Optimization of culture conditions for osteogenically-induced mesenchymal stem cells in β-tricalcium phosphate ceramics with large interconnected channels.
The aim of this study was to optimize culture conditions for human mesenchymal stem cells (hMSCs) in β-tricalcium phosphate ceramics with large interconnected channels. Fully interconnected macrochannels comprising pore diameters of 750 µm and 1400 µm were inserted into microporous β-tricalcium phosphate (β-TCP) scaffolds by milling. Human bone marrow-derived MSCs were seeded into the scaffolds and cultivated for up to 3 weeks in both static and perfusion culture in the presence of osteogenic supplements (dexamethasone, β-glycerophosphate, ascorbate). It was confirmed by scanning electron microscopic investigations and histological staining that the perfusion culture resulted in uniform distribution of cells inside the whole channel network, whereas the statically cultivated cells were primarily found at the surface of the ceramic samples. It was also determined that perfusion with standard medium containing 10% fetal calf serum (FCS) led to a strong increase (seven-fold) of cell numbers compared with static cultivation observed after 3 weeks. Perfusion with low-serum medium (2% FCS) resulted in moderate proliferation rates which were comparable to those achieved in static culture, although the specific alkaline phosphatase (ALP) activity increased by a factor of more than 3 compared to static cultivation. Gene expression analysis of the ALP gene also revealed higher levels of ALP mRNA in low-serum perfused samples compared to statically cultivated constructs. In contrast, gene expression of the late osteogenic marker bone sialoprotein II (BSPII) was decreased for perfused samples compared to statically cultivated samples.
Bernhardt
Anne
A
Max Bergmann Centre of Biomaterials and Institute for Materials Science, Technische Universität Dresden, Germany. abernhardt@nano.tu-dresden.de
Lode
Anja
A
Peters
Fabian
F
Gelinsky
Michael
M
D000328:Adult; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002130:Calcium Phosphates / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002516:Ceramics / Q000494:pharmacology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D058574:Integrin-Binding Sialoprotein / Q000235:genetics / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D010012:Osteogenesis / Q000187:drug effects*; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D010477:Perfusion; D054457:Tissue Scaffolds / Q000737:chemistry
D002130:Calcium Phosphates; D016895:Culture Media, Serum-Free; D058574:Integrin-Binding Sialoprotein; D015676:Osteonectin; C485817:beta-tricalcium phosphate; D053495:Osteopontin; D000469:Alkaline Phosphatase