Molecular Profiling of Oxygen's Impact on Chondrocytes | Insights from Mass Spectrometry Imaging
Brenda Bakker is a PhD student in the department Developmental BioEngineering. (Co)Promotors are prof.dr. H.B.J. Karperien and dr.ing. J.N. Post from the faculty of Sciences & Technolgy and prof.dr. R. Heeren and dr. B. Cillero-Pastor from the University of Maastricht.
Articular cartilage is smooth tissue at the ends of bones, where joints meet, providing protection and enabling pain-free movement. Lacking blood vessels and nerves, cartilage has limited self-repair ability. Cartilage is produced by chondrocytes, and in diseases like osteoarthritis, the tissue breaks down, and chondrocytes are lost. The exact causes of osteoarthritis remain unclear, and treatment options are limited.
One theory is that cartilage damage, whether from injury or aging, alters oxygen levels in the tissue. Oxygen is crucial for cell survival, but both too little and too much can harm tissue. Healthy cartilage has low oxygen levels (1-5%) due to the absence of blood vessels.
This thesis investigates how oxygen influences biochemical changes in chondrocytes (Chapters 2–4), comparing 20% oxygen (normoxia) with 2.5% oxygen (hypoxia). Chondrocytes from osteoarthritis patients undergoing knee replacement were cultured and formed spheroids, mimicking their natural environment to preserve their properties.
Using mass spectrometry (MS) and imaging mass spectrometry (MSI), we measured various molecules in the chondrocytes. These techniques provide precise data on proteins, lipids, metabolites, and nucleic acids. MSI not only identifies molecules but also maps their locations within tissues, enabling a better understanding of chondrocyte responses to oxygen levels.
Chapter 2 focused on lipid changes, showing that sphingomyelin (SM) was more abundant under hypoxia, while cardiolipin (CL) and phosphatidylglycerol (PG) were elevated under normoxia. This suggests that oxygen levels affect lipid distribution, which could indicate mitochondrial changes related to disease processes.
Chapter 3 identified 47 metabolites that differed between normoxia and hypoxia. Under hypoxia, chondrocytes had higher ATP and glutathione (GSH), indicating better energy supply and protection from oxidative stress. Normoxia showed more diverse phospholipids and bioactive lipids, pointing to inflammation and cell membrane changes relevant to osteoarthritis.
Chapter 4 analyzed protein changes during chondrocyte differentiation. Normoxic cells showed more proteins involved in matrix remodeling, while hypoxia stimulated glycolysis and cytoskeleton organization. In later stages, normoxic cells had increased metabolic activity, while hypoxic cells exhibited sustained succinate dehydrogenase (SDH) activity, which helps reduce oxidative stress.
Chapter 5 presents a new protocol for preparing patient-specific organoids for MSI analysis. This method preserves the fragile structure of organoids, enabling accurate histological and molecular imaging. This approach may be valuable for personalized treatments in cancer research.
Laboratory models like chondrocyte spheroids and organoids are crucial for biomedical research and therapy development. Recent advances in these models, along with MS and MSI, help translate laboratory findings into potential treatments.
This research deepens our understanding of how oxygen affects chondrocyte processes, particularly in mitochondrial function, energy production, and oxidative stress. These insights could form the basis for future cartilage repair therapies.
