{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T20:04:47Z","timestamp":1790366687781,"version":"4.1.0"},"reference-count":46,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2020,6,13]],"date-time":"2020-06-13T00:00:00Z","timestamp":1592006400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Rapid vascularization is required for the regeneration of dental pulp due to the spatially restricted tooth environment. Extracellular vesicles (EVs) released from mesenchymal stromal cells show potent proangiogenic effects. Since EVs suffer from rapid clearance and low accumulation in target tissues, an injectable delivery system capable of maintaining a therapeutic dose of EVs over a longer period would be desirable. We fabricated an EV-fibrin gel composite as an in situ forming delivery system. EVs were isolated from dental pulp stem cells (DPSCs). Their effects on cell proliferation and migration were monitored in monolayers and hydrogels. Thereafter, endothelial cells and DPSCs were co-cultured in EV-fibrin gels and angiogenesis as well as collagen deposition were analyzed by two-photon laser microscopy. Our results showed that EVs enhanced cell growth and migration in 2D and 3D cultures. EV-fibrin gels facilitated vascular-like structure formation in less than seven days by increasing the release of VEGF. The EV-fibrin gel promoted the deposition of collagen I, III, and IV, and readily induced apoptosis during the initial stage of angiogenesis. In conclusion, we confirmed that EVs from DPSCs can promote angiogenesis in an injectable hydrogel in vitro, offering a novel and minimally invasive strategy for regenerative endodontic therapy.<\/jats:p>","DOI":"10.3390\/ijms21124226","type":"journal-article","created":{"date-parts":[[2020,6,15]],"date-time":"2020-06-15T03:17:32Z","timestamp":1592191052000},"page":"4226","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":54,"title":["Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6214-3459","authenticated-orcid":false,"given":"Siyuan","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Biohybrid and Medical Textiles (BioTex), AME - Institute of Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University &amp; Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2990-0761","authenticated-orcid":false,"given":"Anja Lena","family":"Thiebes","sequence":"additional","affiliation":[{"name":"Department of Biohybrid and Medical Textiles (BioTex), AME - Institute of Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University &amp; Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franziska","family":"Kreimendahl","sequence":"additional","affiliation":[{"name":"Department of Biohybrid and Medical Textiles (BioTex), AME - Institute of Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University &amp; Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephan","family":"Ruetten","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Institute of Pathology, RWTH Aachen University Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0627-9228","authenticated-orcid":false,"given":"Eva Miriam","family":"Buhl","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Institute of Pathology, RWTH Aachen University Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Wolf","sequence":"additional","affiliation":[{"name":"Department of Orthodontics, RWTH Aachen University Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Jockenhoevel","sequence":"additional","affiliation":[{"name":"Department of Biohybrid and Medical Textiles (BioTex), AME - Institute of Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University &amp; Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1339-1990","authenticated-orcid":false,"given":"Christian","family":"Apel","sequence":"additional","affiliation":[{"name":"Department of Biohybrid and Medical Textiles (BioTex), AME - Institute of Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University &amp; Hospital, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1222","DOI":"10.1177\/0022034514549809","article-title":"Tissue-engineering-based strategies for regenerative endodontics","volume":"93","author":"Albuquerque","year":"2014","journal-title":"J. Dent. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1146\/annurev-bioeng-071812-152428","article-title":"The pivotal role of vascularization in tissue engineering","volume":"15","author":"Auger","year":"2013","journal-title":"Annu. Rev. Biomed. Eng."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.joen.2017.09.003","article-title":"The role of vasculature engineering in dental pulp regeneration","volume":"43","author":"Dissanayaka","year":"2017","journal-title":"J. Endod."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1535750","DOI":"10.1080\/20013078.2018.1535750","article-title":"Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines","volume":"7","author":"Witwer","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1635","DOI":"10.1089\/scd.2014.0316","article-title":"Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts, and enhance angiogenesis in vitro","volume":"24","author":"Shabbir","year":"2015","journal-title":"Stem Cells Dev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"904","DOI":"10.3389\/fphys.2017.00904","article-title":"GMSC-derived exosomes combined with a chitosan\/silk hydrogel sponge accelerates wound healing in a diabetic rat skin defect model","volume":"8","author":"Shi","year":"2017","journal-title":"Front. Physiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1016\/j.joen.2017.12.024","article-title":"Exosomes with highly angiogenic potential for possible use in pulp regeneration","volume":"44","author":"Xian","year":"2018","journal-title":"J. Endod."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.jbiotec.2013.03.013","article-title":"Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection","volume":"165","author":"Takahashi","year":"2013","journal-title":"J. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.jconrel.2014.12.013","article-title":"Biodistribution and delivery efficiency of unmodified tumor-derived exosomes","volume":"199","author":"Smyth","year":"2015","journal-title":"J. Control. Release"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"26316","DOI":"10.3402\/jev.v4.26316","article-title":"Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting","volume":"4","author":"Wiklander","year":"2015","journal-title":"J. Extracell. Vesicles"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1186\/s13287-016-0391-3","article-title":"Exosomes\/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K\/Akt signaling pathway","volume":"7","author":"Zhang","year":"2016","journal-title":"Stem Cell Res. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4430","DOI":"10.1039\/C7NR00352H","article-title":"Integration of stem cell-derived exosomes with in situ hydrogel glue as a promising tissue patch for articular cartilage regeneration","volume":"9","author":"Liu","year":"2017","journal-title":"Nanoscale"},{"key":"ref_13","first-page":"309","article-title":"Biomaterials in tooth tissue engineering: A review","volume":"8","author":"Sharma","year":"2014","journal-title":"J. Clin. Diagn. Res. JCDR"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.archoralbio.2018.11.023","article-title":"Screening of hydrogel-based scaffolds for dental pulp regeneration-A systematic review","volume":"98","author":"Fukushima","year":"2018","journal-title":"Arch. Oral Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2409","DOI":"10.1016\/j.yexcr.2013.06.006","article-title":"In vitro models of angiogenesis and vasculogenesis in fibrin gel","volume":"319","author":"Morin","year":"2013","journal-title":"Exp. Cell Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1080\/14653240600855905","article-title":"Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement","volume":"8","author":"Dominici","year":"2006","journal-title":"Cytotherapy"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.jconrel.2017.08.022","article-title":"Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia","volume":"264","author":"Gangadaran","year":"2017","journal-title":"J. Control. Release"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1619","DOI":"10.1242\/dev.01037","article-title":"Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development","volume":"131","author":"Brachvogel","year":"2004","journal-title":"Development"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1038\/sj.bjp.0703407","article-title":"Apoptosis in the vasculature: Mechanisms and functional importance","volume":"130","author":"Mallat","year":"2000","journal-title":"Br. J. Pharmacol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1007\/s00109-013-1110-5","article-title":"Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model","volume":"92","author":"Bian","year":"2014","journal-title":"J. Mol. Med."},{"key":"ref_21","first-page":"245","article-title":"The angiogenic potential of adipose mesenchymal stem cell-derived extracellular vesicles is modulated by basic fibroblast growth factor","volume":"4","author":"Lopatina","year":"2014","journal-title":"J. Stem Cell Res. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"513","DOI":"10.5966\/sctm.2014-0267","article-title":"Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4\/\u03b2-catenin pathway","volume":"4","author":"Zhang","year":"2015","journal-title":"Stem Cells Transl. Med."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Merckx, G., Hosseinkhani, B., Kuypers, S., Deville, S., Irobi, J., Nelissen, I., Michiels, L., Lambrichts, I., and Bronckaers, A. (2020). Angiogenic effects of human dental pulp and bone marrow-derived mesenchymal stromal cells and their extracellular vesicles. Cells, 9.","DOI":"10.3390\/cells9020312"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1152\/physiol.00036.2016","article-title":"Modeling physiological events in 2D vs. 3D cell culture","volume":"32","author":"Duval","year":"2017","journal-title":"Physiology"},{"key":"ref_25","first-page":"194","article-title":"Alginate nanobeads interspersed fibrin network as in situ forming hydrogel for soft tissue engineering","volume":"3","author":"Deepthi","year":"2018","journal-title":"Bioact. Mater."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1098\/rsif.2008.0327","article-title":"Fibrin gels and their clinical and bioengineering applications","volume":"6","author":"Janmey","year":"2008","journal-title":"J. R. Soc. Interface"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"24641","DOI":"10.3402\/jev.v3.24641","article-title":"Routes and mechanisms of extracellular vesicle uptake","volume":"3","author":"Mulcahy","year":"2014","journal-title":"J. Extracell. Vesicles"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Margolis, L., and Sadovsky, Y. (2019). The biology of extracellular vesicles: The known unknowns. PLoS Biol., 17.","DOI":"10.1371\/journal.pbio.3000363"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ludwig, N., Yerneni, S.S., Azambuja, J.H., Gillespie, D.G., Menshikova, E.V., Jackson, E.K., and Whiteside, T.L. (2020). Tumor-derived exosomes promote angiogenesis via adenosine A 2B receptor signaling. Angiogenesis, 1\u201312.","DOI":"10.1007\/s10456-020-09728-8"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Banfi, A., Holnthoner, W., Martino, M.M., and Yl\u00e4-Herttuala, S. (2018). Editorial: Vascularization for Regenerative Medicine. Front. Bioeng. Biotechnol., 6.","DOI":"10.3389\/fbioe.2018.00175"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1089\/ten.tea.2014.0154","article-title":"The interplay of dental pulp stem cells and endothelial cells in an injectable peptide hydrogel on angiogenesis and pulp regeneration in vivo","volume":"21","author":"Dissanayaka","year":"2014","journal-title":"Tissue Eng. Part A"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Kaessmeyer, S., Sehl, J., In, M., Merle, R., Richardson, K., and Plendl, J. (2017). Subcellular Interactions during Vascular Morphogenesis in 3D Cocultures between Endothelial Cells and Fibroblasts. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18122590"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1089\/ten.tec.2017.0234","article-title":"Three-dimensional printing and angiogenesis: Tailored agarose-type I collagen blends comprise three-dimensional printability and angiogenesis potential for tissue-engineered substitutes","volume":"23","author":"Kreimendahl","year":"2017","journal-title":"Tissue Eng. Part C Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1038\/am.2017.171","article-title":"Novel biomaterial strategies for controlled growth factor delivery for biomedical applications","volume":"9","author":"Wang","year":"2017","journal-title":"Npg Asia Mater."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1016\/j.cell.2011.08.039","article-title":"Basic and therapeutic aspects of angiogenesis","volume":"146","author":"Potente","year":"2011","journal-title":"Cell"},{"key":"ref_36","first-page":"23","article-title":"VEGF coordinates interaction of pericytes and endothelial cells during vasculogenesis and experimental angiogenesis","volume":"230","author":"Hagedorn","year":"2004","journal-title":"Dev. Dyn. Off. Publ. Am. Assoc. Anat."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6390","DOI":"10.1073\/pnas.101564798","article-title":"Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF","volume":"98","author":"Cao","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1007\/s00418-011-0789-z","article-title":"Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs","volume":"135","author":"Ide","year":"2011","journal-title":"Histochem. Cell Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1016\/j.joen.2017.11.018","article-title":"Leptin induces odontogenic differentiation and angiogenesis in human dental pulp cells via activation of the mitogen-activated protein kinase signaling pathway","volume":"44","author":"Ngo","year":"2018","journal-title":"J. Endod."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1007\/s10529-012-1084-3","article-title":"BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells","volume":"35","author":"Bai","year":"2013","journal-title":"Biotechnol. Lett."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1007\/s00441-012-1392-x","article-title":"Effects of VEGF and FGF-2 on proliferation and differentiation of human periodontal ligament stem cells","volume":"348","author":"Lee","year":"2012","journal-title":"Cell Tissue Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/j.devcel.2008.07.002","article-title":"The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis","volume":"15","author":"Wang","year":"2008","journal-title":"Dev. Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1161\/CIRCRESAHA.108.191270","article-title":"MicroRNAs as novel regulators of angiogenesis","volume":"104","author":"Sessa","year":"2009","journal-title":"Circ. Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1093\/ptj\/79.3.308","article-title":"Connective tissues: Matrix composition and its relevance to physical therapy","volume":"79","author":"Culav","year":"1999","journal-title":"Phys. Ther."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1177\/002203458506400405","article-title":"Session II: Cells and Extracellular Matrices of the Dental Pulp\u2014CT Hanks, Chairman: The Extracellular Matrix of the Dental Pulp and Dentin","volume":"64","author":"Linde","year":"1985","journal-title":"J. Dent. Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1023\/B:AGEN.0000021390.09275.bc","article-title":"New insights into the function and regulation of endothelial cell apoptosis","volume":"6","author":"Duval","year":"2003","journal-title":"Angiogenesis"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/12\/4226\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:38:42Z","timestamp":1760175522000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/12\/4226"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,13]]},"references-count":46,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,6]]}},"alternative-id":["ijms21124226"],"URL":"https:\/\/doi.org\/10.3390\/ijms21124226","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,13]]}}}