| Objective The co-culture model has become a necessary condition for obtaining a better understanding of bone homeostasis, which depends on the balance between osteoblasts (OBs) and osteoclasts (OCs). This study aimed to establish a co-culture model that included OB, bone marrow mesenchymal stem cells (BMSCs), and OC. Methods OBs were isolated from the skull of suckling mice. BMSCs were isolated from the tibia and femur of mice. OCs were differentiated from BMSCs. The three types of cells, OBs, BMSCs, and OCs, were inoculated together in a special culture medium at a ratio of 1:1:1, 2:1:1, 4:1:1, 1:1:2, 1:1:4, 1:1:8, and 1:1:16 for co-culture. TRAP staining was used to identify OCs, ALP staining was used to identify OBs, and Alizarin red staining was used to identify BMSCs, on the 7th, 8th, and 9th days of the co-culture. ALP and TRAP activities were detected using ELISA. The ALP/OB and TRAP/OC ratios were calculated. Lactate dehydrogenase (LDH) was used to analyze cell activity in the co-culture system. ELISA was used to analyze the ratio of osteoprotegerin (OPG)/nuclear factor receptor activator (NF)-kB ligand (RANKL) levels in the co-culture system. Results On the 7th, 8th, and 9th day of Scheme six, when the OBs:BMSCs:OCs ratio was 1:1:8, the co-culture system had the highest number of cells, and the ratio of OPG/RANKL, ALP/OBs, and TRAP/OCs tended to stabilize. Conclusion The OB-BMSCs-OC co-culture bone homeostasis model is successfully established in vitro. |