Crosstalk between adipocytes and osteoblasts in regulating bone differentiation.

Bone homeostasis is strongly influenced by adipose tissue, since both cell types coexist in the bone marrow and communicate through soluble mediators. This interaction may create an osteo-adipogenic imbalance that favors adipogenesis over osteogenesis, contributing to conditions such as osteoporosis, often associated with increased marrow adiposity. In this study, we examined the effects of adipocyte-conditioned medium (CM) on osteoblasts. Although CM did not induce cytotoxicity and even increased cell viability, it markedly impaired osteoblastic function. CM-treated cells showed an absence of calcium deposition and significantly reduced alkaline phosphatase (ALP) activity, indicating a blockade in differentiation. Proteomic analysis revealed downregulation of key extracellular matrix proteins, including fibronectin, asporin, and periostin, which are essential for matrix organization and mineralization. Conversely, proteins related to inflammation and oxidative stress, such as GAPDH, vimentin, and the acidic 60S ribosomal protein, were upregulated. STRING network analysis identified clusters enriched in stress response, protein folding, and cytoskeletal remodeling, while matrix-associated proteins appeared fragmented and underrepresented. Long exposure to late CM showed further enrichment of ribosomal and translational proteins, suggesting a shift toward stress adaptation rather than matrix production. Hemoglobin overexpression and reduced histone isoforms indicated NF-κB activation, oxidative stress, and adverse epigenetic alterations. Overall, these findings indicate that the adipocyte secretome creates a microenvironment detrimental to osteoblast differentiation, marked by impaired mineralization, inflammatory signaling, oxidative stress, and cytoskeletal and epigenetic dysfunction. These insights highlight potential molecular pathways that may be targeted to restore osteogenesis under pathological conditions.