Influence of hypoxia and vascularization on the osteogenic differentiation and mineralization capacity of primary osteoporotic human mesenchymal stem cells

Kohll, Céline (2025): Influence of hypoxia and vascularization on the osteogenic differentiation and mineralization capacity of primary osteoporotic human mesenchymal stem cells. Dissertation, LMU München: Medizinische Fakultät

Abstract

The treatment of osteoporotic fractures that are associated with critical bone defects or non-unions is a significant clinical problem. To date, autografts have served as the gold standard for bone grafts. However, autografts have been associated with many risks. Bone tissue engineering (BTE) represents a potential alternative for the treatment of these complicated fractures. Although BTE research has largely improved, the translation into clinical applications has yet to be approved by regulatory agencies. As several companies have received approval for the use of intra-operation cell isolation systems, inadequate vascularization of scaffolds has been identified as a major problem. An additional concern is the reduced osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) in elderly patients. Therefore, many research groups have examined how to improve vascularization and osteogenic differentiation potential. One widespread approach for manufacturing prevascularized scaffolds has been a co-culture of scaffolds with hMSCs and human umbilical vein endothelial cells (hUVECs) combined with the testing of different oxygen levels in cell culture. The aim of the present study was therefore to gain knowledge about the influence of oxygen on the proliferation,growth characteristics, and osteogenic differentiation of hMSCs from different donors, specifically patients with high-energy fracture trauma (HET) and osteoporotic fracture trauma (OFT). Furthermore, co-cultures from donors of the HET group and OFT group with hUVEs, undergoing osteogenic differentiation were analyzed. For this study, hMSCs were isolated from different donor sites and randomized into the two donor groups HET or OFT. Initially, the morphology, growth characteristics, proliferation (CumPD and colony forming units (CFU)) and osteogenic differentiation potential of hMSCs in normoxia and hypoxia were analyzed. Further, the osteogenic differentiation potential of co-cultures (hMSCs:hUVECs) in different ratios was tested in 2D and 3D. Analysis of the morphology aspect in hypoxia and normoxia showed a tendency towards flattening of the cells for both groups (HET and OFT) in normoxia. Following cumulative population doubling in normoxia versus hypoxia, both groups showed a non-significant tendency toward improved proliferation in hypoxia. Further osteogenic differentiation was examined, showing that osteogenic differentiation in hypoxia was non-significantly reduced for both groups (HET and OFT). Comparison of the two groups, HET and OFT, found that the proliferation capacity, CFU capacity, and osteogenic differentiation potential of the HET group had a tendency towards better performance, although that difference was not always significant. Further co-cultures (hMSCs:hUVECs) of different ratios (1:0, 1:2, 1:3) were analyzed to gain a better understanding of the interaction between the cell types and the osteogenic differentiation potential in co-cultures. Most donor samples showed a tendency towards a higher osteogenic differentiation capacity in co-cultures than in hMSCs monocultures. The influence of the co-culture was more pronounced for OFT hMSCs than for the HET hMSCs. Although many results were non-significant, a tendency towards improved proliferation and later senescence in hypoxia, stronger osteogenic differentiation capacity in normoxia and a better osteogenic differentiation capacity in co-cultures demonstrates that hMSCs are very sensitive to oxygen levels and their behavior is influenced by cocultures. The knowledge of the specified pathways behind these interactions and the best conditions for hMSCs and hUVECs still have to be investigated. All these findings will help identify a better solution for non-union fractures and improve BTE.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Osteoporosis, Scaffold, Osteogenic Differentiation
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. Mai 2025
• 1. Bericht­erstatter:in: Böcker, Wolfgang