Effects of Radiation Therapy on Chondrocytes In Vitro

Abstract

The negative irradiation complications of growth loss leading to limb length asymmetry and pathological fracture incurred following radiation therapy in pediatric patients has led to a renewed interest in understanding the specific effects of irradiation on the growth plate and the surrounding bone. In the present report, we examined the radiation therapy effects on primary rat growth cartilage chondrocytes in order to determine the chondrocyte radiosensitivity relative to other bone cell constituents and tumor cells, the postirradiation temporal progression of radiation-induced alterations in chondrocyte function, and the time course for the functional restoration of chondrocyte pathways that drive the eventual recovery in growth function. We employed an in vitro primary rat costochondral growth cartilage cell culture model system to evaluate the radiation therapy effects on proliferative chondrocytes using serial radiation doses (0-20 Gy) that are well within the clinically relevant range. Following irradiation, all of the following occurred in a dose-dependent manner: proliferation decreased, cytotoxicity increased, several markers of apoptosis increased, markers of radiation-induced cellular differentiation increased, and cell synthetic activity was disturbed. Alterations in proliferation, cell death, and induction of apoptosis are likely due to a transient radiation-induced derangement of the parathyroid hormone-related protein-Indian hedgehog proliferation-maturation pathway. Alterations in cellular differentiation and cell synthetic activity are novel observations for chondrocytes. Further, these results correspond very well to our previous work in an in vivo Sprague-Dawley rat model, making this model particularly relevant to researching the radiation therapy effects on longitudinal growth.

SHIP1 Regulates MSC Numbers and Their Osteolineage Commitment by Limiting Induction of the PI3K/Akt/β-Catenin/Id2 Axis

“…Multinucleate OCs were stained with the tartrate-resistant acid phosphatase (TRAP) (Acid Phosphatase, Leukocyte Kit; Sigma) [29]. Multinucleate, TRAP-positive OCs were subsequently counted using a modification of Cavalieri's sampling method and the fractionator to generate an unbiased estimate of the numbers of OCs within each tissue-culture well [32,33]. OCs were also used to detect SHIP expression by western blot.…”

The differential effects of the radioprotectant drugs amifostine and sodium selenite treatment in combination with radiation therapy on constituent bone cells, ewing's sarcoma of bone tumor cells, and rhabdomyosarcoma tumor cells in vitro

“…4 MTT (5 mg/mL (w/v), Sigma, St. Louis, MO) was added to wells, incubated for 2 h, and the cells lysed with DMSO. The formazan product was measured at 570 nm and irradiation effects on cell number were determined by normalizing treated wells to the mean values from nonirradiated wells according to the following: Percent D ¼ 100*[treated/mean control].…”

“…Second, to examine the irradiation effects on changes in endothelial cell, osteoblast, and osteoclast function compared to chondrocytes. 4 Third, to compare the results from the current work with previous in vivo and in vitro work. We hypothesized that irradiation would differentially affect bone cell survival relative to the tumor cells examined.…”

“…Also, we hypothesized that irradiation would alter endothelial cell, osteoblast, and osteoclast function, consistent irradiation-altered chondrocyte function. 4 Further, we hypothesized that radioprotective therapy would differentially protect bone cells relative to the tumor cells.…”

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

“…51 Indeed, we have presented data previously suggesting that hypoxia there may play a significant role in modulating this signal pathway, though this was not examined in this experiment. 52,53 Our laboratory has previously documented a decline in PTHrP expression at 1 week and subsequent recovery by the second week following radiation appears to be concurrent with decreases in hypertrophic cell enlargement and matrix production. 9 This suggests an alteration of the paracrine feedback loop involving signal transduction along the PTHrP/PTHr1 and Indian hedgehog (Ihh) coregulatory pathways.…”