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Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM-MSCs) combined with calcium phosphate microparticles and enveloped in a platelet-rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose-derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM-MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM-MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM-MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un-aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM-MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose-derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.

Introduction Large inter-donor differences exist in human mesenchymal stem cell (hMSC) yield and the response of these cells to osteogenic stimuli. The source of these differences may be clinical differences in stem cell characteristics between individuals or the aspiration procedure itself.Methods From a total of 23 donors, we aimed to take 2 consecutive 10-mL aspirates from the same site in 17 donors and in 6 donors we aimed to take a 5-mL and a 20-mL aspirate. The aspiration was stopped either when the syringe was full or when no more bone marrow came through. Mononuclear cell yield (MNC), MSC yield, and differentiation capacity were analyzed for intra-donor and inter-donor variation. We analyzed the effect of the dilution with peripheral blood by drawing 20 mL at once.Results There was a high correlation between the first and second aspiration volumes, and aspirates with a volume of less than 8 mL showed a large variation in cellular yield. The second 10-mL aspirate, and also 20-mL aspirates, contained a lower concentration of nucleated cells and yielded lower numbers of mesenchymal stem cells. No effect of the aspiration procedure on the biological characteristics of the mesenchymal stem cells was seen.Conclusion We recommend collection volumes of bone marrow aspirates of at least 8 mL to reduce the risk of obtaining aspirates with low cell numbers. From the same site, a second aspiration or an aspirate of > 10 mL can be drawn without any loss of biological quality due to dilution with peripheral blood.

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