Abstract
Introduction: Sufficient quantity of osteogenic cells is an essential aspect for a successful cell therapy in the treatment of difficult bone fractures and defects. At present, this was achieved by culturing bone marrow and bone-derived cells in a relatively long duration. A large number of the non-adherent mesenchymal stem cells were discarded during medium change. We hypothesize that collecting the non-adherent cells and re-plating them may result in more osteogenic cells in the same duration of cell culture. The aim of this study was to investigate the possibility of enhancing number of osteogenic cells by collecting non-adherent cells in the pull-off media and to examine their osteognic potentials.
Methods: Mononuclear cells were isolated by density gradient centrifugation method from bone marrow washouts in the bone samples obtained from 5 patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with αMEM medium and 15% FCS. The first medium change was made at day 7 and every 3 days thereafter. For the first three times of medium change, the removed media were centrifuged at 250 g for 10 minutes and plated in a separate T-75 (first time change) and T-25 flask (for the 2nd and 3rd times change). The non-adherent cells from the second and the third puff-off flasks were also collected and plated in separate T-25 flasks. Thus, 1xT-75 flask and 4xT-25 flasks of non-adherent cells resulted from the original T-75 flask. The cells in all flasks were harvested at 21 days from the day when the original flask was set up. The total number of cells in all pull off flasks were counted and compared with that of the original T-75 flask. Rate of cell proliferation with or without osteogenic growth medium were also examined by MTT method for passage 1 of both cells types. Osteogenic differentiation was defined with immunocytochemistry of bone markers: ALP, type I collagen, Osteocalcin and cbfa1. It is planed that cells of passage 2 will be mixed with HA powders and to be implanted into the SCID mice to examine the in-vivo osteogenic potential of these cells.
Results: Mesenchymal stem cells (MSCs) derived from the non-adherent population of human bone marrow culture have demonstrated having similar cell proliferation and differentiation potential in vitro, when compared to the MSCs derived from the adherent cell population. These cells expressed bone markers such as: ALP, type I collagen, osteocalcin and cbfa1. When the non-adherent cells were collected and cultured accumulatively, the total number of MSCs was increased to an average of 39.7% (36.6%–42.9%), compared to the number of cells obtained from the original T-75 flask.
Conclusions: Collecting the non-adherent cell population in the bone marrow culture appeared to result in more MSCs. This harvesting method may be used as a non-invasive way for enhancing MSC numbers in a given period of time. Further in vitro and in vivo studies of these MSCs of non-adherent origins may provide information for optimizing cell culture protocols for rapid expanding the osteogenic cells in vitro. This will facilitate the clinical applications of human osteogenic cell therapy.
Correspondence should be addressed to Mr Carlos Wigderowitz, Honorary Secretary BORS, University Dept of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School, Dundee DD1 9SY.
