Dilutional and centrifugation factors of lower density gradient separation media on osteoprogenitor cells separated from concentrated bone marrow aspirate

Human bone marrow contains osteoprogenitors capable of differentiating into osteoblasts. Ficoll media is widely used in the isolation of human mesenchymal stem cells. The aim of the study was to compare the effectiveness of the two most commonly used centrifugation protocols for Ficoll-Paque PREMIUM 1.073 lower density gradient centrifugation media in isolating osteoprogenitor cells from aspirated bone marrow. Bone marrow was aspirated from anterior iliac crests, equally divided, with a centrifugal force of 400 g and 1:1 dilution was used for protocol A, while a centrifugal force of 1000 g was used for protocol B after three dilution times with the buffer. After isolation, 106 cells were cultured in 6-well plates. The relative efficacies of each protocol were compared by their ability to produce alkaline phosphatase positive colony forming units-fibroblasts (CFU-F). The cultured monolayers were accessed for osteogenic differentiation and mineralization ability by cytochemical staining with alkaline phosphatase, von Kossa and alizarin red S staining as well as quantitative measurements of alkaline phosphatase activity were taken. The percentage of positive
stromal osteoprogenitor cells marker STRO-1 expression was detected by Flow cytometry. The average numbers of isolated bone marrow mononuclear cells were 6.87x107±4.84x107 and 4.70x107±3.93x107 respectively, which were statistically different. The mean±SD number of alkaline phosphatase positive CFU-Fs in protocol A was 53±6/106 mononuclear cells, which was not significantly different from protocol B (51±8/106 mononuclear cells). Alkaline phosphatase activity did not show any difference between protocols, however, significant increase in enzyme activity was
detected comparing both protocols between day 7 and day 10. Formation of mineralized nodules in osteogenic culture was confirmed by positive alizarin red S and von Kossa staining in both protocols. Assessment of matrix mineralization by optical density measurement revealed increased intensity of alizarin red S on each time interval, however, they did not differ significantly between the protocols. Positive expression of STRO-1 (around 10%) was detected from both protocols. There was no detectable difference between osteogenic differentiation and mineralization ability between protocols. Both protocols produced good results since they contained similar quantity of STRO-1 positive osteoprogenitors, however, using lower centrifugal force produced recovery of more mononuclear cells.