Effects of low dose X-ray irradiation on porcine articular cartilage explants

Abstract

Ionizing radiation therapy is a crucial treatment for cancer, but can damage surrounding normal tissues. Damage to articular cartilage leading to arthropathy can occur at irradiated sites. It is unclear whether this response is due to damaging surrounding skeletal structures or direct effects on cartilage. In this study, we showed that irradiation with 2 Gy of X-rays causes a significant reduction in the stiffness of porcine explants 1 week post-irradiation. By using both microindentation and indentation-type atomic force microscopy, ionizing radiation reduces stiffness in both the superficial zone, and throughout the entire thickness of the tissue. Young's modulus values were 75% and 60% lower in 2 Gy irradiated samples when compared with controls using microindentation and nanoindentation, respectively. Glycosaminoglycans (GAGs) released into the culture media of irradiated samples was nearly 100% greater at 24 h after exposure. While collagen content in the tissue is similar between groups, GAG content is 55% lower in irradiated explants compared with controls 7 days after exposure. Therefore, the irradiated explants are unable to recover from the initial loss of GAGs by 1 week. This acute loss of GAGs is a likely contributor to the reduction in modulus seen after exposure to ionizing radiation.

Keywords: articular cartilage; atomic force microscopy; cartilage mechanics; glycosaminoglycans; radiation exposure.

Figure 1

Young’s modulus values for control (purple) and 2Gy-irradiated samples estimated using the Hertz model tested by a) microindentation and b) nanoindentation. The modulus values for irradiated samples were significantly lower than control samples using both mechanical tests (*, p < 0.001, n = 15). Error bars show ±standard deviation.

Figure 2

Normalized a) sGAG and b) HYP content in the tissue at Day 7, the time point used for mechanical testing. Irradiated samples had significantly lower sGAG content when compared to control groups (*, p < 0.05, n = 9). HYP content was similar between groups. Error bars indicate ±standard deviation.

Figure 3

Normalized sGAG content in the culture media over time (n = 6, error bars ±standard deviation). Significantly higher sGAG was released into the media in irradiated samples when compared to control samples at Day 1 (*, p < 0.001), while there was no significant difference in sGAG released between treatment groups at the Day 0, 3, or 7 time points. Concentrations of sGAG in the media of the irradiated samples were significantly higher at Day 1 when compared to Days 0, 3, and 7 (^#, p < 0.001). Additionally, sGAG content in the media of the control samples was significantly greater at Day 1 when compared with Day 3 (**, p < 0.01).

Figure 4

Histological cross sections of articular cartilage explants stained with H&E, Safranin O, and Masson’s trichrome. Qualitatively, there were no differences seen between control and irradiated samples. Scale bars represent 100µm.