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Aims: The aim of the present report was to evaluate current methods of in vitro analysis of three-dimensional (3D) scaffolds seeded with human bone marrow stromal cells (hBMSCs) from six bone marrow aspirates for tissue engineered growth of bone. Methods: A series of experiments was conducted to compare methods of cell expansion and to validate analysis of proliferation and differentiation of hBMSCs in long term cultures of up to 40 days in 3D scaffolds of calcium carbonate (CaCO3) and mineralized collagen. Proliferation within the seeded scaffolds was monitored using cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), neutral red (NR) and DNA fluorescence assays and compared with empty controls. Differentiation was assessed by means of ELISA for osteocalcin (OC) and real time PCR for OC and collagen I (Coll I). Results: The results showed that the scaffold differed in seeding efficacy (CaCO3: 53.3%, min. Coll.: 83.3%). The precise identification of the number of cells in biomaterials by MTT, NR and DNA assays was problematic, as MTT and NR assay overestimated the number of cells, whereas DNA assay grossly underestimated the number of cells on the scaffolds. Monitoring of changes over time may be biased by unspecific material-dependent background activity that has to be taken into account. Identification of osteogenic differentiation is not reliable by identifying osteogenic markers such as OC in the supernatant but has to be done on the transcriptional level. Conclusions: It is concluded that monitoring of in vitro procedures for the construction of biohybrid scaffolds requires more emphasis in order to make the cell based approach a reliable treatment option in tissue engineering.