Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements

Abstract

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0–30 minutes, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids

“…at 37°C), and then cultured in DMEM with 10% fetal bovine serum and antibiotics (10,000 I.U./mL penicillin and 10000 μg/mL streptomycin) in 5% atmospheric CO 2 . Cells were encapsulated using previously optimized methods [22] at a concentration of ∼500,000 cells/gel (gel diameter = 7mm, gel height = 12.7mm).…”

“…A short loading timescale was used because initial early-time responses can set the trajectory for longer-term behavior. The loading protocol followed previously optimized methods [22] in which homogeneous deformations [20] were applied to the cell-seeded gels using a custom built bioreactor emulating physiological loading conditions: frequency of 1.1 Hz and average sinusoidal compressive strains of 5% with an amplitude of 1.9% based on initial gel height (Supplementary Figure 1). The loading frequency was selected based on the preferred stride rate in humans [23], and the applied strain profile was selected to be in the range of deformations measured in human patients using MRI [24].…”

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

“…Isolated chondrocytes were cultured in DMEM with 10% fetal bovine serum and antibiotics (10,000 I.U./mL penicillin and 10,000 μ g/mL streptomycin) in 5% CO 2 at 37°C. Cells were expanded in monolayer for one passage prior to encapsulation in either 2 or 4.5% (v/v) agarose (Sigma: Type VII-A A0701) using previously established methods (Jutila et al, 2014; Zignego et al, 2015). Chondrocytes were seeded at a concentration of ~500,000 cells/gel (gel diameter=7mm, gel height=12.7mm).…”

“…Cell-seeded constructs from each patient (N=5 patients, n=3 samples per patient) were randomly assigned to one of three experiment groups: unloaded controls (0 minutes of loading), 15, or 30 minutes of cyclical compression for both 2% and 4.5% experimental groups. 24 hours after encapsulation, the gels were placed in fresh antibiotic-free Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and cyclically compressed using a custom built bioreactor (Jutila et al, 2014). This system provides relatively homogeneous deformations with displacement precision of 1% and strain precision of 6.5% (Zignego et al., 2014).…”

“…Metabolite samples were then placed in a centrifuge under vacuum for 6.5 hours for solvent removal. Remaining dried sample pellets were resuspended in 100 μ L of mass spectrometry grade 95:5 water:acetonitrile solution (Jutila et al, 2014). All reagents were mass-spectrometry grade.…”