Faculty of Precision- and Micro-Engineering, Engineering Physics, Munich University of Applied Sciences, Munich, Germany 1. Supervisor: Prof. Dr. rer. nat. Hauke Clausen-Schaumann 2. Supervisor: Prof. Dr. med. Matthias Schieker Mentors: Dr. med. Hans Polzer Dr. rer. nat. Denitsa Docheva Dean: Prof. Dr.-Ing. Alfred Fuchsberger
Abstract I Abstract Despite its apparent disadvantages such as donor site morbidity, limited availability and prolonged hospital stays, autologous bone grafting remains the gold standard of treatment whenever it comes to the repair of large bony defects. The use of human mesenchymal stem cells (hMSCs) seeded on supportive three-dimensional (3D) scaffolds is believed to be a promising alternative for bone substitute. In theory, this technique combines the advantages of autologous grafts (no immune response) with those of synthetic bone substitutes (unlimited supply, no donor site morbidity). In vitro hMSC expansion, long-term growth and differentiation in 2D and 3D are common practice. However, cell survival was shown to be poor in vivo after transplantation of cellularized bone scaffolds, thus hampering the clinical success of this appealing concept. At this stage hMSCs seem to be promising due to their capacity to differentiate into various lineages such as the osteogenic lineage. It has been suggested, that MSCs stay quiescencent in a specific hypoxic stem cell niche at an oxygen level of less than 7% within the bone marrow until activation. Until now most research was performed under standard cell culture conditions (21% O2). Consequently, the present study is designed for comparing oxygen-mediated differences in respect to differentiation potential, surface markers, growth characteristics, morphology, migration and integrin expression. This could possibly influence homing, proliferation and ultimately healing capacity of transplanted hMSCs into critical size bone defects. Cells in both culture conditions (Normoxia: 21% O2, Hypoxia: 2% O2) could be differentiated into the osteogenic and adipogenic lineage. While there was no significant difference in osteogenic potential, the adipogenic differentiation was significantly better in the hypoxic group. The positive mesenchymal surface markers CD73, CD90 and CD105 were highly expressed in both culture conditions (≥ 95%), whereas the hematopoetic markers CD14, CD19, CD45 and HLA-DR were negative (≤ 2%). Cellular growth characteristics were assigned by evaluation of the cumulative population doublings (cum PD) and the population doubling time (PDT). Hypoxic cultured hMSCs were shown to have a nearly doubled cum PD before going into cellular senescence compared to normoxic cultured hMSCs. Concomitantly, PDTs were obviously different, whilst normoxic cells had an immoderate varying PDT, hypoxic cultured cells showed a continuously homogenous PDT. This goes well in line with
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Supplementary data 57 Supp.Fig. 1:
Appendix 59 vacuum pump for cell cu
Abbreviations 61 List of abbreviati
List of figures 63 List of figures
List of figures 65 Fig. 18: Compari
Acknowledgments 67 Acknowledgments