{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T09:18:15Z","timestamp":1787044695502,"version":"build-2736575974"},"reference-count":35,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NL-BioImaging-AM","award":["184.036.012"],"award-info":[{"award-number":["184.036.012"]}]},{"DOI":"10.13039\/501100003246","name":"Dutch Research Council","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004543","name":"China Scholarship Council","doi-asserted-by":"publisher","award":["CSC201808620228"],"award-info":[{"award-number":["CSC201808620228"]}],"id":[{"id":"10.13039\/501100004543","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Osteogenesis imperfecta (OI) and Ehlers\u2013Danlos syndrome (EDS) are inherited connective tissue disorders caused by diverse genetic defects, many of which affect collagen biosynthesis. However, the identified genetic variants do not always fully explain the clinical heterogeneity observed in patients, highlighting the need for advanced models and imaging techniques to assess collagen structure and fibroblast behavior at the microscopic level. In this study, we employed 5-week three-dimensional (3D) dermal fibroblast cultures derived from patients with haploinsufficient (HI) and dominant-negative (DN) OI, EDS, and healthy controls. Using label-free higher harmonic generation microscopy (HHGM), we visualized and quantified secreted collagen fibers and fibroblast morphology in situ. We analyzed fibroblast 3D orientation, collagen fiber diameter, collagen amount per cell, and the spatial alignment between fibroblasts and collagen fibers. HI OI fibroblasts secreted significantly less collagen than both control and EDS-derived cells, while EDS samples exhibited thinner collagen fibers compared to controls. Across all groups, collagen fiber orientation was strongly correlated with fibroblast alignment, in line with the role of fibroblasts in matrix organization. In healthy controls and HI OI samples, we observed a depth-dependent, counterclockwise rotation in fibroblast orientation from the culture bottom to the surface\u2014a pattern that was less prominent in DN OI and EDS samples, potentially reflecting altered matrix guidance in diseased tissues. Overall, the quantity and quality of collagen, as well as fibroblast morphology and organization, were markedly altered in the OI and EDS model systems. These alterations may mirror tissue-level manifestations of the diseases, demonstrating the physiological relevance of patient-derived 3D fibroblast models for OI and EDS, as well as the power of harmonic generation microscopy in probing the cellular and extracellular consequences of disease-related gene defects in collagen or its biosynthetic pathways. Extensions of this methodological approach provide a way towards deeper understanding of tissue-level manifestations of collagen dysregulation in connective tissue disorders.<\/jats:p>","DOI":"10.3390\/ijms262411848","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T14:52:24Z","timestamp":1765205544000},"page":"11848","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Assessment of Collagen and Fibroblast Properties via Label-Free Higher Harmonic Generation Microscopy in Three-Dimensional Models of Osteogenesis Imperfecta and Ehlers-Danlos Syndrome"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2744-3446","authenticated-orcid":false,"given":"Yuanyuan","family":"Ma","sequence":"first","affiliation":[{"name":"Department of Physics, LaserLab Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6551-7196","authenticated-orcid":false,"given":"Qiyu","family":"Bo","sequence":"additional","affiliation":[{"name":"Institute of Modern Optics, Nankai University, Tianjin 300350, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2306-0340","authenticated-orcid":false,"given":"Zhiqing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Modern Optics, Nankai University, Tianjin 300350, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3785-9217","authenticated-orcid":false,"given":"Ludo","family":"van Haasterecht","sequence":"additional","affiliation":[{"name":"Department of Physics, LaserLab Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Kloen","sequence":"additional","affiliation":[{"name":"Department of Orthopedic Surgery and Sports Medicine, Amsterdam University Medical Center, Location Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands"},{"name":"Amsterdam Movement Sciences, 1081 BT Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Rustemeyer","sequence":"additional","affiliation":[{"name":"The Netherlands Department of Dermato-Allergology and Occupational Health, Amsterdam University Medical Center, Location AMC, University of Amsterdam, 1000 GG Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-1732-6618","authenticated-orcid":false,"given":"Laura","family":"Ventura","sequence":"additional","affiliation":[{"name":"Amsterdam Movement Sciences, 1081 BT Amsterdam, The Netherlands"},{"name":"Amsterdam Reproduction & Development, 1105 AZ Amsterdam, The Netherlands"},{"name":"Rare Bone Disease Center Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Department of Human Genetics, Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4682-0402","authenticated-orcid":false,"given":"Lidiia","family":"Zhytnik","sequence":"additional","affiliation":[{"name":"Amsterdam Movement Sciences, 1081 BT Amsterdam, The Netherlands"},{"name":"Amsterdam Reproduction & Development, 1105 AZ Amsterdam, The Netherlands"},{"name":"Rare Bone Disease Center Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Department of Human Genetics, Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5399-676X","authenticated-orcid":false,"given":"Elisabeth M. W.","family":"Eekhoff","sequence":"additional","affiliation":[{"name":"Rare Bone Disease Center Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Department of Endocrinology & Metabolism, Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7890-4411","authenticated-orcid":false,"given":"Dimitra","family":"Micha","sequence":"additional","affiliation":[{"name":"Rare Bone Disease Center Amsterdam, 1081 HV Amsterdam, The Netherlands"},{"name":"Department of Human Genetics, Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5360-6987","authenticated-orcid":false,"given":"Marie Louise","family":"Groot","sequence":"additional","affiliation":[{"name":"Department of Physics, LaserLab Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1210\/endrev\/bnab017","article-title":"Osteogenesis Imperfecta: Mechanisms and Signaling Pathways Connecting Classical and Rare OI Types","volume":"43","author":"Jovanovic","year":"2022","journal-title":"Endocr. 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