There is evidence that bone health is closely linked to a functioning circadian rhythm. Most of the evidence comes from mice, which may exhibit some species-specific differences from humans due to their nocturnal lifestyle. To address the current lack of human model systems, the present study aimed to develop an in vitro model system that can represent diurnal changes in bone metabolism. The model is based on co-cultured SCP-1 and THP-1 cells that serve as osteoblast and osteoclast precursors, respectively. Diurnal effects were induced by replacing the FCS in the differentiation medium with human serum pools (HSPs) obtained in the morning, noon, or evening. The model system was tested for cell viability, gene expression, and osteoblast and osteoclast function. The replacement of the FCS with the HSPs increased viability and induced expression changes in circadian clock genes in the model system. Resulting alterations in osteoblast and osteoclast function led to a gradual increase in mineral density and stiffness when 3D co-cultures were differentiated in the presence of the HSPs collected in the morning, noon, or evening, respectively. Here, we present for the first time an in vitro model that can present diurnal changes in bone metabolism in the form of a snapshot. With the simple use of HSPs, this model can be used as a platform technique to investigate bone function in various situations, taking into account the time of day.

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.

Circadian rhythms are fundamental regulators of skeletal homeostasis, coordinating osteoblast and osteoclast activity through tightly controlled temporal programs. Disruption of these rhythms, whether through environmental misalignment or genetic perturbation of core clock components, alters bone formation, enhances resorption, and contributes to skeletal fragility. This review synthesizes current knowledge on circadian regulation of bone biology across in vivo, ex vivo, and in vitro model systems, highlighting how each platform reveals distinct aspects of rhythmic gene expression, cellular function, and tissue-level remodeling. We critically evaluate the strengths and limitations of these models, outline key controversies such as the interpretation of global clock-gene knockouts, and discuss the emerging relevance of human-derived systems including iPSC-based models, organoids, and microphysiological “bone-on-chip” platforms. Integrative approaches that combine multiple model systems provide the most reliable framework for understanding circadian control of bone and for identifying targets for chronotherapeutic intervention. Advancing human-relevant models and refining temporal experimental design will be essential for translating circadian biology into clinical strategies for metabolic bone diseases.

Circadian rhythms are fundamental regulators of bone remodeling, orchestrating the co-ordinated actions of osteoblasts, osteocytes, and osteoclasts. Recent studies have high-lighted how core clock genes, such as Bmal1, Clock, Per1/2, and Cry1/2, exhibit rhythmic expression in bone tissue and modulate key markers of bone formation and resorption. Disruptions in circadian regulations, whether caused by environmental factors or genetic alterations, have been linked to osteoporosis, impaired fracture healing, and increased risk of bone fragility. This review provides a comprehensive evaluation of current experi-mental models used to study circadian regulation in skeletal biology, including in vivo, ex vivo, and in vitro approaches. We summarize their respective advantages and limitations and outline the molecular and cellular markers employed to assess circadian function in bone cells. We also discuss the emerging co-culture models and human-relevant plat-forms, for their potential to bridge the gaps between mechanistic research and transla-tional applications. By comparing model characteristics and highlighting integrated re-search strategies, this review aims to advance circadian bone research and inform future investigations into potential temporal aspects of skeletal health.