Microarray Analysis of Irradiated Growth Plate Zones following Laser Microdissection Shows Later Importance of Differentially Expressed Genes during Radiorecovery

Abstract

Purpose: Potential targets for selective radiorecovery modulation were investigated via the identification of late upregulated genes and pathways during growth plate chondrocyte recovery. Methods and Materials: Three groups of six 5-week-old male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days totaling 17.5 Gy and were then killed at 7, 11, and 16 days following the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibiae using RAE230 2.0 GeneChip microarray, compared between zones and time points, and subjected to functional pathway cluster analysis with real-time polymerase chain reaction (PCR) to confirm selected results. Results: The reserve zone showed the greatest number of differentially expressed genes and enriched pathways: 259 and 134, respectively. Differentially expressed genes included: Timp3, Gpx1, Gas6, Notch2, VEGF, and HIF-1. Enriched pathways included the developmental processes of regeneration, antiapoptosis, developmental growth, tissue regeneration, mesenchymal cell proliferation, negative regulation of immune response, and determination of symmetry. The reserve zone late upregulation of genes was validated using real-time PCR for Mgp, Gas6, and Eef1a1. Conclusions: A significant difference in late upregulated genes between growth plate zones exists. The reserve zone shows the greatest change, containing a 10-fold increase in the total number of genes differentially expressed between days 7 and 16. These findings suggest that reserve zone chondrocytes may play a later role in growth plate recovery response following irradiation.

Effect of X-ray Irradiation on Articular Cartilage Mechanical Properties

“…The effects of radiation on cartilage may be concealed by the considerable delay existing between radiation therapy and joint symptoms [24,31]. Compressive modulus values for equine dorsal cartilage are similar to previous reported E. Cicek values [8,18,20,21,23]. Our results show that the lower doses of the ionizing radiation can significantly affect compressive modulus values in equine articular cartilage.…”

“…Ionizing radiation is known to lower proteoglycan content and compressive stiffness [8,[22][23][24][25]. Previous studies display that reactive oxygen radicals induced by ionizing radiation may be responsible for the degradation of the physiologically important glycosaminoglycan hyaluronan [10,26,27].…”

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

“…During the course of radiation therapy (RT) for tumor treatment, the incidental irradiation of non-cancerous (normal) skeletal tissue in joints can be fairly substantial (Konski & Sowers 1996; Luxton et al 2004). While various reports demonstrate the extent and nature of bone and growth plate cartilage damage following exposure (Damron et al 2008; Kwon et al 2008; Pritchard et al 2010; Willey et al 2010; Jia et al 2011; Alwood et al 2012), radiation effects on articular cartilage within the joint structure are undefined and relatively unstudied. Progressive degeneration and arthritis have been reported in various joints exposed to radiation (Kolar et al 1967).…”

Total-body irradiation produces late degenerative joint damage in rats

“…In the musculoskeletal system, the clinical concerns in children have been altered growth, bone fracture, spinal deformity, osteochondroma, and secondary bone malignancies (Leiper et al 1987; Chemaitilly et al 2007; Shido et al 2012). Metaphyseal trabecular bone changes, osteonecrosis of the femoral head, growth plate cartilage manifestations, including slipped capital femoral epiphysis, and platyspondylyl of the spine have been well studied and documented (Kaste et al 2004; Damron et al 2008; Damron et al 2009; Miyazaki et al 2009; Pritchard et al 2010; Mostoufi-Moab et al 2012; Mostoufi-Moab et al 2013). …”

“…Radiation exposure has been shown to cause acute degenerative alterations of the cartilage matrix composition and metabolism in vitro ; specifically, radiation is known to lower proteoglycan (PGs) content and compressive stiffness (Cornelissen & de Ridder 1990; Cornelissen et al 1993, 1996; Pritchard et al 2010; Lindburg et al 2013; Willey et al 2013). Biological changes in the joint are also likely influenced by radiation-induced bone changes and biomechanical alterations across the joint that disrupt the normal distribution of forces.…”