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

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.