| Objective:To evaluate the effects of low level laser irradiation(LLLI) on osteogenic differentiation of periodontal ligament cells in tension area during orthodontic tooth movement in rats, and on MAPKs signaling pathway during this process.Methods:1.Establishment of improved tooth movement models and LLLI 43 healthy male Wistar rats of 6-week-old were selected for the experiment. Three were for blank control, and both sides of the others maxillary molars were randomly divided into 4groups: group 0s,group 5s,group 10 s and group 20 s. The upper two incisors of the rats(except the rats in blank control group)were fixed as a whole secure anchorage to move the first maxillary molars mesially at an orthodontic force of 40 g. Low level laser(wavelength 650 nm,100mW) irradiation at energy densities of 15.92J/cm2, 31.84J/cm2 or 63.68J/cm 2 was applied towards the buccal, palatal and mesial mucosa of the experimental molars’ roots as soon as the tooth movement models were established.Irradiation was performed once a day from day 0 to 6. Rats in group 0s received no irradiation.2. Anesthetized 8 rats were killed on day 1, 3, 5, 7and 14 respectively using 4%neutral formaldehyde solution via heart perfusion. The maxilla containing the fist molar was made into sparaffin-embedded ections. We stained the selected sections with hematoxylin and eosin to count the numbers of osteoblasts on the tension side of the moved molrs. SPSS17.0 software was used to carry out statistical treatment.3.Paraffin section were taken for immunohistochemical staining. Under 400× light microscope, 5 regionins from each slice was selected randomly in the tension area of the periodontal ligament. Image-Pro Plus 6 image analysis software was used to measure the integrating optical density value. SPSS17.0 was used for statistical analysis to comparethe expression of ALP and Runx2 among group 0s, group 5s and group 10 s, as well as p-ERK1/2 and p-JNK among group 0s and group 5s. The correlation analysis was also done.Results:1.In tension area, on day 3, we found the number of osteoblasts increased significantly in four groups. There were more osteoblats in group 5s and 10 s than that in group 0s and 20 s, and there was statistical significance(P < 0.05); On day 5,the number of osteoblasts in each group further increased, group 5s>group 10 s >group 20 s >group 0s;osteoblasts of group 5s increased the fastest,while group 0s the slowest; no statistical significance was found between group 0s and group 20s(P=0.165),but between the other groups. On day 7, mean numbers of osteoblasts in group 5s and group 10 s reached their peak, group 5s>group 10 s, and there was statistical significance between them(P < 0.05).Group 0s and group 20 s grew fast on day 7 day 14; osteoblasts in group 5s and group 10 s began to decrease, while group 0s and group 20 s reached the maximum, but still lower than that of group 5s and group 10s; osteoblasts number was sill group 5s>group10s >group 20 s >group 0s.There was statistical significance between group 5s, group 10 s and group 0s(P < 0.05); group 5s, group 10 s and group 20 s had no statistical significance compared to each other(P < 0.05).2. ALP was observed in the cytoplasm of osteoblasts and cementoblasts,as well as intercellular substance. On day 3 and day 5, group 0s expressed more ALP than the other days and the most on day 5. Group 5s and group 10 s expressed much more ALP on day 1,3 and 5. And like group 0s, group 5s expressed the most on day 5, while group 10 s expressed the least. The difference between them was statistically significant(P<0.05).Runx2 mainly expressed in dental pulp cells and periodontal ligament cells. On day 1,3,5and 7, expression of Runx2 in three groups increased, and decreased on day 14. IOD values of Runx2 of three groups had little difference On day 3 and day 5. On day 5, IOD values of Runx2 of group 0s and 5s were higher than that of group 0s(P<0.05);no significant difference between group5 s and group 10 s. On day 7 and 14, IOD values from largest to smallest were:group 5s >group 10s>group 0s, but the difference was not statistically significant on day 7 opposite to that on day 14.3. The p-ERK1/2 and p-JNK mainly expressed in the nucleus and cytoplasm of periodontal ligament cells. The p-ERK was expressed much more on day 5 than that on day 1 and day 3. Group 5s expressed the most on day 5, while group 0s on day 7. On day14, expression of p-ERK in both groups was significantly decreased. IOD values of group 5s were higher than that of group 0s on day 3 and 5, and the difference was statistically significant. On day 1, 3 and 14, p-JNK expressed little in group 0s, and morein group 5s. On day 5, positive staining became deeper; in group 5s, p-JNK expressed most on day 7, and group 0s on day 5. on day 1,3,7,14, IOD values of group 5s were larger than that of group 0s with statistical significance(p<0.05).4.There was a positive correlation between the expression of ALP, Runx2 and p-ERK1/2(r=0.399, 0.799, P=0.024,0.000<0.01); Runx2 was positively correlatad with p-JNK(r=0.794, P=0.000<0.01); no significantly correlation were observed between ALP and p-JNK(r=0.262, P=0.148>0.05).Conclusions:1. LLLI at energy density of 15.92J/cm and 31.84J/cm promotes periodontal tissue remodeling and induces osteogenic differentiation of periodontal ligament cells on the tension side during orthodontic tooth movement in rats; and the role of the former is more obvious.2.LLLI at energy density of 15.92J/cm and 31.84J/cm can enhance the expression of ALP, Runx2 in the tension area of the periodontal ligament of the moving tooth.; and the role of the former is more obvious.3.LLLI at energy density of 15.92J/cm promotes the expression of p-ERK1/2, p-JNK by periodontal ligament cells under tension strain. ERK1/2 and JNK may both be involved in periodontal tissue remodeling and osteogenic differentiation of periodontal ligament cells induced by orthodontic force under LLLI. |
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