Co-culture of Schwann cells and endothelial cells for synergistically regulating dorsal root ganglion behavior on chitosan-based anisotropic topology for peripheral nerve regeneration

Abstract

Background: Anisotropic topologies are known to regulate cell-oriented growth and induce cell differentiation, which is conducive to accelerating nerve regeneration, while co-culture of endothelial cells (ECs) and Schwann cells (SCs) can significantly promote the axon growth of dorsal root ganglion (DRG). However, the synergistic regulation of EC and SC co-culture of DRG behavior on anisotropic topologies is still rarely reported. The study aims to investigate the effect of anisotropic topology co-cultured with Schwann cells and endothelial cells on dorsal root ganglion behavior for promoting peripheral nerve regeneration. Methods: Chitosan/artemisia sphaerocephala (CS/AS) scaffolds with anisotropic topology were first prepared using micro-molding technology, and then the surface was modified with dopamine to facilitate cell adhesion and growth. The physical and chemical properties of the scaffolds were characterized through morphology, wettability, surface roughness and component variation. SCs and ECs were co-cultured with DRG cells on anisotropic topology scaffolds to evaluate the axon growth behavior. Results: Dopamine-modified topological CS/AS scaffolds had good hydrophilicity and provided an appropriate environment for cell growth. Cellular immunofluorescence showed that in contrast to DRG growth alone, co-culture of SCs and ECs could not only promote the growth of DRG axons, but also offered a stronger guidance for orientation growth of neurons, which could effectively prevent axons from tangling and knotting, and thus may significantly inhibit neurofibroma formation. Moreover, the co-culture of SCs and ECs could promote the release of nerve growth factor and vascular endothelial growth factor, and up-regulate genes relevant to cell proliferation, myelination and skeletal development via the PI3K-Akt, MAPK and cytokine and receptor chemokine pathways. Conclusions: The co-culture of SCs and ECs significantly improved the growth behavior of DRG on anisotropic topological scaffolds, which may provide an important basis for the development of nerve grafts in peripheral nerve regeneration.

Keywords

Schwann cells / Endothelial cells / Dorsal root ganglion behavior / Co-culture / Anisotropic topology / Regulation mechanism / Nerve / Regeneration

References

[1] Zhang Y, Yang S, Liu C, Han X, Gu X, Zhou S. Deciphering glial scar after spinal cord injury. Burn Trauma. 2021; 9: 35. https://doi.org/10.1093/burnst/tkab035.

[2] Yi S, Zhang Y, Gu X, Huang L, Zhang K, Qian T, et al. Application of stem cells in peripheral nerve regeneration. Burn Trauma. 2020; 8: 2.

[3] Pop NL, Nan A, Urda-Cimpean AE, Florea A, Toma VA, Moldovan R, et al. Chitosan functionalized magnetic nanoparticles to provide neural regeneration and recovery after experimental model induced peripheral nerve injury. Biomol Ther. 2021; 11: 676.

[4] Gordon T.Peripheral nerve regeneration and muscle reinnervation. Int J Mol Sci. 2020; 21: 1-24.

[5] Kornfeld T, Vogt PM, Radtke C. Nerve grafting for peripheral nerve injuries with extended defect sizes. Wien Med Wochenschr. 2018; 169: 240-51.

[6] Isaacs J. Treatment of Acute Peripheral Nerve Injuries: Current Concepts. J Hand Surg Am. 2010; 35: 491-7.

[7] Meek MF, Coert JH. US Food and Drug Administration /Conformit Europe-approved absorbable nerve conduits for clinical repair of peripheral and cranial nerves. Ann Plast Surg. 2008; 60: 466-72.

[8] Moskow J, Ferrigno B, Mistry N, Jaiswal D, Bulsara K, Rudraiah S, et al. Review: Bioengineering approach for the repair and regeneration of peripheral nerve. Bioact Mater. 2019; 4: 107-13.

[9] Lyu Y, Azevedo HS. Supramolecular hydrogels for protein delivery in tissue engineering. Molecules. 2021; 26: 873.

[10] Carvalho CR, Oliveira JM, Reis RL. Modern Trends for Peripheral Nerve Repair and Regeneration: Beyond the Hollow Nerve Guidance Conduit. Front Bioeng Biotechnol. 2019; 7: 337.

[11] Liu C, Fan L, Tian Z, Wen H, Zhou L, Guan P, et al. Self-curling electroconductive nerve dressing for enhancing peripheral nerve regeneration in diabetic rats. Bioact Mater. 2021; 6: 3892-903.

[12] Chen X, Ge X, Qian Y, Tang H, Song J, Qu X, et al. Electrospinning Multilayered Scaffolds Loaded with Melatonin and Fe₃O₄ Magnetic Nanoparticles for Peripheral Nerve Regeneration. Adv Funct Mater. 2020; 30: 2004537.

[13] Wang P, Sun Y, Shi X, Shen H, Ning H, Liu H. Bioscaffolds embedded with regulatory modules for cell growth and tissue formation: A review. Bioact Mater. 2021; 6: 1283-307.

[14] Li G, Zhao X, Zhang L, Yang J, Cui W, Yang Y, et al. Anisotropic ridge/groove microstructure for regulating morphology and biological function of Schwann cells. Appl Mater Today. 2020; 18: 100468.

[15] Sobral JM, Malheiro VN, Clyne TW, Harris J, Rezk R, O’Neill W, et al. An accelerated buoyancy adhesion assay combined with 3-D morphometric analysis for assessing osteoblast adhesion on microgrooved substrata. J Mech Behav Biomed Mater. 2016; 60: 22-37.

[16] Li G, Xue C, Wang H, Yang X, Zhao Y, Zhang L, et al. Spatially featured porous chitosan conduits with micropatterned inner wall and seamless sidewall for bridging peripheral nerve regeneration. Carbohydr Polym. 2018; 194: 225-35.

[17] El-Metwally SA, Abou-El-Regal MM, Eissa IH, Mehany ABM, Mahdy HA, Elkady H, et al. Discovery of thieno[2,3-d]pyrimidine-based derivatives as potent VEGFR-2 kinase inhibitors and anti-cancer agents. Bioorg Chem. 2021; 112: 104947. https://doi.org/10.1016/j.bioorg.2021.104947.

[18] Muangsanit P, Roberton V, Costa E, Phillips JB. Engineered aligned endothelial cell structures in tethered collagen hydrogels promote peripheral nerve regeneration. Acta Biomater. 2021; 126: 224-37. https://doi.org/10.1016/j.actbio.2021.03.039.

[19] Mompéo B, Engele J, Spanel-Borowski K. Endothelial cell influence on dorsal root ganglion cell formation. J Neurocytol. 2003; 32: 123-9.

[20] Yuan Q, Li JJ, An CH, Sun L. Biological characteristics of rat dorsal root ganglion cell and human vascular endothelial cell in mono-and co-culture. Mol Biol Rep. 2014; 41: 6949-56.

[21] Zhou M, Hu M, He S, Li B, Liu C, Min J, et al. Effects of RSC96 schwann cell-derived exosomes on proliferation, senescence, and apoptosis of dorsal root ganglion cells in Vitro. Med Sci Monit. 2018; 24: 7841-9.

[22] Yuan Q, Sun L, Yu H, An C. Human microvascular endothelial cell promotes the development of dorsal root ganglion neurons via BDNF pathway in a co-culture system. Biosci Biotechnol Biochem. 2017; 81: 1335-42.

[23] Miyoshi S, Sekiguchi M, Konno SI, Kikuchi SI, Kanaya F. Increased expression of vascular endothelial growth factor protein in dorsal root ganglion exposed to nucleus pulposus on the nerve root in rats. Spine (Phila Pa 1976). 2011; 36: E1-6.

[24] Grasman JM, Kaplan DL. Human endothelial cells secrete neurotropic factors to direct axonal growth of peripheral nerves. Sci Rep. 2017; 7: 4092.

[25] Ruiz-Soto M, Riancho J, Tapia O, Lafarga M, Berciano MT. Satellite Glial Cells of the Dorsal Root Ganglion: A New “Guest/Physiopathological Target” in ALS. Front Aging Neurosci. 2020; 12: 1-15.

[26] Bolívar S, Navarro X, Udina E. Schwann Cell Role in Selectivity of Nerve Regeneration. Cell. 2020; 9: 2131.

[27] Zhang L, Zheng T, Wu L, Han Q, Chen S, Kong Y, et al. Fabrication and characterization of 3D-printed gellan gum/starch composite scaffold for Schwann cells growth. Nanotechnol Rev. 2021; 10: 50-61.

[28] Tricaud N.Myelinating schwann cell polarity and mechanically-driven myelin sheath elongation. Front Cell Neurosci. 2018; 11: 414.

[29] Xie J, Macewan MR, Liu W, Jesuraj N, Li X, Hunter D, et al. Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system. ACS Appl Mater Interfaces. 2014; 6: 9472-80.

[30] Qu WR, Zhu Z, Liu J, Song DB, Tian H, Chen BP, et al. Interaction between Schwann cells and other cells during repair of peripheral nerve injury. Neural Regen Res. 2021; 16: 93-8.

[31] Gordon T, Sulaiman O, Boyd JG. Experimental strategies to promote functional recovery after peripheral nerve injuries. J Peripher Nerv Syst. 2003; 8: 236-50.

[32] Endo T, Kadoya K, Kawamura D, Iwasaki N. Evidence for cell-contact factor involvement in neurite outgrowth of dorsal root ganglion neurons stimulated by Schwann cells. Exp Physiol. 2019; 104: 1447-54.

[33] Richardson JA, Rementer CW, Bruder JM, Hoffman-Kim D. Guidance of dorsal root ganglion neurites and Schwann cells by isolated Schwann cell topography on poly(dimethyl siloxane) conduits and films. J Neural Eng. 2011; 8: 46015.

[34] Yang J, Lou Q, Huang R, Shen L, Chen Z. Dorsal root ganglion neurons induce transdifferentiation of mesenchymal stem cells along a Schwann cell lineage. Neurosci Lett. 2008; 445: 246-51.

[35] Hynds DL, Rangappa N, Ter Beest J, Snow DM, Rabchevsky AG. Microglia Enhance Dorsal Root Ganglion Outgrowth in Schwann Cell Cultures. Glia. 2004; 46: 218-23.

[36] Päiväläinen S, Nissinen M, Honkanen H, Lahti O, Kangas SM, Peltonen J, et al. Myelination in mouse dorsal root ganglion/Schwann cell cocultures. Mol Cell Neurosci. 2008; 37: 568-78.

[37] Zhang H, Lin F, Huang J, Xiong C. Anisotropic stiffness gradient-regulated mechanical guidance drives directional migration of cancer cells. Acta Biomater. 2020; 106: 181-92.

[38] Dong X, Liu S, Yang Y, Gao S, Li W, Cao J, et al. Aligned microfiber-induced macrophage polarization to guide schwann-cell-enabled peripheral nerve regeneration. Biomaterials. 2021; 272: 120767. https://doi.org/10.1016/j.biomaterials.2021.120767.

[39] Li G, Zhao X, Zhao W, Zhang L, Wang C, Jiang M, et al. Porous chitosan scaffolds with surface micropatterning and inner porosity and their effects on Schwann cells. Biomaterials. 2014; 35: 8503-13.

[40] Werner M, Blanquer SBG, Haimi SP, Korus G, Dunlop JWC, Duda GN, et al. Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation. Adv Sci. 2017; 4: 1600347.

[41] Priemel T, Palia G, Förste F, Jehle F, Sviben S, Mantouvalou I, et al. Microfluidic-like fabrication of metal ion-cured bioadhesives by mussels. Science. 2021; 374: 206-11.

[42] Li G, Li S, Zhang L, Chen S, Sun Z, Li S, et al. Construction of Biofunctionalized Anisotropic Hydrogel Micropatterns and Their Effect on Schwann Cell Behavior in Peripheral Nerve Regeneration. ACS Appl Mater Interfaces. 2019; 11: 37397-410.

[43] Yan J, Wu R, Liao S, Jiang M, Qian Y. Applications of Polydopamine-Modified Scaffolds in the Peripheral Nerve Tissue Engineering. Front Bioeng Biotechnol. 2020; 8: 590998.

[44] Shen YAA, Chen Y, Dao DQ, Mayoral SR, Wu L, Meijer D, et al. Phosphorylation of LKB1/Par-4 establishes Schwann cell polarity to initiate and control myelin extent. Nat Commun. 2014; 5: 4991.

[45] Szulczewski JM, Inman DR, Proestaki M, Notbohm J, Burkel BM, Ponik SM. Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers. Acta Biomater. 2021; 129: 96-109. https://doi.org/10.1016/j.actbio.2021.04.053.

[46] Li G, Xiao Q, Zhang L, Zhao Y, Yang Y. Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration. Carbohydr Polym. 2017; 171: 39-49.

[47] Castillo X, Melo Z, Varela-Echavarría A, Tamariz E, Aroña RM, Arnold E, et al. Vasoinhibin Suppresses the Neurotrophic Effects of VEGF and NGF in Newborn Rat Primary Sensory Neurons. Neuroendocrinology. 2018; 106: 221-33.

[48] Marro BS, Skinner DD, Cheng Y, Grist JJ, Dickey LL, Eckman E, et al. Disrupted CXCR2 Signaling in Oligodendroglia Lineage Cells Enhances Myelin Repair in a Viral Model of Multiple Sclerosis. J Virol. 2019; 93: 154-5.

[49] Li G, Zheng T, Wu L, Han Q, Lei Y, Xue L, et al. Bionic microenvironment-inspired synergistic effect of anisotropic micro-nanocomposite topology and biology cues on peripheral nerve regeneration. Sci Adv. 2021; 7: 5812.

[50] Carroll SL, Stonecypher MS. Tumor suppressor mutations and growth factor signaling in the pathogenesis of NF1-associated peripheral nerve sheath tumors. I. The role of tumor suppressor mutations. J Neuropathol Exp Neurol. 2004; 63: 1115-23.