Hierarchically aligned fibrin nanofiber hydrogel accelerated axonal regrowth and locomotor function recovery in rat spinal cord injury

Abstract

Background: Designing novel biomaterials that incorporate or mimic the functions of extracellular matrix to deliver precise regulatory signals for tissue regeneration is the focus of current intensive research efforts in tissue engineering and regenerative medicine.

Methods and results: To mimic the natural environment of the spinal cord tissue, a three-dimensional hierarchically aligned fibrin hydrogel (AFG) with oriented topography and soft stiffness has been fabricated by electrospinning and a concurrent molecular self-assembling process. In this study, the AFG was implanted into a rat dorsal hemisected spinal cord injury model to bridge the lesion site. Host cells invaded promptly along the aligned fibrin hydrogels to form aligned tissue cables in the first week, and then were followed by axonal regrowth. At 4 weeks after the surgery, neurofilament (NF)-positive staining fibers were detected near the rostral end as well as the middle site of defect, which aligned along the tissue cables. Abundant NF- and GAP-43-positive staining indicated new axon regrowth in the oriented tissue cables, which penetrated throughout the lesion site in 8 weeks. Additionally, the abundant blood vessels marked with RECA-1 had reconstructed within the lesion site at 4 weeks after surgery. Basso-Beattie-Bresnahan scoring showed that the locomotor performance of the AFG group recovered much faster than that of blank control group or the random fibrin hydrogel (RFG) group from 2 weeks after surgery. Furthermore, diffusion tensor imaging tractography of MRI confirmed the optimal axon fiber reconstruction compared with the RFG and control groups.

Conclusion: Taken together, our results suggested that the AFG scaffold provided an inductive matrix for accelerating directional host cell invasion, vascular system reconstruction, and axonal regrowth, which could promote and support extensive aligned axonal regrowth and locomotor function recovery.

Keywords: aligned structure; fibrin hydrogel; nerve regrowth; soft stiffness; spinal cord injury.

Figure 1

Fibrin hydrogel scaffold and rat T9 dorsal hemisected SCI model. (A) The AFG fabrication process. (B) (a1–a2) and (b1–b2) SEM images of the cross sections of AFG and RFG scaffold reveal the hierarchically aligned structure of AFG and random fibrous structure of RFG, respectively; the insert pictures in (a1) and (b1) display gross images of the implanted AFG and RFG scaffolds; the arrows in (a2) and (b2) show the alginate film in the surface of the hydrogel for adhesive prevention. (a3 and b3) SEM images of the longitudinal sections of AFG and RFG scaffolds, demonstrating the aligned and random nanoscale fibrous structure, respectively. (C) Schematic illustration of the rat T9 dorsal hemisected model, and RFG and AFG scaffolds implantation (D and E). Abbreviations: AFG, aligned fibrin hydrogel; RFG, random fibrin hydrogel; SCI, spinal cord injury; SEM, scanning electron microscope.

Figure 2

Vascularization in the dorsal hemisected site. (A–C) Representative sagittal sections of control, RFG, and AFG groups stained by DAPI show the tissue regeneration at 2 weeks post-surgery, and the lesion site region is labeled by dashed line. (D) Quantification of RECA-1 positive vessels at the middle site of injury at 1, 2, and 4 weeks after spinal cord injury. *p < 0.05, **p < 0.01 for the comparison of RFG and AFG. Four individual rats per group were used for statistical analysis (n = 4), and five confocal images per rats were used for the vessel counts. Immunofluorescence staining images of the T8–T10 sagittal sections in RFG (E–G) and AFG (H–J) groups labeled by RECA-1 (green). Abbreviations: AFG, aligned fibrin hydrogel; DAPI, 4′,6-diamidino-2-phenylindole; RFG, random fibrin hydrogel.

Figure 3

Aligned tissue cables formed by the endogenous cell invasion promote extensive axonal regeneration. (a1–a3) F-actin immunofluorescence staining at 1 week after surgery. The representative immunofluorescence staining images of β-tubulin III (red), NF (green), and DAPI (blue) at 2 weeks (b1–b3) and 4 weeks (c1–c3) after surgery. The dashed lines indicate the long axis of the aligned AFG and cell cables. The arrows in b3 show the cells (neurons) labeled by β-tubulin III migrate into the regrowth tissue. Abbreviations: AFG, aligned fibrin hydrogel; DAPI, 4′,6-diamidino-2-phenylindole; NF, neurofilament; RFG, random fibrin hydrogel.

Figure 4

Nerve regeneration in the dorsal hemisected site at 8 weeks after surgery. Shown in Figure 5A, (a1–c1) are the immunofluorescence staining images of T8–T10 sagittal sections with NF (green), GAP43 (red), and DAPI (blue). Higher magnification images of the areas circled by the dotted white squares in the left images are shown in the right columns ([a2–a3], [b2–b3] and [c2–c3], respectively). The quantification of NF-positive fibers at the injury site is shown in (B), (C), and (D): (B) the density of NF-positive fibers at the injury site; the frequency distribution of NF-positive fibers of RFG (C) and AFG (D). Four individual rats per group were used for statistical analysis (n = 4), and five confocal images per rats were used for the NF-positive fiber counts and length calculation. Abbreviations: AFG, aligned fibrin hydrogel; DAPI, 4′,6-diamidino-2-phenylindole; NF, neurofilament; RFG, random fibrin hydrogel.

Figure 5

Transmission electron micrographs of the middle lesion site. (A) (a1–a2) Axons with loose myelin sheaths and abundant collagen regenerated in the area of lesion site in the control group; compared to the control group, a great deal of myelinated axons with relatively compact myelin sheaths (Ax) are seen in the middle region of lesion site (c1–c2), and accompanying blood vessels (V) in the RFG group (b1–b2). The arrows show the axons. (B and C) The number and myelin thickness of axons in the middle lesion site are summarized and analyzed. Data are expressed as mean ± standard deviation. Statistical comparisons were performed using ANOVA, and *p < 0.05, **p < 0.01. Four individual rats per group were used for statistical analysis (n = 4), and five TEM images per rat were used for the axon counts and myelin thickness calculation. Abbreviations: AFG, aligned fibrin hydrogel; ANOVA, analysis of variance; RFG, random fibrin hydrogel; TEM, transmission electron microscopy.

Figure 6

Representative FA maps, tractography images, and FA values of the spinal cord at 16 weeks after the implantation. (A) The bright region indicates high anisotropy in the representative FA maps (a1–a3, b1–b3 and c1–c3, where A, P, L, and R represent anterior, posterior, left, and right, respectively). In the images of (a4–a5), (b4–b5), and (c4–c5), yellow stands for fibers traced from the ROI rostral to the epicenter, where blue corresponds to fibers traced from the ROI caudal to the epicenter. (B) FA value versus location (mm) reveals statistically significant differences between the AFG group and the RFG group at the lesion site (n = 4), *p < 0.05, **p < 0.01. Furthermore, the FA value of AFG revealed statistically significant differences from control group animals at all locations along the spinal cord (p < 0.01). Abbreviations: AFG, aligned fibrin hydrogel; FA, fractional anisotropy; RFG, random fibrin hydrogel; ROI, region of interest.

Figure 7

BBB score of the experimental groups. Data are expressed as mean ± standard error of the mean (n $ 8 for each group). ^#p < 0.05, ^##p < 0.01 (AFG vs control at the same time point); **p < 0.01 (RFG vs control at the same time point); &p < 0.05, ^&&p < 0.01 (AFG vs RFG at the same time point). Abbreviations: AFG, aligned fibrin hydrogel; BBB, Basso-Beattie-Bresnahan; RFG, random fibrin hydrogel.

Figure 8

Summarized schematic of regrowth process with AFG implantation in rat dorsal semi-section SCI model. For the T9 dorsal hemisection SCI (A), the AFG scaffold was implanted into the injury site to bridge the gap (B). Then, the host cells migrated into the AFG scaffold to replace the aligned structure (C) which supports the axons’ regeneration (D). Abbreviations: AFG, aligned fibrin hydrogel; SCI, spinal cord injury.