In vitro evaluation of bone graft materials is generally performed by analyzing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give specific first information on the performance of those materials. 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 paracrine signaling 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 and . Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP ( ) and expression and decreased TRAP activity. and expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 ( ) was unchanged; however, osteocalcin ( ) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.