Investigation of peripheral rat blood immune phenotype to evaluate the biocompatibility of graphene nanoparticles and xenografted mesenchymal stem cells

Abstract

Aim:Peripheral nerve injuries (PNIs) often result in a diminished quality of life for those affected and are the most common nervous system injury, with limited treatment options. Regenerative medicine presents novel biomaterial and cell-based therapies to repair the damaged tissue. Graphene oxide (GO), and mesenchymal stem cells (MSCs) have the potential to serve as components to treat PNI. This study evaluates the systemic toxicity of GO and xenogenic human MSCs by analyzing the peripheral blood immune phenotype when a novel nerve guidance conduit (NGC) is implanted in a rat model for six months. Methods:A 10-mm long sciatic nerve defect model was created in 8–10-week-old Lewis rats. Four treatment groups were generated: autograft (positive control), poly (lactic- co-glycolic acid) (PLGA) NGC, PLGA NGC with 0.25% GO, and PLGA/GO NGC seeded with 1 × 10⁶ human adipose-derived MSCs. Tail blood was collected before surgery, and at 24 hours, 2 weeks, 2, 3, 5, and 6 months after surgery. Hematological analyses were carried out to evaluate systemic changes, if any, in peripheral immune cell types, namely, T lymphocytes, B lymphocytes, natural killer cells, and macrophages. The treated and contralateral sciatic nerves were excised, paraffin embedded, sectioned, and H&E stained, to identify any local foreign body rejection. Results:Treatment groups with GO and MSCs displayed percent total values of peripheral immune cells equivalent to the autograft at each time point. There was no evidence of an inflammatory response in the histological samples. Conclusions:The lack of changes in immune phenotype demonstrates a lack of nanotoxicity of the graphene nanoparticles and no evidence of adverse effects due to the MSCs. This was further supported by a lack of local foreign body response at the site of implantation. Overall, the PLGA/GO NGC + MSCs construct is biocompatible for six months in a rat PNI model, exhibiting a potential for clinical translation.

Keywords

graphene oxide / mesenchymal stem cells / systemic toxicity / biocompatibility / immunology

References

[1] Modrak M, Talukder MAH, Gurgenashvili K, Noble M, Elfar JC. Peripheral nerve injury and myelination: Potential therapeutic strategies. J Neurosci Res. 2020; 98: 780-95.

[2] Lopes B, Sousa P, Alvites R, Branquinho M, Sousa AC, Mendonça C, et al. Peripheral Nerve Injury Treatments and Advances: One Health Perspective. Int J Mol Sci. 2022; 23: 918.

[3] Wang ML, Rivlin M, Graham JG, Beredjiklian PK. Peripheral nerve injury, scarring, and recovery. Connect Tissue Res. 2019; 60: 3-9.

[4] Houshyar S, Bhattacharyya A, Shanks R. Peripheral Nerve Conduit: Materials and Structures. ACS Chem Neurosci. 2019; 10: 3349-65.

[5] Doblado LR, Martínez-Ramos C, Pradas MM. Biomaterials for Neural Tissue Engineering. Front Nanotechnol. 2021; 3: 643507.

[6] Li X, Guan Y, Li C, Zhang T, Meng F, Zhang J, et al. Immunomodulatory effects of mesenchymal stem cells in peripheral nerve injury. Stem Cell Res Ther. 2022; 13: 18.

[7] MacDonald AF, Harley-Troxell ME, Newby SD, Dhar MS. 3D-Printing Graphene Scaffolds for Bone Tissue Engineering. Pharmaceutics. 2022; 14: 1834.

[8] Tupone MG, Panella G, d'Angelo M, Castelli V, Caioni G, Catanesi M, et al. An Update on Graphene-Based Nanomaterials for Neural Growth and Central Nervous System Regeneration. Int J Mol Sci. 2021; 22: 13047.

[9] Maleki M, Zarezadeh R, Nouri M, Sadigh AR, Pouremamali F, Asemi Z, et al. Graphene Oxide: A Promising Material for Regenerative Medicine and Tissue Engineering. Biomol Concepts. 2020; 11: 182-200.

[10] Joseph G, Orme RP, Kyriacou T, Fricker RA, Roach P. Effects of Surface Chemistry Interaction on Primary Neural Stem Cell Neurosphere Responses. ACS Omega. 2021; 6: 19901-10.

[11] Rhazouani A, Gamrani H, Achaby ME, Aziz K, Gebrati L, Uddin MS, et al. Synthesis and Toxicity of Graphene Oxide Nanoparticles: A Literature Review of In Vitro and In Vivo Studies . Biomed Res Int. 2021; 2021: 5518999.

[12] Hui Y, Yan Z, Yang H, Xu X, Yuan W, Qian Y. Graphene Family Nanomaterials for Stem Cell Neurogenic Differentiation and Peripheral Nerve Regeneration. ACS Appl Bio Mater. 2022; 5: 4741-59.

[13] Zhang D, Yao Y, Duan Y, Yu X, Shi H, Nakkala JR, et al. Surface-Anchored Graphene Oxide Nanosheets on Cell-Scale Micropatterned Poly(d,l-lactide- co-caprolactone) Conduits Promote Peripheral Nerve Regeneration . ACS Appl Mater Interfaces. 2020; 12: 7915-30.

[14] Harley-Troxell ME, Steiner R, Advincula RC, Anderson DE, Dhar M. Interactions of Cells and Biomaterials for Nerve Tissue Engineering: Polymers and Fabrication. Polymers (Basel). 2023; 15: 3685.

[15] Rhazouani A, Gamrani H, Ed-Day S, Lafhal K, Boulbaroud S, Gebrati L, et al. Sub-acute toxicity of graphene oxide (GO) nanoparticles in male mice after intraperitoneal injection: Behavioral study and histopathological evaluation. Food Chem Toxicol. 2023; 171: 113553.

[16] Zhao Y, Wu P, Zhao Z, Chen F, Xiao A, Yue Z, et al. Electrodeposition of chitosan/graphene oxide conduit to enhance peripheral nerve regeneration. Neural Regen Res. 2023; 18: 207-12.

[17] Yunus MA, Ramli MM, Osman NH, Mohamed R. Stimulation of Innate and Adaptive Immune Cells with Graphene Oxide and Reduced Graphene Oxide Affect Cancer Progression. Arch Immunol Ther Exp (Warsz). 2021; 69: 20.

[18] Palmieri V, Perini G, De Spirito M, Papi M. Graphene oxide touches blood: in vivo interactions of bio-coronated 2D materials. Nanoscale Horiz. 2019; 4: 273-90.

[19] Yi S, Zhang Y, Gu X, Huang L, Zhang K, Qian T, et al. Application of stem cells in peripheral nerve regeneration. Burns Trauma. 2020; 8: tkaa002.

[20] Ławkowska K, Pokrywczyńska M, Koper K, Kluth LA, Drewa T, Adamowicz J. Application of Graphene in Tissue Engineering of the Nervous System. Int J Mol Sci. 2021; 23: 33.

[21] Kubiak CA, Grochmal J, Kung TA, Cederna PS, Midha R, Kemp SWP. Stem-cell-based therapies to enhance peripheral nerve regeneration. Muscle Nerve. 2020; 61: 449-59.

[22] Kaminska A, Radoszkiewicz K, Rybkowska P, Wedzinska A, Sarnowska A. Interaction of Neural Stem Cells (NSCs) and Mesenchymal Stem Cells (MSCs) as a Promising Approach in Brain Study and Nerve Regeneration. Cells. 2022; 11: 1464.

[23] Lv B, Zhang X, Yuan J, Chen Y, Ding H, Cao X, et al. Biomaterial-supported MSC transplantation enhances cell-cell communication for spinal cord injury. Stem Cell Res Ther. 2021; 12: 36.

[24] Priester C, MacDonald A, Dhar M, Bow A. Examining the Characteristics and Applications of Mesenchymal, Induced Pluripotent, and Embryonic Stem Cells for Tissue Engineering Approaches across the Germ Layers. Pharmaceuticals (Basel). 2020; 13: 344.

[25] Cofano F, Boido M, Monticelli M, Zenga F, Ducati A, Vercelli A, et al. Mesenchymal Stem Cells for Spinal Cord Injury: Current Options, Limitations, and Future of Cell Therapy. Int J Mol Sci. 2019; 20: 2698.

[26] Drela K, Stanaszek L, Nowakowski A, Kuczynska Z, Lukomska B. Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes. Stem Cells Int. 2019; 2019: 7012692.

[27] Dąbrowski B, Żuchowska A, Brzózka Z. Graphene oxide internalization into mammalian cells - a review. Colloids Surf B Biointerfaces. 2023; 221: 112998.

[28] Sensharma P, Madhumathi G, Jayant RD, Jaiswal AK. Biomaterials and cells for neural tissue engineering: Current choices. Mater Sci Eng C Mater Biol Appl. 2017; 77: 1302-15.

[29] Harley-Troxell ME, Pedersen AP, Newby SD, Christoph E, Stephenson S, Masi TJ, et al. 3D-Printed Poly(Lactic-Co-Glycolic Acid) and Graphene Oxide Nerve Guidance Conduit with Mesenchymal Stem Cells for Effective Axon Regeneration in a Rat Sciatic Nerve Defect Model. Int J Nanomedicine. 2025; 20: 3201-17.

[30] Bow A, Newby S, Rifkin R, Jackson BK, Matavosian A, Griffin C, et al. Evaluation of a Polyurethane Platform for Delivery of Nanohydroxyapatite and Decellularized Bone Particles in a Porous Three-Dimensional Scaffold. ACS Appl Bio Mater. 2019; 2: 1815-29.

[31] Newby SD, Masi T, Griffin CD, King WJ, Chipman A, Stephenson S, et al. Functionalized Graphene Nanoparticles Induce Human Mesenchymal Stem Cells to Express Distinct Extracellular Matrix Proteins Mediating Osteogenesis. Int J Nanomedicine. 2020; 15: 2501-13.

[32] MacDonald AF, Trotter RD, Griffin CD, Bow AJ, Newby SD, King WJ, et al. Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene. J Nanobiotechnology. 2021; 19: 285.

[33] Alghazali KM, Newby SD, Nima ZA, Hamzah RN, Watanabe F, Bourdo SE, et al. Functionalized gold nanorod nanocomposite system to modulate differentiation of human mesenchymal stem cells into neural-like progenitors. Sci Rep. 2017; 7: 16654.

[34] Wachs RA, Wellman SM, Porvasnik SL, Lakes EH, Cornelison RC, Song YH, et al. Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap. Cells Tissues Organs. 2023; 212: 512-22.

[35] Li C, Zhang M, Liu S, Zhang F, Wan T, Ding Z, et al. Chitin Nerve Conduits with Three-Dimensional Spheroids of Mesenchymal Stem Cells from SD Rats Promote Peripheral Nerve Regeneration. Polymers (Basel). 2021; 13: 3957.

[36] Wu W, Dong Y, Liu H, Jiang X, Yang L, Luo J, et al. 3D printed elastic hydrogel conduits with 7,8-dihydroxyflavone release for peripheral nerve repair. Mater Today Bio. 2023; 20: 100652.

[37] Onode E, Uemura T, Takamatsu K, Yokoi T, Shintani K, Hama S, et al. Bioabsorbable nerve conduits three-dimensionally coated with human induced pluripotent stem cell-derived neural stem/progenitor cells promote peripheral nerve regeneration in rats. Sci Rep. 2021; 11: 4204.

[38] Ma Y, Dong L, Zhou D, Li L, Zhang W, Zhen Y, et al. Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats. J Cell Mol Med. 2019; 23: 2822-35.

[39] Li A, Pereira C, Hill EE, Vukcevich O, Wang A. In Vitro, In Vivo and Ex Vivo Models for Peripheral Nerve Injury and Regeneration . Curr Neuropharmacol. 2022; 20: 344-61.

[40] Harley-Troxell ME, Steiner R, Newby SD, Bow AJ, Masi TJ, Millis N, et al. Electrospun PCL Nerve Wrap Coated with Graphene Oxide Supports Axonal Growth in a Rat Sciatic Nerve Injury Model. Pharmaceutics. 2024; 16: 1254.

[41] Brown C. Blood collection from the tail of a rat. Lab Anim (NY). 2006; 35: 24-5.

[42] Bio-Rad Laboratories. Direct Immunofluorescence Staining of Surface Epitopes of Cells and Blood V1.1.206. Bio-Rad Laboratories ; 2016.

[43] Nikzamir M, Akbarzadeh A, Panahi Y. An overview on nanoparticles used in biomedicine and their cytotoxicity. J Drug Deliv Sci Technol. 2021; 61: 102316.

[44] Wang J, Wang NS. Nanoparticles in Biomedical Applications and Their Safety Concerns. InTech; 2011.

[45] Zhang M, Li L, An H, Zhang P, Liu P. Repair of Peripheral Nerve Injury Using Hydrogels Based on Self-Assembled Peptides. Gels. 2021; 7: 152.

[46] Salthouse D, Novakovic K, Hilkens CMU, Ferreira AM. Interplay between biomaterials and the immune system: Challenges and opportunities in regenerative medicine. Acta Biomater. 2023; 155: 1-18.

[47] Lock A, Cornish J, Musson DS. The Role of In Vitro Immune Response Assessment for Biomaterials. J Funct Biomater. 2019; 10: 31.

[48] Whitaker R, Hernaez-Estrada B, Hernandez RM, Santos-Vizcaino E, Spiller KL. Immunomodulatory Biomaterials for Tissue Repair. Chem Rev. 2021; 121: 11305-35.

[49] Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol. 2008; 20: 86-100.

[50] Shen P, Chen Y, Luo S, Fan Z, Wang J, Chang J, et al. Applications of biomaterials for immunosuppression in tissue repair and regeneration. Acta Biomater. 2021; 126: 31-44.

[51] Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation. 2011; 8: 110.

[52] Dudek I, Skoda M, Jarosz A, Szukiewicz D. The Molecular Influence of Graphene and Graphene Oxide on the Immune System Under In Vitro and In Vivo Conditions. Arch Immunol Ther Exp (Warsz). 2016; 64: 195-215.