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Emerging evidence suggests that disturbances in hepatic vitamin D metabolism may contribute to these skeletal impairments. However, the hepatic mechanisms driving bone deterioration remain poorly defined. This study aimed to establish a human 3D in vitro model of MASLD and demonstrate that hepatic vitamin D dysregulation adversely affects bone homeostasis. Liver spheroids composed of HepaRG cells, LX-2 stellate cells, and HUVECs were stimulated with 600 \u00b5M free fatty acids (2:1 oleic: palmitic acid) to induce MASLD-like features, validated by BODIPY staining and gene expression. MASLD model induction led to downregulation of hepatic genes regulating lipid and vitamin D metabolism. ELISA confirmed significantly reduced 25-hydroxyvitamin D levels, aligning with downregulation of\n                    <jats:italic>CYP2R1<\/jats:italic>\n                    and\n                    <jats:italic>CYP27A1<\/jats:italic>\n                    . Transcriptomic profiling of human MASLD liver biopsies validated these molecular changes. To evaluate MASLD\u2019s systemic impact on bone, THP-1-derived macrophages and SCP-1 mesenchymal stem cells were seeded onto bone scaffolds and co-cultured with spheroids. Bone scaffolds co-cultured with MASLD spheroids showed impaired mineralization and elevated expression of bone resorption markers. These findings mirror skeletal dysfunction observed in MASLD patients and suggest a mechanistic link between hepatic vitamin D dysregulation and bone pathology. This study introduces a pioneering 3D human liver-bone co-culture model that reveals MASLD-driven disruption of hepatic vitamin D metabolism as a direct contributor to bone deterioration. This 3D model develops a powerful translational platform for decoding systemic disease mechanisms and targeting the liver-bone axis therapeutically.\n                  <\/jats:p>","DOI":"10.1007\/s00204-026-04431-x","type":"journal-article","created":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T07:14:27Z","timestamp":1778656467000},"page":"4151-4169","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Free Fatty acid-induced disruption of hepatic vitamin D metabolism impairs bone homeostasis in an in vitro 3D human liver\u2013bone model"],"prefix":"10.1007","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-6679-2495","authenticated-orcid":false,"given":"Mohammad Majd","family":"Hammour","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa","family":"Herzberger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4865-8787","authenticated-orcid":false,"given":"Yuxuan","family":"Xin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7860-8154","authenticated-orcid":false,"given":"Guanqiao","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0528-6680","authenticated-orcid":false,"given":"Melike","family":"Tombaz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4347-1702","authenticated-orcid":false,"given":"Sabrina","family":"Ehnert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabian","family":"Springer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2104-8076","authenticated-orcid":false,"given":"Georg","family":"Damm","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5924-4366","authenticated-orcid":false,"given":"Massoud","family":"Vosough","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1427-5246","authenticated-orcid":false,"given":"Jan G.","family":"Hengstler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ursula","family":"M\u00fcller-Vieira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6666-6791","authenticated-orcid":false,"given":"Andreas K.","family":"N\u00fcssler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5160-2111","authenticated-orcid":false,"given":"Romina H.","family":"Aspera-Werz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,13]]},"reference":[{"key":"4431_CR1","doi-asserted-by":"publisher","first-page":"1001281","DOI":"10.37349\/emed.2025.1001281","volume":"6","author":"LE Adinolfi","year":"2025","unstructured":"Adinolfi LE, Marrone A, Rinaldi L, Nevola R, Izzi A, Sasso FC (2025) Metabolic dysfunction-associated steatotic liver disease (MASLD): a systemic disease with a variable natural history and challenging management. 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