Abstract
Bone tissue engineering has the intent to grow bone copies in the laboratory that could be used either for bone regeneration or as model systems to study bone physiology and pathology. Bone marrow- or adipose derived derived mesenchymal stromal cells are commonly used as they have been shown to be capable to differentiate into osteoblasts and depositing a calcium phosphate rich extracellular matrix. However, real bone is more than that: there are commonly three cell types described that are essential contributors to the tissue's native function: osteoblasts, osteocytes and osteoclasts. While all three cell types are being investigated separately, co-cultures of them including their precursors and inactive forms still provide a huge challenge these days, both in terms of culturing and (quantitative) evaluation. In addition, the matrix deposited by the osteoblasts in vitro is still far from bone's hierarchical organization in vivo that contributes to bone's impressive mechanical properties. Using a large set of microscopic tools (micro-computed tomography, SEM, 3D FIB/SEM, TEM and fluorescence), combined with spectroscopic (FTIR) and molecular tools (qPCR) we show that our 3D model system develops the main features of bone by human stromal cells differentiating first into osteoblasts who further embed themselves to become osteocytes. In their right environment and when stimulated mechanically, the cells are embedded within a collagenous matrix which is mineralized with carbonated hydroxyapatite. While this system still needs the addition of osteoclasts to represent ‘real’ bone, it allows to study the interaction between osteoblasts and osteocytes and to invest parameters contributing to collagen mineralization in high resolution and cryogenic conditions.
