{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T22:07:35Z","timestamp":1784758055255,"version":"3.55.0"},"reference-count":52,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2024,2,28]],"date-time":"2024-02-28T00:00:00Z","timestamp":1709078400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health","award":["RC 2022\u20132023"],"award-info":[{"award-number":["RC 2022\u20132023"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast\/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease\u2019s pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.<\/jats:p>","DOI":"10.3390\/ijms25052780","type":"journal-article","created":{"date-parts":[[2024,2,28]],"date-time":"2024-02-28T06:14:22Z","timestamp":1709100862000},"page":"2780","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Fibroblasts and Endothelial Cells in Three-Dimensional Models: A New Tool for Addressing the Pathogenesis of Systemic Sclerosis as a Prototype of Fibrotic Vasculopathies"],"prefix":"10.3390","volume":"25","author":[{"given":"Caterina","family":"Bodio","sequence":"first","affiliation":[{"name":"Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandra","family":"Milesi","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Neurobiology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 28824 Piancavallo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paola Adele","family":"Lonati","sequence":"additional","affiliation":[{"name":"Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3787-9632","authenticated-orcid":false,"given":"Cecilia Beatrice","family":"Chighizola","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences and Community Health, University of Milan, 20122 Milan, Italy"},{"name":"U.O.C. Clinica Reumatologica Pediatrica, ASST G. Pini\u2014CTO, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9072-7454","authenticated-orcid":false,"given":"Alessandro","family":"Mauro","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Neurobiology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 28824 Piancavallo, Italy"},{"name":"Department of Neuroscience, University of Turin, 10124 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1103-1964","authenticated-orcid":false,"given":"Luca Guglielmo","family":"Pradotto","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Neurobiology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 28824 Piancavallo, Italy"},{"name":"Department of Neuroscience, University of Turin, 10124 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pier Luigi","family":"Meroni","sequence":"additional","affiliation":[{"name":"Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8967-9678","authenticated-orcid":false,"given":"Maria Orietta","family":"Borghi","sequence":"additional","affiliation":[{"name":"Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy"},{"name":"Department of Clinical Sciences and Community Health, University of Milan, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Raschi","sequence":"additional","affiliation":[{"name":"Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Jensen, C., and Teng, Y. (2020). Is It Time to Start Transitioning From 2D to 3D Cell Culture?. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.00033"},{"key":"ref_2","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_3","first-page":"22","article-title":"Why animal model studies are lost in translation","volume":"2","author":"Frangogiannis","year":"2022","journal-title":"J. Cardiovasc. Aging"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"218","DOI":"10.2174\/1570163813666160114093140","article-title":"From in vitro experiments to in vivo and clinical studies; pros and cons","volume":"12","author":"Saeidnia","year":"2015","journal-title":"Curr. Drug Discov. Technol."},{"key":"ref_5","first-page":"199","article-title":"Ethical Considerations in Animal Research: The Principle of 3R\u2019s","volume":"73","author":"Zambrano","year":"2020","journal-title":"Rev. Investig. Clin."},{"key":"ref_6","first-page":"12","article-title":"Ethical Considerations in Animal Studies","volume":"2","author":"Ghasemi","year":"2009","journal-title":"J. Med. Ethics Hist. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"275","DOI":"10.14573\/altex.2013.3.275","article-title":"Food for Thought Look Back in Anger-What Clinical Studies Tell Us About Preclinical Work","volume":"30","author":"Hartung","year":"2013","journal-title":"ALTEX"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.15252\/embr.201744428","article-title":"Expanding the 3R Principles","volume":"18","author":"Aske","year":"2017","journal-title":"EMBO Rep."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Habanjar, O., Diab-Assaf, M., Caldefie-Chezet, F., and Delort, L. (2021). 3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222212200"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1177\/1087057117696795","article-title":"Three-Dimensional Cell Cultures in Drug Discovery and Development","volume":"22","author":"Fang","year":"2017","journal-title":"SLAS Discov."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/apm.13168","article-title":"Characterization of in vitro 3D cultures","volume":"129","author":"Mousavi","year":"2021","journal-title":"APMIS"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-60761-984-0_1","article-title":"3D cell culture: A review of current approaches and techniques","volume":"695","author":"Haycock","year":"2011","journal-title":"Methods Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1016\/j.biotechadv.2016.11.002","article-title":"3D Tumor Spheroids: An Overview on the Tools and Techniques Used for Their Analysis","volume":"34","author":"Costa","year":"2016","journal-title":"Biotechnol. Adv."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.actbio.2018.05.034","article-title":"Design of Spherically Structured 3D in Vitro Tumor Models\u2014Advances and Prospects","volume":"75","author":"Ferreira","year":"2018","journal-title":"Acta Biomater."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1002\/biot.201400004","article-title":"A Novel Dual-Flow Bioreactor Simulates Increased Fluorescein Permeability in Epithelial Tissue Barriers","volume":"9","author":"Giusti","year":"2014","journal-title":"Biotechnol. J."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Paolillo, M., Colombo, R., Serra, M., Belvisi, L., Papetti, A., Ciusani, E., Comincini, S., and Schinelli, S. (2019). Stem-Like Cancer Cells in a Dynamic 3D Culture System: A Model to Study Metastatic Cell Adhesion and Anti-Cancer Drugs. Cells, 8.","DOI":"10.3390\/cells8111434"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2129","DOI":"10.1002\/bit.23164","article-title":"A Flexible Bioreactor System for Constructing in Vitro Tissue and Organ Models","volume":"108","author":"Vozzi","year":"2011","journal-title":"Biotechnol. Bioeng."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1172\/JCI31139","article-title":"Systemic sclerosis: A prototypic multisystem fibrotic disorder","volume":"117","author":"Varga","year":"2007","journal-title":"J. Clin. Investig."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1002\/kjm2.12505","article-title":"Pathophysiology of systemic sclerosis (scleroderma)","volume":"38","author":"Rosendahl","year":"2022","journal-title":"Kaohsiung J. Med. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Patnaik, E., Lyons, M., Tran, K., and Pattanaik, D. (2023). Endothelial Dysfunction in Systemic Sclerosis. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241814385"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1186\/s13075-018-1689-6","article-title":"Immune Complexes Containing Scleroderma-Specific Autoantibodies Induce a Profibrotic and Proinflammatory Phenotype in Skin Fibroblasts","volume":"20","author":"Raschi","year":"2018","journal-title":"Arthritis Res. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1186\/s13075-020-02360-3","article-title":"Scleroderma-Specific Autoantibodies Embedded in Immune Complexes Mediate Endothelial Damage: An Early Event in the Pathogenesis of Systemic Sclerosis","volume":"22","author":"Raschi","year":"2020","journal-title":"Arthritis Res. Ther."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"102954","DOI":"10.1016\/j.jaut.2022.102954","article-title":"Immune Complexome Analysis of a Rich Variety of Serum Immune Complexes Identifies Disease-Characteristic Immune Complex Antigens in Systemic Sclerosis","volume":"134","author":"Kutsuna","year":"2023","journal-title":"J. Autoimmun."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1002\/jbm.a.31785","article-title":"Primary Sequence of Ionic Self-assembling Peptide Gels Affects Endothelial Cell Adhesion and Capillary Morphogenesis","volume":"87A","author":"Sieminski","year":"2008","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1038\/nmeth.3839","article-title":"A Practical Guide to Hydrogels for Cell Culture","volume":"13","author":"Caliari","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.ceb.2019.10.003","article-title":"The Golgi Apparatus and Cell Polarity: Roles of the Cytoskeleton, the Golgi Matrix, and Golgi Membranes","volume":"62","author":"Ravichandran","year":"2020","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1007\/s12016-021-08889-8","article-title":"Current Concepts on the Pathogenesis of Systemic Sclerosis","volume":"64","author":"Truchetet","year":"2023","journal-title":"Clin. Rev. Allergy Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1016\/S0140-6736(22)01692-0","article-title":"Systemic sclerosis","volume":"401","author":"Volkmann","year":"2023","journal-title":"Lancet"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1007\/s12016-022-08946-w","article-title":"Systemic Sclerosis-Specific Antibodies: Novel and Classical Biomarkers","volume":"64","author":"Cavazzana","year":"2023","journal-title":"Clin. Rev. Allergy Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1747","DOI":"10.1136\/annrheumdis-2013-204424","article-title":"2013 classification criteria for systemic sclerosis: An American college of rheumatology\/European league against rheumatism collaborative initiative","volume":"72","author":"Khanna","year":"2013","journal-title":"Ann. Rheum. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1520","DOI":"10.1002\/acr.22304","article-title":"Early Systemic Sclerosis: Analysis of the Disease Course in Patients with Marker Autoantibody and\/or Capillaroscopic Positivity","volume":"66","author":"Valentini","year":"2014","journal-title":"Arthritis Care Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1097\/BOR.0000000000000331","article-title":"Animal Models of Scleroderma: Recent Progress","volume":"28","author":"Marangoni","year":"2016","journal-title":"Curr. Opin. Rheumatol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1225","DOI":"10.1016\/j.autrev.2018.06.013","article-title":"Recent Advances in Mouse Models for Systemic Sclerosis","volume":"17","author":"Yue","year":"2018","journal-title":"Autoimmun. Rev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1186\/s13075-022-02896-6","article-title":"Both T and B Cells Are Indispensable for the Development of a PBMC Transfer-Induced Humanized Mouse Model for SSc","volume":"24","author":"Shu","year":"2022","journal-title":"Arthritis Res. Ther."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Vazquez-Armendariz, A.I., Barroso, M.M., El Agha, E., and Herold, S. (2022). 3D In Vitro Models: Novel Insights into Idiopathic Pulmonary Fibrosis Pathophysiology and Drug Screening. Cells, 11.","DOI":"10.3390\/cells11091526"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Paradiso, F., Quintela, M., Lenna, S., Serpelloni, S., James, D., Caserta, S., Conlan, S., Francis, L., and Taraballi, F. (2022). Studying Activated Fibroblast Phenotypes and Fibrosis-Linked Mechanosensing Using 3D Biomimetic Models. Macromol. Biosci., 22.","DOI":"10.1002\/mabi.202100450"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5898","DOI":"10.1038\/s41598-018-24273-7","article-title":"3D Artificial Round Section Micro-Vessels to Investigate Endothelial Cells under Physiological Flow Conditions","volume":"8","author":"Sfriso","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1007\/978-1-0716-0255-3_6","article-title":"3D Cell-Culture Models for the Assessment of Anticoagulant and Anti-Inflammatory Properties of Endothelial Cells","volume":"2110","author":"Sfriso","year":"2020","journal-title":"Methods Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"39","DOI":"10.31557\/APJCP.2022.23.1.39","article-title":"In 2D and 3D Cell Culture Models, Effects of Endothelial Cells on E-Cadherin\/\u03b2-Catenin Expression Levels and Spheroid Sizes in Ishikawa Cells","volume":"23","author":"Kalender","year":"2022","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1038\/nrm3176","article-title":"Molecular Control of Endothelial Cell Behaviour during Blood Vessel Morphogenesis","volume":"12","author":"Herbert","year":"2011","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1515\/BC.2009.115","article-title":"Ancestral Vascular Tube Formation and its Adoption by Tumors","volume":"390","author":"Lammert","year":"2009","journal-title":"Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1161\/CIRCRESAHA.107.148932","article-title":"Endothelial Precursor Cell Migration during Vasculogenesis","volume":"101","author":"Schmidt","year":"2007","journal-title":"Circ. Res."},{"key":"ref_43","first-page":"671","article-title":"Involvement of Endothelial PECAM-1\/CD31 in Angiogenesis","volume":"151","author":"DeLisser","year":"1997","journal-title":"Am. J. Pathol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"C1181","DOI":"10.1152\/ajpcell.00524.2001","article-title":"Involvement of Human PECAM-1 in Angiogenesis and in Vitro Endothelial Cell Migration","volume":"282","author":"Cao","year":"2002","journal-title":"Am. J. Physiol.-Cell Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1161\/01.RES.0000259593.07661.1e","article-title":"Endothelial Cell Migration during Angiogenesis","volume":"100","author":"Lamalice","year":"2007","journal-title":"Circ. Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.1002\/jcb.20614","article-title":"Mechanotransduction in Endothelial Cell Migration","volume":"96","author":"Li","year":"2005","journal-title":"J. Cell Biochem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4373","DOI":"10.1039\/D0LC00738B","article-title":"Endothelial Cell Polarization and Orientation to Flow in a Novel Microfluidic Multimodal Shear Stress Generator","volume":"20","author":"Sonmez","year":"2020","journal-title":"Lab. Chip"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e45253","DOI":"10.15252\/embr.201745253","article-title":"PAR-3 Controls Endothelial Planar Polarity and Vascular Inflammation under Laminar Flow","volume":"19","author":"Hikita","year":"2018","journal-title":"EMBO Rep."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1038\/s41536-021-00133-3","article-title":"Scaffold-Free Cell-Based Tissue Engineering Therapies: Advances, Shortfalls and Forecast","volume":"6","author":"Rochev","year":"2021","journal-title":"NPJ Regen. Med."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"De Pieri, A., Korman, B.D., J\u00fcngel, A., and Wuertz-Kozak, K. (2021). Engineering Advanced In Vitro Models of Systemic Sclerosis for Drug Discovery and Development. Adv. Biol., 5.","DOI":"10.1002\/adbi.202000168"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"16930","DOI":"10.1038\/s41598-022-21468-x","article-title":"High-Throughput 3D Microvessel-on-a-Chip Model to Study Defective Angiogenesis in Systemic Sclerosis","volume":"12","author":"Kramer","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.actbio.2017.03.035","article-title":"Human Lung Fibroblast-Derived Matrix Facilitates Vascular Morphogenesis in 3D Environment and Enhances Skin Wound Healing","volume":"54","author":"Du","year":"2017","journal-title":"Acta Biomater."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/5\/2780\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:06:19Z","timestamp":1760105179000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/5\/2780"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,28]]},"references-count":52,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,3]]}},"alternative-id":["ijms25052780"],"URL":"https:\/\/doi.org\/10.3390\/ijms25052780","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,28]]}}}