Castro Vázquez, DavidGarcía López, IvanWirsig, KatharinaArribas Castaño, PaulaPérez Gomáriz, Rosa MaríaMartínez Mora, María Del CarmenJuarranz Moratilla, YasminaBernhardt, AnneCarrión Caballo, Mar2026-05-082026-05-082026-04-19Castro-Vazquez, D., García-Lopez, I., Wirsig, K., Arribas-Castaño, P., Gomariz, R. P., Martínez, C., Juarranz, Y., Bernhardt, A., & Carrion, M. (2026). Osteoprotective effects of the neuropeptide VIP: insights from triple cultures of human osteocytes, osteoblasts, and osteoclasts. Translational Research, 292, 40–51. https://doi.org/10.1016/j.trsl.2026.04.0041931-524410.1016/j.trsl.2026.04.004https://hdl.handle.net/20.500.14352/136623Acknowledgements We thank the Genomics and Fluorescence Microscopy Centres of Complutense University for the use of their facilities. We are grateful to all donors and the collaborating clinicians for their participation in this study. We also appreciate the assistance of Isabel Montero, for her assistance with histology techniques. Funding This research was funded by grants RD24/0007/0014 and RD21/0002/0004 from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), co-funded by European regional development fund (ERDF) and by the UCM grants PR12/24-31568 and PR12/24-31572. DC-V was awarded with an EMBO Scientific Exchange Grant (SEG 10287). KW was awarded a scholarship funded by and in cooperation with the Saxon Ministry for Higher Education and Arts and the Studentenwerk Dresden.Bone remodelling is a dynamic process of osteoblastic bone formation and osteoclastic bone resorption, regulated by local, paracrine and endocrine factors, in which osteocytes act as orchestrators of bone homeostasis. Among these regulatory factors, the neuropeptide VIP (vasoactive intestinal peptide) has demonstrated osteoprotective effects by inhibiting osteoclastogenesis and promoting osteoblast differentiation. However, its potential role in osteocyte biology remains unexplored. In this study, we investigated the effect of VIP on the differentiation of primary human osteocytes. We describe for the first time the expression of VIP and its receptors during in vitro osteocyte differentiation. Our results show that VIP promotes osteocytogenesis, accompanied by a reduction in the RANKL/OPG ratio, thereby supporting its role as an anti-osteoclastogenic factor. To better mimic the complexity of bone tissue, we performed triple co-cultures of osteoblasts and simultaneously differentiating osteocytes and osteoclasts, in the presence or absence of VIP. Our results confirmed that VIP supports the osteoblast-to-osteocyte transition and promotes an osteoprotective phenotype, characterized by a reduced RANKL/OPG ratio and decreased SOST expression. The downregulation of sclerostin may attenuate osteocyte-mediated inhibitory signalling toward osteoblasts and, in turn, contribute to the reduction of the osteoblastic RANKL/OPG ratio. Collectively, our findings reinforce the anti-osteoclastogenic actions of VIP, supporting its role as a potential osteoprotective factor.engAttribution-NonCommercial 4.0 Internationalhttp://creativecommons.org/licenses/by-nc/4.0/journal article1878-1810https://doi.org/10.1016/j.trsl.2026.04.00442013997open access577.1:611.71In vitro bone modelOsteocyte differentiationRANKL/OPG ratioSclerostinVIPBiología celular (Biología)Bioquímica (Biología)FisiologíaSistema musculoesquelético2407 Biología Celular2410.10 Fisiología Humana2403 Bioquímica