Various clinical and experimental investigations showed that irradiation could affect osteoblastic activity, including proliferation decrease, cell cycle arrest, increased sensitivity to apoptosis, and reduce of osteoblast differentiation, which was tightly associated with fracture union delay and osteoradionecrosis. However, radiation dose selected above was mainly moderate or high [16
]. This study focused on effects of low dose X-irradiation on proliferation, differentiation and mineralization of osteoblasts (MC3T3-E1 cells) in vitro.
Above all, we tried to determine the nontoxic doses of radiation on MC3T3-E1 osteoblastic cells. We found that cell proliferation measured by MTT and BrdU was significantly decreased when cells were exposed to 2.0
Gy, which is consistent with some other studies [25
], ruling out the possible severe toxicity of radiation used in the present study at low dose X-irradiation (≤ 1
Cell proliferation dropped significantly on Day 8. Coincidently, we found that cell apoptosis increased instead at this time. Furthermore, we considered that cell reached confluence and contact inhibition of cell-to-cell had initiated at this stage. On the other hand, cell proliferation decreased in 0.5
Gy and 1.0
Gy groups on Day 8. MC3T3-E1 cells showed alkaline phosphatase activity here and began to differentiation. Some studies reported that proliferation activity was inversely related to differentiation of osteoblasts [27
]. Thus, we supposed that proliferation decrease might be associated with their promoting cell differentiation.
It was widely accepted that ALP was the early phenotypic marker and in accordance with the differentiation of osteoblasts [29
]. Formation of mineralized nodules was the ultimate expression of the osteogenic phenotype in vitro and the characteristic marker of mineralization [31
]. Our results showed that ALP activity and mineralized nodules exposed to 0.1
Gy were not statistically different from those of nonirradiated group, which may represent a threshold effect for X-ray irradiation. But in LDI of 0.5
Gy and 1.0
Gy groups, ALP activity and mineralized nodules were positively correlated with the dosage of irradiation.
To further ascertain the effects of LDI on differentiation of MC3T3-E1 cells, we examined the expression of genes and proteins associated with osteoblastic differentiation. OCN was generally considered as a late marker in the mineralization stage, which bond with calcium and hydroxyapatite closely [32
]. Our results showed that OCN expression was increased in 0.5 and 1.0
Gy groups compared with that of control. We also analyzed gene expression of collagen I. Surprisingly, no change was found in collagen I (data not shown). Wang reported that MC3T3 subclones with both high and low differentiation potential produced similar amounts of collagen in culture [34
]. Variety of researches demonstrated that osteoblasts expressed the nuclear protein Cbfα1, which could act as a transcriptional factor and bind with certain cis-acting elements of OCN genes to further enhance their transcriptional activities [35
]. The skeletal systems of the mice with a homozygous mutation in Cbfα1 showed a complete lack of ossification [37
]. Cbfα1-deficient calvarial cells did not acquire osteoblastic phenotypes [38
]. Thus, Cbfα1 was a critical gene not only for osteoblast differentiation but also for osteoblast function. Consequently, X-irradiation of 0.5
Gy and 1.0
Gy could increase the expression of Cbfα1, which might further activate the transcriptional activities of OCN in MC3T3-E1 cells.
Taking all results into consideration, low dose X-irradiation promoted differentiation of osteoblasts, but without impairing proliferation. As multipotential cells, mesenchymal stem cells (MSCs) could be induced into osteoblasts and had been long taken as important subjects of research. Among studies of moderate and high dose irradiation, some showed that radiation mainly suppressed the proliferation or cell cycle progression [39
], while some showed that only the process of differentiation was suppressed [40
], as well as some showed that proliferation and differentiation of MSCs were both suppressed [42
]. Multipotential cells are heterogeneous in differentiation potential and comprise both progenitors and relative mature cells. These controversial conclusions may be associated with MSCs themselves besides the diversity of radiation dose and research models.
Few available literature described the effects of low-dose irradiation on osteoblasts in vitro. Dare [44
] reported that ≤400
mGy of X-irradiation had no significant changes in proliferation and differentiation of MC3T3-E1 cells. Ahmad [26
] showed ≤2
Gy of 137Cs irradiator had no effects on proliferation and ALP activity of human fetal osteoblast 1.19 cells. Kurpinski [45
] reported that 1
Gy X-ray perturbed DNA replication and DNA binding activity of MSCs, without impairing their osteogenic differentiation process in vitro. The discrepancy needed further study and the difference in radio sources, cell types and timing was also undoubtedly important like Kurpinski and Jin reported [45
Interestingly, irradiation also induced terminal differentiation in some other culture systems, such as, human skin fibroblasts [47
] and erythroid progenitor cells [48
]. While some reported ionizing radiation greater than 2
Gy promoted osteoblasts terminal differentiation [18
]. Different from the effects of moderate and high dose irradiation, LDI had no impacts on the process of proliferation of osteoblasts in our study. We thought that increasing differentiation of osteoblasts by LDI might be regarded as a kind of promotion of tissue repair on the condition that osteoblasts have the normal ability of proliferation. Furthermore, the more differentiated osteoblasts can help callus formation and callus calcification in vivo.