Regulation of bone homeostasis by denosumab and genistein in a human quadruple culture bone model identifies osteocytes as important mediators.

Osteoporosis is a prevalent disease in the aging population worldwide. However, treatment options have drawbacks, such as increased bone turnover after denosumab discontinuation which is not yet fully understood. Therefore, this study investigates the effects of denosumab or genistein on a novel in vitro quadruple culture bone model, comprising primary human osteoblasts, osteocytes, osteoclasts, and endothelial cells. Denosumab, an antiresorptive monoclonal antibody, inhibited osteoclastogenesis but also altered osteocyte differentiation by reducing the gene expression and protein levels of the late osteocyte marker sclerostin, a WNT inhibitor. This explains the unexpected, induced osteoblast differentiation observed in the presence of denosumab in quadruple cultures. Genistein, a phytoestrogen, allowed for osteoclast formation but also stimulated osteogenesis alongside reduced sclerostin levels. Both drugs indirectly affected bone cells through cellular signaling, identifying osteocytes as important regulators. The quadruple culture model provides a promising platform for further drug testing, offering insights into cellular crosstalk and drug mechanisms in bone metabolism without the reliance on animal testing.