Trypsin vs IL-1β Mediated GAG Depletion

Background

One of the main characteristics of osteoarthritis is the degradation of the ECM. The Bonassar Group performs most of its experiments using neonatal bovine joints. Since neonatal bovines experience very little metabolic stress throughout their lifetimes, the samples are perfectly intact and have very dense ECMs. Therefore, external agents must be used to degrade the samples and create a more realistic imitation of the conditions seen in arthritis. Two proteins that could be used for this purpose are trypsin (fast-acting) and IL-1β (slow-acting). 

Glycosaminoglycans (GAGs) mediate resistance to compression throughout the ECM. GAG depletion can be measured both quantitatively and qualitatively via Safranin-O staining. Combining both quantitative and qualitative observations can then be used to determine whether trypsin or IL-1β cause GAG depletion that more closely mimics the depletion seen in osteoarthritis. 

Aim

Determine which length of 50 µg/mL trypsin treatment leads to similar GAG depletion depth to 4 days of 10 ng/mL IL-1β treatment. 

Methods

Two experiments were conducted to determine the magnitude of GAG depletion caused by different concentrations of IL-1β (10 ng/mL, 30 ng/mL) administered for 4 days to 50 µg/mL trypsin administered for different amounts of time (2 min, 10 min). Safranin-O staining was performed on all samples. Images were taken for qualitative comparison and mean GAG depletion depth was measured on ImageJ for quantitative comparison. 

Results

The only statistically significant relationship in experiment 1 was the Trypsin-10 min group having a greater mean GAG depletion depth than the IL-1β-30 ng/mL group (p = 0.028). There was very high variability in both control and IL-1β-10 ng/mL groups, with standard deviations of 100.8 µm and 88.8 µm, respectively. Although the data from experiment 2 showed less variability than experiment 1, the only statistically significant relationship was the Trypsin-10 group having a greater mean GAG depletion depth than the IL-1β-10 ng/mL group (p = 0.048). 

Significance

The Bonassar Group currently uses IL-1β as a degrading agent for most of its experiments that require ECM depletion. However, IL-1β takes a week to degrade the ECM, causing experiments to take longer than they should. My experiments seem to suggest that trypsin is capable of degrading the ECM just as effectively as IL-1β. Therefore, some students may consider switching to trypsin for future experiments, as its shorter administration period improves experimental logistics.