Combined Strategy Of α9-Integrin Transduction and AEIDGIEL Peptide-Functionalized Fibrin Gel Biomaterials to Promote Mature DRG Neurite Growth

Combined Strategy Of α9-Integrin Transduction and AEIDGIEL Peptide-Functionalized Fibrin Gel Biomaterials to Promote Mature DRG Neurite Growth | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Combined Strategy Of α9-Integrin Transduction and AEIDGIEL Peptide-Functionalized Fibrin Gel Biomaterials to Promote Mature DRG Neurite Growth Anda Cimpean, Lars Roll, Jacqueline Reinhard, Jessica Kwok, Andreas Faissner, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5289110/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 can offer three-dimensional support for axonal extension through the cavitated area, preventing the formation of aberrant paths and connections that occur in the absence of a suitable scaffold. The aim of this study was to investigate how combining α9-integrin expression with the use of a fibrin gel as an extracellular microenvironment affects mature DRG neurites growth in vitro . Additionally, we sought to functionalize fibrin with AEIDGIEL peptide, the active domain of tenascin-C, to ensure α9-integrin activation. Our results indicate that fibrin gels are a suitable biomaterial for promoting neurite growth and that AEIDGIEL peptide effectively activates the integrin. In conclusion, 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. Biological sciences/Cell biology Biological sciences/Neuroscience α9-integrin fibrin gel AEIDGIEL peptide Tenascin-C dorsal root ganglion Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Spinal cord injury (SCI) can result from either traumatic events such as vertebral fractures or non-traumatic causes like infections or vascular damage, and has a poor prognosis with sensory, motor and autonomic disfunction, which leads to disability and imposes a heavy burden on healthcare systems and society 1 . SCI creates a complex pathological situation with many barriers to recovery, including the inability of the adult central nervous system (CNS) axons to regenerate, cavitation and excessive scarring at the lesion site 2 . Therefore, treatment requires a combinatorial approach with different therapies acting in a synergistic manner. Currently, achieving complete recovery from severe SCI remains challenging due to the absence of successful translational therapies 3 , 4 . Dorsal root ganglion (DRG) neurons are the afferent neurons that relay sensory information from the periphery to the brain. Injured central dorsal root axons are unable to regenerate in the spinal cord resulting in loss of sensorimotor functions and neuropathic pain. Previous studies reported that α9-integrin expression and activation in mature DRG neurons allow regeneration of injured axons into the spinal cord and enable functional recovery of sensation and locomotion 5 – 7 . The major ligand of the integrin is tenascin-C (TN-C), the main extracellular matrix inhibitory glycoprotein upregulated in the CNS environment after SCI. The bioactive domain of TN-C that activates α9-integrin is the AEIDGIEL sequence, contained in the B-C loop of the third subunit of fibronectin type III protein 8 . Integrin expression provides a proper adhesion receptor to control migration of axon growth through the unfavorable environment, and triggers an intracellular genetic program specific to CNS regeneration, with upregulation of genes related to ubiquitination, autophagy, endoplasmic reticulum, casein kinases, transport/trafficking and other signaling molecules 9 . However, the massive tissue disruption and extensive scarring formation at the injury side hinder axons capable of regeneration and prevent the formation of physiological meaningful connections after injury. Hydrogel materials can expand to fill the entire wound site, providing a surface and scaffold through which nerves can regenerate, avoid aberrant growth and protect themselves from the surrounding fibrosis. Many studies have demonstrated their beneficial role in SCI, however, hydrogel treatment alone doesn’t completely restore neurological function 10 . Hydrogel materials contain extracellular matrix (ECM) proteins such as fibrin, which plays an important role in natural wound healing. Because of its affordability, cytocompatibility and ability to modulate angiogenesis and inflammation, fibrin has been used clinically for wound coverings, surgical glues and cell delivery 11 . The aim of this study was to examine how the combination of α9-integrin expression and the use of a fibrin-gel as an extracellular microenvironment affects the growth of mature DRG neurites in vitro . Additionally, we aimed to improve the biomaterial properties by incorporating the bioactive TN-C domain, AEIDGIEL, to ensure the integrin activation. We assessed neurite growth of un-transduced, GFP-transduced and α9-integrin-transduced DRG neurons cultured on coverslips with various coatings, fibrin-gels and AEIDGIEL-modified fibrin-gels (Fig. 1 ). We concluded that fibrin-gels serve as a suitable biomaterial to promote neurite growth and that AEIDGIEL peptide is sufficient to activate the integrin. The intriguing finding that α9-integrin-transduced neurons grow long neurites irrespective of the surface motivated us to further investigations which led to the corroboration of the autocrine signaling loop of α9-integrin and TN-C produced by neurons. Results Expression of α9-integrin in adult DRG neurons enables neurite growth on tenascin-C and its motif peptide AEIDGIEL Previous studies have shown that α9-integrin expression in adult DRG neurons enhances neurite outgrowth and overcomes the growth inhibition by TN-C, a major extracellular inhibitory protein that is upregulated after SCI 5 , 6 . α9-integrin recognizes a peptide in the third fibronectin type III domain of TN-C, AEIDGIEL 8 . First, we confirmed that TN-C inhibits neurite outgrowth from adult DRG neurons, and then determined whether the presence of the AEIDGIEL peptide creates an inhibitory environment similar to wild-type TN-C. We cultured adult DRG neurons on glass coverslips coated with PDL alone, with chicken TN-C, or with the AEIDGIEL peptide. Cells were transduced with AAV-CAG-GFP and AAV-CAG-α9-V5 (Fig. 2 a). On PDL coated coverslips, un-transduced and GFP-transduced DRG neurons grew to an average length of 150 µm (Fig. 2 b), whereas on coverslips coated with TN-C or AEIDGIEL the average neurite outgrowth was 90 µm (Fig. 2 c,d). Next, we asked whether AAV-mediated expression of α9-integrin would promote outgrowth on TN-C and AEIDGIEL. Cells with α9-transduced neurites grew significantly longer than controls on the surface of both TN-C and AIDGIEL, with an average length of approximately 300 µm (Fig. 2 c,d). However, the length of neurites grown by α9-transduced neurons on coverslips coated with PDL alone also increased compared to the controls, and the length was not significantly different to that of neurons grown on TN-C or AEIDGIEL (Fig. 2 b). α9-integrin thus, promoted growth on all surfaces. Because α9-integrin is a specific receptor for TN-C it is likely that axons respond to TN-C in all three situations. The TN-C protein has been shown to be produced by several glial cell types, as well as by DRG neurons, where it is located on their membrane and the surrounding culture surface 5 . Therefore, from this set of experiments, it is not possible to determine whether α9-transduced neurons are responding to the TN-C or AEIDGIEL that has been applied to the culture surface, or the TN-C secreted by DRG neurons. α9-integrin activation relies on neuronal tenascin-C In the above results, we showed that α9-integrin-transduced DRG neurons grew on coverslips coated with TN-C or AEIDGIEL to an average neurite length of 300 µm, but they also grew to a similar distance on PDL surfaces without added TN-C or peptide. This suggests that cells in culture produce TN-C protein, that adheres to PDL and provides a ligand for α9-integrin. Therefore, to confirm that α9-integrin-transduced DRG neurites can grow on AEIDGIEL alone, we had to prevent TN-C production by cultured DRGs and therefore repeated the neurite outgrowth experiments with dissociated cultures of TN-C knockout DRGs 12 . The three experimental surfaces were coverslips coated with PDL, followed by one group coated with AEIDGIEL and the other with chicken TN-C. On these three surfaces, un-transduced and GFP- or α9-transduced adult DRG neurons from TN-C knockout mice were grown (Fig. 3 a). Un-transduced, GFP-transduced and α9-transduced TN-C knockout cells growing on PDL coated coverslips showed equal and small neurite outgrowth (Fig. 3 b). Integrin-transduced TN-C knockout cells grew on TN-C and AEIDGIEL coated coverslips neurites two to three times longer than un-transduced or GFP-transduced (Fig. 3 c,d). This demonstrates that α9-transduced TN-C knockout neurons do not show enhanced axon growth in the absence of ligand on the culture surface. However, in TN-C knockout neurons, TN-C or AEIDGIEL coated on the surface of the PDL can strongly promote the growth of α9-integrin-transduced neurites. These results demonstrate that AEIDGIEL or TN-C coated on coverslips both act as a ligand for α9-integrin, and enable strong neurite outgrowth from α9-expressing DRG neurons. Comparing these results with those presented in the previous paragraph, we can conclude that WT DRGs produce enough TN-C to promote the neurite outgrowth from α9-transduced neurons. In the absence of secreted TN-C from knockout neurons, both AEIDGIEL and TN-C coated surfaces provide a good ligand for α9-integrin. Fibrin gels and modified fibrin gels with AEIDGIEL promote the neurite outgrowth of α9-transduced cells In SCI, cavitation of the injured area occurs, which physically prevents regenerating axons from passing through the lesion. Furthermore, it induces remodeling of the microenvironment of the affected area, dysregulating the ECM to a state of scarring which represents one of the major obstacles to axonal regeneration in the CNS. Biomaterials are a promising tool to overcome these limitations by providing a scaffold through which axons can grow and mimicking the natural cellular environment to prevent obstruction of neurite outgrowth. We investigated whether fibrin gels and fibrin gels containing AEIDGIEL were able to promote neurite growth driven by transduced α9-integrin (Fig. 4 a,b). The results show that un-transduced and GFP-transduced cells grow neurites on fibrin gel with an average length of 230 µm for both WT and TNC-KO DRG neurons (Fig. 4 c,e), suggesting that this biomaterial promotes physiological neurite outgrowth. As expected, α9-transduced neurons grew longer neurites in WT experiments, averaging 420 µm, due to the production of TN-C which activates the integrin (Fig. 4 c), but we no longer observed this effect in the TNC-KO neurons, and un-transduced, GFP-transduced and α9-transduced groups did not differ in their neurite growth (Fig. 4 e). AEIDGIEL containing fibrin-gels created a slightly inhibitory environment for un-transduced and GFP-transduced cells in both WT and TNC-KO conditions, with an average neurite length of 180 µm (Fig. 4 d,f). However, when DRG neurons expressed α9-integrin they grew significantly longer neurites, reaching lengths of 450 µm in WT conditions (Fig. 4 d) and 300 µm in TN-C KO conditions (Fig. 4 f). The satisfactory neurite length found on fibrin gels indicates that they are a favorable material for neurite outgrowth of DRG neurons. The outgrowth of WT axons is likely mediated by one of the several integrins spontaneously expressed by DRG neurons 13 , 14 . This growth was enhanced by the expression of α9-integrin, likely because of interaction with TN-C secreted by DRG, as discussed above. TN-C KO neurons growing on fibrin gels behaved similarly to WT except that lack of neuronal TN-C abolished the ability of α9-integrin to increase neurite length. The AEIDGIEL peptide slightly inhibited the growth of neurons without transduction and with GFP-transduction but promoted outgrowth of α9-integrin expressing neurites. If used as a biomaterial, the growth of α9-integrin transduced axons would be enhanced by AEIDGIEL-modified gels. This would be significant, because α9-transduced axons have the ability to re-enter CNS tissue and regenerate over long distances 6 , 7 . Discussion SCI affects millions of people around the world and conventional surgical and medical treatments fail to fundamentally cure it because of the complex pathological mechanisms involved that requires a multidimensional approach 2 . To persuade axons to regenerate along the lesion we need to provide a bridge through which they can grow and trigger an appropriate intracellular response for axonal elongation. The present study investigates how a combined strategy involving the promotion of intracellular neurite growth through α9-integrin expression and the creation of an external favorable environment using fibrin gels affect DRG neurite length in vitro . Neurite growth depends on the adhesion of the growth cone to the ECM and active intracellular signaling for the growth machinery. Integrins can facilitate both processes, but the ECM in an injured spinal cord lacks ligands compatible with DRG integrins, making it unfavorable for axon growth. After SCI, the ECM upregulates the TN-C protein, whose primary receptor, α9-integrin, is developmentally downregulated. Consequently, adult neurons lack this receptor to match the TN-C ligand in the injured environment. Forced expression of α9-integrin via AAV vectors has been shown to significantly promote the regeneration of adult sensory axons in the CNS after dorsal root crush 6 and dorsal column crush 7 . Axons expressing α9-integrin and its activator, kindlin, can grow through the nonpermissive microenvironment around the lesion, which is rich in upregulated TN-C and inhibitory chondroitin sulfate proteoglycans (CSPGs). However, tissue cavitation and fibrosis obstruct the regenerating axons from following their usual pathways, forcing them to find alternative growth routes rich in TN-C, so they extend along the meningeal connective tissue around the lesion. Some of these axons re-enter the CNS, but their regeneration pathway differs from the typical path taken by sensory axons, and they often get lost in the meninges 7 . Hydrogels can provide a three-dimensional spatial support for axonal extension, thereby preventing the aberrant paths and connections through meninges that axons are forced to form in the absence of a suitable scaffold. The utility of hydrogels for SCI has been extensively studied and demonstrated to be able to fill the lesion area by mimicking the natural ECM, improving the microenvironment at the lesion site and promoting reconnection of damaged tissue 10 . Fibrin gel is an FDA approved, biocompatible material for filling the spinal cord lesion area that prevents cavitation and facilitates DRG axons regeneration 15 – 18 . Adult DRG neurons spontaneously express several integrins that bind to ECM molecules (α3β1, α4β1, α5β1 α6β1 and α7β1), the α5β1-integrin particularly is responsible for the fibrin attachment 13 , 14 , 19 . Fibrin is formed by the enzymatic polymerization of fibrinogen monomers with thrombin to form non-covalent cross-linked chains 11 . This feature causes fibrin-gel to degrade rapidly in the body, an inconvenience that limits its usefulness. However, it has been described that the addition of chemical crosslinkers and proteolysis inhibitors can improve and provide better control over the biomaterial degradation rate. The proteolytic enzyme inhibitor aprotinin has been shown to reduce fibrinolysis by inhibiting trypsin, plasmin and kallikreins, without affecting other cell functions 20 . Moreover, the addition of Factor XIII and CaCl2 solution can covalently cross-link the polymer network providing greater stability and resistance to degradation. Factor XIII has also been shown to covalently cross-link other proteins or peptides into the gel, further extending the utility of the biomaterial to include the desired bioactive domains 21 . ECM molecules are notably large and challenging to construct and incorporate into the gel but short peptide domains can also act as ligands to produce the desired functions 22 – 24 . Moreover, functionalization of fibrin with integrin ligand peptides has been shown to facilitate regeneration of neuronal precursor axons through gels 25 . To improve the stability of the fibrin-gel we added aprotinin, Factor XIII and CaCl2, and to ensure the activation of α9-integrin, we modified the fibrin-gel with AEIDGIEL peptide, described as the bioactive domain of TN-C 8 . We first assessed the neurite growth of adult DRG neurons on glass coverslips coated with PDL, AEIDGIEL peptide or TN-C. The results indicate that the peptide is sufficient to create the same effect as the TN-C protein, as reflected by the absence of differences in neurite growth between the two conditions: both the TN-C protein and the AEIDGIEL peptide created the expected inhibitory environment for neurite outgrowth in control cells, and α9-integrin was able to overcome it, resulting in higher average neurite length (Fig. 2 c,d). When DRG neurons were cultured on fibrin gels, we observed twice the neurite length in control cells than on the neutral coverslips, demonstrating that the fibrin-gel is a suitable biomaterial for promoting physiological neurite growth (Fig. 4 c). Fibrin-gel containing AEIDGIEL created a slightly inhibitory environment, which was again overcome by integrin expression (Fig. 4 d). The striking results showing that α9-transduced neurons grew to greater neurite length under all conditions, including a coverslip coated only with PDL (Fig. 2 b) or an unmodified fibrin gel (Fig. 4 c) wouldn’t allow us to conclude that the AEIDGIEL peptide can activate the integrin. Previous studies examining DRG neurites growth also contributed to this unexpected result, cells transfected with α9-integrin showed longer neurite outgrowth, even when grown on uncoated plastic, suggesting that the cultured cells possess α9-ligand. Andrews et al., treated neurons with TN-C siRNA, and observed a significant reduction in neurite outgrowth on uncoated plastic, but this had no effect on neurite outgrowth on TN-C or laminin coated surfaces. Furthermore, the study further demonstrated TN-C immunoreactivity on DRGs bodies and processes, and TN-C deposited on surface adjacent to neurons 5 . We also observed robust outgrowth of neurites when α9-integrin is expressed regardless of the surface, leading us to conclude that neuronal TN-C activates the integrin and prevents interaction with the AEIDGIEL peptide. We therefore decided to repeat the experiments using TN-C knockout DRGs. In the knock-out condition we no longer observe significant difference in neurite growth between controls or α9-transduced cells when grown on PDL or fibrin-gel, but when cells are grown on surfaces where TN-C or the AEIDGIEL peptide are exogenously provided, α9-transduced neurons show significantly higher neurite growth (Fig. 3 , 4 e,f). This finding demonstrates that the presence of the AEIDGIEL peptide is sufficient for integrin activation and supports previous findings of an autocrine signaling loop of α9-integrin and TN-C produced by neurons, in addition to integrin interaction with extracellular ligands. In summary, the proposed combinational therapy involving α9-integrin and fibrin gel biomaterials incorporating the AEIDGIEL peptide holds promise for addressing the complex challenges of SCI and promoting effective neural regeneration, laying the foundation for further in vivo research. The introduction of fibrin gel with AEIDGIEL peptide to fill the lesion site could prevent cavity expansion, protect neurons from toxic substances at the lesion site, stabilize the spinal cord and reduce its susceptibility to tissue collapse, and serve as a scaffold to facilitate the migration of regenerating axons along the lesion. Axons with activated α9-integrin could thus acquire both the adhesion necessary for growth cone growth and the signals for the growth mechanism, which will be provided through α9-integrin expression and activation. Fibrin gel has been widely used in in vivo murine models for SCI repair, demonstrating excellent biocompatibility and non-toxicity of the gel or its degradation products. Our in vitro study involved a 2D culture where primary cells were seeded on the gel surface as a proof of concept of the compatibility of this combinatorial strategy and the incorporation of the AEIDGIEL peptide. For in vivo use, further optimization of the fibrin gel's properties is necessary, including achieving the appropriate stiffness (matching the 1.2 kPa of spinal tissue) and a suitable porous microstructure to support 3D axonal extension. Accurately adjusting the physicochemical properties of the biomaterial is crucial, as these properties influence repair cellular responses such as immunomodulation and innate neurotrophic signaling 26 . The stiffness and porosity can be easily adjusted by varying the concentrations of fibrinogen and thrombin 27 . A major advantage of the fibrin gel is its ability to be injected as a liquid and solidify in situ, conforming to the lesion site’s shape. In our study, the fibrin gel remained intact for 5 days in vitro before beginning to degrade. For in vivo applications, optimizing the gel’s chemical composition with novel strategies as for example engineered aprotinin 28 , 29 to control the degradation rate is crucial to ensure it remains effective for approximately six weeks, the necessary period for axons to cross the lesion site. Methods Cell culture Adult three months old WT C57BL/6 and TN-C KO mice (kindly provided by Andreas Faissner, Ruhr University Bochum, Bochum, Germany) were placed in a close box and fully anesthetized with 3% isoflurane in air flow (Aerrane, Baxter). Once overdosed, mice were decapitated with guillotine (Ježek s.r.o., Czech Republic) and DRGs were extracted. All experiments were performed in accordance with the European Communities council directive of 22nd of September 2010(2010/63/EU), follow the ARRIVE guidelines ( https://arriveguidelines.org/ ) and were approved by the Ethics Committee of the Institute of Experimental Medicine ASCR, Prague, Czech Republic. DRGs were dissociated in 0.2% collagenase (Sigma-Aldrich) for 2h and 0.1% trypsin (Sigma-Aldrich) for 10 min in DMEM at 37°C, followed by trituration and 15% BSA (Sigma-Aldrich) gradient centrifugation. Cells were plated and grown on coated glass coverslips and fibrin hydrogel matrices at a density of 1x10^4 cells/condition in the following culture medium: 1% ITS (Sigma-Aldrich), 1% PSF (Sigma-Aldrich), 10 ng/ml NGF (Sigma-Aldrich) and 0.5 µg/ml Mitomycin-C (Sigma-Aldrich) in DMEM. Coating of coverslips and fibrin hydrogel matrices preparation Coverslips were coated with either 20 µg/ml PDL (Sigma-Aldrich) or 1 µg/ml PDL + 10 µg/ml chicken TN-C (Sigma-Aldrich) or 1 µg/ml PDL + 5 µg/ml AEIDGIEL peptide (Sigma-Aldrich). Preparation of fibrin hydrogel matrices consisted of mixing a fibrinogen-based solution and an enzymatic solution in equal proportions, as described previously 21 . The enzyme solution contained HEPES buffer, 2mM CaCl2, 2 U/ml thrombin (Sigma-Aldrich) and 5 U/ml Factor XIII (Sigma-Aldrich). The enzyme solution was incubated for 10 min at 37°C on a glass coverslip, followed by the addition of the fibrinogen solution. The fibrinogen solution contained HEPES buffer, 7.5 mg/ml fibrinogen (Sigma-Aldrich), 10 µg/ml aprotinin (Sigma-Aldrich), without or with 1 mg/ml AEIDGIEL peptide. The mixture of enzyme and fibrinogen solution was incubated for 1 hour at 37°C to form a polymerized gel. Cell transduction Dissociated DRG neurons were incubated with 1x 10^9 tu/ml of AAV-CAG-GFP and AAV-CAG-α9-V5. The plasmids carrying GFP or human α9 integrin were amplified and sequenced before packaging into AAV serotype 5 (AAV5) using HEK293T cells as described previously 30 . Cells were incubated with the virus for 72h at 37°C to induce viral transduction. On the third day, the media was replaced, and the cells were kept for additional 48h to ensure protein expression. Microscopy, neurite outgrowth assay and statistical analysis Cells were analyzed by fluorescent microscopy. Imaging was performed using a Zeiss Axio Observer D1 fluorescence microscope with inverted phase contrast and 40x magnification. To assess viral transduction, cells were identified and categorized based on their fluorescence signals: green for AAV-GFP-transduced cells, red for AAV-α9-V5-transduced cells, and only blue for un-transduced cells. The longest neurite length per neuron was measured using ImageJ software (National Institutes of Health). Neurite outgrowth was quantified from at least three separate experiments, with data representing individual neurites. Comparisons were made between un-transduced (NT), AAV-GFP transduced (GFP) and AAV-α9-integrin-transduced (A9) DRG cultures under different growth conditions. Statistical analysis was performed using a one-way ANOVA test followed by Tuckey analysis using GraphPad Prism 9 software. Declarations Author contributions statement All authors were involved in the study's conception and design. Material preparation, data collection, and analysis were carried out by AC. The first draft of the manuscript was written by AC and all authors commented on previous versions of the manuscript. All authors reviewed and approved the final manuscript. Additional information Competing interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution All authors were involved in the study's conception and design. Material preparation, data collection, and analysis were carried out by AC. The first draft of the manuscript was written by AC and all authors commented on previous versions of the manuscript. PJ secured the funding. All authors reviewed and approved the final manuscript. Acknowledgement We would like to acknowledge helpful discussions with Priscilla S Briquez and Jeffrey A Hubbell from University of Chicago. Data Availability The datasets generated and/or analyzed during the current study are available in the “A9-FibrinGel-TNC-KO project” repository, https://doi.org/10.5281/zenodo.13142847 References Ding, W. et al. Spinal Cord Injury: The Global Incidence, Prevalence, and Disability From the Global Burden of Disease Study 2019. Spine . 47 , 1532–1540 (2022). Ahuja, C. S. et al. Traumatic spinal cord injury. Nat. Rev. Dis. Primer . 3 , 17018 (2017). Fawcett, J. W. The Struggle to Make CNS Axons Regenerate: Why Has It Been so Difficult? Neurochem Res. 45 , 144–158 (2020). Hu, X. et al. Spinal cord injury: molecular mechanisms and therapeutic interventions. Signal. Transduct. Target. Ther. 8 , 245 (2023). Andrews, M. R. et al. α9 Integrin Promotes Neurite Outgrowth on Tenascin-C and Enhances Sensory Axon Regeneration. J. Neurosci. 29 , 5546–5557 (2009). Cheah, M. et al. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord. J. 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Biomaterials . 32 , 430–438 (2011). Sacchi, V. et al. Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF 164 . Proc. Natl. Acad. Sci. 111, 6952–6957 (2014). Hermens, W. T. J. M. C. et al. Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System. Hum. Gene Ther. 10 , 1885–1891 (1999). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5289110","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":369960580,"identity":"bc7d6c1f-a321-4ec3-b13d-7903360a654a","order_by":0,"name":"Anda Cimpean","email":"","orcid":"","institution":"Institute of Experimental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Anda","middleName":"","lastName":"Cimpean","suffix":""},{"id":369960581,"identity":"43a72549-4182-43c8-bca1-ab68fc1a33df","order_by":1,"name":"Lars Roll","email":"","orcid":"","institution":"RuhrUniversity Bochum","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Roll","suffix":""},{"id":369960582,"identity":"e7b7ee79-7ac9-481a-ac60-bf6320ff38ed","order_by":2,"name":"Jacqueline Reinhard","email":"","orcid":"","institution":"RuhrUniversity Bochum","correspondingAuthor":false,"prefix":"","firstName":"Jacqueline","middleName":"","lastName":"Reinhard","suffix":""},{"id":369960583,"identity":"2d2c963a-7eb5-4e82-aca9-f8044ad5f81e","order_by":3,"name":"Jessica Kwok","email":"","orcid":"","institution":"Institute of Experimental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Kwok","suffix":""},{"id":369960584,"identity":"d1c4d6ed-84c6-4bf2-9494-12433c7b70ce","order_by":4,"name":"Andreas Faissner","email":"","orcid":"","institution":"RuhrUniversity Bochum","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Faissner","suffix":""},{"id":369960585,"identity":"f8377902-ba16-4287-91e3-9043c0b4a2e9","order_by":5,"name":"James W. Fawcett","email":"","orcid":"","institution":"Institute of Experimental Medicine","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"W.","lastName":"Fawcett","suffix":""},{"id":369960586,"identity":"6966c21a-beda-43a6-abd9-17d611248a3a","order_by":6,"name":"Pavla Jendelová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABH0lEQVRIie2QsWrDMBBAzxjkRW1WD8X+hQsZgpd+i4yhWWy6GhqcdlGHuuma/oX6BzICZ3Ho6jGhkKmFdCkeQqlMSaFg04wd9AbpkO7dSQdgMPxPLAlA9M7aADxyjHNQ2gBGxyrwo4TXf6WPnQplA6fe2ImkfOfnk7lzx6CZZjB4kJ1KkMdY5EBGQb5lxSOPEk5XwspLBa7sVlBeMEmBhKKOUdHKTribCBuIBLS634jPuvgeyEzUlzutzCbEf9PKZwZod/8F60gq3YVhHYOiqWLEPRG2xW3AntkFixepzpAMRbXFYpEuh5wmorifK+pWPRMbhDeb17T0cRlt1ju88v3b1dO6+ci8vonh91r+Pm1zaXePgwLTvnuDwWAwAHwBtuNiB95xCtAAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Experimental Medicine","correspondingAuthor":true,"prefix":"","firstName":"Pavla","middleName":"","lastName":"Jendelová","suffix":""}],"badges":[],"createdAt":"2024-10-18 11:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5289110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5289110/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67924909,"identity":"ff785708-eee4-457a-a7d3-670e7b13f2b3","added_by":"auto","created_at":"2024-10-31 08:48:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":532433,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram illustrating the combined strategy integrating biomaterial scaffolding with molecular activation strategies and genetic manipulation of adult neurons as a novel and promising therapeutic approach for spinal cord injuries.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5289110/v1/3be43e258596df7c56091f21.png"},{"id":67924913,"identity":"5cba59c7-6c4e-470f-bd72-39b2eb61b2c0","added_by":"auto","created_at":"2024-10-31 08:48:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":318852,"visible":true,"origin":"","legend":"\u003cp\u003eWT DRG neurite growth on treated coverslips. a) Un-transduced, GFP-transduced and α9-integrin-transduced neurons growing on PDL-only, TN-C or AEIDGIEL coated coverslips. Quantification of the longest neurite (µm) growing on b) PDL-coated coverslips, c) TN-C coated coverslips, d) AEIDGIEL coated coverslips. The longest neurite of each neuron was quantified from at least three separate experiments, with data representing individual neurites. Statistical analysis was performed using one-way ANOVA. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Error bars indicate SD. Scale bars, 70 µm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5289110/v1/f46d730bdc7c608c0cd36d50.jpeg"},{"id":67924910,"identity":"b0a0c0b1-0c50-4a8e-9f33-c6d6765d43c6","added_by":"auto","created_at":"2024-10-31 08:48:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":356530,"visible":true,"origin":"","legend":"\u003cp\u003eTN-C KO DRG neurite growth on treated coverslips. a) Un-transduced, GFP-transduced and α9-integrin-transduced TN-C knockout neurons growing on PDL-only, TN-C or AEIDGIEL coated coverslips. Quantification of the longest neurite (µm) growing on b) PDL-coated coverslips, c) TN-C coated coverslips, d) AEIDGIEL coated coverslips. The longest neurite of each neuron was quantified from at least three separate experiments, with data representing individual neurites. Statistical analysis was performed using one-way ANOVA. ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Error bars indicate SD. Scale bars, 70 µm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5289110/v1/bf9549364a0df2406b3fb558.jpeg"},{"id":67924912,"identity":"39925f4a-a46a-4c03-9cea-54072c531de6","added_by":"auto","created_at":"2024-10-31 08:48:12","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":333421,"visible":true,"origin":"","legend":"\u003cp\u003eWT and TN-C KO DRG neurons growing on fibrin gel and AEIDGIEL-modified fibrin gel. a) Un-transduced, GFP-transduced and A9-transduced WT neurons growing on fibrin gel and fibrin gel containing AEIDGIEL peptide. b) Un-transduced, GFP-transduced and A9-transduced TN-C KO neurons growing on fibrin gel and fibrin gel containing AEIDGIEL peptide. c) Longest neurite length quantification (µm) of WT DRG neurons growing on fibrin gel or on d) AEIDGIEL-containing fibrin gel. e) Longest neurite length quantification (µm) of TN-C KO neurons growing on fibrin gel or f) AEIDGIEL-containing fibrin gel. Data represents individual neurites from at least three different experiments. Statistical analysis was performed using one-way ANOVA. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001. Error bars indicate SD. Scale bars, 70 µm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5289110/v1/458ba0723808ee3fe442b08e.jpeg"},{"id":70311542,"identity":"f948bb7b-a828-4a56-a128-c862679a53e7","added_by":"auto","created_at":"2024-12-02 04:24:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1958281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5289110/v1/7233d418-f5d8-40aa-8f6f-9b056d769a43.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combined Strategy Of α9-Integrin Transduction and AEIDGIEL Peptide-Functionalized Fibrin Gel Biomaterials to Promote Mature DRG Neurite Growth","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) can result from either traumatic events such as vertebral fractures or non-traumatic causes like infections or vascular damage, and has a poor prognosis with sensory, motor and autonomic disfunction, which leads to disability and imposes a heavy burden on healthcare systems and society\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. SCI creates a complex pathological situation with many barriers to recovery, including the inability of the adult central nervous system (CNS) axons to regenerate, cavitation and excessive scarring at the lesion site\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, treatment requires a combinatorial approach with different therapies acting in a synergistic manner. Currently, achieving complete recovery from severe SCI remains challenging due to the absence of successful translational therapies\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Dorsal root ganglion (DRG) neurons are the afferent neurons that relay sensory information from the periphery to the brain. Injured central dorsal root axons are unable to regenerate in the spinal cord resulting in loss of sensorimotor functions and neuropathic pain. Previous studies reported that α9-integrin expression and activation in mature DRG neurons allow regeneration of injured axons into the spinal cord and enable functional recovery of sensation and locomotion\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The major ligand of the integrin is tenascin-C (TN-C), the main extracellular matrix inhibitory glycoprotein upregulated in the CNS environment after SCI. The bioactive domain of TN-C that activates α9-integrin is the AEIDGIEL sequence, contained in the B-C loop of the third subunit of fibronectin type III protein\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Integrin expression provides a proper adhesion receptor to control migration of axon growth through the unfavorable environment, and triggers an intracellular genetic program specific to CNS regeneration, with upregulation of genes related to ubiquitination, autophagy, endoplasmic reticulum, casein kinases, transport/trafficking and other signaling molecules\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the massive tissue disruption and extensive scarring formation at the injury side hinder axons capable of regeneration and prevent the formation of physiological meaningful connections after injury. Hydrogel materials can expand to fill the entire wound site, providing a surface and scaffold through which nerves can regenerate, avoid aberrant growth and protect themselves from the surrounding fibrosis. Many studies have demonstrated their beneficial role in SCI, however, hydrogel treatment alone doesn\u0026rsquo;t completely restore neurological function\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Hydrogel materials contain extracellular matrix (ECM) proteins such as fibrin, which plays an important role in natural wound healing. Because of its affordability, cytocompatibility and ability to modulate angiogenesis and inflammation, fibrin has been used clinically for wound coverings, surgical glues and cell delivery\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe aim of this study was to examine how the combination of α9-integrin expression and the use of a fibrin-gel as an extracellular microenvironment affects the growth of mature DRG neurites \u003cem\u003ein vitro\u003c/em\u003e. Additionally, we aimed to improve the biomaterial properties by incorporating the bioactive TN-C domain, AEIDGIEL, to ensure the integrin activation. We assessed neurite growth of un-transduced, GFP-transduced and α9-integrin-transduced DRG neurons cultured on coverslips with various coatings, fibrin-gels and AEIDGIEL-modified fibrin-gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We concluded that fibrin-gels serve as a suitable biomaterial to promote neurite growth and that AEIDGIEL peptide is sufficient to activate the integrin. The intriguing finding that α9-integrin-transduced neurons grow long neurites irrespective of the surface motivated us to further investigations which led to the corroboration of the autocrine signaling loop of α9-integrin and TN-C produced by neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExpression of α9-integrin in adult DRG neurons enables neurite growth on tenascin-C and its motif peptide AEIDGIEL\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that α9-integrin expression in adult DRG neurons enhances neurite outgrowth and overcomes the growth inhibition by TN-C, a major extracellular inhibitory protein that is upregulated after SCI\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. α9-integrin recognizes a peptide in the third fibronectin type III domain of TN-C, AEIDGIEL\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. First, we confirmed that TN-C inhibits neurite outgrowth from adult DRG neurons, and then determined whether the presence of the AEIDGIEL peptide creates an inhibitory environment similar to wild-type TN-C. We cultured adult DRG neurons on glass coverslips coated with PDL alone, with chicken TN-C, or with the AEIDGIEL peptide. Cells were transduced with AAV-CAG-GFP and AAV-CAG-α9-V5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). On PDL coated coverslips, un-transduced and GFP-transduced DRG neurons grew to an average length of 150 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), whereas on coverslips coated with TN-C or AEIDGIEL the average neurite outgrowth was 90 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d). Next, we asked whether AAV-mediated expression of α9-integrin would promote outgrowth on TN-C and AEIDGIEL. Cells with α9-transduced neurites grew significantly longer than controls on the surface of both TN-C and AIDGIEL, with an average length of approximately 300 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d). However, the length of neurites grown by α9-transduced neurons on coverslips coated with PDL alone also increased compared to the controls, and the length was not significantly different to that of neurons grown on TN-C or AEIDGIEL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). α9-integrin thus, promoted growth on all surfaces. Because α9-integrin is a specific receptor for TN-C it is likely that axons respond to TN-C in all three situations. The TN-C protein has been shown to be produced by several glial cell types, as well as by DRG neurons, where it is located on their membrane and the surrounding culture surface\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, from this set of experiments, it is not possible to determine whether α9-transduced neurons are responding to the TN-C or AEIDGIEL that has been applied to the culture surface, or the TN-C secreted by DRG neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eα9-integrin activation relies on neuronal tenascin-C\u003c/h2\u003e \u003cp\u003eIn the above results, we showed that α9-integrin-transduced DRG neurons grew on coverslips coated with TN-C or AEIDGIEL to an average neurite length of 300 \u0026micro;m, but they also grew to a similar distance on PDL surfaces without added TN-C or peptide. This suggests that cells in culture produce TN-C protein, that adheres to PDL and provides a ligand for α9-integrin. Therefore, to confirm that α9-integrin-transduced DRG neurites can grow on AEIDGIEL alone, we had to prevent TN-C production by cultured DRGs and therefore repeated the neurite outgrowth experiments with dissociated cultures of TN-C knockout DRGs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The three experimental surfaces were coverslips coated with PDL, followed by one group coated with AEIDGIEL and the other with chicken TN-C. On these three surfaces, un-transduced and GFP- or α9-transduced adult DRG neurons from TN-C knockout mice were grown (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Un-transduced, GFP-transduced and α9-transduced TN-C knockout cells growing on PDL coated coverslips showed equal and small neurite outgrowth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Integrin-transduced TN-C knockout cells grew on TN-C and AEIDGIEL coated coverslips neurites two to three times longer than un-transduced or GFP-transduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). This demonstrates that α9-transduced TN-C knockout neurons do not show enhanced axon growth in the absence of ligand on the culture surface. However, in TN-C knockout neurons, TN-C or AEIDGIEL coated on the surface of the PDL can strongly promote the growth of α9-integrin-transduced neurites. These results demonstrate that AEIDGIEL or TN-C coated on coverslips both act as a ligand for α9-integrin, and enable strong neurite outgrowth from α9-expressing DRG neurons. Comparing these results with those presented in the previous paragraph, we can conclude that WT DRGs produce enough TN-C to promote the neurite outgrowth from α9-transduced neurons. In the absence of secreted TN-C from knockout neurons, both AEIDGIEL and TN-C coated surfaces provide a good ligand for α9-integrin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFibrin gels and modified fibrin gels with AEIDGIEL promote the neurite outgrowth of α9-transduced cells\u003c/h3\u003e\n\u003cp\u003eIn SCI, cavitation of the injured area occurs, which physically prevents regenerating axons from passing through the lesion. Furthermore, it induces remodeling of the microenvironment of the affected area, dysregulating the ECM to a state of scarring which represents one of the major obstacles to axonal regeneration in the CNS. Biomaterials are a promising tool to overcome these limitations by providing a scaffold through which axons can grow and mimicking the natural cellular environment to prevent obstruction of neurite outgrowth. We investigated whether fibrin gels and fibrin gels containing AEIDGIEL were able to promote neurite growth driven by transduced α9-integrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). The results show that un-transduced and GFP-transduced cells grow neurites on fibrin gel with an average length of 230 \u0026micro;m for both WT and TNC-KO DRG neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,e), suggesting that this biomaterial promotes physiological neurite outgrowth. As expected, α9-transduced neurons grew longer neurites in WT experiments, averaging 420 \u0026micro;m, due to the production of TN-C which activates the integrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), but we no longer observed this effect in the TNC-KO neurons, and un-transduced, GFP-transduced and α9-transduced groups did not differ in their neurite growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAEIDGIEL containing fibrin-gels created a slightly inhibitory environment for un-transduced and GFP-transduced cells in both WT and TNC-KO conditions, with an average neurite length of 180 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,f). However, when DRG neurons expressed α9-integrin they grew significantly longer neurites, reaching lengths of 450 \u0026micro;m in WT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and 300 \u0026micro;m in TN-C KO conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThe satisfactory neurite length found on fibrin gels indicates that they are a favorable material for neurite outgrowth of DRG neurons. The outgrowth of WT axons is likely mediated by one of the several integrins spontaneously expressed by DRG neurons\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This growth was enhanced by the expression of α9-integrin, likely because of interaction with TN-C secreted by DRG, as discussed above. TN-C KO neurons growing on fibrin gels behaved similarly to WT except that lack of neuronal TN-C abolished the ability of α9-integrin to increase neurite length. The AEIDGIEL peptide slightly inhibited the growth of neurons without transduction and with GFP-transduction but promoted outgrowth of α9-integrin expressing neurites. If used as a biomaterial, the growth of α9-integrin transduced axons would be enhanced by AEIDGIEL-modified gels. This would be significant, because α9-transduced axons have the ability to re-enter CNS tissue and regenerate over long distances\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSCI affects millions of people around the world and conventional surgical and medical treatments fail to fundamentally cure it because of the complex pathological mechanisms involved that requires a multidimensional approach\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To persuade axons to regenerate along the lesion we need to provide a bridge through which they can grow and trigger an appropriate intracellular response for axonal elongation. The present study investigates how a combined strategy involving the promotion of intracellular neurite growth through α9-integrin expression and the creation of an external favorable environment using fibrin gels affect DRG neurite length \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNeurite growth depends on the adhesion of the growth cone to the ECM and active intracellular signaling for the growth machinery. Integrins can facilitate both processes, but the ECM in an injured spinal cord lacks ligands compatible with DRG integrins, making it unfavorable for axon growth. After SCI, the ECM upregulates the TN-C protein, whose primary receptor, α9-integrin, is developmentally downregulated. Consequently, adult neurons lack this receptor to match the TN-C ligand in the injured environment. Forced expression of α9-integrin via AAV vectors has been shown to significantly promote the regeneration of adult sensory axons in the CNS after dorsal root crush\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and dorsal column crush\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Axons expressing α9-integrin and its activator, kindlin, can grow through the nonpermissive microenvironment around the lesion, which is rich in upregulated TN-C and inhibitory chondroitin sulfate proteoglycans (CSPGs). However, tissue cavitation and fibrosis obstruct the regenerating axons from following their usual pathways, forcing them to find alternative growth routes rich in TN-C, so they extend along the meningeal connective tissue around the lesion. Some of these axons re-enter the CNS, but their regeneration pathway differs from the typical path taken by sensory axons, and they often get lost in the meninges\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Hydrogels can provide a three-dimensional spatial support for axonal extension, thereby preventing the aberrant paths and connections through meninges that axons are forced to form in the absence of a suitable scaffold. The utility of hydrogels for SCI has been extensively studied and demonstrated to be able to fill the lesion area by mimicking the natural ECM, improving the microenvironment at the lesion site and promoting reconnection of damaged tissue\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Fibrin gel is an FDA approved, biocompatible material for filling the spinal cord lesion area that prevents cavitation and facilitates DRG axons regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Adult DRG neurons spontaneously express several integrins that bind to ECM molecules (α3β1, α4β1, α5β1 α6β1 and α7β1), the α5β1-integrin particularly is responsible for the fibrin attachment\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Fibrin is formed by the enzymatic polymerization of fibrinogen monomers with thrombin to form non-covalent cross-linked chains\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This feature causes fibrin-gel to degrade rapidly in the body, an inconvenience that limits its usefulness. However, it has been described that the addition of chemical crosslinkers and proteolysis inhibitors can improve and provide better control over the biomaterial degradation rate. The proteolytic enzyme inhibitor aprotinin has been shown to reduce fibrinolysis by inhibiting trypsin, plasmin and kallikreins, without affecting other cell functions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, the addition of Factor XIII and CaCl2 solution can covalently cross-link the polymer network providing greater stability and resistance to degradation. Factor XIII has also been shown to covalently cross-link other proteins or peptides into the gel, further extending the utility of the biomaterial to include the desired bioactive domains\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ECM molecules are notably large and challenging to construct and incorporate into the gel but short peptide domains can also act as ligands to produce the desired functions\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, functionalization of fibrin with integrin ligand peptides has been shown to facilitate regeneration of neuronal precursor axons through gels\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To improve the stability of the fibrin-gel we added aprotinin, Factor XIII and CaCl2, and to ensure the activation of α9-integrin, we modified the fibrin-gel with AEIDGIEL peptide, described as the bioactive domain of TN-C\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. We first assessed the neurite growth of adult DRG neurons on glass coverslips coated with PDL, AEIDGIEL peptide or TN-C. The results indicate that the peptide is sufficient to create the same effect as the TN-C protein, as reflected by the absence of differences in neurite growth between the two conditions: both the TN-C protein and the AEIDGIEL peptide created the expected inhibitory environment for neurite outgrowth in control cells, and α9-integrin was able to overcome it, resulting in higher average neurite length (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d). When DRG neurons were cultured on fibrin gels, we observed twice the neurite length in control cells than on the neutral coverslips, demonstrating that the fibrin-gel is a suitable biomaterial for promoting physiological neurite growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Fibrin-gel containing AEIDGIEL created a slightly inhibitory environment, which was again overcome by integrin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The striking results showing that α9-transduced neurons grew to greater neurite length under all conditions, including a coverslip coated only with PDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) or an unmodified fibrin gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) wouldn\u0026rsquo;t allow us to conclude that the AEIDGIEL peptide can activate the integrin. Previous studies examining DRG neurites growth also contributed to this unexpected result, cells transfected with α9-integrin showed longer neurite outgrowth, even when grown on uncoated plastic, suggesting that the cultured cells possess α9-ligand. Andrews et al., treated neurons with TN-C siRNA, and observed a significant reduction in neurite outgrowth on uncoated plastic, but this had no effect on neurite outgrowth on TN-C or laminin coated surfaces. Furthermore, the study further demonstrated TN-C immunoreactivity on DRGs bodies and processes, and TN-C deposited on surface adjacent to neurons\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. We also observed robust outgrowth of neurites when α9-integrin is expressed regardless of the surface, leading us to conclude that neuronal TN-C activates the integrin and prevents interaction with the AEIDGIEL peptide. We therefore decided to repeat the experiments using TN-C knockout DRGs. In the knock-out condition we no longer observe significant difference in neurite growth between controls or α9-transduced cells when grown on PDL or fibrin-gel, but when cells are grown on surfaces where TN-C or the AEIDGIEL peptide are exogenously provided, α9-transduced neurons show significantly higher neurite growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f). This finding demonstrates that the presence of the AEIDGIEL peptide is sufficient for integrin activation and supports previous findings of an autocrine signaling loop of α9-integrin and TN-C produced by neurons, in addition to integrin interaction with extracellular ligands.\u003c/p\u003e \u003cp\u003eIn summary, the proposed combinational therapy involving α9-integrin and fibrin gel biomaterials incorporating the AEIDGIEL peptide holds promise for addressing the complex challenges of SCI and promoting effective neural regeneration, laying the foundation for further \u003cem\u003ein vivo\u003c/em\u003e research. The introduction of fibrin gel with AEIDGIEL peptide to fill the lesion site could prevent cavity expansion, protect neurons from toxic substances at the lesion site, stabilize the spinal cord and reduce its susceptibility to tissue collapse, and serve as a scaffold to facilitate the migration of regenerating axons along the lesion. Axons with activated α9-integrin could thus acquire both the adhesion necessary for growth cone growth and the signals for the growth mechanism, which will be provided through α9-integrin expression and activation. Fibrin gel has been widely used in \u003cem\u003ein vivo\u003c/em\u003e murine models for SCI repair, demonstrating excellent biocompatibility and non-toxicity of the gel or its degradation products. Our \u003cem\u003ein vitro\u003c/em\u003e study involved a 2D culture where primary cells were seeded on the gel surface as a proof of concept of the compatibility of this combinatorial strategy and the incorporation of the AEIDGIEL peptide. For \u003cem\u003ein vivo\u003c/em\u003e use, further optimization of the fibrin gel's properties is necessary, including achieving the appropriate stiffness (matching the 1.2 kPa of spinal tissue) and a suitable porous microstructure to support 3D axonal extension. Accurately adjusting the physicochemical properties of the biomaterial is crucial, as these properties influence repair cellular responses such as immunomodulation and innate neurotrophic signaling\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The stiffness and porosity can be easily adjusted by varying the concentrations of fibrinogen and thrombin\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. A major advantage of the fibrin gel is its ability to be injected as a liquid and solidify in situ, conforming to the lesion site\u0026rsquo;s shape. In our study, the fibrin gel remained intact for 5 days in vitro before beginning to degrade. For \u003cem\u003ein vivo\u003c/em\u003e applications, optimizing the gel\u0026rsquo;s chemical composition with novel strategies as for example engineered aprotinin\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e to control the degradation rate is crucial to ensure it remains effective for approximately six weeks, the necessary period for axons to cross the lesion site.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eAdult three months old WT C57BL/6 and TN-C KO mice (kindly provided by Andreas Faissner, Ruhr University Bochum, Bochum, Germany) were placed in a close box and fully anesthetized with 3% isoflurane in air flow (Aerrane, Baxter). Once overdosed, mice were decapitated with guillotine (Ježek s.r.o., Czech Republic) and DRGs were extracted. All experiments were performed in accordance with the European Communities council directive of 22nd of September 2010(2010/63/EU), follow the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org/\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and were approved by the Ethics Committee of the Institute of Experimental Medicine ASCR, Prague, Czech Republic. DRGs were dissociated in 0.2% collagenase (Sigma-Aldrich) for 2h and 0.1% trypsin (Sigma-Aldrich) for 10 min in DMEM at 37\u0026deg;C, followed by trituration and 15% BSA (Sigma-Aldrich) gradient centrifugation. Cells were plated and grown on coated glass coverslips and fibrin hydrogel matrices at a density of 1x10^4 cells/condition in the following culture medium: 1% ITS (Sigma-Aldrich), 1% PSF (Sigma-Aldrich), 10 ng/ml NGF (Sigma-Aldrich) and 0.5 \u0026micro;g/ml Mitomycin-C (Sigma-Aldrich) in DMEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCoating of coverslips and fibrin hydrogel matrices preparation\u003c/h2\u003e \u003cp\u003eCoverslips were coated with either 20 \u0026micro;g/ml PDL (Sigma-Aldrich) or 1 \u0026micro;g/ml PDL\u0026thinsp;+\u0026thinsp;10 \u0026micro;g/ml chicken TN-C (Sigma-Aldrich) or 1 \u0026micro;g/ml PDL\u0026thinsp;+\u0026thinsp;5 \u0026micro;g/ml AEIDGIEL peptide (Sigma-Aldrich).\u003c/p\u003e \u003cp\u003ePreparation of fibrin hydrogel matrices consisted of mixing a fibrinogen-based solution and an enzymatic solution in equal proportions, as described previously\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The enzyme solution contained HEPES buffer, 2mM CaCl2, 2 U/ml thrombin (Sigma-Aldrich) and 5 U/ml Factor XIII (Sigma-Aldrich). The enzyme solution was incubated for 10 min at 37\u0026deg;C on a glass coverslip, followed by the addition of the fibrinogen solution. The fibrinogen solution contained HEPES buffer, 7.5 mg/ml fibrinogen (Sigma-Aldrich), 10 \u0026micro;g/ml aprotinin (Sigma-Aldrich), without or with 1 mg/ml AEIDGIEL peptide. The mixture of enzyme and fibrinogen solution was incubated for 1 hour at 37\u0026deg;C to form a polymerized gel.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell transduction\u003c/h3\u003e\n\u003cp\u003eDissociated DRG neurons were incubated with 1x 10^9 tu/ml of AAV-CAG-GFP and AAV-CAG-α9-V5. The plasmids carrying GFP or human α9 integrin were amplified and sequenced before packaging into AAV serotype 5 (AAV5) using HEK293T cells as described previously\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Cells were incubated with the virus for 72h at 37\u0026deg;C to induce viral transduction. On the third day, the media was replaced, and the cells were kept for additional 48h to ensure protein expression.\u003c/p\u003e\n\u003ch3\u003eMicroscopy, neurite outgrowth assay and statistical analysis\u003c/h3\u003e\n\u003cp\u003eCells were analyzed by fluorescent microscopy. Imaging was performed using a Zeiss Axio Observer D1 fluorescence microscope with inverted phase contrast and 40x magnification. To assess viral transduction, cells were identified and categorized based on their fluorescence signals: green for AAV-GFP-transduced cells, red for AAV-α9-V5-transduced cells, and only blue for un-transduced cells. The longest neurite length per neuron was measured using ImageJ software (National Institutes of Health). Neurite outgrowth was quantified from at least three separate experiments, with data representing individual neurites. Comparisons were made between un-transduced (NT), AAV-GFP transduced (GFP) and AAV-α9-integrin-transduced (A9) DRG cultures under different growth conditions. Statistical analysis was performed using a one-way ANOVA test followed by \u003cem\u003eTuckey\u003c/em\u003e analysis using GraphPad Prism 9 software.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor contributions statement\u003c/h2\u003e \u003cp\u003eAll authors were involved in the study's conception and design. Material preparation, data collection, and analysis were carried out by AC. The first draft of the manuscript was written by AC and all authors commented on previous versions of the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003e \u003cb\u003eAdditional information\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors were involved in the study's conception and design. Material preparation, data collection, and analysis were carried out by AC. The first draft of the manuscript was written by AC and all authors commented on previous versions of the manuscript. PJ secured the funding. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to acknowledge helpful discussions with Priscilla S Briquez and Jeffrey A Hubbell from University of Chicago.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the \u0026ldquo;A9-FibrinGel-TNC-KO project\u0026rdquo; repository, https://doi.org/10.5281/zenodo.13142847\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDing, W. et al. Spinal Cord Injury: The Global Incidence, Prevalence, and Disability From the Global Burden of Disease Study 2019. \u003cem\u003eSpine\u003c/em\u003e. \u003cb\u003e47\u003c/b\u003e, 1532\u0026ndash;1540 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhuja, C. S. et al. Traumatic spinal cord injury. \u003cem\u003eNat. Rev. Dis. 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Gene Ther.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1885\u0026ndash;1891 (1999).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"α9-integrin, fibrin gel, AEIDGIEL peptide, Tenascin-C, dorsal root ganglion","lastPublishedDoi":"10.21203/rs.3.rs-5289110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5289110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal 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 can offer three-dimensional support for axonal extension through the cavitated area, preventing the formation of aberrant paths and connections that occur in the absence of a suitable scaffold. The aim of this study was to investigate how combining α9-integrin expression with the use of a fibrin gel as an extracellular microenvironment affects mature DRG neurites growth \u003cem\u003ein vitro\u003c/em\u003e. Additionally, we sought to functionalize fibrin with AEIDGIEL peptide, the active domain of tenascin-C, to ensure α9-integrin activation. Our results indicate that fibrin gels are a suitable biomaterial for promoting neurite growth and that AEIDGIEL peptide effectively activates the integrin. In conclusion, 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 \u003cem\u003ein vivo\u003c/em\u003e research.\u003c/p\u003e","manuscriptTitle":"Combined Strategy Of α9-Integrin Transduction and AEIDGIEL Peptide-Functionalized Fibrin Gel Biomaterials to Promote Mature DRG Neurite Growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-31 08:48:07","doi":"10.21203/rs.3.rs-5289110/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4562927a-f7ba-43e9-8b75-0147e7112a99","owner":[],"postedDate":"October 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39371835,"name":"Biological sciences/Cell biology"},{"id":39371836,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2024-12-02T04:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-31 08:48:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5289110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5289110","identity":"rs-5289110","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}