gms | German Medical Science

Objectives: Large bone defects after e.g. open long bone fractures remain a challenge for orthopedic surgeons. Several approaches have been described involving autographs, allographs and growth factors. Usually autologous bone acts as a scaffold to bridge the existing gap and cancellous bone harvested form the patient itself combined with growth factors are added to it. Due to the fact that the amount of autogenous graft usually is small in comparison to the defect, efforts have been made to integrate scaffolds with stem cells to treat such defect. Traditionally mesenchymal stem cells are chosen for those kind of procedures. However, harvesting and cultivation of mesenchymal stem cells is a tedious process. Induced pluripotent stem cells (iPSC) represent an intriguing alternative source of stem cells. Unfortunately, the genetic modification associated with their creation precludes them from direct use. Nevertheless, they create extracellular matrix, which can be used to enhance scaffolds. Scaffolds created in that manner would be easily sterilized and would no longer be bound to the original donor, making their clinical application more feasible.

Methods: Secondary mouse embryonic fibroblasts were reprogrammed to induced pluripotent stem cells by activating c-Myc, Klf-4, Oct-4 and Sox-2. Proof of pluripotency was provided by letting the cells build complete teratoma after implantation. Under fluorescence microscopy, SSEA1 and Oct-4 positivity could be demonstrated. Osteogenic differentiation was conducted for a total of 21 days using two different osteoinductive media. RNA samples were taken at d0, d3, d7, d10, 14, 21 and alizarin red staining was performed on d3, d7, d14, d17 and d21. An in-vivo mouse model is performed comparing fracture healing in mice with critical size defects with (1) plating, (2) plating + PLGA scaffold, (3) plating + PLGA scaffold loaded with iPSC and (4) plating + PLGA scaffold loaded with iPSC extracellular matrix.

Results and conclusion: Preliminary in-vitro results show significantly higher calcium deposits under the commercial standardized osteogenic medium compared to regular DMEM/FCS/glutamine + Beta-glycerol/ascorbic acid/dexamethasone. Furthermore, IPS differentiation with subsequent alizarin positivity could already be observed at day 3 when compared to day 21 in mesenchymal stem cells. The decellurized and iPSC-scaffolds were implanted in a mouse model critical size defects and the differences between them quantified by µCT and immunohistochemical analysis.