| Bone development and homeostasis are maintained through the balance betweenbone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are the chiefbone-making cells that are responsible for the production of bone extracellular matrixduring the remodeling or healing of bone. It is well known that high-dose irradiationdelivers deleterious effects to bone tissue, including osteoradionecrosis, sclerosis, loss ofbone mass and bone fracture, in a dose-and time-dependent manner. However, the effectsof low-dose irradiation on bone responses and healing have rarely been described in theliterature. Orthopedic patients are more often subjected to radiation exposure, such asradiography, computed tomography or fluoroscopy during surgery, where the exposure toionizing radiation is usually at low-dose levels. Our preliminary studies surprisinglydemonstrated that low dose X-irradiation promoted callus formation and mineralization ina rat model, and this type of radiation was also shown to have different effects on theproliferation and differentiation of osteoblasts in vitro. However, the underlyingmechanism has not been evaluated in osteoblasts exposed to low-dose irradiation. In thisstudy, we investigated how low-dose X-ray irradiation influences the proliferation anddifferentiation of osteoblasts and promotes fracture healing. The expression patterns ofosteogenic genes were evaluated to explore the possible mechanisms involved inirradiation-stimulated osteoblast differentiation. Our findings provide a greaterunderstanding of the biologic responses of osteoblasts exposed to low dose X-irradiationand highlight the potential positive effects of such treatment.Part I Low-dose X-irradiation promotes osteoblast proliferation, differentiation andfracture healing in vitro and in vivoThere is a great controversy regarding the biologic responses of osteoblasts toX-irradiation, and the mechanisms are poorly understood. In this study, the biologicaleffects of low-dose radiation in stimulating osteoblast proliferation, differentiation andfracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5Gy) X-irradiation induced significant increases in the cell viability andproliferation of MC3T3-E1cells. However, high-dose (5Gy) X-radiation inhibited theviability and proliferation of osteoblasts. In addition, dynamic variations in osteoblastdifferentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterixand osteocalcin, were observed after both low-dose and high-dose irradiation by Westernblot analysis. Second, fracture healing was evaluated via histology and gene expressionafter single-dose X-irradiation, and low-dose X-irradiation was found to accelerate fracturehealing of closed femoral fractures in rats. In low-dose X-ray-irradiated fractures,increased proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation, andfracture callus were observed. In addition, we observed more rapid completion ofendochondral and intramembranous ossification, which was accompanied by alteredexpression of gene involved in bone remodeling and fracture callus mineralization.Although several osteoblast differentiation gene expression levels were increased in thefracture callus of high-dose irradiated rats, the callus formation and fracture union weredelayed compared to the control and low-dose irradiated fractures. These results revealbeneficial effects of low-dose irradiation, including the stimulation of osteoblastproliferation, differentiation and fractures healing, and highlight its potential translationalapplication in novel therapies against bone-related diseases.Part II The effects of low-dose X-ray radiation on the profile of gene expression inMC3T3-E1cellsIn order to evaluate global differences of expression between low-dose irradiationgroup and the control, we conducted microarray analyses using mouse Roche NimbleGen12x135K chips. MC3T3-E1cells were irradiated0.5Gy X-irradiation. Cells withoutirradiation were regarded as control group. Cells were cultured for7days after irradiationand collected. Total RNA were extracted and RNA integrity was assessed by standarddenaturing agarosegel electrophoresis. About5μg total RNA of each sample was used forlabeling and array hybridization. The experiment of collecting fluorescent information wasperformed by scanning images. They were then imported into software and expression datawere analyzed. Differentially expressed genes with statistical significance were identifiedthrough Volcano Plot filtering and the threshold was fold change≥1.3. Pathway Analysisand GO analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical clustering wasperformed to show distinguishable gene expression profiling among samples. We identified1412differentially expressed transcripts in the radiated group compared with the control.Among the identified genes,559transcripts were up-and853transcripts weredown-regulated after irradiation. These identified transcripts were further analyzed byPathway analysis and GO analysis. The overrepresented genes were associated withosteoblast differentiation and ossification. Many genes belonged to focal adhesion,extracellular matrix space, cytoskeletal protein binding and angiogenesis. Some transcriptspossessing growth factor activity such as epidermal growth factor receptor, fibroblastgrowth factor, insulin-like growth factor were statistically overpresented. The studyindicated low-dose X-ray radiation promoted some genes of osteoblast differentiationup-regulated which might be involved extracellular matrix, actin cytoskeleton, focaladhension and growth factor activities.Part III0.5Gy X-ray radiation promoted osteoblast proliferation and differentiationby Wnt/-Catenin signalingIn order to investigate the important role of Wnt signaling in the processes of0.5GyX-ray promote osteoblast differentiation, and make clear the molecular mechanismsinvolved. Flow cytometry was employed to detect the apoptosis after osteoblast exposureto0.5Gy X-ray radiation. The protein expression level of osteoblast differentiationmarkers, such as Col1α, ALP, OCN, were detected and ALP activity staining wasperformed. Real-time PCR and Western blot were utilized to evaluate the variations of keyfactors in Wnt signaling pathways, while specific inhibitor of Wnt/-Catenin, XAV939wasused to block the Wnt signaling. Our results showed that the apoptosis of MC3T3-E1significantly declined at three days after0.5Gy X-ray radiation. The protein levels ofCol1α, ALP and OCN had increase during osteoblast differentiation except a temporary fallat four days of radiation exposure. At the same time,0.5Gy X-ray radiation can activateWnt/GSK-3/-Catenin signaling, and specific inhibitor XAV939completely abrogatedthe increase in ALP expression and activaty induced by0.5Gy X-ray radiation. This studydemonstrated that low dose X-ray radiation promoted osteoblast early survival, andstimulated middle and late osteoblast differentiation, in which Wnt signaling participatedthe regulation processes. Part IV0.5Gy X-ray radiation stimulates TGF-1/Smad2/3signaling, osteoblastproliferation and differentiation in vitroTo investigate the changes of TGF-1/Smad2/3signaling pathway in osteoblastproliferation and differentiation induced by0.5Gy X-ray radiation. Clone formationefficiency and cell cycle analysis were performed to detect the ability osteoblast divisionand proliferation after radiation exposure. The effects of irradiation on osteoblastdifferentiation and mineralization were assessed at different times. Real-time PCR wasused to detect the mRNA expression level of cyclin D1, Id2, TGF-1, ALK5afterirradiation, and TGF-1secretory activity was evaluated by ELISA. Western blot wasutilized to detect the expression of phosphorylated Smad2, Runx2and Osterix that wereimportant signaling molecules in TGF-1signaling pathways. The results revealed that0.5Gy X-ray radiation increased clone formation rate and G2/M proportion in osteoblast. Theincreases in mRNA expression levels of CyclinD1, Id2, TGF-1and ALK5were found inosteoblast after radiation exposure. Osteoblast differentiation markers presented dynamicchanges after0.5Gy X-ray radiation. And an accelerated mineralization was detected at thesame time.0.5GyX-ray radiation also activated TGF-1/Smad2/3/Osterix signalingpathwaysin osteoblast. Thus, low dose X-ray radiation can increase the expression ofrelated osteogenesis genes, and promote osteoblast differentiation, mature, mineralizationand bone formation ability. In this process, TGF-1/Smad2/3/Osterix may be involved inthe important regulation mechanism.Part V IGF-1/Akt signaling involves in the processes of differentiation regulationinduced by0.5Gy X-ray radiationThis part highlighted the effects of low dose X-ray radiation on IGF-1signaling inosteoblast, and the cross-talk between IGF-1signaling, and Wnt signaling and TGF-1signaling. Our purpose was to understand the biological effects of low dose irradiation onosteoblast and the related mechanism more deeply. We constructed IRS-1shRNAlentivirus firstly. Then the shRNA lentivirus infected MC3T3-E1in ordor to silence IRS-1gene. After osteoblast exposure to0.5Gy X-ray radiation, real-time PCR detected themRNA expression of IGF-1and membrane receptor IGF-1R. Western blot was used toinvestigate activity of Akt which was a key regulation molecular in IGF-1signalingpathway. Moreover, after irradiation, the changes in osteoblast differentiation, Wnt/-Catenin signaling, TGF-1/Smad2/3signaling were detected after IGF-1/Aktsignaling pathway was blocked by IGF-1R RNA interference. We successfully constructedIRS-1mRNA interference vector and established stable transfection clones. The resultsshowed that low dose X-ray acradiation activated IGF-1/Akt signaling pathway andpromoted the osteoblast differentiation at the same time. IRS-1gene silencing blockedIGF-1/Akt signaling. That also eliminated the effects of low dose X-ray radiation onosteoblast differentiation, mature and mineralization. At early stage after low dose X-rayradiation, IGF-1signaling adjusted Wnt signaling pathways by activating GSK-3, andthere had no significant effects on the expression level of phosphorylated Smad2whichwas an important molecular mediator in TGF-1signaling pathway. Thus it can be seenthat0.5Gy X-ray radiation promoted osteoblast differentiation and mineralization byactivating IGF-1/Akt signal pathway, and IGF-1/Akt signaling might be cross-talk withWnt signaling and TGF-1after low dose radiation.Together, our experiments revealed dynamic phenotypic expression changes inosteoblastic cells after X-ray radiation. Low-dose (0.5Gy) X-ray radiation had stimulatoryeffects on osteoblast proliferation, differentiation and fracture healing in vitro and vivo.Multiple signaling pathways including Wnt/-Catenin signaling, TGF-1/Smad2/3signaling and IGF-1/Akt signaling, mediated these stimulatory effects of radiation onosteoblast. Our findings provide a better understanding of low-dose radiation-inducedbiologic responses during bone formation and may lead to the development of improvedstrategies with translational applications for bone-related diseases in humans. |
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