Effects Of Low-dose X-irradiation On Callus Formation In Fracture Healing And Osteoblast-like Cells In Vitro

Ionizing radiation is a common therapy in the treatment of cancer, especially the headand neck carcinomas. Despite of its therapeutic value, osteoradionecrosis is considered tobe one of the most serious clinical complications after radiation therapy. Classical theoriesbelieved that the pathogenesis of osteoradionecrosis is associated with the progression ofirradiation, trauma, infection and compromised vasculature. Recently, increasingresearches indicated that alterations of osteoblasts activity, including proliferation, cellcycle arrest, increased sensitivity to agents that induce apoptosis, and reduces collagenproduction might play important roles in the process of osteoradionecrosis. As we known,the dosage of ionizing radiation is usually moderate or high in the application of cancertherapy. However, in clinic, patients with fractures should receive CT scan before surgeryand fluoroscopy during operation, which means that injured bone tissues often wereexposed to low-dose irradiation (LDI). To our knowledge, LDI had several biologic effectsof increasing expression of vascular endothelial growth factor (VEGF) and mobilization ofprogenitor cells. Whether LDI could also affect osteoblasts so as to change the healing andremodeling processes of bone tissue is relatively little concerned to date. Our preliminarydata showed that LDI could promote callus mineralization in Sprague-Dawley rat model. Itwas generally considered that ordered proliferation and differentiation of osteoblast wasindispensable for mineralization of extracellular matrix in bone formation during woundhealing. In the light of these, the major objective of this study was to examine whether andto what extent LDI could influence the proliferation and differentiation of osteoblastic-likecell in vitro and callus formation in fracture healing. As a consequence, our findings wouldbe beneficial for further understanding the potential roles of LDI on healing andremodeling of bone in fracture healing and the underlying cellular and molecularmechanisms.There are four major parts in this research．1. Construct the model of osteoblastic-like cell culture in vitro In order to harvest abundant vital osteoblasts, MC3T3-E1subclone14, a clonalosteoblastic cell line, was cultured in a-MEM,10%FBS,5mM β-glycerophosphate,50μM L-ascorbic acid and100nM of dexamethasone. The cell line was identified by alkalinephosphotase staining, Von Kossa staining of mineralized nodule and expression of themarker associated to cell differentiation by RT-PCR and western blot. The results showedcell staining of ALP and Von Kossa was positive and expression of osteocalcin was found.MC3T3-E1subclone14cells can differentiate along the osteoblast pathway and expressmineralization, which met the experimental need.2. Effects of low-dose X-irradiation (LDI) on the proliferation and differentiationof MC3T3-E1cells in vitroThe clonal osteoblastic cell line (MC3T3-E1) were exposed to single dose ofX-irradiation with0(the control),0.1,0.5and1Gy respectively. The cultures wereevaluated in terms of methylthiazoletetrazolium (MTT) assay, bromodeoxyuridine (BrdU)incorporation, flow cytometery, annexin V-FITC/PI apoptosis detection, assay of ALPactivity and ALP staining, Von Kossa staining, gene and protein expression of some factorsrelated to differentiation using real-time PCR and western bolt. Experimental findingsrevealed that activities of proliferation, cell cycle and apoptosis were not significantlyaffected by LDI. Although no simple linear relationship between the irradiation dose anddifferentiation of MC3T3-E1cells existed, LDI of0.5and1Gy increase ALP activities andmineralized nodules in MC3T3-E1cell line(p<0.05). Expressions of collagen type I,osteocalcin (OCN) and core-binding factor α1(Cbfα1) in0.5and1Gy groups wereincreased and had significant difference compare with the control group (p<0.05). Inaddition, the ratio of OPG/RANKL mRNA expression in0.5and1Gy groups wassignificantly increased with OPG up-regulated and RANKL down-regulated. Cellsdifferentiation in0.1Gy groups had no significant difference compared with the control.The study indicated that LDI of0.5and1Gy could enhance the differentiation ofMC3T3-E1cells in vitro.3. Effects of low-dose X-irradiation on callus formation and osteogenic marker infracture healing128male Sprague–Dawley rats were subjected to standard closed fracture on rightfemur shaft. Rats were averagely divided into four groups (32per group) and local area offracture was exposed to single dose of X-irradiation with0(the control group),0.1,0.5and1Gy respectively.8rats of each group were sacrificed and the fracture specimens were collected at day10, week2, week3, week4after operation. Four specimens from eachgroup were fixed in formalin and decalcified in EDTA. Then, the specimens wereembedded in paraffin, and sectioned at a thickness of5μm. The sections were stained usinghematoxylin and eosin for observation of callus and cartilage and stained usingimmunohistochemisty for observation of the expression of OCN and Cbfα1. Geneexpression of ALP, collage I, collagen II, OCN, Cbfa1, OPG, RANKL was evaluated usingother four specimens by real-time PCR. Experimental findings revealed that LDI of1.0Gyincreased callus area and accelerated endochondral bone formation. By week4, morecallus and the medullary cavity of the callus site were found. Cartilage tissue wasessentially absent in the callus in1.0Gy group compared with the control group.Immunohistochemistry of callus sections revealed that positive expression of OCNgradually increased over time. More cells of OCN positive staining in0.5Gy and1.0Gygroups could be seen compared with the control and0.1Gy group (p<0.05). Expression ofCbfα1in fracture site continuously increased and peaked at3weeks, then decreased at4weeks. At2and3weeks, cells of Cbfα1positive staining in0.5Gy and1.0Gy were morethan those of control groups (p<0.05). Cells of positive staining in0.5Gy and1.0Gy groupswere less than those in control groups at4weeks (p<0.05). Quantitative real-time PCRanalysis showed the expression of collagen II mRNA associated with endochondral boneformation in the fracture callus tissue were significantly higher in1.0Gy group at2and3weeks. There were some discrepancies of gene expression involved in osteoblastdifferentiation in the callus tissue. Consistently, the expression of genes was notsignificantly difference among LDI and control groups on day10. And expression of ALP,collage I, OCN, Cbfa1peaked earlier and was significantly higher in0.5Gy and1.0Gygroups compared with the control at2and3weeks. The study indicated that LDI of0.5and1Gy accelerated callus formation and osteoblast differentiation in the callus tissue offractures4. Effects of1.0Gy X-irradiation on gene expression profiling of MC3T3-E1cellsin vitroIn 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 irradiated1.0Gy X-irradiation. Cells withoutirradiation were regarded as control group. Cells were cultured for14days after irradiationand collected. Total RNA of six samples (3per group) are extracted and purified by QIAGEN'S RNAeasy mini kit. RNA integrity was assessed by standard denaturing agarosegel electrophoresis. About5μg total RNA of each sample was used for labeling and arrayhybridization as the following steps:1) Reverse transcription;2) ds-cDNA labeling;3)Array hybridization and wash;4) Array scanning. Scanned images were then imported intosoftware and expression data were analyzed. Differentially expressed genes with statisticalsignificance were identified through Volcano Plot filtering and the threshold is FoldChange>=1.3, P-value <=0.05. Pathway Analysis and GO analysis were applied todetermine the roles of these differentially expressed genes played in these biologicalpathways or GO terms. Finally, Hierarchical Clustering was performed to showdistinguishable gene expression profiling among samples. We identified740differentiallyexpressed transcripts in1.0Gy group compared with the control. Among the identifiedgenes,384transcripts were up-and356transcripts were down-regulated after irradiation.These identified transcripts were further analyzed by Pathway Analysis and GO analysis.The overrepresented genes were associated with osteoblast differentiation and ossification.Many genes belonged to focal adhesion, extracellular matrix space, cytoskeletal proteinbinding and angiogenesis. Some transcripts possessing growth factor activity such asepidermal growth factor receptor, fibroblast growth factor, insulin-like growth factor werestatistically overpresented. The study indicated1.0Gy X-irradiation promoted some genesof osteoblast differentiation up-regulated which might be involved extracellular matrix,actin cytoskeleton, focal adhension and growth factor activities.In conclusion, this research focused on effects of low-dose X-irradiation (LDI) oncallus formation in fracture healing and further explored effects of LDI on proliferation,differentiation and signal pathway of osteoblastic cells on cellular and molecular level invitro. The results showed LDI of0.1Gy didn't accelerate callus formation and had noeffect on proliferation and differentiation of osteoblasts, which may represent a thresholdeffect for x-ray irradiation. But LDI of0.5and1.0Gy promoted callus formation anddifferentiation of osteoblasts. The study provided theoretical basis to elucidate themechanism of LDI on bone.