INTRODUCTION: Spinal cord injury involves complex pathobiological mechanisms, necessitating a multidimensional approach for its cure. Previous studies have shown that α9-integrin expression and activation in mature dorsal root ganglion neurons enable the regeneration of injured axons within the spinal cord. However, tissue cavitation and fibrosis impede the regenerating axons from following their usual pathways, forcing them to seek alternative routes rich in tenascin-C, the primary ligand of the integrin. Fibrin gel, an FDA-approved and biocompatible material, can offer three-dimensional support for axonal extension through the cavitated area, thus preventing the formation of aberrant paths and connections that occur in the absence of a suitable scaffold. METHODS: The aim of this study was to investigate how combining α9-integrin expression by adeno-associated virus with the use of a fibrin gel as an extracellular microenvironment affects the growth of mature DRG neurites in vitro. Additionally, we sought to functionalize fibrin with integrin ligand peptides, specifically AEIDGIEL, the active domain of tenascin-C, to ensure α9-integrin activation. RESULTS: Our results indicate that fibrin gels are a suitable biomaterial for promoting neurite growth and that AEIDGIEL peptide effectively activates the integrin. Furthermore, we corroborate an autocrine signaling loop of α9-integrin and TN-C produced by neurons. DISCUSSION: the proposed combination therapy of α9-integrin and fibrin gel biomaterials incorporating AEIDGIEL peptide shows promise for addressing the complex challenges of spinal cord injury and promoting effective neural regeneration, laying the foundation for further in vivo research.

2nd Faculty of Medicine Charles University Prague Czechia

Department of Cell Morphology and Molecular Neurobiology Faculty of Biology and Biotechnology Ruhr University Bochum Bochum Germany

Department of Clinical Neurosciences John Van Geest Centre for Brain Repair University of Cambridge Cambridge United Kingdom

Faculty of Biological Sciences University of Leeds Leeds United Kingdom

Institute of Experimental Medicine Czech Academy of Science Prague Czechia

Neuroregeneration Research Group Netherlands Institute for Neuroscience Amsterdam Netherlands

Zobrazit více v PubMed

Ahuja C. S., Wilson J. R., Nori S., Kotter M. R. N., Druschel C., Curt A., et al. (2017). Traumatic spinal cord injury. Nat. Rev. Dis. Primer 3:17018. 10.1038/nrdp.2017.18 PubMed DOI

Andrews M. R., Czvitkovich S., Dassie E., Vogelaar C. F., Faissner A., Blits B., et al. (2009). α9 integrin promotes neurite outgrowth on tenascin-c and enhances sensory axon regeneration. J. Neurosci. 29 5546–5557. 10.1523/jneurosci.0759-09.2009 PubMed DOI PMC

Cai M., Chen L., Wang T., Liang Y., Zhao J., Zhang X., et al. (2023). Hydrogel scaffolds in the treatment of spinal cord injury: A review. Front. Neurosci. 17:1211066. 10.3389/fnins.2023.1211066 PubMed DOI PMC

Cheah M., Andrews M. R., Chew D. J., Moloney E. B., Verhaagen J., Fassler R., et al. (2016). Expression of an activated integrin promotes long-distance sensory axon regeneration in the spinal cord. J. Neurosci. 36 7283–7297. 10.1523/jneurosci.0901-16.2016 PubMed DOI PMC

Cheah M., Cheng Y., Petrova V., Cimpean A., Jendelova P., Swarup V., et al. (2023). Integrin-driven axon regeneration in the spinal cord activates a distinctive CNS regeneration program. J. Neurosci. 43 4775–4794. 10.1523/jneurosci.2076-22.2023 PubMed DOI PMC

Coffin S. T., Gaudette G. R. (2016). Aprotinin extends mechanical integrity time of cell-seeded fibrin sutures. J. Biomed. Mater. Res. A 104 2271–2279. 10.1002/jbm.a.35754 PubMed DOI PMC

Ding W., Hu S., Wang P., Kang H., Peng R., Dong Y., et al. (2022). Spinal cord injury: The global incidence, prevalence, and disability from the global burden of disease study 2019. Spine 47 1532–1540. 10.1097/BRS.0000000000004417 PubMed DOI PMC

Fawcett J. W. (2020). The struggle to make CNS axons regenerate: Why has it been so difficult? Neurochem. Res. 45 144–158. 10.1007/s11064-019-02844-y PubMed DOI PMC

Gardiner N. J. (2011). Integrins and the extracellular matrix: Key mediators of development and regeneration of the sensory nervous system. Dev. Neurobiol. 71 1054–1072. 10.1002/dneu.20950 PubMed DOI

He X., Yang L., Dong K., Zhang F., Liu Y., Ma B., et al. (2022). Biocompatible exosome-modified fibrin gel accelerates the recovery of spinal cord injury by VGF-mediated oligodendrogenesis. J. Nanobiotechnol. 20:360. 10.1186/s12951-022-01541-3 PubMed DOI PMC

Herbert C. B., Nagaswami C., Bittner G. D., Hubbell J. A., Weisel J. W. (1998). Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels. J. Biomed. Mater. Res. 40 551–559. PubMed

Hu X., Xu W., Ren Y., Wang Z., He X., Huang R., et al. (2023). Spinal cord injury: Molecular mechanisms and therapeutic interventions. Signal Transduct. Target. Ther. 8:245. 10.1038/s41392-023-01477-6 PubMed DOI PMC

Jarrell D. K., Vanderslice E. J., Lennon M. L., Lyons A. C., VeDepo M. C., Jacot J. G. (2021). Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering. PLoS One 16:e0239242. 10.1371/journal.pone.0239242 PubMed DOI PMC

Ju Y.-E., Janmey P. A., McCormick M. E., Sawyer E. S., Flanagan L. A. (2007). Enhanced neurite growth from mammalian neurons in three-dimensional salmon fibrin gels. Biomaterials 28 2097–2108. 10.1016/j.biomaterials.2007.01.008 PubMed DOI PMC

Lorentz K. M., Kontos S., Frey P., Hubbell J. A. (2011). Engineered aprotinin for improved stability of fibrin biomaterials. Biomaterials 32 430–438. 10.1016/j.biomaterials.2010.08.109 PubMed DOI

Matthews J., Surey S., Grover L. M., Logan A., Ahmed Z. (2021). Thermosensitive collagen/fibrinogen gels loaded with decorin suppress lesion site cavitation and promote functional recovery after spinal cord injury. Sci. Rep. 11:18124. 10.1038/s41598-021-97604-w PubMed DOI PMC

Pan D., Sayanagi J., Acevedo-Cintrón J. A., Schellhardt L., Snyder-Warwick A. K., Mackinnon S. E., et al. (2021). Liposomes embedded within fibrin gels facilitate localized macrophage manipulations within nerve. J. Neurosci. Methods 348:108981. 10.1016/j.jneumeth.2020.108981 PubMed DOI PMC

Schense J. C., Hubbell J. A. (1999). Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug. Chem. 10 75–81. 10.1021/bc9800769 PubMed DOI

Silva J., Bento A. R., Barros D., Laundos T. L., Sousa S. R., Quelhas P., et al. (2017). Fibrin functionalization with synthetic adhesive ligands interacting with α6β1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors. Acta Biomater. 59 243–256. 10.1016/j.actbio.2017.07.013 PubMed DOI

Steindler D., Settles D., Erickson H., Laywell E., Yoshiki A., Faissner A., et al. (1995). Tenascin knockout mice: Barrels, boundary molecules, and glial scars. J. Neurosci. 15 1971–1983. 10.1523/jneurosci.15-03-01971.1995 PubMed DOI PMC

Stepankova K., Smejkalova B., Machova-Urdzikova L., Haveliková K., Verhaagen J., De Winter F., et al. (2023). Alpha 9 integrin expression enables reconstruction of the spinal cord sensory pathway. bioRxiv [Preprint] 10.1101/2023.03.24.534172 DOI

Urech L., Bittermann A. G., Hubbell L., Jeffrey A., Hall H. (2005). Mechanical properties, proteolytic degradability and biological modifications affect angiogenic process extension into native and modified fibrin matrices in vitro. Biomaterials 26 1369–1379. 10.1016/j.biomaterials.2004.04.045 PubMed DOI

Verhaagen J., Hobo B., Ehlert E. M. E., Eggers R., Korecka J. A., Hoyng S. A., et al. (2018). “Small scale production of recombinant adeno-associated viral vectors for gene delivery to the nervous system,” in Retinal Gene Therapy, eds Boon C. J. F., Wijnholds J. (New York, NY: Springer New York; ), 3–17. 10.1007/978-1-4939-7522-8_1 PubMed DOI

Werner A., Willem M., Jones L. L., Kreutzberg G. W., Mayer U., Raivich G. (2000). Impaired axonal regeneration in α7 integrin-deficient mice. J. Neurosci. 20 1822–1830. 10.1523/JNEUROSCI.20-05-01822.2000 PubMed DOI PMC

Woods I., O’Connor C., Frugoli L., Kerr S., Gutierrez Gonzalez J., Stasiewicz M., et al. (2022). Biomimetic scaffolds for spinal cord applications exhibit stiffness-dependent immunomodulatory and neurotrophic characteristics. Adv. Healthc. Mater. 11:2101663. 10.1002/adhm.202101663 PubMed DOI

Yokosaki Y., Matsuura N., Higashiyama S., Murakami I., Obara M., Yamakido M., et al. (1998). Identification of the ligand binding site for the integrin α9β1 in the third fibronectin type III repeat of tenascin-C. J. Biol. Chem. 273 11423–11428. 10.1074/jbc.273.19.11423 PubMed DOI

Yu Z., Li H., Xia P., Kong W., Chang Y., Fu C., et al. (2020). Application of fibrin-based hydrogels for nerve protection and regeneration after spinal cord injury. J. Biol. Eng. 14:22. 10.1186/s13036-020-00244-3 PubMed DOI PMC