gms | German Medical Science

Objectives: Bone Tissue Engineering (BTE) approaches hold great promise for promoting bone healing and regeneration while overcoming some of the drawbacks of current clinical techniques. These approaches typically combine bone forming stem cells with scaffolds, which restore missing bone volume and signaling molecules, which control cell-cell and cell-scaffold interactions in the bone defect. Clinical BTE approaches have demonstrated encouraging early outcomes, however, availability of stem cells is a bottleneck of the whole procedure. BTE approaches require significant resources and time to isolate, characterize, and expand autologous mesenchymal stem cells (MSCs). Heterogeneous transplantation of pooled MSCs has been shown to be safe in patients with acute graft-versus-host disease. We hypothesized that random-donor-derived MSCs could be immobilized onto osteoconductive matrices and then frozen and that such BTE products could be released ready-to-use for permanent implantation during surgery after thawing.

Methods: The human MSCs (hMSCs), generated from pooled mononuclear cells from eight random bone marrow donors, were seeded on b-TCP scaffold granules and cultured for 24 hrs. The scaffold granules with seeded cells were frozen by mean of "air-dry" protocol (without freezing medium), thawed and cultured for two weeks in either growth or osteogenic supplemented medium (experimental groups). In the control group, cells were treated the same way as in experimental group except for freeze-thawing. The effects of cryopreservation and different cryostorage temperatures on cell metabolic activity after thaw were analyzed by mean of AlamarBlue assay. Expression of osteogenic- and hypoxia- marker genes was evaluated by mean of RT-qPCR.

Results and conclusion: While hMSCs lost some metabolic activity at early time points after thawing, they quantitatively regained it later on. We have shown that cells seeded on b-TCP scaffold and stepwise cryopreserved could not be stored at -20C, but could be stored at least for 3 days on dry ice without an apparent impact on post-thaw cell activity.

According to the osteogenic marker gene expression analysis, hMSCs osteogenic differentiation was accelerated after freezing/thawing procedure, as most of the analyzed markers were expressed earlier in experimental cells than in control cells. Cryopreservation has no effect on the expression of major signaling pathway genes (Wnt3A, MAPK8, MAPK14 and SMAD5), but significantly altered expression pattern of hypoxia related genes. Expression of HIF1a gene and its target genes PDK1, SLC2A1, EGLN1 and BNIP3 was up regulated in experimental group cells on day 7. We speculate that the cryopreservation procedure affects HIF1a expression, and this mechanism stimulates osteogenesis.

In conclusion, our findings show that hMSCs can be freeze-thawed on b-TCP scaffold through a technically simple procedure and thus provide a critical step towards the development of clinical-grade BTEs for functional bone augmentation.