Anisotropic microtopography surface of chitosan scaffold regulating skin precursor-derived Schwann cells towards repair phenotype promotes neural regeneration

Abstract

For repairing peripheral nerve and spinal cord defects, biomaterial scaffold-based cell-therapy was emerged as an effective strategy, requiring the positive response of seed cells to biomaterial substrate and environment signals. Previous work highlighted that the imposed surface properties of scaffold could provide important guidance cues to adhered cells for polarization. However, the insufficiency of native Schwann cells and unclear cellular response mechanisms remained to be addressed. Given that, this study aimed to illuminate the micropatterned chitosan-film action on the rat skin precursor-derived Schwann cells (SKP-SCs). Chitosan-film with different ridge/groove size was fabricated and applied for the SKP-SCs induction. Results indicated that SKP-SCs cultured on 30 μm size microgroove surface showed better oriented alignment phenotype. Induced SKP-SCs presented similar genic phenotype as repair Schwann cells, increasing expression of c-Jun, neural cell adhesion molecule, and neurotrophic receptor p75. Moreover, SKP-SC-secretome was subjected to cytokine array GS67 assay, data indicated the regulation of paracrine phenotype, a panel of cytokines was verified up-regulated at secreted level and gene expression level in induced SKP-SCs. These up-regulated cytokines exhibit a series of promotive neural regeneration functions, including cell survival, cell migration, cell proliferation, angiogenesis, axon growth, and cellular organization etc. through bioinformatics analysis. Furthermore, the effectively polarized SKP-SCs-sourced secretome, promoted the proliferation and migration capacity of the primarily cultured native rat Schwann cells, and augmented neurites growth of the cultured motoneurons, as well as boosted axonal regrowth of the axotomy-injured motoneurons. Taken together, SKP-SCs obtained pro-neuroregeneration phenotype in adaptive response to the anisotropic topography surface of chitosan-film, displayed the oriented parallel growth, the transition towards repair Schwann cell genic phenotype, and the enhanced paracrine effect on neural regeneration. This study provided novel insights into the potency of anisotropic microtopography surface to Schwann-like cells phenotype regulation, that facilitating to provide promising engineered cell-scaffold in neural injury therapies.

Keywords: anisotropic microtopography; chitosan-film; neural regeneration; phenotype regulation; skin precursor-derived Schwann cells.

Graphical abstract

Scheme 1.

A flowchart design of experiments in this study.

Figure 1.

Characterization of SKPs and SKP-derived SCs. (A) Cultured SKPs showed floating spherical clone morphology. (B) SKP-derived SCs displayed long-spindle shape and side-by-side alignment. (C) GFP (green)-positive SKP-SCs showed expression of SC marker S100-β (red) with Hoechst (blue) labeled cell nuclei. Scale bar, 50 μm.

Figure 2.

Optical images and SEM images of micropatterned chitosan films and adhered SKP-SCs. (A) Representative optical images of micropatterned chitosan films with different size of ridge/groove, flat film served as control. (B) Representative optical images of SKP-SCs cultured on different chitosan film surface groups for 36 h, showing the random arranged SKP-SCs on flat film surface obtained the regular arrangement on micropatterned film. (C) Representative SEM images of the microstructure of chitosan film in four groups, and the simulation maps of the cross-sections of different chitosan films. (D) Representative SEM images of adhered SKP-SCs showing best arrangement on chitosan film surface with 30 μm size of ridge/groove, and improved arrangement of SKP-SCs in ‘10 μm’ and ‘50 μm’ group than that in ‘flat’ group. Scale bar, 50 μm.

Figure 3.

Morphological indexes and proliferation of SKP-SCs on different film surface. (A) Cell area, (B) cell aspect ratio and (C) cell angle of SKP-SCs was statistically analysed and showed significant improvement in micropatterned chitosan film groups especially in ‘30 μm’ surface group, with smaller cell area, more cell aspect ratio, and narrower cell angle, comparing to ‘flat’ group. (D) Representative fluorescence microscope images showed SKP-SCs morphology after culture on different surface samples for 36 h. The rectangle box at top-right was the magnification of the typical field of images. Scale bar, 50 μm. (E) The CCK-8 cell counting assay showed better proliferation status of SKP-SCs on ‘30 μm’ surface group and ‘flat’ surface group than that on ‘10 μm’ surface group, and no significant difference when compare ‘50 μm’ group with other groups. (F) The SKP-SCs from ‘30 μm’ surface group was further detected via qRT-PCR, showing increased mRNA expression level of repair-type Schwann cell phenotype markers c-Jun, NCAM and p75NTR, comparing to cells in ‘flat’ surface group. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Detection and informative analysis of cytokines in SKP-SC-secretome. (A) Heat-map displayed the expression difference of 67 cytokines in SKP-SC-secretome from the ‘30 μm’ and ‘flat’ surface groups by cytokine array assay. (B) Volcano plot showing nine significantly up-regulated and three down-regulated cytokines in ‘30 μm-CM’ group compared with ‘flat-CM’ group according to log₂FC > 0.58 and −log₁₀ (P-value) > 0.89. (C) Nine up-regulated cytokines were subjected to further validation by qRT-PCR, results showed consistent expression except for CTACK, eight cytokines up-regulated in SKP-SCs from ‘30 μm’ surface group compared with that from ‘flat’ surface group, including galectin-3, eotaxin, Notch-2, neuropilin-2, galectin-1, CNTF, VEGF-A and β-NGF. (D) Histograms showed distinct pro-neuroregeneration-associated processes involved with the eight up-regulated cytokines via GO clusters analysis, with different functional terms on the ordinate and the number of genes within each cluster on the abscissa. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

The SKP-SC-secretome from ‘30 μm’ surface group promoted primary SCs proliferation and migration. (A) Primary SCs showed positive expression of SC marker S100-β (red) with Hoechst labeled nuclei (blue). scale bar, 100 μm. (B) Representative images of EdU (red) labeled nuclei with Hoechst labeled nuclei (blue) background of SCs treated with ‘30 μm-CM’, ‘flat-CM’, and ‘MN-medium’ for 24 h, respectively. Scale bar, 100 μm. (C) Representative images of SCs scratch healing showed different migration area ratio of SCs in three groups after treatment for 8 h. Scale bar, 100 μm. (D) Statistical analysis showing the percentage of EdU positive SCs in ‘30 μm-CM’ group were remarkedly more than that in ‘flat-CM’ and ‘MN-medium’ group. (E) Statistical analysis showing the wound healing percentage of SCs in ‘30 μm-CM’ group were significantly higher than that in ‘flat-CM’ and ‘MN-medium’ group. n = 3, *P < 0.05, **P < 0.01.

Figure 6.

The SKP-SC-secretome from ‘30 μm’ surface group promoted MNs neurite growth. (A) Primary cultured MNs positively expressed NF200 (green) and ChAT (red) with Hoechst (blue) labeled nuclei. Scale bar, 25 μm. (B) Representative images of MNs showing better neurite growth of MNs in ‘30 μm-CM’ group than that in ‘flat-CM’ and ‘MN-medium’ groups after incubation for 24 h. Scale bar, 25 μm. (C, D) Statistical analysis showing the average length of the longest neurite and the number of branches per hundred neurons of MNs in ‘30 μm-CM’ group and ‘flat-CM’ group was increased compared with that in ‘MN-medium’ group, and the promoted growth was more significant in ‘30 μm-CM’ group than that in ‘flat-CM’ group. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

The SKP-SC-secretome from ‘30 μm’ surface group promoted MNs axonal regrowth. (A) Schematic of axonal outgrowth from MNs soma chamber to axon chamber through grooves for 4 days, followed by the axotomy of the sprouting axons and axonal regrowth for 24 h. (B) Representative images of MNs showed different length of regenerated axons of damaged MNs after treatment with ‘30 μm-CM’, ‘flat-CM’, and ‘MN-medium’, respectively. Scale bar, 100 μm. (C) Statistical analysis showed that the average length of MNs regenerated axons in ‘30 μm-CM’ group and ‘flat-CM’ group was longer than that in ‘MN-medium’ group, and the promoted axonal regrowth was more significant in ‘30 μm-CM’ group than that in ‘flat-CM’ group. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.