Dual-bionic regenerative microenvironment for peripheral nerve repair

Abstract

Autologous nerve grafting serves is considered the gold standard treatment for peripheral nerve defects; however, limited availability and donor area destruction restrict its widespread clinical application. Although the performance of allogeneic decellularized nerve implants has been explored, challenges such as insufficient human donors have been a major drawback to its clinical use. Tissue-engineered neural regeneration materials have been developed over the years, and researchers have explored strategies to mimic the peripheral neural microenvironment during the design of nerve catheter grafts, namely the extracellular matrix (ECM), which includes mechanical, physical, and biochemical signals that support nerve regeneration. In this study, polycaprolactone/silk fibroin (PCL/SF)-aligned electrospun material was modified with ECM derived from human umbilical cord mesenchymal stem cells (hUMSCs), and a dual-bionic nerve regeneration material was successfully fabricated. The results indicated that the developed biomimetic material had excellent biological properties, providing sufficient anchorage for Schwann cells and subsequent axon regeneration and angiogenesis processes. Moreover, the dual-bionic material exerted a similar effect to that of autologous nerve transplantation in bridging peripheral nerve defects in rats. In conclusion, this study provides a new concept for designing neural regeneration materials, and the prepared dual-bionic repair materials have excellent auxiliary regenerative ability and further preclinical testing is warranted to evaluate its clinical application potential.

Keywords: Electrospun; Extracellular matrix; Peripheral nerve regeneration; Tissue engineering; Umbilical cord mesenchymal stem cells.

Fig. 1

Characterization of hUMSCs-ECM modified PCL-SF electrospinning materials. (A) Scanning electron microscopy test. a represent the surface topography of APS material; b, c represent the morphology of APS material modified by hUMSCs-ECM before decellularization; d represent the surface topography of MAPS materials; e represent the surface topography of RPS material; f, g represent the morphology of RPS material modified by hUMSCs-ECM before decellularization; h represent the surface topography of MRPS material. (B) Immunofluorescence staining of hUMSCs-ECM modified electrospinning materials. a and b represent the MAPS materials before acellular treatment; c, d represent the immunofluorescence staining image of MAPS material after acellular treatment; e, f represent the MRPS material before acellular treatment; g, h represent MRPS material after acellular treatment (Scale bar = 100 μm). (C) AFM detection results. a, b, c and d represent the surface topography of APS, MAPS, RPS and MRPS materials respectively. (D) Results of elastic modulus of different substrates. ECM: hUMSCs-ECM; EF: PCL/SF electrospinning material; TCP: tissue culture plate. (E) Statistical results of diameters of four electrospun nanofibers. [<0.01 (**), <0.001 (***), <0.0001(****)].

Fig. 2

Protein quantitative analysis of HUMSCS-ECM and Acellular nerve allograft (ANA) matrix. (A–D) Differences between hUMSCs-ECM and ANA proteins. (A) Quantitative heat maps of all proteins were processed by Log10. The darker the color, the higher the expression level, the more yellow the expression level, and the white the protein was not present in the sample. T represented the hUMSCs-ECM and CK represented the ANA group. (B) Sample relationship Venn diagram, screening genes according to protein abundance, to find common or unique proteins between groups. (C) Volcanic diagram of hUMSCs -ECM and ANA protein differences. The abscissa represents the log value of the multiple of difference between the two groups, the ordinate represents the negative Log10 value of the FDR of the two groups, the different colors represent the differentially up-regulated and down-regulated proteins screened according to the threshold, and the blue dots represent no difference. (D) The differential protein heat map between HUMSCS-ECM and ANA, in which each column represents a sample and each row represents a protein. Z-score was used to normalize the protein expression level of the row. The redder the default color, the higher the protein expression level; The bluer, the lower the protein expression. (E) GO differential enrichment bubble diagram. (F) KEGG differential pathway enrichment bubble diagram. (G) The protein composition characteristics of HUMSCS-ECM were compared with the data of MatrisomeDB database. The percentage of each matrix protein subclass in the total ECM protein was represented by the rising sun diagram, and the gene identifications of the significantly expressed proteins in each subclass were given respectively.

Fig. 3

In vitro mimic of axon growth on the material surface. (A) Immunofluorescence representative picture of DRG cultured on each group (Schwann Cells: S100-β; Nerve axons: NF200; Nucleus: DAPI). (B) The rose diagram of axon growth of each group. Each dorsal root ganglion image is centered on the central ganglion tissue mass, which is radially cut into 24 parts (each 15°). For the ganglion cultured on the Aligned fiber, parallel to the fiber direction is defined as 0°, perpendicular to the fiber direction is defined as 90°; The other groups were randomly assigned. The sector length represents the average length of axon growth (n = 8), and the direction corresponds to the region division. The inner circle represents the axon growth length of 1000 μm, and the outer circle represents 2000 μm. (C) Each DRG selected four longest axons for length statistics. (D) Image of PC-12 cells cultured on each group (scale bar = 50 μm). (E) Statistical results of the length of PC-12 cells(n = 20). [<0.01 (**), <0.001 (***), <0.0001(****)].

Fig. 4

Material-related behaviors of Schwann cells. (A) Immunofluorescence of Schwann cell morphology (S-100β: Schwann cells; F-actin: cytoskeleton; DAPI: nucleus. scale bar = 50 μm). (B) SEM images of Schwann cells (scale bar = 100 μm). (C) Length of Schwann cells grow on each material. (D) Real-time fluorescence dynamic screenshots of Schwann cells on each group of materials were taken by the Harmony 4.8 system every 75 min. The camera window is fixed in position to continuously track the dynamic changes of a cell (scale bar = 50 μm). (E) Vector trajectory diagram of Schwann cells. Real-time, dynamic images of the Schwann cells in the material plane are captured by the Harmony 4.8 system and visualized as a collection of cell trajectories. The starting point of each tracked cell is labeled as (0,0). The red arrow indicates the orientation of the fiber. [<0.05(*), <0.01(**), <0.001(***), <0.0001(****)].

Fig. 5

Transcriptome study of Schwann cells. (A) Scatter diagram of GO enrichment analysis. The most significant 30 terms were selected from the GO enrichment analysis results to draw a scatter diagram for display, in which the abscissa is the ratio of the number of differential genes annotated to GO Term to the total number of differential genes, the ordinate is GO Term, and the size of points represents annotation to GO. The number of genes at Term, red to purple, represents the magnitude of significance of enrichment. (B) The 20 most significant KEGG pathways were selected from the KEGG enrichment results to draw a scatter map for display. The abscissa is the ratio of the number of genes annotated to the KEGG pathway to the total number of genes, the ordinate is the KEGG pathway, the size of the dots represents the number of genes annotated to the KEGG pathway, and the color from red to purple represents the significance of enrichment. (C) GSEA analysis results of GO dataset. (D) GSEA analysis results of KEGG dataset.

Fig. 6

Early-stage histological evaluation of sciatic nerve repair in vivo. (A) In vivo experimental design of sciatic nerve repair in rats. Three groups of nerve catheter grafts were involved, namely MAPS, APS and ANA (Acellular allogeneic nerve). (B) Critical evaluation nodes after in vivo nerve repair. (C) Representative images of IF staining of longitudinal section of graft (Schwann Cells: S100-β; Axons: NF200; Nucleus: DAPI) (Scale bar = 500 μm); The white dotted line indicates the length of regeneration from the Proximal end of the axon, and both sides display the Distal and Distal magnified renderings (Scale bar = 100 μm). (D) Representative images of IF staining of transection in the middle of the nerve graft (Scale bar = 200 μm). (E) Statistical results of axon regeneration length in each group. (F) Statistical results of axon density in the transverse section of the midsection of the graft. [<0.05 (*), <0.01 (**), <0.001 (***)].

Fig. 7

Early-stage histological evaluation of Angiogenesis. (A) Vascular infiltration of regenerated nerve tissue inside the nerve catheter grafts in each group. (α-smooth muscle actin: α-SMA; endothelial cells: CD31; Nucleus: DAPI) (Scale bar = 100 μm). (B) The proportion of α-SMA⁺ CD31⁺ region in the proximal longitudinal section. (C) The proportion of α-SMA⁺ CD31⁺ region in the distal longitudinal section. (D) Immunofluorescence staining of vessels in transverse sections of the graft (Scale bar = 100 μm). (E) Proportion of α-SMA⁺ CD31⁺ region in the proximal transverse section. (F) Proportion of α-SMA⁺ CD31⁺ region in the distal transverse section. [<0.05 (*), <0.01 (**), <0.001 (***)].

Fig. 8

Behavioral and functional evaluation after sciatic nerve repair. (A) 12 weeks after surgery, 3D stress on the toes of rats in each group is displayed. 2D plane photos of footprints of corresponding groups are shown on the right. LH is the left hind, namely the control side, and RH is the right hind, namely the operative side. (B) Statistical results of sciatic function index (SFI) of rats in each group at 4, 8 and 12 weeks after bridging “*” represents the significant difference between MAPS group and APS group; “&” represents the significant difference between the AUTO group and MAPS group. (C) Masson staining for cross sections of gastrocnemius muscle of each group (Scale Bar = 500 μm). (D) Wet weight recovery rate of gastrocnemius muscle in each group (operative side/healthy side). (E) Average cross sectional area of gastrocnemius midsection abdominal muscle fiber in each group. (F) Neuroelectrophysiological evaluation results, representative compound muscle action potential (cMAP) results of each group. (G) Statistical results of cMAP delay time ratio in each group (operative side/control side). (H) Statistical results of cMAP peaks ratio in each group (operative side/control side). [<0.05 (*), <0.01 (**), <0.001 (***)].