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80Justyna Szymańska et al.gle, double and triple exposure to radiation wasapplied.First five experiments (1-5) revealed 15 thousandcells within a single hole, 10 thousand in 6 th experiment,5 thousand in 7 th and 8 th experiment. There werefreshly isolated cells used in the experiments, exceptfor the 6 th sample in which the cells were previouslyfrozen. The cells of 1 st -4 th and 6 th experiment weresubjected to single irradiation. In 7 th experiment thecells were irradiated three times, two times in case of5 th and 6 th experiment. The interval between exposuresto radiation amounted to 24 hours. The laser irradiationparameters applied in the experiments are presented inTable I. Using the colorimetric detection set of livingcells, the proliferation of investigated group (subjectedto irradiation) as well as the control group was determined.The cells proliferation was denoted indirectlyby means of determining the absorption value measuredby ELISA factor.Achieved absorption values are directly proportionalto the number of living cells. The final relativepercentage values were received after correlating particularresults with mean value achieved within thecontrol group.Table 1. Laser irradiation parameters applied in the experimentsNumber Wavelenght Power Eds – Power DensityExperiment1 660 nm 40 mW 1 - 5.5 J/cm 2Experiment 2 670 nm 20 mW 0.28 – 5.71 J/cm 2Experiment 3 670 nm 20 mW 2.86 - 11.43 J/cm 2Experiment 4 670 nm 10mW i 20 mW 2.86 - 11.43 J/cm 2Experiment 5 670 nm 20 mW 2 - 8 J/cm 2Experiment 6 670 nm 10mW i 20 mW 2 - 8 J/cm 2Experiment 7 820 nm 100 mW 2 - 8 J/cm 2Experiment 8 808 nm 1500 mW 2 - 8 J/cm 2RESULTSThe experiments were to be carried out in a form ofeight examinations. There were three trials of irradiationof energy dose of 1-5.5 J/cm 2. power of 40 mWand wavelength of 660 nm introduced in the first experiment.The results are shown in Table II. It wasaffirmed that each energy dose by the constant powerof 40mW, stimulates the cells proliferation, in comparisonto the control group which was not subjected toirradiation. The most significant increase of proliferation(196.6%) was observed at the energy dose of 4.5J/cm 2. , the slightest proliferation (104%) was achievedby the energy dose of 5.5 J/cm 2 in the case of the controlgroup.Table II. Results of experiment 1Tabela II. Wyniki eksperymentu 1Parameters40 mWEds [J/cm 2 ] 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5t [s] 10 15 20 25 30 35 40 45 50 55Test 1 0.84 0.739 0.939 0.891 1.01 0.978 1.021 0.892 1.301 0.853Test 2 1.663 1.655 1.039 1.789 1.091 1.776 1.784 1.911 1.144 0.314Test 3 0.977 1.781 1.279 1.723 1.572 1.412 1.423 1.99 1.611 1.365Medium 1.16 1.392 1.086 1.468 1.224 1.389 1.409 1.598 1.352 0.844Control 1.265 0.857 0.767 0.751 0.748 0.796 0.871 0.736 0.681 0.654% 142.8 171.3 133.6 181 151 171 173.4 196.6 166.4 103.9There were five trials of irradiation with energydoses within the range of 0.28 – 5.71 J/cm 2 and powerdecreased by a half (20 mW) and wavelength of 670nm introduced in the second experiment. The resultsare shown in table III. In the case of energy dose of0.28 J/cm 2. the proliferation was not as significant as itwas noticed in the control group, which was not subjectedto irradiation. Energy doses of 1.43 J/cm 2 and5.71 J/cm 2 produced not large increase of proliferationof cells, which was more significant at the energy doseof 5.71 J/cm 2 . After decreasing power of laser irradiation,the proliferation of cells decreased significantly.Table III. Results of experiment 2Tabela III. Wyniki eksperymentu 2Parametry20 mWEds [J/cm 2 ] 0.28 1.43 5.71 Controlt [s] 5s 25s 100sTest 1 1.82 1.95 2.021 1.876Test 2 1.915 1.943 2.101 2.06Test 3 1.835 2.043 2.025 1.801Test 4 1.664 1.922 1.948 1.942Test 5 1.389 1.75 1.944 1.784Medium 1.72 1.92 2.01 1.89Control % 91.25 101.67 106.23p NS NS NSp – statistical importanceNS – non importantBasing on the results of the previous examinations,the energy intensity increased from 2.86 to 11.43 J/cm 2in the third one, with the same power as well as thewavelength. Four trials of irradiations were carried out.The results are shown in Table IV. The application ofenergy dose of 2.86 J/cm 2 caused the decrease of proliferationwith reference to the control group, which
Effect of Low Level Laser Therapy and High Intensity Laser Therapy on endothelial cell proliferation in vitro... 81was not subjected to irradiation. Energy doses of 5.71J/cm 2 and 11.43 J/cm 2 caused not a large increase ofthe cells proliferation. Achieved results were similarand amounted in turn to about 104% and 107%. So, theincrease of energy intensity by the constant power didnot influence the increase of proliferation – the resultscame out to be worse than it was noticed in the firstexperiment. The change of wavelength from 660 nm to670 nm may also modify the stimulation of epitheliumcells.Table IV. Results of experiment 3Tabela IV. Wyniki eksperymentu 3Parameters20 mWEds [J/cm 2 ] 2.86 5.71 11.43 Controlt [s] 50s 100s 200sTest 1 1.083 1.566 1.661 1.311Test 2 1.257 1.573 1.509 1.624Test 3 1.314 1.596 1.781 1.773Test 4 1.339 1.642 1.589 1.407Medium 1.248 1.594 1.635 1.529Control% 81.64 104.27 106.93p 0.0119 NS NSThere were five trials of irradiation of energy doseswithin the range of 2.86-11.43 J/cm 2. power of 10 mWand 20 mW and the wavelength of 670 nm introducedin the fourth experiment. The results are shown inTable V.Table V. Results of experiment 4Tabela V. Wyniki eksperymentu 4Parameters 10 mW 20mWEds [J/cm 2 ] 2.86 5.71 11.43 Control 2.86 5.71 11.43t [s] 100s 200s 400s 50s 100s 200sControlTest 1 1.056 1.247 1.331 1.36 0.763 1.453 1.588 0.848Test 2 1.355 1.792 1.711 1.293 1.373 1.86 1.846 1.297Test 3 1.122 1.78 1.763 1.072 1.412 1.84 1.99 1.267Test 4 0.935 1.926 1.86 1.017 1.451 1.827 1.53 1.096Test 5 - 1.668 1.814 - 1.367 1.673 1.671 0.939Medium 1.117 1.6826 1.6958 1.1855 1.2732 1.7306 1.725 1.0894Control % 94.22 141.93 143.05 116.87 158.86 158.34p NS 0.037 0.0039 NS 0.005 0.004The application of energy dose of 2.86 J/cm 2 and apower of 10 mW caused the decrease of proliferationwith reference to the control group. However the sameenergy dose but the power of 20 mW caused the increaseof cells proliferation – 117% in comparison tothe control. The results were not as significant as it wasnoticed at the power of 40 mW. Energy doses of 5.71J/cm 2 and 11.43 J/cm 2 caused the increase of the cellproliferation by the power of 10 mW as well as 20mW. However, best result was achieved in case of 20mW – 160% of control group. So, the number of cellswithin the culture exposed to radiation increased alongwith the increase of the power of laser irradiation.Another examination (5) was anticipated to be carriedout in a form of double irradiation of the cell culture,using the energy doses within the range of 2-8J/cm 2 with a one-day interval. The results are shown inTable VI.Table VI. Results of experiment 5Tabela VI. Wyniki eskperymentu 5Parameters20 mWEds [J/cm 2 ] 2 4 8 Controlt [s] 35s 70s 140sTest 1Test 2Test 3Test 4Test 5Test 6Medium1.657 1.531 1.912 1.2751.801 1.792 1.926 1.5281.952 1.574 1.771 1.6121.553 1.755 1.671 1.5711.517 1.382 1.547 1.7631.475 1.219 1.097 1.111.659 1.542 1.654 1.477Control %112.37 104.45 112.02p NS NS NSThe results were similar to those from examinationno. 3. i.e. the one with comparable intensity doses ofenergy but single exposure to irradiation. So, the influenceof double irradiation on the increase of the cellsproliferation of HUVEC line was not revealed. Themost significant increase of proliferation (112%) wasobserved at the energy dose of 2 J/cm 2 and 8 J/cm 2 .The application of energy dose of 4 J/cm 2 caused theincrease of cell proliferation, which amounted 104% incase of the control group.There were six trials of irradiation of energy doseswithin the range of 2-8 J/cm 2. at the power of 10 mWand 20 mW performed in the sixth experiment, howeverthe researches were carried out using frozen cells.The results are shown in Table VII. A significant differencewithin the living cell culture after the terminationof test was observed – the number of cells decreased.Decreased number of cells used in the experimentsto 10 000 – greater distances between cells andsmaller possibility of interaction – could influence theresults. It was the only test on cells which were previ-
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