Abstract
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 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.
Highlights
After osteocytic differentiation for 2 weeks, osteoblasts and osteoclasts were seeded to the apical side of the porous membrane and the triple cultures were maintained for another 7 days again under low serum conditions
Osteoclasts and osteoblasts can be combined in triple cultures in vitro, maintaining their typical morphology
Typical markers for osteoblasts, osteoclasts and osteocytes were expressed during triple culture of the three cell types and were detected on mRNA and, in some cases, on protein level
Summary
Bone is a complex dynamic tissue being permanently remodeled through bone forming osteoblasts and bone resorbing osteoclasts orchestrated by osteocytes. In vitro testing of novel bone graft materials is restricted to biocompatibility analysis using osteoblasts or osteoblast precursors It has become clear, that osteoclasts [1] and osteocytes [2] play an important role in bone healing and should be considered for in vitro investigations of bone graft materials. Several approaches have been published combining osteoblasts and osteoclasts in different in vitro co-culture systems [7,8]. The intention behind these coculture approaches is to better understand the interaction of osteoblasts and osteoclasts during bone remodeling or to test drugs and biomaterials/scaffolds in an environment, which is closer to human bone. This is mainly because of the increasing complexity of those systems, as each cell species requires special media composition [21] and environment
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
