PRIMARY HUMAN CELL-BASED IN VITRO BONE MODELS

In vitro experiments analysing the impact of bioactive molecules and ions on tissue regeneration are often restricted to one cell species. In case of bone tissue, experiments are mostly performed with osteoblasts or their progenitor cells. To mimic the situation in native bone tissue, in vitro bone models involving all major bone cell species - osteoblasts (OB), osteoclasts (OC) and osteocytes (OCY)- are beneficial. However, the simultaneous cultivation and differentiation of cells with discrete requirements concerning media composition is challenging. Furthermore, primary OCY are hard to isolate from bone tissue and cannot be expanded in vitro due to their post-mitotic state. The generation of OCY-containing in vitro bone models therefore mainly relied on murine OCY cell lines. Another challenge is the separation of the different (human) cell species for analysis. We have been working a couple of years on the creation of OCY-containing in vitro bone models comprising solely primary human cells. Starting with co-cultures of OCY and other bone cells, we went further to basic and advanced triple cultures of bone cells. We identified a suitable 3D co-culture setup, allowing simultaneous differentiation of OB, OC and OCY. To reach this goal we optimized the osteocytic differentiation of human osteoblasts in 3D collagen hydrogels finally obtaining cells with typical osteocytic morphology and stable gene expression of late OCY markers like sclerostin and dentin matrix protein protein 1. Furthermore, the differentiation of OC from their precursor cells was accomplished in the presence of the other cell species and even under sub optimal conditions concerning serum concentration in the common co-culture medium. Since OB and OC are separated from OCY by a porous membrane, signal molecules can be exchanged by the different cell species and cell species can be analysed separately after co-cultivation. After having created a functional in vitro triple culture model, we implemented human endothelial cells (EC) to perform quadruple cultures, since vascularization is crucial for bone regeneration. While the external addition of receptor activator of NF-kB Ligand was not necessary to obtain osteoclastic differentiation in triple cultures, the vascular endothelial growth factor (VEGF) concentrations, produced by the OB were not sufficient to induce tube formation in EC. However, with external addition of VEGF we are able to generate quadruple cultures of human OB, OC, OCY and EC, which displayed all typical markers as well as the formation of tube-like structures from EC.