Defended by Rebecca LANDON on December 19, 2023
PhD director: Fani ANAGNOSTOU
Abstract
Type 2 diabetes mellitus (T2DM) is a major health problem. It affects more than 5% of the world’s population, and its prevalence continues to rise. T2DM is a metabolic disease characterized by chronic hyperglycaemia, leading to severe complications (heart disease, nephropathy, retinopathy, neuropathy, etc.) that are costly and time-consuming to treat. T2DM does not spare bone tissue, increasing the risk of fracture and limiting bone repair potential. The underlying mechanisms are complex, but the T2DM microenvironment remains one of the determining factors. Chronic hyperglycaemia leads to the exacerbated formation of advanced glycation products (AGEs) and chronic oxidative stress modifying the matrix and cellular functionality, including that of bone marrow mesenchymal stem cells (MSCs). MSCs are essential actors in bone homeostasis, bone repair and promising candidates for tissue engineering, yet the impact of AGEs and oxidative stress on these cells remains only partially characterized. Furthermore, the use of antioxidants on MSCs to regulate intracellular oxidative stress has been little investigated to date.
The aim of this PhD work was to understand the impact of the T2DM microenvironment on mesenchymal stem cells and the therapeutic potential of antioxidants for the regulation of oxidative stress induced by AGEs. The Zucker Diabetic Fatty (ZDF) rat was used as an animal model of T2DM and its healthy counterpart the Zucker Lean (ZL) rat as a control. We have shown that ZDF-MSCs derived from a long-standing T2DM microenvironment and cultured in vitro under normoglycaemia display changes in several functions relevant to bone repair (adhesion, clonogenicity, proliferation, migration, adipocyte differentiation) compared with those derived from control animals. As such, the use of autologous MSCs seems compromised for bone repair therapies.
To better understand the impact of chronic hyperglycaemia on MSCs, we studied the effects of AGEs. The results showed that AGEs alter the viability, proliferation, migration and gene expression profile of MSCs. The effects of AGEs on ZDF-MSCs were more pronounced than those observed on ZL-MSCs. In both cell types, they lead to oxidative stress through the excess production of intracellular reactive oxygen species (ROS); which is very rapid, remains constant for 60min and does not induce the formation of excess superoxide (O2-) by the mitochondria. To regulate intracellular ROS induced by AGEs or other inducers, we studied the potential of N-acetylcysteine and Astaxanthin, two powerful antioxidants, on MSCs. The results showed that intracellular ROS levels are similar in ZDF- and ZL-MSCs, and are modulated differently in response to antioxidants. Co-exposure to antioxidants was more effective than pre-exposure in regulating intracellular oxidative stress in MSCs. ZDF-MSCs also showed a gene expression profile, in response to exposure to antioxidants, that differed from that of ZL-MSCs. Finally, in vivo implantation of hydrogels containing Astaxanthin in a subcritical bone defect model in healthy rats appears to promote bone neoformation.
Overall, this thesis contributes to a better understanding of the impact of the T2DM microenvironment on bone tissue and highlights the negative impact of long-term diabetes, AGEs generated by hyperglycaemia and excess oxidative stress on the functionality of MSCs. The results call into question the use of autologous bone marrow MSCs in T2DM individuals as part of cell therapy and highlight the importance of using antioxidants to regulate oxidative stress as part of bone tissue repair.