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Objectives and questions: Hepatic osteodystrophy (HOD) is a frequent but underdiagnosed complication of chronic liver disease that compromises bone microarchitecture and increases fracture risk. Alcohol-related liver disease is one of the leading causes of cirrhosis in Europe and North America, yet the impact of liver dysfunction on skeletal homeostasis via the liver–bone axis remains poorly understood. A major limitation has been the lack of physiologically relevant human in vitro models that capture long-term liver–bone crosstalk and bone morphogenetic protein (BMP) dysregulation characteristic of HOD.

Material and methods: We established a long-term human 3D liver–bone co-culture system that integrates HepaRG hepatocytes, LX-2 hepatic stellate cells, and HUVECs as liver micro-organoids with platelet-rich plasma bone scaffolds seeded with osteoblast (SCP-1) and osteoclast (THP-1) precursors. After independent validation of liver and bone compartments, the combined system was exposed to 50 mM alcohol daily for up to 28 days to mimic chronic excessive alcohol intake. We assessed liver viability and function (resazurin, UGT, CYP activities), fibrogenic markers (CYP2E1, FAPα, TGF-β1, EMT markers), BMP ligand secretion (BMP2, BMP9, BMP13), BMP pathway activation in bone progenitors (p-Smad1/5, p-p38, luciferase BRE-reporter), lineage transcription factors (RUNX2, PPARγ, SOX9), and bone remodeling readouts (AP, TRAP, PINP, NTX, quantitative CT-based mineral content, and mechanical stiffness testing). Statistical analysis was performed using the Kruskal–Wallis test on 3 biological replicates, each with at least 2 technical replicates.

Results: Chronic alcohol exposure induced a fibrogenic liver phenotype with CYP2E1 upregulation, stellate cell activation, EMT-related gene expression (SNAIL1/2), matrix formation (↑PINP, ↓MMP9), and elevated TNAP, indicating liver damage. At the signaling level, alcohol selectively decreased hepatic BMP2 and increased BMP13, while BMP9 remained unchanged. Conditioned medium from alcohol-exposed liver micro-organoids activated both canonical (p-Smad1/5) and non-canonical (p-p38) BMP signaling in SCP-1 cells and increased BMP-responsive luciferase activity, demonstrating functional transmission of liver-derived cues to bone progenitors. This was associated with marked downregulation of RUNX2 and PPARγ and upregulation of SOX9, indicating a shift from osteogenic/adipogenic to chondrogenic lineage commitment. In the bone compartment, osteoblast AP activity and collagen synthesis (PINP) were significantly reduced, whereas osteoclast TRAP activity and NTX levels remained largely unchanged. These alterations resulted in reduced bone-scaffold mineral density and stiffness, recapitulating key skeletal features of alcohol-induced HOD.

Discussion and conclusions: This human 3D liver–bone co-culture mimics alcohol-induced BMP imbalance and downstream bone remodeling defects, providing an organoid-based platform to dissect liver–bone crosstalk and to screen BMP-targeted and hepatoprotective therapies for HOD.