Abstract
Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy) X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy) X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.
Highlights
Bone development and homeostasis are maintained through the balance between bone-forming osteoblasts and bone-resorbing osteoclasts
The results indicated that low-dose (0.5 Gy) X-ray irradiation promoted cell proliferation on days 2, 4 and 6 post-irradiation compared with the 0 Gy control (p,0.05), whereas high-dose (5 Gy) irradiation inhibited MC3T3-E1 cell proliferation (Fig. 2A)
Cell cycle analysis by flow cytometry revealed that single, low-dose (0.5 Gy) X-ray irradiation induced a decrease in the proportion of cells at the sub-G1 phase, which indicates active apoptosis (6.75%60.21%, p,0.05 vs. control), whereas increasing levels of cell death by apoptosis were observed in the high-dose irradiation group (14.26%60.41%, p,0.05 vs. control)
Summary
Bone development and homeostasis are maintained through the balance between bone-forming osteoblasts and bone-resorbing osteoclasts. Previous studies have suggested that irradiation can impair bone formation by impeding osteoblast proliferation and differentiation, inducing cell-cycle arrest, reducing collagen production and increasing the sensitivity to apoptotic agents [7,8]. The effects of low-dose irradiation, especially at levels less than 1Gy, on bone responses and healing have rarely been described in the literature. A previous study demonstrated that 2 Gy X-ray ionizing radiation induced time-dependent cell cycle arrest and had no significant effects on osteoblast proliferation and differentiation in an osteoblastic cell line [16,17]. Park et al [18] reported that 2 Gy X-ray irradiation increased differentiation and mineralization of the cells and upregulated the expression of ALP, Col, OPN and OCN in the early stage of differentiation
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