Purpose of the study: Several studies have demonstrated the usefulness of mesenchymatous stem cells (MSC) for cell therapy aimed at favoring bone tissue healing. Bone morphogenesis proteins (BMP) orient MSC towards osteoblastic differentiation. Since they are rapidly degraded in the organism, these proteins require a continuous release system to potentialize their biological activity in a controlled localized manner. We evaluated the usefulness of using the electroporation technique to insert a BMP transgene into the MSC of rats to enable sufficient transient expression of BMP genes to enable satisfactory bone healing. We first developed electroporation conditions for rat MSC and checked cell viability after the electric shock. Secondly, in order to obtain quantitative and/or temporal BMP expression, we tested the influence of different promoters on transcription actvity.

Material and methods: To determine the electroporation parameters, MCS were transfected with the pCMV-LacZ plasmid using two electric impulsions: a series of eight 100 impulsions/μs at high voltage (900-170V/cm) followed or not by a series of eight 12.5 ms low-voltage impulsions (60 V/cm). After determining the electroporation conditions, six plasmids carrying different promoters were electroporated.

Results: The best transfection rate in rat MSC was obtained with a series of 8 impulsions at 1500 V/cm. Before the electrical shock, the suspended rat MSC had to be incubated at ambient temperature to favor cell survival. Proliferation of electroporated cells was comparable to that of non electroporated cells. Surprisingly, addition of low-voltage pulses significantly decreased the efficacy of transfection. In addition, MSC transfected with the promoters GAPDH and beta-actin presented a beta-galactoside activity (at 48 h) superior to that obtained with the pCMV promoter.

Discussion: After optimization of these parameters, we demonstrated that MCS can be effectively transfected by electroporation. The following steps will be to check for long-term expression of beta-galactoside by electroporated MSC, transfection of MSC with plasmids or the BMP-2 gene controlled by these same promoters and monitoring promoter activity as a function of the stage of MSC differentiation.

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.