摘要
背景:
牙周炎可造成牙龈炎症、牙周袋形成、牙槽骨吸收以及牙齿松动,最终可导致牙齿脱落。据世界卫生组织(WHO)的调查表明牙周病的患病率高达75%,我国某些地区更高达93%。现已证实牙菌斑中细菌及其产物是牙周病的始动因子,尤其是龈下菌斑与牙周组织的破坏关系密切。对于牙周病发生、发展有关的特异性细菌的控制是牙周病治疗的关键。在各种治疗方法中,激光作为近四十年来发展起来的一种新型光源,具有普通光源无可比拟的特点:如亮度高、单色性、方向性和相干性好等。从1960年第一台红宝石激光问世以来,激光已经被广泛应用于医学的各个领域。在口腔医学使用的各类型激光中,Nd:YAG激光的应用最为广泛。使用脉冲Nd : YAG激光,由光纤传导进入牙周袋内,光能转换成热能,凝固及汽化上皮衬里及部分炎症浸润的软组织。又由于激光被血红蛋白特异性吸收,胶原纤维蛋白变性及胶原链收缩,毛细血管封闭,因而术区出血少或不出血,术野清楚。激光照射创口甚小,不用切开牙龈和翻瓣,加上激光有电磁场、生物刺激等效应,使术后反应很轻,术后牙龈退缩不明显,患者乐于接受。然而脉冲Nd:YAG激光治疗牙周炎尚缺乏深入、系统的研究。
目的:
本研究首先通过体外实验比较5种不同剂量脉冲Nd:YAG激光照射对人牙周膜成纤维细胞的增殖作用,选出有效的实验剂量,为脉冲Nd:YAG激光对慢性牙周炎的治疗供重要理论依据。建立慢性牙周炎动物模型,探讨5种不同剂量参数脉冲Nd:YAG激光用于牙周炎治疗的有效性和安全性,并获得激光对牙周组织作用的有效方式、激光的适合剂量参数,探讨脉冲Nd:YAG激光在临床应用的适用性和可靠性,以期为该激光在临床慢性牙周炎治疗中的应用提供理论和实验依据。
材料和方法:
1、HPDLFs的培养、鉴定及不同剂量激光照射对HPDLFs增殖力和形态的影响酶消化法培养人牙周膜成纤维细胞,利用免疫组化ABC法对原代培养的HPDLFs进行波形丝蛋白和角蛋白多克隆抗体染色,作细胞来源鉴定。取生长良好的第5代HPDLFs,在含10﹪胎牛血清的DMEM培养液中培养24 h后,1.0W 20pps组、1.5W 20pps组、2.0W 20pps组、2.5W 20pps组和3.0W 20pps组激光照射,正常对照组只进行常规培养,72 h后MTT法用酶标仪检测各组OD值,评定激光对细胞增殖的影响。将不同激光剂量照射的96孔板HPDLFs ,每孔加入100μl 0.2% TritonX-100 ,考马斯亮兰染色,检测OD值,探讨该激光对HPDLFs蛋白含量的影响。取在标准环境下培养24 h的96孔培养板HPDLFs,根据分组进行激光照射后,每孔加入1μCi3H-TdR,在液闪记数仪上测量每分钟闪烁记数(cpm),评定激光对HPDLFs DNA合成的影响。
2、将180～220 g,雌雄各半,健康的SD大鼠采用牙颈部结扎法进行建模,8周后随机取8只模型大鼠和正常大鼠,观察检测各项牙周指标,判断建模是否成功。将建模成功的48只SD大鼠随机分为6组,另外8只健康SD大鼠作为正常对照组,在牙周洁治的基础上按照1.0W 20pps、1.5W 20pps、2.0W 20pps、2.5W 20pps、3.0W 20pps实验剂量给予激光照射。末次治疗24 h后处死大鼠,对比各组大鼠的实验选用牙牙周指标(牙周袋深度PD,菌斑指数PLI,牙龈指数GI )及牙周组织中SOD、MDA、NO水平,验证脉冲Nd:YAG激光在SD大鼠牙周袋内的治疗作用。3、建立慢性牙周炎动物模型,先取24只随机分为:牙周炎对照组(P组)、激光治疗组(L组)、药物治疗组(D)组,另取8只大鼠作为正常对照组(N组)。建模成功后P组、L组、D组进行牙周洁治,L组给予2.0W 20pps剂量激光照射,D组按照200 mg/kg的剂量给SD大鼠灌胃甲硝唑。末次治疗24 h后,测量牙周指标并收集实验选用牙GCF上机检测,各组间进行比较。治疗结束30 d后取实验选用牙的牙周组织病理切片观察组织的愈合情况。
结果:
1、酶消化法原代培养HPDLFs,免疫组化染色表明,波形丝蛋白染色阳性,角蛋白染色阴性,这表明为来自中胚层的HPDLFs。经单因素方差分析的结果显示脉冲Nd:YAG激光剂量为1.0W 20pps-2.0W 20pps时,与正常对照组比较能明显促进HPDLFs增殖,且激光剂量参数为1.5W 20pps时作用效应最大。N组及1.0W 20pps-2.5W 20pps剂量组蛋白含量明显高于3.0W 20pps剂量组(P < 0. 01),其中2.0 W 20pps剂量组的蛋白含量最高。与N组比较1.0W 20pps-2.5W 20pps剂量组DNA显著增高, 3.0W 20pps剂量组有所降低(P < 0. 01)。
2、在脉冲Nd:YAG激光治疗牙周炎动物体内实验中,激光照射剂量为1.5W 20pps-2.0W 20pps时可使牙周组织SOD活性、MDA含量达到正常水平,NO含量接近于正常对照组的水平。同时在此剂量范围内在大鼠牙周袋内进行提拉式扫描照射能有效减少牙周袋的深度,降低PLI、GI指数。
3、在脉冲Nd:YAG激光与药物对比治疗慢性牙周炎动物体内实验中激光组(L组)、药物组(D组)的牙周各项指标较牙周炎对照组均有所好转。治疗后L组龈沟液中GSH-Px活性高于P组( P < 0. 05 ),与N组及D组无显著性差异( P > 0. 05 )。治疗后L组龈沟液中ALP活性高于N组低于P组( P < 0. 05) ,与D组无显著性差异( P > 0. 05 )。在镜下,L组与D组相似,与牙周炎组(P组)比较,L、D组牙周软组织炎症细胞浸润明显减轻,大量成骨细胞生成,牙周袋变浅,牙槽骨破坏处于静止状态。
结论:
1、1.0W 20pps-2.0W 20pps激光剂量照射能促进HPDLFs增殖,提高蛋白质含量,可见脉冲Nd:YAG激光能促进HPDLFs的增殖而起到治疗牙周炎的作用。
2、动物体内实验部分表明激光剂量为1.5W 20pps-2.0W 20pps时对大鼠慢性牙周炎能起到较好的治疗效果。综合实验各项指标初步确定该激光牙周袋内作用的有效、安全剂量为2.0W 20 pps。
3、激光与药物治疗对比实验表明2.0W 20 pps剂量激光照射能达到药物治疗的效果,能有效的降低牙周组织的炎症程度和破坏程度。
Background
Periodontitis is one of the common diseases of mankind, and the pathogenic factors of this chronic disease are always complicated. Periodontitis can cause gingival inflammation, periodontal pocket formation and alveolar bone resorption, as a result the affected teeth become mobility and eventually fall off. According to WHO investigations, the overall morbidity of periodontal disease is 75%. In some parts of China, this rate can be as high as 93%. It has been proved that bacterium and its products are the causes of parodontopathy in bacteria plaque, and subgingival plaque is closely related to the destruction of periodontal tissues. Control of specific bacteria relating to the occurring and developing of parodontopathy is the key approach to effective treatment. Fortunately, the pulsed Nd:YAG laser was introduced in 1960. Studies have revealed that the laser had the abilitities of solidify and vaporize both the endepidermis and the inflammatory infiltration soft tissue. Because of the laser can be specificity merging by hemoglobin, fibroglia fibrils fibrinous degeneration , collogen chain shrinkage at the same time seal off the micrangium so by this way it can reduce bleed or not bleed . The laser profit a lot to the patients who suffer from periodontopathy due the good quality of it’s electromagnetic field and biostimulation effect there is only lightly reaction after the gingivoectomy or gingival flap operation.The wound of entry of the laser operation is tiny most of the patients can accept . But these studies were more in vitro experiments in which irradiation parameters and experimental condition varied greatly and provided little information for in vivo application. Few investigation at present has been found on the safety and feasibility in clinical treatment.
Objective
This study is to obtain an effective energy by comparing the generation effect of 5 different laser energy levels radiation on HPDLFs by cells experiment, and thus to provide theoretical support for the chronic periodontitis therapy by the pulsed Nd:YAG laser. Establish experimental periodontitis animal model similar to periodontitis of mankind in SD rats to approach the 5 energy levels of the pulsed Nd:YAG laser was used to establish the relationship between energy parameters irradiation in periodontal pocket, their safety to periodontal tissue, then the proper parameters may obtain and apply for chronic periodontitis therapy to evaluate the feasibility, reliability and superiority in the healing of periodontitis
Materials and methods
1. Cultivation, verification the HPDLFs and the effect of the laser radiation on HPDLFs .
Taking use of immunohistochemistry ABC method to human periodontal ligament fibroblast to colour the undee silk protein and cell keratin monoclone antibody coloration and identify the origin of cells .Choosing the five generation well growth ligament fibroblast and then confect hang cell solution at 3×104/L concentration. Cultivation the cells 24h by putting into DMEM which contained 15﹪fetal calf serum .Then radiation on HPDLFs on the energy levels of .OW 20pps,1.5W 20pps,2.0W 20pps,2.5W 20pps and 3.0W 20pps , normal control conventional culture. Taking use of empirical method MTT to test the OD value of each group to evaluate effects of the laser on multiplication of cells . Taking use of 96 holes with the cells have been radiation on by laser ,putting 100ul 0.2% TritonX-100 into each hole. Putting over 20ul solution from each hole into 200ul dying fluid, keeping on surging 10 minutes, testing the OD value at 570 wavelength .Chosing a culture board with 96 holes cultivated for 24 hours under standard condition, and radiation by the laser and putting 1μCi3H-TdR into each holes . Testing glittering count in one minute by glitter counting apparatus to evaluate effects of laser on HPDLFs DNA synthesis .
2. Dental cervix ligation was used with the healthy SD rats (half male and half female, 180~220g each) to perform modeling. 8 weeks later, 8 modeling rats and 8normal rats were randomly selected to test the periodontal specifications to conform whether the modeling was successful. Then 48 rats of successful modeling and 8 normal rats were divided into 7 groups to receive experimental treatment of oral prophylaxis and radiation by laser with the energy level of1.0W 20pps、1.5W 20pps、2.0W 20pps、2.5W 20pps、3.0W 20pps. 24 hours later they were sacrificed to test the PD,PLI,GI and SOD、MDA、NO in periodontal to verify the treatment effects of the pulsed Nd:YAG laser.
3. 32 Sprague - Dawley rates were randomly assigned to the control group, periodontitis control group, periodontitis with pules Nd:YAG laser iatreusis group and drug treatment group . Radiation on the periodontal pocket of rats of group L by 2.0W 20pps laser energy , Intragastric administration the rats by 200mg/kg arilin to group D.GCF samples were collected with strips of filter paper 24 hours after the last treatment. The activity of GSH - Px and the ALP were measured by automatic biochemistry analyzer. 15 days later they were sacrificed to test the histopathological change of the 4 groups of rats, and to verify the treatment effects of the pules Nd:YAG laser.
Results
1. The primary cell is long fusiform shape, and it’s cytoplam is uniform, with nucleus round or ellipse, chromatospherite clear. Immunohistochemistry coloretur indicated that the undee silk protein is masculine, keratin negative.By analysis of variance of single factor, it’s showed that the pules Nd:YAG laser energy level between 1.0 W 20pps-2.0 W 20pps can promote HPDLFs generation,and 1.5 W 20pps most effective. The protein level of N group and 1.0 W 20pps-2.5 W 20pps group was significantly higher than the 3.0 W 20pps group(P < 0. 01),the 2.0 W 20pps group is maxlmum. The DNA’s synthesis of 1.0W 20pps-2.5W 20pps groups was significantly higher than the control group but 3.0W 20pps group was significantly lower than N group (P < 0. 01).
2. Level of SOD in 1.5W 20pps-2.0W 20pps 20pps groups w as signif icantly higher than the Pgroup (P < 0.05) ; L evel of MDA in 1.5W、2.0W、2.5W 20pps groups were significantly lower than the 0W group (P < 0. 05).Level of NO in each energy level group was signif icantly lower than the P group (P < 0.05) . The NO content of the periodontitis with pules Nd:YAG laser iatreusis group and the drug group is (0.53±0.02)they were significantly higher than the control group, was significantly lower than the periodontitis control group ( P < 0. 05). By radiation on the rats periodontal pocket the PD,PLI,GI can be improved with the proper energy level of pules Nd:YAG laser.
3. The GSH-Px activityof the periodontitis with pules Nd:YAG laser iatreusis group is(40.71±6.35)was significantly higher than the periodontitis control group ( P < 0. 05),had no statistic difference to the control group and drug treatment group ( P > 0. 05). The ALP activityof the periodontitis with pules Nd:YAG laser iatreusis group is(16.75±1.47)was significantly higher than the control group, was significantly lower than the periodontitis control group ( P < 0. 05), had no statistic difference to the drug treatment group ( P > 0. 05).The clinical exponents were statistic difference to the three groups( P < 0. 01).In microscopical study, the group of laser was similar to the positive drug group of. In comparison with the periodontitis group, the cellular infiltration of soft tissues in the group of laser was greatly alleviated, periodontal pockets became much shallower, and alveolar resorption ceased.
Conclusions
1. 1.0W 20pps-2.0W 20pps laser radiation on the HPDLFs can promote the cells generation ,increase protein content.The pules Nd:YAG laser can be effective to treat periodontitis.
2. 1.5W 20pps-2.0W 20pps of pulsed Nd :YAG laser irradiation on periodontal pocket of rats is effective to treat chronic periodontitis therapeutic efficacy. Based on the above results, a preliminary parameters both safety and efficacy of the laser could be 2.0 W 20pps with initiator.
3. Contrast experiment of two different way treat chronic periodontitis: with laser and with drug the result approved 2.0 W 20 pps laser irradiation periodontitis can be controlled and healing well.
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