Hydrogel containing decellularized spinal cord tissue induces M2 polarization in macrophages

Hydrogel containing decellularized spinal cord tissue induces M2 polarization in macrophages | 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 Hydrogel containing decellularized spinal cord tissue induces M2 polarization in macrophages Marcelo Garrido dos Santos, Fernanda Stapenhorst Franca, Augusto Vargas Pessi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5334346/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 Study design Basic cellular study. Objectives The use of biomaterials for regeneration is a promising approach in the tissue engineering field, but some materials can cause an inflammatory response when introduced into an organism. Macrophages play a central role in controlling inflammation, and when exposed to a stimuli, they polarize into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In this study, an evaluation was madeof macrophage polarization and inflammatory response when exposed to a hydrogel, used to produce a bioink, containing Decellularized Spinal Cord Tissue (DSCT) using RAW 264.7 macrophages. Setting Stem Cells Research Institute, Brazil. Methods Macrophages were exposed to the hydrogel for 24 hours. Cell viability was analyzed by MTT and Live/Dead assay. Flow cytometry was used to evaluate macrophage polarization and mitochondrial membrane potential. Cytokines, ROS (reactive oxygen species) production, sulfhydryl compounds and TBARS were quantified to assess inflammatory response. PC12 cells were exposed to macrophage conditioned medium and MTT was used to analyze cell viability. Results Although macrophage exposure to the hydrogel altered its metabolic activity, no cytotoxic effect nor oxidative damage was shown. Hydrogel induced an increase in IL-10 levels. The presence of DSCT in the biomaterial induced a decrease in CD45 and CD69 expression as well as an increase in CD206, a classic M2 marker, in comparison to the controls and the hydrogel without DSCT. Conclusion The hydrogel induced M2 polarization in the macrophages and a decrease in the inflammatory response. This biomaterial may be a suitable alternative for spinal cord regeneration after SCI. Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations There is no duality of interest 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5334346","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":371316362,"identity":"ad154994-70bc-4305-b4c5-462d6bb9c486","order_by":0,"name":"Marcelo Garrido dos Santos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYNACA2YGhgPMB4AsCRlStLAlgLTwEGsNSAuPAYhFWItu/+JnnysKrOX4zp/5/OpGjQUPA/vhoxvwaTG78cx45hmDdGPJG7nbrHOOAR3Gk5Z2A7+WA8aMDQaHEzfc4N1mnMMG1CLBY0ZAy/HPEC3nzzwzzvlHjJbzPVBbDuQwP85tI8oWnmKgFpBf0syYc/skeNgI+uX88c2MDX9AIXb48eecb3Vy/OyHj+HVwiCRAGeySYBJvMpBgP8AnMn8gaDqUTAKRsEoGJEAAJ9bS1ClRhY7AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Marcelo","middleName":"Garrido dos","lastName":"Santos","suffix":""},{"id":371316363,"identity":"bf59688e-354b-43ae-8b61-1df2a59ffe8c","order_by":1,"name":"Fernanda Stapenhorst Franca","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"Stapenhorst","lastName":"Franca","suffix":""},{"id":371316364,"identity":"e4df333f-2aed-4280-91e6-7ee7b02725af","order_by":2,"name":"Augusto Vargas Pessi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Augusto","middleName":"Vargas","lastName":"Pessi","suffix":""},{"id":371316365,"identity":"5daa68e4-fab3-4344-862b-d6cc51fd7629","order_by":3,"name":"Patrick Turck","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Turck","suffix":""},{"id":371316366,"identity":"6a265404-0054-4137-a524-c154226b246c","order_by":4,"name":"Juliete Scholl","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Juliete","middleName":"","lastName":"Scholl","suffix":""},{"id":371316367,"identity":"a089af1f-09d0-4e8d-8fa2-c95a7e3e3053","order_by":5,"name":"Adriane Bello klein","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adriane","middleName":"Bello","lastName":"klein","suffix":""},{"id":371316368,"identity":"0d325853-444f-40e5-b649-c9859ff35a52","order_by":6,"name":"Fabricio Figueiro","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fabricio","middleName":"","lastName":"Figueiro","suffix":""},{"id":371316369,"identity":"f20d6169-e9c4-440a-83d2-86d532e7189d","order_by":7,"name":"Alessandra Peres","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Peres","suffix":""},{"id":371316370,"identity":"28b1e97b-39c8-43d7-9db0-9dffb266b0ca","order_by":8,"name":"Patricia Pranke","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Pranke","suffix":""}],"badges":[],"createdAt":"2024-10-25 19:10:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5334346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5334346/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69568191,"identity":"3d25c35c-9c63-447a-8e6a-fdb79bebe1f5","added_by":"auto","created_at":"2024-11-21 18:13:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":442135,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability assays. Live/Dead assay analyzing the presence of live (stained in green) and dead (stained in red) macrophages when cultured with standard cultivated medium (A-C), challenged with H2O2 (D-F) and exposed to the hydrogel (G-I). (J) Cell counts of live and dead cells. There was no difference in the live cells in the hydrogel group when compared to the control but both groups showed a higher number of live cells in comparison to the H2O2 group. N=4. MTT assay indicated a reduction in cell viability in the hydrogel group compared to the control and an increase in comparison to the H2O2 group (K). Results are expressed as a percentage of the control. N=3. * = p\u0026lt;0.05; ** = p\u0026lt;0.005; *** = p\u0026lt;0.0005; **** = p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5334346/v1/5da2c6da2805f702bbc63b84.png"},{"id":69568190,"identity":"c99134f4-ea86-4807-9696-539875374076","added_by":"auto","created_at":"2024-11-21 18:13:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":236982,"visible":true,"origin":"","legend":"\u003cp\u003eMacrophage polarization. CD45 expression was quantified by flow cytometry (A-D, M). Hydrogel containing DSCT (C) decreased CD45 expression when compared to the cells cultivated in standard medium (control) (A), H2O2 group (B) and the hydrogel without DSCT (D). CD69 expression was quantified by flow cytometry (E-H, N). Hydrogel containing DSCT (G) and without DSCT (H) decreased CD69 expression when compared to the control (E) and H2O2 group (F). CD206 expression was quantified by flow cytometry (I-L, O). Hydrogel containing DSCT (K) increased CD206 expression when compared to the control (I), H2O2 group (J) and hydrogel without DSCT (L). N=5. p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5334346/v1/11838980c8c65f53eec2343f.png"},{"id":69568192,"identity":"f50dcd56-a321-42a2-a75d-b1c902dde4b3","added_by":"auto","created_at":"2024-11-21 18:13:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":265779,"visible":true,"origin":"","legend":"\u003cp\u003eCellular behavior modulation by DSCT. There was no difference in ROS production (A) and sulfhydryl compound concentration (B). The H2O2 group presented a higher lipid peroxidation than the control and the cells exposed to the hydrogel (C). N=3. Hydrogel exposure did not increase IL-6(D) and IL-1 (F) levels, but significantly increased IL-10 (E) levels. IL-1 concentration was significantly higher in the H2O2 group when compared to the medium and hydrogel groups. N=3. Conditioned medium from the macrophages stressed with LPS and treated with the hydrogel increased PC12 cell viability (G). N=3. Hydrogel exposure did not alter mitochondrial membrane potential when compared to the cells cultivated with standard medium. Both the groups aforementioned showed higher mitochondrial membrane potential when compared to the H2O2 group (H). N=3 * = p\u0026lt;0.05; ** = p\u0026lt;0.005; *** = p\u0026lt;0.0005\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5334346/v1/67f4b99686a37b928ba71e21.png"},{"id":87295856,"identity":"a26c1682-cb2f-4390-ac9e-af332781c4bc","added_by":"auto","created_at":"2025-07-22 12:23:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":602706,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.docx.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5334346/v1_covered_acedfec9-d1a1-4680-952c-d00a88023c00.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Hydrogel containing decellularized spinal cord tissue induces M2 polarization in macrophages","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-5334346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5334346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStudy design\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBasic cellular study.\u003c/p\u003e\n\u003cp\u003eObjectives\u003c/p\u003e\n\u003cp\u003eThe use of biomaterials for regeneration is a promising approach in the tissue engineering field, but some materials can cause an inflammatory response when introduced into an organism. Macrophages play a central role in controlling inflammation, and when exposed to a stimuli, they polarize into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In this study, an evaluation was madeof macrophage polarization and inflammatory response when exposed to a hydrogel, used to produce a bioink, containing Decellularized Spinal Cord Tissue (DSCT) using RAW 264.7 macrophages.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSetting\u003c/p\u003e\n\u003cp\u003eStem Cells Research Institute, Brazil.\u003c/p\u003e\n\u003cp\u003eMethods\u003c/p\u003e\n\u003cp\u003eMacrophages were exposed to the hydrogel for 24 hours. Cell viability was analyzed by MTT and Live/Dead assay. Flow cytometry was used to evaluate macrophage polarization and mitochondrial membrane potential. Cytokines, ROS (reactive oxygen species) production, sulfhydryl compounds and TBARS were quantified to assess inflammatory response. PC12 cells were exposed to macrophage conditioned medium and MTT was used to analyze cell viability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eAlthough macrophage exposure to the hydrogel altered its metabolic activity, no cytotoxic effect nor oxidative damage was shown. Hydrogel induced an increase in IL-10 levels. The presence of DSCT in the biomaterial induced a decrease in CD45 and CD69 expression as well as an increase in CD206, a classic M2 marker, in comparison to the controls and the hydrogel without DSCT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusion\u003c/p\u003e\n\u003cp\u003eThe hydrogel induced M2 polarization in the macrophages and a decrease in the inflammatory response. 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