Effects of ECM proteins (laminin, fibronectin, and type IV collagen) on the biological behavior of Schwann cells and their roles in the process of remyelination after peripheral nerve injury

Abstract

Introduction: It is important to note that complete myelination and formation of myelinated fibers are essential for functional nerve regeneration after peripheral nerve injury (PNI). However, suboptimal myelin regeneration is common and can hinder ideal nerve regeneration. Therefore, it is important to closely monitor and support myelin regeneration in patients with PNI to achieve optimal outcomes. Methods: This study analyzed the effects of three extracellular matrix (ECM) proteins on Schwann cells (SCs) in the nerve regeneration environment, including their adhesion, proliferation, and migration. The study also explored the use of composite sodium alginate hydrogel neural scaffolds with ECM components and investigated the effects of ECM proteins on remyelination following peripheral nerve injury. Results: The results showed that laminin (LN), fibronectin (FN), and collagen Ⅳ (type IV Col) promoted the early adhesion of SCs in 2-dimensional culture but the ratios of early cell adhesion were quite different and the maintenance of cells' morphology by different ECM proteins were significantly different. In transwell experiment, the ability of LN and FN to induce the migration of SCs was obviously higher than that of type IV Col. An vitro co-culture model of SCs and dorsal root ganglia (DRG) neurons showed that LN promoted the transition of SCs to a myelinated state and the maturation of the myelin sheath, and increased the thickness of neurofilaments. Animal experiments showed that LN had superior effects in promoting myelin sheath formation, axon repair, and reaching an ideal G-ratio after injury compared to FN and Col IV. The situation of gastrocnemius atrophy was significantly better in the LN group. Notably, the thickness of the regenerated myelin sheaths in the type IV Col group was the thickest. Conclusion: In this experiment, we analyzed and compared the effects of LN, FN, and type IV Col on the biological behavior of SCs and their effects on remyelination after PNI and further clarified their unique roles in the process of remyelination. Further research is necessary to explore the underlying mechanisms.

Keywords: ECM proteins; Schwann cells; myelination; peripheral nerve injury; regeneration.

FIGURE 1

Animal modeling diagram. The ECM solution and alginate hydrogel were mixed and introduced into a silicone tube, followed by the addition of a Ca²⁺ salt solution at 37°C to form a gel. The tube was then used to bridge a 1 mm sciatic nerve gap in adult SD rats by suturing.

FIGURE 2

Adhesion and cell morphology of SCs in early 2D culture conditions. The adhesion of SCs at 2 h after seeding (A) and the morphology of SCs at 24 h after seeding (B): axial pseudopodia (red arrow) and radial pseudopodia (green arrow). The adhesion ratio in the LN group was significantly higher than that in the other groups (p < 0.01) [(C), n = 3]. Major axis of SCs (the maximum length of a single cell) in the LN group was significantly larger than that in the FN group and type IV Col group (p < 0.0001) [(D), n = 9]. The number of radial pseudopodia of SCs in the LN group was significantly less than that of SCs in the FN group and IV Col group (p < 0.01) [(E), n = 8]. **p < 0.01, ***p < 0.001, ****p < 0.0001, ^ns p > 0.05. One-way ANOVA with Tukey’s post hoc test. Scale bar (A) = 10 µm. Scale bar (B) = 1 µm.

FIGURE 3

The proliferation of SCs under 2D culture conditions (EdU staining). Twenty-four hours after seeding SCs, the proliferation of SCs in the LN, FN and type IV Col groups were observed under a fluorescence microscope(A), and the proportion of proliferating cells was quantified [(B), n = 5]. The proportion of proliferating cells (red) in the LN group and FN group was significantly greater than that in the type IV Col group and Control group (p < 0.0001). ***p < 0.001, ****p < 0.0001, ^ns p > 0.05. One-way ANOVA with Tukey’s post-hoc test. Scale bar = 4 µm.

FIGURE 4

Immunofluorescence staining of SC DRG cocultures: NF-200 (green) and MBP (red) were used to identify neurofilaments and myelin sheaths, respectively. The neurofilaments in the LN group were the thickest, and a large number of SCs were arranged around the neurofilaments (A). Quantitative analysis showed that the number of SCs around neurofilaments per 150 μm length in the LN group was significantly more than that in the FN group and IV Col group (p < 0.0001) [(B), n = 6]. In the supernatant, the absorbance of MAG (Y-axis) significantly increased and NCAM-1 significantly decreased (p < 0.05) [(C), n = 3], suggesting a key role of LN in promoting myelin formation. In the FN group, both MAG and NCAM-1 levels were low, a finding that may be related to the local aggregation of FN, thus inhibiting myelination. The average relative area [(D), n = 5] of MBP-positive regions (colored red) and the average extension length [(E), n = 5] of DRG neuronal processes were measured. The Western blot analysis results (F) revealed the presence of transcription factors associated with myelin sheath formation. The expression of myelin-related transcription factors (n = 3): Krox 20 (G), Sox 10 (H) and Oct 6 (I). *p < 0.05, **p < 0.01, ****p < 0.0001, ^ns p > 0.05. One-way ANOVA with Tukey’s post-hoc test. Scale bar = 20 μm.

FIGURE 5

Transwell migration assay of SCs and SCs morphology under 3D culture conditions. The results of the transwell experiment showed that the numbers of SCs that migrated in the LN, FN, and IV Col groups were significantly greater than that in the Control group (A). Growth conditions of SCs under 3D culture conditions were observed using an optical microscope. Cells in the LN and FN groups showed good cell morphology, with elongated processes (black arrows), and cells in the type IV Col group showed mass-like aggregation (B). Live/dead staining analysis of live cells (green)/dead cells (red) in each group (C). Quantitative analysis of cell migration (number of cells in the Control group was taken as reference value 1) [(D), n = 5]: the LN group and FN group had similar relative cell migration numbers (p > 0.05), and both were significantly more than that in the type IV Col group (p < 0.0001). The proportion of SCs with filamentous processes among the total number of cells was assed under 3D culture conditions [(E), n = 25]. The proportion of SCs with filamentous processes in the LN group was significantly higher than that in the FN group and type IV Col groups (p < 0.0001). Cell survival was assessed [(F), n = 3]. The proportion of viable cells in the LN group, FN group and type IV Col group was similar (p > 0.05). ****p < 0.0001, ^ns p > 0.05. Scale bar = 100 µm.

FIGURE 6

Axon and myelin regeneration at the fourth week after model construction. TEM images (A–E) and TB staining images (F–J) of axon regeneration at the implanted graft. Quantitative analysis of the proportion of myelinated nerve fibers [(K), n = 3]. Quantitative analysis of myelin sheath thickness [(L), n = 5]. G-ratio index and nerve function recovery: Quantitative analysis of the G-ratio of axons with a diameter > 2 μm [(M), n = 50] and axons with a diameter of < 2 μm [(N), n = 50) and a scatter plot based on axon diameter (X-axis) and G-ratio (Y-axis) [(O), n = 100]: the G-ratio of the LN group is closer to the best G-ratio (0.6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^ns p > 0.05. One-way ANOVA with Tukey’s post-hoc test. Scale bar (A–E) = 2μm, Scale bar (F–J) = 700 μm.

FIGURE 7

Immunofluorescence assessment of nerve regeneration and remyelination at 4 weeks after model construction. Neurofilaments (NF-200, green) in the LN group were thicker than those in the FN group and type IV Col group, and myelin sheaths were mostly distributed along the neurofilaments (yellow). The neurofilaments in the FN group were significantly thinner than those in the LN group, and although the number of myelin sheaths was greater in the FN group than in the LN group, they were mostly scattered. The numbers of neurofilaments and myelin sheaths in the type IV Col group were significantly less than those in the LN and FN groups (A). The red arrow points from the proximal end of the nerve stump to the distal end. The relative areas of green [neurofilament, (B)], red [myelin sheath, (C)] and yellow [myelin sheath along the neurofilament, (D)] (n = 10) in the image were quantified. The level of EphA4 in regenerating nerves was measured at 2 weeks post-surgery (E) and at 4 weeks post-surgery (F). Additionally, the level of Ninj2 was measured in regenerating nerves at 4 weeks post-surgery (G) (n = 5). **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. Scale bar = 100 μm.

FIGURE 8

Atrophy of innervated muscles at 4 weeks after model construction. Representative gross view of gastrocnemius muscle in each group (A): the injured side is on the left, and the normal side is on the right. Masson staining of the gastrocnemius muscle on the injured side of the nerve (B). Wet weight ratio of gastrocnemius muscle (injured side/normal side) [(C), n = 5]. Average cross-sectional area of muscle fibers in each group [(D), n = 5]. Sciatic functional index (SFI) was measured in each group after surgery [(E), n = 3]. Scale bar (A) = 5 mm. Scale bar (B) = 50 μm ***p < 0.001, ****p < 0.0001, ^ns p > 0.05. One-way ANOVA with Tukey’s post-hoc test.