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Objectives and questions: Metabolic dysfunction-associated steatotic liver disease (MASLD) is linked to reduced bone mineral density and elevated fracture risk. This study examined whether MASLD-related hepatic dysfunction impairs bone mineralization and early biomechanical strength by disrupting hepatic vitamin D 25-hydroxylation, thereby directly compromising skeletal health.

Material and methods: A human 3D co-culture system was established by co-culturing liver spheroids (HepaRG/LX-2/HUVEC) with mineralized human platelet-rich plasma (hPRP) bone scaffolds. These scaffolds were seeded with THP-1–derived macrophages (osteoclast precursors) and SCP-1 mesenchymal stem cells (osteogenic lineage). MASLD was modeled by exposing liver spheroids to free fatty acids (FFAs; oleic:palmitic 2:1) for 7 days. This was followed by up to 28 days of co-culture with bone scaffolds. Bone endpoints included viability and stress (DNA content, resazurin); osteogenic and osteoclastic activity (alkaline phosphatase [AP], carbonic anhydrase II [CA II], Tartrate-Resistant Acid Phosphatase [TRAP]); CT-based mineral density; and uniaxial compression stiffness. Hepatic CYP27A1/CYP2R1 expression and 25-hydroxyvitamin D [25(OH)D₃] formation were quantified to connect liver function to skeletal outcomes. Statistical analysis used the Kruskal–Wallis test.

Results: Under MASLD conditions, bone scaffolds retained DNA content but exhibited significantly reduced mineral density by CT at day 28 compared to controls (p < 0.05). Stiffness remained unchanged, indicating an early deficit in bone mineralization that precedes structural failure. Increased mitochondrial activity reflected cellular stress, without evidence of cytotoxicity. AP activity was unchanged, while CA II and TRAP levels trended upward without reaching statistical significance, suggesting mild osteoclast activation. In the liver compartment, FFAs suppressed CYP27A1/CYP2R1 expression and decreased 25(OH)D₃ formation. Collectively, these findings establish a link between hepatic dysfunction and impaired bone mineralization.

Discussion and conclusions: In conclusion, this human organ-crosstalk model reveals that the MASLD-conditioned liver secretome compromises bone mineral deposition without immediate biomechanical deterioration, mirroring the clinical trajectory toward osteodystrophy and heightened fracture risk. While the model provides mechanistic insight, it does not fully capture the systemic complexity of MASLD in vivo. This platform supports preclinical evaluation of interventions to preserve bone mineral density and mechanical properties in the context of metabolic liver disease.