Advancing Biomaterials Evaluation: A Human Quadruple Bone Cell Culture Reveals Molybdenum-Driven Pro-Osteogenic and...

The increasing prevalence of bone-related diseases and the desire to improve patient outcomes are driving research into bone replacement materials that overcome the limits of current bone substitutes. Molybdenum (Mo) is a promising candidate as an implant and degradable bone replacement material because it combines three key properties: mechanical strength, biocompatibility, and resorbability. However, little is known about the cellular mechanisms induced by Mo on bone regeneration. This study exposed a complex in vitro bone model as quadruple culture with primary human osteoblasts, osteocytes, osteoclasts, and endothelial cells, to Mo powder extracts to understand cell-material interactions in a multicellular system. Extracts with a final concentration of 1 mM Mo in quadruple cultures induced osteogenic differentiation by stimulation of ALPL gene expression and ALP activity, BMP-2 and BGLAP gene expression, as well as enhanced calcium deposition of osteoblasts. Furthermore, VEGFA expression of osteoblasts increased significantly and network formation of HUVEC with stimulated VWF expression occurred. However, CD31 (PECAM1) expression and endothelial network density were reduced, indicating a complex, mixed angiogenic response. In contrast, Mo inhibited osteoclast formation and slowed down osteocyte differentiation, reducing SOST, DMP1, and MEPE gene expression. Additionally, the RANKL (TNFSF11)/OPG (TNFRSF11B) ratio of osteocytes was shifted toward OPG after Mo treatment. Cellular effects are most likely caused by the presence of molybdate anions. In summary, Mo extracts stimulated early bone healing factors involved in osteogenesis, vascularization, and mineralization, while osteoclastogenesis was inhibited. These dual effects in vitro provide mechanistic evidence supporting the potential of Mo as a growth factor-free bone replacement material and establish a cellular foundation for further preclinical development.