Effect of donor characteristics, technique of harvesting and in vitro processing on culturing of human marrow stroma cells for tissue engineered growth of bone.

The aim of this study was to assess the effect of donor characteristics and the technique of harvesting and in vitro processing on the efficacy of culturing of human mesenchymal stem cells (hMSCs) for tissue engineered growth of bone. Cultures of hMSCs were derived from iliac crest bone marrow aspirates (21 donors, age 11-76) and from cancellous bone grafting material (32 donors, age 13-84). Age had no significant effect on the ability to isolate and culture hMSCs, although the failure rate was 55.6% in donors beyond the age of 60, while it varied between 14.3% and 22.2% in donors under 60 years of age. Male and female donors had comparable failure rates (27.3% and 28.6%, respectively). Culturing of hMSCs was successful in 90.4% of marrow aspirates from 21 donors and in 62.5% of cancellous bone specimens from 35 donors. This difference was statistically significant (P=0.023). Regression analysis confirmed that at simultaneous testing of the three variables, only the source of cells significantly affected the result (P=0.043). Morphological evaluation of the unfractionated primary population showed a change in cell shape of the adherent cells from a triangular into thin spindle-shaped elongated form, which remains until confluence. When the cultures were exposed to osteoinductive medium, various morphotypes expressing different levels of alkaline phosphatase and secreting different amounts of mineral were evident. Morphology of marrow stroma cells (MSCs) from marrow aspirates was not different from MSCs derived from cancellous bone specimens. Expression of osteogenic markers in MSCs as shown by PCR as well, did not differ between the two sources. It is concluded that marrow aspirates and cancellous bone specimens produce comparable populations of MSCs. However, bone marrow aspirates from donors under the age of 60 years rather than cancellous bone chips are favourable for isolation and expansion of hMSCs for tissue engineered growth of bone.