A Three-Dimensional in vitro Bone Model – Triple Culture of Primary Human Osteoblasts, Osteoclasts and Osteocytes

In vitro evaluation of bone graft materials is generally performed by analysing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give more specific first information on the performance of those materials after being implanted. In the present study, a 3D co-culture model was established comprising primary human osteoblasts, osteoclasts and osteocytes. Osteocytes were differentiated from osteoblasts embedded in collagen gels and were cultivated with osteoblast and osteoclasts seeded in patterns on a porous membrane. This experimental setup allowed the exchange of soluble factors between the cells as well as separation of the different cell types for final analysis. After 7 days of co-culture the three cell species showed their typical morphology and gene expression of typical markers like ALPL, BSPII, BLGAP, E11, PHEX, MEPE, RANKL, ACP5, CAII and CTSK. Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP ( ACP5 ) and CAII expression and decreased TRAP activity. ALP and BSPII expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 ( PDPN ) was unchanged, however, osteocalcin ( BGLAP ) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.