Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Abstract

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1.

Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours.

Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy.

Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.

Figure 1

Cell viability of human and pig chondrocytes following irradiation with 2 or 10 Gy γ-rays. “NR” represents “non-irradiated”. A). Live (green) versus dead (red) human chondrocytes at 1 day following exposure to 2 and 10 Gy of radiation using a LIVE/DEAD assay, with and without IGF-1 stimulation (100X magnification). 70% MeOH was used as a control for dead cell imaging. B) and C). Counts are from n = 2 independent studies of irradiated human (B) and pig (C) chondrocytes.

Figure 2

Glycosaminoglycans (GAG) released into the media from explants of human (A) and pig (B) cartilage. GAG content was quantified after exposing explants to 2 and 10 Gy of radiation. Media was harvested at 48 hours after exposure or at 96 hours (48 hours after an exchange of fresh media). Plots represent fold change versus control (0 Gy). Data are n = 3 independent experiments from both humans and pigs, each using data from two explants per treatment. Error bars indicate SEM. The asterisk (*) indicates a significant main radiation effect of 10 Gy regardless of time relative to 0 Gy control following a two-way ANOVA of raw data and a Tukey’s Post Hoc Test (α = 0.05). The (#) indicates a significant difference relative to control at 48 hours after exposure.

Figure 3

Effect of radiation on MMP production in human and pig chondrocytes from media collected at one day after irradiation with 2 Gy or 10 Gy γ-Rays. “NR” represents “non-irradiated”. A). Representative blots from human chondrocytes for ADAMTS 5 (aggrecanase-2), MMP-1, and MMP-13. Results are representative of n = 3 independent experiments. B). Results from an ELISA for MMP-13 concentration in conditioned media of human chondrocytes one day after irradiation (n = 2 independent experiments). Error bars indicate SEM. Statistics were performed on raw data. The asterisk (*) indicates significant difference from 0 Gy following a two-way ANOVA (main effects were individual and dose) and a Tukey’s Post Hoc Test indicating difference between groups (α = 0.05). C). ADAMTS 5 concentration from the conditioned media of pig explants irradiated with either 2 or 10 Gy doses. Data represent n = 3 studies.

Figure 4

Proteoglycan synthesis from human (A) and pig (B) chondrocytes at one day after irradiation with 2 or 10 Gy and IGF-1 stimulation. “NR” represents “non-irradiated”. Data are representative of n = 2 (human) and n = 3 (pig) independent studies per treatment condition. Error bars indicate SEM. These data are determined from [35S] incorporation into newly formed proteoglycans, and calculated as counts per minute relative to DNA content of the cells. Data are plotted as the average fold change for individuals relative to NR, No IGF. Statistics were performed on raw data. The symbols above horizontal lines indicates significant difference between groups following a two-way ANOVA (main effects were individual and dose) and a Tukey’s Post Hoc Test indicating difference between groups (α = 0.05). The topmost lines indicate differences between IGF-1 and non-IGF-1 treated groups. (*P < 0.01; #P < 0.05).

Figure 5

Effects of radiation on IGF-1 activation of Akt and IGF-1R in human (A and B) and pig (C) chondrocytes. “NR” represents “non-irradiated”. One hour after irradiation using 2 or 10 Gy γ-Rays, groups of cells were treated with IGF-1 overnight (100ng/ml). Immunoblots are representative results from a minimum of n = 3 independent experiments. Error bars indicate SEM. Lysates were immunoblotted for: A) p-Akt (S473 and T308), total Akt, p-IGF-1R, total IGF-1R, and β actin. Densitometry for each protein is identified by the figures on the right. For p-Akt blots, total Akt was used as the referent. For IGF-1R blots, β actin density was used as the referent. B) Immunoblot for pIGF-1R were overexposed to identify differences in the 2 Gy and 10 Gy lanes versus non-irradiated (NR) lanes. Densitometry is not presented for overexposed blots. C). P-Akt (S473) and total Akt blots from pig lysates. Densitometry of the blot is identified by a graph on the right.