Oxygen regulates lipid profiles in human primary chondrocyte cultures

Abstract

Purpose: Articular cartilage is generally exposed to a finely regulated gradient of relatively low oxygen percentages (from 8% at the surface to 1% in the deepest layers). While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype and cartilage-specific matrix formation. Although hypoxiainducible factor-1α is identified as a key mediator of these beneficial effects on chondrogenesis, the underlying mechanisms remain unclear. A recent study in primary chondrocytes and chondrogenically differentiating mesenchymal stem cells showed that the cholesterol metabolism is altered in hypoxic conditions. Moreover the lipid metabolism is changed in osteoarthritic cartilage and intracellular lipid accumulation is correlated with osteoarthritis (OA) severity. In this study we assessed whether healthy and OA chondrocytes have distinct responses in normoxia or hypoxia with respect to their lipid composition. Methods: Human primary chondrocytes were isolated from cartilage knee biopsies of patients (n = 5) undergoing total knee replacement. From each donor cells were isolated from macroscopically healthy and OA damaged areas. Cells were expanded in monolayer in normoxia (21% oxygen) or hypoxia (2.5% oxygen) and subsequently cultured in 3D pellets in normoxia or hypoxia for 7 days. Lipid profiles were assessed with Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI MSI). Cell pellets were cryosectioned (10 μm section) and sprayed with α-Cyano-4-hydroxycinnamic acid (CHCA) matrix 5 mg/ml in methanol/water/trifluoroacetic acid (70:30:0.01) using the Suncollect (Sunchrom). A Synapt HDMS MALDI-Q-TOF (Waters) instrument was used to perform the MSI experiments with a spatial raster size of 100 μm. Principal component analysis (PCA) and discriminant analysis (DA) were used to search for spectral similarities and differences between the conditions. Biomap software was used to visualize molecular distributions. The Lipid Maps database was used for lipid assignment and tandem MS for the identification. Results: Chondrocyte cells cultured under different oxygen tensions were discriminated by MALDI MSI followed by discriminant analysis. Discriminant function 1 (DF1) described the lipid profiles specific to each oxygen tension (Figure 1). In hypoxic pellets the intensity of phosphatidylcholine (PC) 16:0/18:1 (m/z 798.5) and sphingomyelin (SM) d18:1/16:0 (m/z 741.5 and 725.5) (Figure 1A)was higher compared to normoxic pellets. Moreover, while various phosphatidylinositol (PI) were present at both oxygen tensions, the intensity of the PI's in normoxia (m/z 865.5 and 885.5) was much higher (Figure 1B). The molecular distribution of SM d18:1/16:0 at m/z 741.5 and PC 18:0/18:1 (m/z 810.6) showed a higher expression at the edges of the pellet (Figure 2). Interestingly, we found that the lipid profiles of chondrocytes harvested from either OA or healthy cartilage showed a more pronounced difference when cultured in hypoxia. In both positive and negative ion modes the second DF separated healthy from OA chondrocytes in hypoxic conditions. Conclusions: Our MALDI-MSI data show that oxygen tension modulated the lipid composition of chondrocytes. Furthermore, culturing OA or healthy chondrocytes in hypoxic conditions resulted in more pronounced differences in lipid profiles as compared to culturing in normoxia. Since glycerophospholipids, including PC, SM, PS, and PI, are key components of the lipid bilayer and involved in metabolism and cell signaling, we will next investigate how these lipids influence chondrocyte metabolism and cell signaling. (Figure presented).