Smoking is the most significant and modifiable risk factor for a range of conditions, including cancer, cardiovascular and respiratory diseases. Furthermore, it significantly reduces bone mass and increases the risk of fragility fractures due to its detrimental effects on bone metabolism and regeneration. Moreover, smoking is a known cause of chronic systemic inflammation, leading to an imbalance of cytokines. Comprehending the pathological mechanisms that underlie cytokine production and its impact on post-surgical healing is essential to prevent post-surgical complications. The present study recruited a total of 1144 patients, including 897 patients, among them non-smokers (N = 413), current smokers (N = 201) and ex-smokers (N = 283). Human proteome profiler arrays were used to screen for smoking-dependent differences in the serum cytokine and protein profiles, after matching samples for age, gender, body mass index (BMI), alcohol use, and diabetes risk. Cytokines and immune checkpoint proteins such as CD28, B7-1, MIG, TGFb2 and IL-1a/b were quantified by ELISA. Our study demonstrates a comprehensive understanding of the relationship between smoking, the development of complications, the systemic immune inflammation index (SII) and cytokine/protein levels. We found that a comparison of non-smokers, former smokers, and active smokers in our study cohort did not exhibit significantly altered cytokine and protein serum levels although other studies reported differences between smokers and non-smokers. We were unable to identify single blood circulating markers that could predict complications in smokers after trauma. However, we found the ratio of women to men to be inverted between non-smokers and active smokers resulting in a ratio of 0.62 in smokers. Furthermore, we demonstrate a higher complication rate, longer hospitalizations and elevated SII values among smokers, indicating an involvement of the immune system.

Drug toxicity is an important cause of chronic liver damage, which in the long term can lead to impaired bone homeostasis through an imbalance in the liver-bone axis. For instance, non-steroidal anti-inflammatory drugs (e.g., diclofenac), which are commonly used to control pain during orthopaedic interventions, are known to reduce bone quality and are the most prevalent causes of drug-induced liver damage. Therefore, we used human cell lines to produce a stable, reproducible, and reliable in vitro liver-bone co-culture model, which mimics the impaired bone homeostasis seen after diclofenac intake in vivo. To provide the best cell culture conditions for the two systems, we tested the effects of supplements contained in liver and bone cell culture medium on liver and bone cell lines, respectively. Additionally, different ratios of culture medium combinations on bone cell scaffolds and liver spheroids’ viability and function were also analysed. Then, liver spheroids and bone scaffolds were daily exposed to 3–6 µM diclofenac alone or in co-culture to compare and evaluate its effect on the liver and bone system. Our results demonstrated that a 50:50 liver:bone medium combination maintains the function of liver spheroids and bone scaffolds for up to 21 days. Osteoclast-like cell activity was significantly upregulated after chronic exposure to diclofenac only in bone scaffolds co-cultured with liver spheroids. Consequently, the mineral content and stiffness of bone scaffolds treated with diclofenac in co-culture with liver spheroids were significantly reduced. Interestingly, our results show that the increase in osteoclastic activity in the system is not related to the main product of diclofenac metabolism. However, osteoclast activation correlated with the increase in oxidative stress and inflammation associated with chronic diclofenac exposure. In summary, we established a long-term stable liver-bone system that represents the interaction between the two organs, meanwhile, it is also an outstanding model for studying the toxicity of drugs on bone homeostasis.

Hepatic osteodystrophy (HOD) is a frequent complication of chronic liver disease, marked by impaired osteogenesis and elevated fracture risk, particularly under sustained alcohol exposure. Bone morphogenetic proteins (BMPs), which play a crucial role in maintaining bone homeostasis, are dysregulated in alcoholic liver disease. Specifically, decreased BMP2 and increased BMP13 have been linked to impaired osteogenesis and cartilage-like shifts in bone progenitors. A human in vitro system that recapitulates this hepatic BMP imbalance is needed to dissect mechanisms and identify targets. To address this, we established a long-term human three-dimensional liver–bone co-culture model that integrates hepatocytes (HepaRG), hepatic stellate cells (LX-2), and human umbilical vein endothelial cells (HUVECs) with bone scaffolds seeded with osteoblast precursors (SCP-1) and osteoclast precursors (THP-1). This study aimed to characterize the effects of chronic 50 mM alcohol exposure on hepatic fibrogenic activation and BMP ligand secretion, and to investigate the associated BMP-responsive signaling involved in bone cell lineage differentiation and functional activity. The results demonstrated alcohol-induced hepatic CYP2E1 activation and fibrogenic remodeling with EMT signatures, as well as a decrease in BMP2 and an increase in BMP13, without affecting BMP9. Liver-derived factors activated both canonical and non-canonical BMP signaling in bone progenitors, reduced osteoblast activity and mineralization, preserved osteoclast TRAP activity, and shifted the lineage toward chondrogenesis (SOX9↑, RUNX2↓). Notably, this BMP profile and skeletal phenotype reflect clinical observations in chronic liver disease, indicating that the model recapitulates key in vivo pathological features. This human liver micro-organoid co-culture reproduces alcohol-induced hepatic BMP dysregulation and downstream bone defects, offering an organoid-centric, microengineered platform for mechanistic studies and BMP-targeted therapeutic screening in HOD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic condition associated with compromised bone integrity. 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 µM 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 CYP2R1 and CYP27A1 . Transcriptomic profiling of human MASLD liver biopsies validated these molecular changes. To evaluate MASLD’s 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.

Metabolic dysfunction-associated steatotic liver disease (MASLD) involves metabolic, inflammatory, and fibrotic processes which are not fully reproducible in vitro. However, MASLD is increasingly investigated using human-relevant in vitro models. This study models the steatosis component using a harmonized free fatty acid (FFA; oleic/palmitic acid) exposure across four human hepatic platforms: 2D primary human hepatocyte (PHH) suspension cultures, 2.5D PHH sandwich cultures, 3D PHH spheroids, and HepaRG/LX-2/HUVEC micro-organoids. Steatosis and function were assessed via lipid imaging, albumin/LDH release, mRNA profiling of drug metabolizing enzymes (DMEs) and transporters using Multiplex technology, and LC–MS/MS-based CYP1A2/CYP2C9 activity. Untreated cultures served as controls; FFA-treated cultures were designated “FFA-induced steatotic”, reflecting lipid accumulation rather than full MASLD. PHH suspensions, classified a priori as putatively healthy or MASH based on medical records of the donor, showed rapid functional decline and substantial donor variability. In contrast, 2.5D sandwiches, 3D spheroids, and micro-organoids remained stable for 10–21 days and developed robust FFA-induced steatotic. Spheroids and micro-organoids maintained CYP activity and exhibited distinct metabolic responses; micro-organoids partially recovered after FFA-stimulus withdrawal. Cross-platform differences arise from both biological responses and model-intrinsic characteristics. 3D spheroids and micro-organoids were most suitable for long-term metabolic remodeling studies, while 2.5D sandwich cultures provided a practical system for transporter and enzyme induction research. Despite functional instability, 2D suspensions retained value for rapid short-term screening. These findings support use-case-driven selection of hepatic in vitro models for toxicology and ADME applications under FFA-induced steatosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern associated with various metabolic disorders and a high prevalence of vitamin D deficiency. Current animal models face limitations in accurately mimicking human metabolic pathways and are constrained by ethical considerations. This study aimed to develop an in vitro MASLD-induced liver micro-organoid model to investigate the impact of MASLD on vitamin D metabolism. The model incorporates HepaRG cells as hepatocytes, LX-2 cells as stellate cells, and human umbilical vein endothelial cells (HUVECs). Fatty liver characteristics were induced by treating the micro-organoids with palmitic/oleic acids. fatty acid accumulation was quantified and visualized using Bodipy staining. The expression of genes involved in drug and vitamin D metabolism was evaluated by RT-PCR. Dot blot analysis assessed secreted proteins indicative of liver damage. ELISA was used to examine vitamin D metabolism. The results confirmed the successful induction of MASLD, evidenced by altered cytochrome P450 gene expression, elevated markers of liver damage, and reduced vitamin D metabolism. These findings align with clinical studies and RNA-sequencing data from liver biopsies of MASLD patients. This innovative micro-organoid platform offers a robust tool for studying the molecular mechanisms of MASLD and represents a promising alternative to animal experiments.