Lipid profiles as biomarker for chondrogenesis
Healthy articular cartilage is preserved by chondrocytes by a balance between the formation and degeneration of cartilage matrix molecules. In osteoarthritis this delicate balance is disturbed towards more cartilage matrix breakdown. Leading to degradation of articular cartilage and loss of its protective function of the bone. The regenerative capacity of cartilage is limited mainly due to the absence of blood vessels, which makes the chondrocytes rely on diffusion for their supply of oxygen and nutrition. Scientists have tried to repair cartilage defects by expanding chondrocytes in culture and replacing them into the joint. Unfortunately, this was not very successful, because the cartilage tends to calcify and form bone. Recently, we discovered that this might be due to the oxygen (O2) levels at which the chondrocytes were cultured. In cartilage research, chondrocytes are often cultured at atmospheric O2 levels (normoxia) which is much higher (21%) than the in vivo situation (0.5-5% depending on depth)[1, 2]. When chondrocytes are expanded at oxygen levels that more closely resembles the in vivo situation (2.5%), the cartilage is of better quality and does not form bone when implanted in vivo .
Little is known about how oxygen levels influence chondrogenesis. Studies with mesenchymal stromal cells (MSCs) showed that the cartilage specific transcription factor SOX9 is upregulated in hypoxia. Moreover, gene expression and protein deposition of the main extracellular matrix molecule collagen type 2 is also upregulated in hypoxia. Next to effects on chondrogenesis, we found that culturing MSCs and chondrocytes in hypoxia altered the lipid profiles of the cells. Cell pellets cultured in 2.5% O2 contained less neutral lipids compared to cell pellets cultured in normoxia. Imaging mass spectrometry analysis revealed that, in the early stage of chondrogenesis, cholesterol was the major lipid that changed in hypoxia. We postulate that the cholesterol levels and other lipid profiles are not static, but are dynamic over time and can be correlated to specific stage of chondrogenesis.
To get a better insight in the dynamics of lipid profiles during early and late stages of chondrogenesis this project will study lipid profiles using matrix assisted laser desorption mass spectrometry (MALDI-MS).
During this project you will culture human primary chondrocytes and bone marrow derived mesenchymal stem cells and follow them in time to assess chondrogenic (re-)differentiation and lipid deposition. You will do so by using histological staining techniques, quantitative PCR (qPCR) and MALDI- MS.
Cell and pellet cultures, mass spectrometry (MALDI), histology and qPCR
- 1Brighton, C.T. and R.B. Heppenstall, Oxygen tension of the epiphyseal plate distal to an arteriovenous fistula. Clin Orthop Relat Res, 1971. 80: p. 167-73.
- Lund-Olesen, K., Oxygen tension in synovial fluids. Arthritis Rheum, 1970. 13(6): p. 769-76.
- Leijten, J., et al., Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate. Proc Natl Acad Sci U S A, 2014. 111(38): p. 13954-9.
Daily supervisor: Brenda Bakker, MSc
If you are interested in this assignment, please contact: Dr. Janneke Alers, contact person for all DBE assignments