In vitro experiment of stem cell engineered two-sided anisotropic electrospun membranes for promoting dural repair

doi:10.12307/2024.248

Abstract

Abstract: BACKGROUND: Currently, the dura mater is clinically repaired using autologous tissue or materials such as gelatin sponge, but all of them have their inherent defects. Therefore, there is an urgent need for a biomaterial that can promote dural repair.
OBJECTIVE: The two-sided anisotropic electrospun membrane was constructed by using directional electrospinning technology and collagen self-assembly technology, and was used as a carrier for bone marrow mesenchymal stem cells to investigate various physicochemical properties and biological characteristics of the artificial dura mater.
METHODS: Ordered polylactic acid electrospun fibers with double-sided (collagen protein on one side and polylactic acid on the other side) anisotropic electrospun membranes (collagen group), disordered polylactic acid electrospun membranes (disordered fiber group), and ordered oriented polylactic acid electrospun membranes (ordered fiber group) were prepared by electrospinning technique as well as collagen self-assembly technique. Scanning electron microscopy, mechanical stretching, water contact angle testing, and degradation experiments were used to characterize the physicochemical properties of the electrospun membranes. Electrospun membranes in the collagen group (bone marrow mesenchymal stem cells were inoculated on the collagen surface to obtain the stem cell-engineered electrospun membranes), disordered fiber group and ordered fiber group were cocultured with bone marrow mesenchymal stem cells. The biocompatibility of electrospun membranes was evaluated using CCK-8 assay and live/dead staining. Integrin β1 immunofluorescence staining was used to evaluate the adhesion characteristics of electrospun membranes. The stem cell-engineered electrospun membrane and the electrospun membrane in the collagen group were cocultured with bone marrow macrophages respectively. Immunomodulatory properties were assessed by detecting the expression of inflammation-related genes using inducible nitric oxide synthase (M1 type), CD206 (M2 type) immunofluorescence staining, and qRT-PCR.
RESULTS AND CONCLUSION: (1) The oriented electrospun fiber membrane could mimic the structure of the longitudinally aligned natural dura mater, and the addition of collagen increased the hydrophilicity of the fiber membrane by about 2-fold and the mechanical properties by 1.2-fold. (2) When cocultured with bone marrow mesenchymal stem cells, CCK-8 assay and live/dead staining suggested that the cellular bioactivity in the collagen group was significantly higher than that in the disordered fiber group and ordered fiber group. Immunofluorescence staining revealed that the expression of integrin β1 in the collagen group was about 2.6 times higher than that of the disordered and ordered fiber groups, and the cell spreading morphology was good. (3) When cocultured with bone marrow macrophages, immunofluorescence staining exhibited that the fluorescence intensity of M1 type macrophages in the stem cell-engineered electrospun membrane group was lower than that in the collagen group (P < 0.01), and the fluorescence intensity of M2 type macrophages was higher than that in the collagen group (P < 0.01). qRT-PCR demonstrated that proinflammatory gene tumor necrosis factor α and interleukin-1β mRNA expression in the stem cell-engineered electrospun membrane group was lower than that of the collagen group (P < 0.001); anti-inflammatory genes such as interleukin-10 and transforming growth factor β mRNA expressions were higher than those in the collagen group (P < 0.001). (4) The above results suggest that the stem cell-engineered amphipathic artificial dura mimics the directional structure of normal dura, with the inner surface facilitating cell growth and adhesion and the outer edge avoiding tissue adhesion, while the polarization of macrophages to the M2 subtype is promoted and the local inflammatory microenvironment is regulated through the mesenchymal stem cell paracrine component.

Key words: dural repair, artificial dura, electrospinning, collagen, bone marrow mesenchymal stem cell, polylactic acid

CLC Number:

Xu Jingzhi, Wang Wenbo, Sun Huiwen, Gu Yong. In vitro experiment of stem cell engineered two-sided anisotropic electrospun membranes for promoting dural repair[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(10): 1540-1546.

Figures/Tables 11

References

[1] GUERIN P, EL FEGOUN AB, OBEID I, et al. Incidental durotomy during spine surgery: incidence, management and complications. A retrospective review. Injury. 2012;43(4):397-401.
[2] LUSZCZYK MJ, BLAISDELL GY, WIATER BP, et al. Traumatic dural tears: what do we know and are they a problem? Spine J. 2014;14(1):49-56.
[3] WEBER C, PIEK J, GUNAWAN D. Health care costs of incidental durotomies and postoperative cerebrospinal fluid leaks after elective spinal surgery. Eur Spine J. 2015;24(9):2065-2068.
[4] HEO DH, HA JS, LEE DC, et al. Repair of Incidental Durotomy Using Sutureless Nonpenetrating Clips via Biportal Endoscopic Surgery. Global Spine J. 2022;12(3): 452-457.
[5] SKOVSTED YDE S, BRUNBJERG ME, GUDMUNDSDOTTIR G, et al. Dural repair using porcine ADM: two cases and a literature review. Case Reports Plast Surg Hand Surg. 2017;4(1):5-8.
[6] STENDEL R, DANNE M, FISS I, et al. Efficacy and safety of a collagen matrix for cranial and spinal dural reconstruction using different fixation techniques. J Neurosurg. 2008;109(2):215-521.
[7] BOHOUN C A, GOTO T, MORISAKO H, et al. Skull Base Dural Repair Using Autologous Fat as a Dural Substitute: An Efficient Technique. World Neurosurg. 2019;127:e896-e900.
[8] FAN B, WEI Z, YAO X, et al. Microenvironment Imbalance of Spinal Cord Injury. Cell Transplant. 2018;27(6):853-866.
[9] VENKATALAXMI A, PADMAVATHI BS, AMARANATH T. A general solution of unsteady Stokes equations. Fluid Dyn Res. 2004;35(3):229-236.
[10] LI Q, MA L, GAO C. Biomaterials for in situ tissue regeneration: development and perspectives. J Mater Chem B. 2015;3(46):8921-8938.
[11] SCHMALZ P, GRIESSENAUER C, OGILVY CS, et al. Use of an Absorbable Synthetic Polymer Dural Substitute for Repair of Dural Defects: A Technical Note. Cureus. 2018;10(1):e2127.
[12] LI J, TIAN J, LI C, et al. A hydrogel spinal dural patch with potential anti-inflammatory, pain relieving and antibacterial effects. Bioact Mater. 2022;14:389-401.
[13] LI Q, MU L, ZHANG F, et al. A novel fish collagen scaffold as dural substitute. Mater Sci Eng C Mater Biol Appl. 2017;80:346-351.
[14] WANG Z, CUI W. Two Sides of Electrospun Fiber in Promoting and Inhibiting Biomedical Processes. Advanced Therapeutics. 2020;4(1): 2000096.
[15] VIGANI B, ROSSI S, SANDRI G, et al. Design and criteria of electrospun fibrous scaffolds for the treatment of spinal cord injury. Neural Regen Res. 2017;12(11): 1786-1790.
[16] XU Y, CUI W, ZHANG Y, et al. Hierarchical Micro/Nanofibrous Bioscaffolds for Structural Tissue Regeneration. Adv Healthc Mater. 2017;6(13). doi: 10.1002/adhm.201601457.
[17] CHEN AY, ZHONG C, LU TK. Engineering living functional materials. ACS Synth Biol. 2015;4(1):8-11.
[18] TANG TC, AN B, HUANG Y, et al. Materials design by synthetic biology. Na Rev Mater. 2020;6(4):332-350.
[19] SHANG L, SHAO C, CHI J, et al. Living Materials for Life Healthcare. Acc Mater Res. 2020;2(1):59-70.
[20] WANG S, QU X, ZHAO RC. Clinical applications of mesenchymal stem cells. J Hematol Oncol. 2012;5:19.
[21] BRUNELLE AR, HORNER CB, LOW K, et al. Electrospun thermosensitive hydrogel scaffold for enhanced chondrogenesis of human mesenchymal stem cells. Acta Biomater. 2018;66:166-176.
[22] LV B, ZHANG X, YUAN J, et al. Biomaterial-supported MSC transplantation enhances cell-cell communication for spinal cord injury. Stem Cell Res Ther. 2021;12(1):36.
[23] MOTHE AJ, TATOR CH. Advances in stem cell therapy for spinal cord injury . J Clin Invest. 2012;122(11): 824-3834.
[24] MARDPOUR S, HAMIDIEH AA, TALEAHMAD S, et al. Interaction between mesenchymal stromal cell-derived extracellular vesicles and immune cells by distinct protein content. J Cell Physiol. 2019;234(6):8249-8258.
[25] ANTON K, BANERJEE D, GLOD J. Macrophage-associated mesenchymal stem cells assume an activated, migratory, pro-inflammatory phenotype with increased IL-6 and CXCL10 secretion. PLoS One. 2012;7(4):e35036.
[26] CARTY F, MAHON BP, ENGLISH K. The influence of macrophages on mesenchymal stromal cell therapy: passive or aggressive agents? Clin Exp Immunol. 2017; 188(1):1-11.
[27] CHUNG E, SON Y. Crosstalk between mesenchymal stem cells and macrophages in tissue repair. Tissue Eng Regen Med. 2014;11(6):431-438.
[28] DAFFORD EE, ANDERSON PA. Comparison of dural repair techniques. Spine J. 2015;15(5):1099-1105.
[29] XI K, GU Y, TANG J, et al. Microenvironment-responsive immunoregulatory electrospun fibers for promoting nerve function recovery. Nat Commun. 2020; 11(1):4504.
[30] WU L, GU Y, LIU L, et al. Hierarchical micro/nanofibrous membranes of sustained releasing VEGF for periosteal regeneration. Biomaterials. 2020;227:119555.
[31] TANG J, XI K, CHEN H, et al. Flexible Osteogenic Glue as an All‐In‐One Solution to Assist Fracture Fixation and Healing. Adv Funct Mater. 2021;31(38):2102465.1-2102465.16.
[32] BAI J, WANG H, CHEN H, et al. Biomimetic osteogenic peptide with mussel adhesion and osteoimmunomodulatory functions to ameliorate interfacial osseointegration under chronic inflammation. Biomaterials. 2020;255:120197.
[33] LEE HG, KANG MS, KIM SY, et al. Dural Injury in Unilateral Biportal Endoscopic Spinal Surgery. Global Spine J. 2021;11(6):845-851.
[34] NAGEL SJ, REDDY CG, FRIZON LA, et al. Spinal dura mater: biophysical characteristics relevant to medical device development. J Med Eng Technol. 2018;42(2):128-139.
[35] SHIN JK, YOUN MS, SEONG YJ, et al. Iatrogenic dural tear in endoscopic lumbar spinal surgery: full endoscopic dural suture repair (Youn’s technique). Eur Spine J. 2018;27(Suppl 3):544-548.
[36] SHIMADA Y, HONGO M, MIYAKOSHI N, et al. Dural substitute with polyglycolic acid mesh and fibrin glue for dural repair: technical note and preliminary results. J Orthop Sci. 2006;11(5):454-458.
[37] IWATA A, TAKAHATA M, KADOYA K, et al. Effective Repair of Dural Tear Using Bioabsorbable Sheet With Fibrin Glue. Spine (Phila Pa 1976). 2017;42(18):1362-1366.
[38] PENG Z, GAO W, YUE B, et al. Promotion of neurological recovery in rat spinal cord injury by mesenchymal stem cells loaded on nerve-guided collagen scaffold through increasing alternatively activated macrophage polarization. J Tissue Eng Regen Med. 2018;12(3):e1725-e736.
[39] LUZ-CRAWFORD P, JORGENSEN C, DJOUAD F. Mesenchymal Stem Cells Direct the Immunological Fate of Macrophages. Results Probl Cell Differ. 2017;62:61-72.
[40] ORR MB, GENSEL JC. Spinal Cord Injury Scarring and Inflammation: Therapies Targeting Glial and Inflammatory Responses. Neurotherapeutics. 2018;15(3):541-553.