Low-Stiffness Hydrogels Promote Peripheral Nerve Regeneration Through the Rapid Release of Exosomes

Abstract

A hydrogel system loaded with mesenchymal stem cell-derived exosome (MSC-Exos) is an attractive new tool for tissue regeneration. However, the effect of the stiffness of exosome-loaded hydrogels on tissue regeneration is unclear. Here, the role of exosome-loaded hydrogel stiffness, during the regeneration of injured nerves, was assessed in vivo. The results showed that the photocrosslinkable hyaluronic acid methacrylate hydrogel stiffness plays an important role in repairing nerve injury. Compared with the stiff hydrogels loaded with exosomes, soft hydrogels loaded with exosomes showed better repair of injured peripheral nerves. The soft hydrogel promoted nerve repair by quickly releasing exosomes to inhibit the infiltration of macrophages and the expression of the proinflammatory factors IL-1β and TNF-α in injured nerves. Our work revealed that exosome-loaded hydrogel stiffness plays an important role in tissue regeneration by regulating exosome release behavior and provided important clues for the clinical application of biological scaffold materials.

Keywords: exosome release behavior; hydrogel stiffness; mesenchymal stem cell–derived exosomes; nerve injury inflammation; sciatic nerve injury.

FIGURE 1

Hydrogel-loaded exosomes for the treatment of sciatic nerve injury. (A) Characterization of human umbilical cord–derived mesenchymal stem cell–derived exosomes (left: TEM of exosomes, bar = 200 nm; right: Western blot characterization of exosome marker proteins). (B) Fluorescence 3D imaging of hydrogels loaded with PKH26-labeled exosomes (red: PKH26, bar = 50 μm). (C) Rat SNCI model. Different concentrations of HAMA solution mixed with exosomes were injected into the injured nerve, and after curing with ultraviolet light, the hydrogels with different mechanical properties were formed at the nerve injury site (I, uninjured rat sciatic nerve; II, injured sciatic nerve; and III, injured nerve embedded in hydrogel).

FIGURE 2

Soft hydrogel–loaded exosomes can better repair injured nerves. (A) Rat hind footprints were collected after surgery, and the SFI was used to evaluate the functional recovery of the injured sciatic nerve (left: typical footprint pattern, 7 days, bar = 1 cm; right: SFI score, 14 days). Con: surgery group, sham: sham operation group, con vs. sham p = 1.71649E-4; soft: soft hydrogel treatment group, soft vs. con p = 0.01813); and stiff: stiff hydrogel treatment group, soft vs. stiff p = 0.02697. (B) Wet weight of the gastrocnemius muscle and the cross-sectional area of the gastrocnemius muscle fibers. (left: photographs of rat gastrocnemius muscles on the operative and nonoperative sides and HE-stained images of the transverse section of the gastrocnemius muscle on the operating side, bar = 1 cm black, bar = 100 μm yellow).

FIGURE 3

Soft hydrogels loaded with exosomes better repair nerve damage by inhibiting inflammation. (A) H&E staining imaging of the injured nerves (left: 14 days, bar = 1,000 μm black, bar = 100 μm yellow). Immunohistochemistry imaging of IL-1β and TNF-α (right: 14 days, bar = 50 μm red). (B) Immunofluorescence imaging of the injured nerves (1 day, bar = 50 μm, green: CD68, blue: DAPI).

FIGURE 4

Rapid release of exosomes mediated by soft hydrogels inhibits macrophage inflammation. (A) After 24 h, the hydrogel-embedded sciatic nerves were collected, and the frozen sections were imaged under a fluorescence microscope. Image analysis showed that compared with the stiff hydrogel–embedded sciatic nerves, the sciatic nerves embedded in the soft hydrogels had a larger fluorescent area (p = 0.00162, n = 5) and higher IOD (p = 0.01827, n = 5; bar = 200 μm). (B) Hydrogels with different stiffnesses were loaded with PKH26-labeled exosomes and incubated with THP1 cells and PMA (100 ng/ml). After 24 h, the cells were imaged under a laser confocal microscope (bar = 250 μm). Image analysis shows that compared with the cells grown on stiff hydrogels, the cells grown on soft hydrogels had a larger fluorescent area (p = 0.00101, n = 15) and higher IOD (p = 0.00201, n = 15). (C) After PMA (100 ng/ml) induced THP1 to adhere to the wall (con), LPS (100 ng/ml) was used to induce the cells to differentiate into M1 for 12 h (LPS). Then, the cells were treated with different concentrations of exosomes to detect the expression of the related genes, IL-1β and TNF-α (IL-1β: con vs. LPS p = 6.57496E-5, LPS vs. 10 μg/ml p = 7.66959E-4, 10 μg/ml vs. 100 μg/ml p = 3.2624E-4; TNF-α: con vs. LPS p = 9.737936E-4, LPS vs. 10 μg/ml p = 0.00355, 10 μg/ml vs. 100 μg/ml p = 1.63632E-4; n = 3). (D) Schematic diagram of the mechanism by which exosome release affects damaged nerve repair. The rapid release of exosomes from the soft hydrogel can reduce macrophages and the expression of proinflammatory IL-1β and TNF-α in M1 macrophages, thereby promoting the repair of the injured nerve. *p < 0.05; **p < 0.01; and ***p < 0.001, t test.