Purpose of the study: Cell therapy proposes to fill gaps left by bone stock loss using osteocompetent cells (mesenchymatous stem cells, MSC). Preclinical results have been promising but still require improvement particularly concerning stress to the MSC during in vivo implantation. Stress results from sudden transfer i) from oxygen medium (21% O2) to a hypoxic medium (0–5% O2 because O2 diffusion is limited to 200 mm from a blood vessel), ii) a cell support to an osteoconductor support, et iii) a rich medium (fecal calf serum, FCS) to a medium with a limited supply of nutrients, hormones and growth factors diffusing from the environing biological fluids. The purpose of this study was to evaluate in vitro the impact of these different factors on MSC survival.
Material and methods: Human MSC(hMSC) harvested from bone marrow (n= 5 donors) and sheep MSC (sMSC) obtained with a preclinical model (n = 5 animal donors) were exposed for 48 h(hMSC) or 72h (sMSC) to the following transfers: i) rich medium (10% FCS) to poor medium (1% FCS), ii) plastic support to osteo-conductor supports (alumina, calcium carbonate), and iii) oxygen medium (21% O2) to hypoxic medium (6% O2). sMSC were also exposed to prolonged hypoxia (48–120h). Cell death was determined using image analysis after live/dead cell staining.
Results: The results demonstrated that MSC are: i) sensitive to a decrease from 10% to 0% FCS; 14% death of hMSC and 17% death of sMSC), ii) sensitive to transfer onto osteoconductor supports (sMSC on calcium carbonate: 23%), iii) very sensitive to prolonged hypoxia (120h) when combined with decreased FCS (sMSC: 23%; hMSC: 98%). A complementary study on the influence of hypoxia on differentiation properties of surviving sMSC is under way.
Conclusion: If the in vivo results concord with the in vitro results, i.e. if massive cell death is observed 4 days after implantation due to hypoxia, the current transplantation conditions will have to be revisited. Acceleration of neovascularization of in vivo implants which would shorten the period of hypoxia should allow better survival of implanted sMSC.
