3). The horizontal holes of the DON scaffold were evenly distributed, and the longitudinal channels were straight. Additionally, there were a few pore structures connecting the individual longitudinal channels (Figure 1G1 and H1). In the DSN scaffold, the distribution of the channel and hole structures in longitudinal and transverse sections was relatively non-uniform (Figure 1G2 and H2), and in the CS scaffold, they were random and disordered (Figure 1G3 and H3). These results suggest that the DON scaffold has the structural basis for guiding the directional outgrowth of neuronal axons, and at the same time, to allow the exchange of information (cues) between channels, which is conducive to axonal communication and cell migration.

    Figure 2|ECM proteins and their impact on the viability of NSCs in the scaffolds.

    Before assessing the effects of the three kinds of scaffolds on DRG neurite outgrowth, we quantitatively measured the levels of relevant ECM proteins on each scaffold. To this end, we selected three major ECM proteins associated with neurite growth—CSPGs, LN and COL4. CSPGs were rarely detected on the DON and CS scaffolds, whereas the DSN scaffold exhibited the highest immunofluorescence for CSPGs (Figure 2A–C and Additional Figure 1A–C). The distributions of LN and COL4 were notably different among the scaffolds. The DON had the highest fluorescence staining for LN and COL4, followed by the DSN, with the CS having the least staining (Figure 2D–I and Additional Figure 1D–I). These results indicate that the DON scaffold contains abundant LN and COL4 (Figure 2J), which may promote neurite growth, but barely any CSPGs, which inhibit neurite growth.

    Before being seeded onto the scaffolds, Nestin immunoreactivity was confirmed for all neurospheres. To determine whether the NSCs on each scaffold were viable, they were seeded and cultured for 7 days. The Cell Counting Kit-8 analyses showed that NSCs grew better and displayed better viability on the DON and CS scaffolds than on the DSN scaffold (n = 9 in each group, P < 0.05; Figure 2K). These results suggest that the DON and CS scaffolds can provide better structural support for NSCs adherence and growth.

    Figure 3|Effects of DON, DSN and CS scaffolds on neurite outgrowth and myelination.

    Next, the ability of each scaffold to support neurite extension was examined using the DRG culture model. All DON slices were placed at the bottom of a 24-well culture plate, and freshly isolated DRG cells were seeded on them and cultured for 3 days. DSN and CS slices were used as controls. The cultures were stained for NF by immunofluorescence, and the maximum distance of DRG neurite extension was measured (Figure 3A–C). All scaffolds supported robust NF-positive neurite extension (Figure 3D–F), but the neurites on the DON scaffold were the longest (Figure 3A and G), followed by the DSN (Figure 3B and G) and then the CS (Figure 3C and G; n = 5 in each group, P < 0.01). The total area showing DRG NF-positive neurite extension had similar characteristics (Figure 3G and H), and the DON and DSN scaffolds showed the best directional growth of neurites (n = 5 in each group, P < 0.01; Figure 3I). These results indicate that the DON and DSN scaffolds promote the directional growth of DRG neurites.

    Next, we evaluated the potential of each scaffold to support neurite myelination using the DRG culture system. To distinguish between DRG neurites and SCs, the cultures were immunostained for NF and MBP. Most SCs adhered to NF-positive neurites on the DON scaffold, followed by the DSN and CS scaffolds (n = 5 in each group, P < 0.05; Figure 3J). Next, the wrapping of SCs around DRG neurites was investigated by transmission electron microscopy. After culture for 2 weeks, SCs were observed on the DON scaffold, which appeared to feature different stages of myelination, including a multilayer myelin sheath structure and early myelinated neurites (Figure 3K and L). These results suggest that the DON scaffold may be better able to promote myelination of SCs on neurites, compared with the DSN and CS scaffolds.

    Figure 4|DON functional scaffold construction and transplantation.


    Figure 5|Axonal regeneration and remyelination in lesions of the dorsal white matter of the spinal cord after transplantation of the functional scaffolds.


    Figure 6|Impact of functional scaffolds on newborn myelin sheaths in lesions in the spinal cord.

    In the present study, both the DON and CS scaffolds contained SCNTs. The transfection rate of the NT-3 gene in cultured SCNTs exceeded 80%, and the SCNTs were positive for S100 protein (Figure 4A and B), indicating that the modified SCNTs were suitable for loading onto these scaffolds. Next, these scaffolds were transplanted into SCI lesions of the dorsal white matter (Figure 4C). Four weeks later, the distribution of grafted SCNTs in lesions better paralleled the longitudinal axis of the spinal cord in the DON group (Figure 5A and C1), compared with the CS group (Figure 5B and D1). In addition, the directional regeneration of host axons in lesions was consistent with the longitudinal axis of the spinal cord (Figure 5C, C2 and G). In contrast, in the CS group, the regenerated axons in lesions grew in a disordered manner (Figure 5D, D2 and G). At the same time, the quantification of axons and myelin revealed more NF and MBP expression in lesions in the DON group (Figure 5C–F), compared with the CS group. To verify the effects of the functional scaffolds on axon remyelination, myelin sheath analysis was performed. In lesions in the DON group, most regenerated axons were wrapped with new myelin sheaths, whereas in the CS group, few axons were wrapped in such a manner (Figure 6A–D). Transmission electron microscopy analyses confirmed these results (Figure 6E and F).

    Figure 7|Effects of functional scaffolds on the inflammatory cells and CSPGs levels in the injured spinal cord.

    At 4 weeks after SCI, IBA-1-positive cells and CSPGs level were evaluated in the DON and CS groups (Figure 7A). There were more IBA-1-positive cells in lesions in the CS group, compared with the DON group (P < 0.05; Figure 7B1–C3). Transplantation with the DON scaffold decreased the number of IBA-1-positive cells in the areas rostral and caudal to the lesion, as well as within the lesion, compared with the CS group (P < 0.05; Figure 7F). CSPGs levels in these areas were lower in the DON group than in the CS group (P < 0.05; Figure 7D1–E3 and G). Therefore, these results indicate that the transplanted DON scaffold containing SCNTs attenuates inflammation and lowers CSPGs levels.


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  • 发布日期: 2021-04-21  浏览: 545
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