Investigating the Effects of Polytrauma and Acute Ethanol Intoxication on the Bone-Liver Crosstalk In Vitro

Severe trauma represents a major clinical challenge, often involving multiple organs and systemic inflammation. Among the most commonly affected systems, the liver is a frequent target of remote organ damage, while the bone is the most frequently injured organ in trauma. Recent evidence has highlighted the existence of a crosstalk between liver and bone, especially relevant in chronic inflammatory conditions. However, the extent to which these organs influence each other in trauma settings and how acute ethanol intoxication (AEI), a frequent comorbidity in trauma patients, modulates this interaction remains largely unexplored. To address this gap, a 3D bone-liver co-culture model was established, with pre-osteoblasts (SCP-1) and pre-osteoclasts (THP-1) seeded on platelet-rich plasma (PRP) scaffolds, combined with HepG2 spheroids. The model was exposed to 200 mM ethanol to mimic AEI and to a polytrauma cocktail (PTC) to reproduce systemic post-traumatic inflammation. Cellular viability, inflammatory responses, organ-specific analysis, and inter-organ communication were assessed at different days post-stimulation, providing an integrated view on how polytrauma- and AEI-like stimuli shape the interaction between liver and bone. The model proved to be robust, as co-culture preserved liver and bone viability. Stimulation with PTC induced a strong pro-inflammatory profile, characterized by increased interleukin 6 (IL-6) and interleukin 1β (IL-1β) levels, while also promoting a compensatory anti-inflammatory response, resembling the post-traumatic immune profile observed in polytrauma patients. Additional ethanol stimulation transiently attenuated the pro-inflammatory response and enhanced the anti-inflammatory mediators transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10), suggesting short-term immunomodulation. Beyond inflammation, PTC and AEI influenced osteoblast and osteoclast activity and altered hepatic function in a stimulus- and time-dependent manner. It is noteworthy that although ethanol reduced the inflammation caused by PTC, it did not restore bone-liver crosstalk but rather caused further dysregulation. Analysis of key mediators of bone-liver crosstalk revealed that AEI strongly increased fibroblast growth factor 23 (FGF23) secretion, whereas PTC also induced transient elevation of fibroblast growth factor 21 (FGF21) and osteopontin (OPN). These findings suggest that both polytrauma and AEI disrupt bone-liver crosstalk with different mechanisms of action, potentially influencing regeneration and organ dysfunction after trauma. Overall, this work provides the first in vitro evidence for a direct interaction between liver and bone under trauma-like conditions and AEI. While findings are limited by in vitro constraints, this study establishes a model that can be further exploited to clarify molecular mechanisms governing bone-liver crosstalk in the context of polytrauma.