TBO-PDT对大鼠口腔混合菌生物膜抑制作用的实验研究
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摘要
目的:探讨不同光敏剂浓度和不同光照时间的TBO-PDT对大鼠口腔混合菌菌斑生物膜的抑制效果,确定最佳光敏剂参数和时间参数,并在此基础上探讨TBO-PDT在抑制混合菌菌斑生物膜的作用优势,为PDT应用于龋病预防提供理论与实验依据。方法:以Wistar大鼠构建混合菌致龋模型。将其随机分组:A组为生理盐水阴性处理组;B组为洗必泰阳性对照组;其余组别根据光敏剂浓度的不同,分为C1组60μg/m L、D1组80μg/m L、E1组100μg/m L、F1组120μg/m L、G1组140μg/m L,避光孵育5min后用波长为630nm,输出强度为105m W/cm2的二极管激光器照射9min;根据照射时间的不同,分为C2组3min、D2组6min、E2组9min、F2组12min、G2组15min,光敏剂浓度100mg/L避光孵育5min后用波长为630nm,输出强度为105m W/cm2的二极管激光器照射;实验处理后取样,通过平板菌落计数法观察各组对大鼠口腔混合菌生物膜的抑制作用,原子吸收分光光度计测定光动力疗法作用后大鼠口腔混合菌生物膜的钙离子溶出量,评价TBO-PDT对大鼠口腔混合菌菌斑生物膜的抑制作用。结果:不同光敏剂浓度下TBO-PDT对大鼠口腔混合菌菌斑生物膜抑菌效果的影响。平板菌落计数结果显示:不同光敏剂浓度的各组抑菌率随光敏剂浓度的增加而增高。与阴性对照组相比,C1组、D1组、E1组、F1组和G1组的抑菌率显著提高,E1组、F1组和G1抑菌率高于阳性对照组,而F1和G1两组间抑菌率没有明显差异。不同光照时间下TBO-PDT对大鼠口腔混合菌菌斑生物膜抑菌效果的影响。平板菌落计数结果显示:不同光照时间的各组抑菌率随时间的增加而增高。与阴性对照组相比,C2组、D2组、E2组、F2组和G2组的抑菌率显著提高,E2组、F2组和G2抑菌率高于阳性对照组,而F2和G2两组间抑菌率没有明显差异。原子吸收分光光度计测定钙离子溶出量结果显示,在48h内,各组钙离子溶出量(ug/mL)随时间的延长而增加。与阴性对照组相比PDT组各时间点的钙离子溶出量均显著减小(P<0.05)。与阴性对照组相比PDT组与阳性对照组均能使钙离子溶出量减小(P<0.05),但二者之间无显著性差异(P>0.05)。结论:TBO-PDT对大鼠混合菌菌斑生物膜有明显的抑制作用,实验证实TBO-PDT最佳光敏剂浓度为100μg/mL,最佳光照时间为9min。光动力疗法能有效抑制大鼠混合菌菌斑生物膜中的致龋菌,可减少早期龋病的钙离子溶出量,不但阻止了釉质继续脱矿,而且促进了釉质的再矿化。PDT在龋病的预防中具有广阔的临床应用前景。
Objective: To Study the inhibitory effect of TBO-PDT in different photosensitizer concentration and different lighting time on the cariogenic bacteria in dental plaque biofilms of rats,and determine the optimal photosensitizer parameters and time parameters. On the basis of this,to discuss the advantages of TBO-PDT in inhibiting the biofilm of mixed bacterial plaque,and provided theoretical and experimental evidence for the application of PDT in the prevention of dental caries and to provid theoretical and experimental basis for the application of PDT for dental caries prevention. Method: The caries model was constructed with Wistar rats. All the rats in accordance with the principle of randomization were divided into groups as follows: Group A: a saline-negative treatment group; Group B: a positive control group for chlorhexidine. The other groups were based on the concentration of the photosensitizer as group C1: 60 mg/L,group D1: 80 mg/L,group E1:100 mg/L,group F1: 120 mg/L,group G1: 140 mg/L. Treated group C1-G1 with incubation for 5 minutes in dark place and exposed 9 min by a semiconductor laser with a wavelength of 630 nm. The output strength was105 m W/cm2. The rest groups were based on the exposure time: group C2: 3 min; group D2: 6 min; group E2:9 min; group F2: 12 min; group G2: 15 min. Treated group C2-G2 with incubation for 5 minutes in dark place and exposed by a semiconductor laser with a wavelength of 532 nm. The photosensitizer concentration was100 mg/L. The output strength was 105 m W/cm2. According to the different exposure time,it was divided into C2 group 3 min,D2 group 6 min,E2 group 9 min,F2 group 12 min,G2 group 15 min,and the photosensitizer concentration was 100 mg/L after incubating 5 min,and the output intensity was 105 diode laser. The inhibitory effect of TBO-PDT on oral mixed bacteria biofilm of rat oral mixed bacteria was evaluated by atomic absorption spectrophotometer,and the inhibitory effect of TBO-PDT on oral mixed bacterial plaque biofilm was evaluated by atomic absorption spectrophotometer. Resultes: The bacteriostatic effect of TBO-PDT on bacterial plaque in rat oral cavity was studied in different concentration of photosensitizer. The tablet colony count results showed with the increase of concentration of photosensitizer stress the bacteria inhibitive rate was increased.Compared to the negative control group and positive control group,bacteria inhibitive rate of group C1,group D1,group E1,group F1 and group G1 changed most obviously. Bacteria inhibitive rate of group E1,group F1 and group G1 were increased than positive control group. The bacteriostatic effect of TBO-PDT on bacterial plaque in rat oral cavity was studied in different exposure time. The tablet colony count results showed with the increase of exposure time the bacteria inhibitive rate was increased. Compared to the negative control group,bacteria inhibitive rate of group C2,group D2,group E2,group F2 and group G2 changed most obviously.Bacteria inhibitive rate of group E2,group F2 and group G2 were increased than positive control group. Results of the calcium ion dissolution detected by atomic absorption spectrophotometer showed with the increase of exposure time the calcium ion dissolution was increased in each group in 48 hours. Compared to the negative control group,the calcium ion dissolution of each group was decreased significantly( P < 0.05). Compared to the negative control group,the calcium ion dissolution of each group was decreased. However,there was no significant difference between them( P > 0. 05). Conclusion: TBO-PDT has obvious inhibitory effect on the biofilm of mixed bacteria plaque in rats. The results showed that the best photosensitizer concentration of TBO-PDT was 100 g/m L and the best illumination time was 9 min. Photodynamic therapy can effectively inhibit the cariogenic bacteria in the bacterial plaque biofilm of rats,reduce the amount of calcium ion dissolution in early caries,not only prevent the continued demineralization of the enamel,but also promote the remineralization of the enamel. PDT has broad clinical application prospects in the prevention of dental caries.
Objective: To Study the inhibitory effect of TBO-PDT in different photosensitizer concentration and different lighting time on the cariogenic bacteria in dental plaque biofilms of rats,and determine the optimal photosensitizer parameters and time parameters. On the basis of this,to discuss the advantages of TBO-PDT in inhibiting the biofilm of mixed bacterial plaque,and provided theoretical and experimental evidence for the application of PDT in the prevention of dental caries and to provid theoretical and experimental basis for the application of PDT for dental caries prevention. Method: The caries model was constructed with Wistar rats. All the rats in accordance with the principle of randomization were divided into groups as follows: Group A: a saline-negative treatment group; Group B: a positive control group for chlorhexidine. The other groups were based on the concentration of the photosensitizer as group C1: 60 mg/L,group D1: 80 mg/L,group E1:100 mg/L,group F1: 120 mg/L,group G1: 140 mg/L. Treated group C1-G1 with incubation for 5 minutes in dark place and exposed 9 min by a semiconductor laser with a wavelength of 630 nm. The output strength was105 m W/cm2. The rest groups were based on the exposure time: group C2: 3 min; group D2: 6 min; group E2:9 min; group F2: 12 min; group G2: 15 min. Treated group C2-G2 with incubation for 5 minutes in dark place and exposed by a semiconductor laser with a wavelength of 532 nm. The photosensitizer concentration was100 mg/L. The output strength was 105 m W/cm2. According to the different exposure time,it was divided into C2 group 3 min,D2 group 6 min,E2 group 9 min,F2 group 12 min,G2 group 15 min,and the photosensitizer concentration was 100 mg/L after incubating 5 min,and the output intensity was 105 diode laser. The inhibitory effect of TBO-PDT on oral mixed bacteria biofilm of rat oral mixed bacteria was evaluated by atomic absorption spectrophotometer,and the inhibitory effect of TBO-PDT on oral mixed bacterial plaque biofilm was evaluated by atomic absorption spectrophotometer. Resultes: The bacteriostatic effect of TBO-PDT on bacterial plaque in rat oral cavity was studied in different concentration of photosensitizer. The tablet colony count results showed with the increase of concentration of photosensitizer stress the bacteria inhibitive rate was increased.Compared to the negative control group and positive control group,bacteria inhibitive rate of group C1,group D1,group E1,group F1 and group G1 changed most obviously. Bacteria inhibitive rate of group E1,group F1 and group G1 were increased than positive control group. The bacteriostatic effect of TBO-PDT on bacterial plaque in rat oral cavity was studied in different exposure time. The tablet colony count results showed with the increase of exposure time the bacteria inhibitive rate was increased. Compared to the negative control group,bacteria inhibitive rate of group C2,group D2,group E2,group F2 and group G2 changed most obviously.Bacteria inhibitive rate of group E2,group F2 and group G2 were increased than positive control group. Results of the calcium ion dissolution detected by atomic absorption spectrophotometer showed with the increase of exposure time the calcium ion dissolution was increased in each group in 48 hours. Compared to the negative control group,the calcium ion dissolution of each group was decreased significantly( P < 0.05). Compared to the negative control group,the calcium ion dissolution of each group was decreased. However,there was no significant difference between them( P > 0. 05). Conclusion: TBO-PDT has obvious inhibitory effect on the biofilm of mixed bacteria plaque in rats. The results showed that the best photosensitizer concentration of TBO-PDT was 100 g/m L and the best illumination time was 9 min. Photodynamic therapy can effectively inhibit the cariogenic bacteria in the bacterial plaque biofilm of rats,reduce the amount of calcium ion dissolution in early caries,not only prevent the continued demineralization of the enamel,but also promote the remineralization of the enamel. PDT has broad clinical application prospects in the prevention of dental caries.
引文
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[2]常秀明,邹朝晖,阴慧娟,等.光动力疗法对牙菌斑生物膜内致龋菌的影响及作用机制的研究[J].口腔医学研究,2012,28(4):303~309.
[3]Souza L,Brito P,Oliveira J,et al.Photodynamic therapy with two different photosensitizers as supplement to instrumentation irrigation procedures in promoting intracanal reduction of Enterococcus faecalis.[J]Endod,2010,36(2):292~296.
[4]Michael Wilson.L et al photosensitization of oral bacteria and its potential application in the photodynamic therapy of oral in fections[J]Photochem Photobiol Sci,2004,3(5):412~418.
[5]Soukos NS,Wilson M,Burns T,et al.Photodynamic effects of toluidine blue on human oral keratinocytes and fibroblasts and Streptococcus sanguis evaluated in vitro[J].Lasers Surg Med.1996;18(3):253~259.
[6]Soares B M,da-Silva D L,Sousa G R et al.In vitro photodynamic in activation of Candida spp.growth and adhesion to buccal epithelial cells[J].Photochem Photobiol B,2009,94(1):65~70.
[7]许小辉,林居红,刘明方.光动力学疗法影响人工菌斑生物膜形成的扫描电镜观察[J]第三军医大学学报,2010,32(21):2314~2317.