Immunohistological and morphometric analysis of inflammatory cells in rapidly progressive periodontitis and adult periodontitis

The purpose of this study was to localize, characterize, and quantify in situ the inflammatory cells in the gingival connective tissue prior and subsequent to the initial therapy of ten patients with rapidly progressive periodontitis (RPP) and five patients with adult periodontitis (AP). Using immunohistological techniques, the amount of T lymphocytes, alphabeta-T lymphocytes, gammadelta-T lymphocytes, B lymphocytes, and plasma cells was determined at the beginning of the periodontal therapy (baseline) and at the time of periodontal surgery. Furthermore, the distribution of collagen types I, III, V, and VI was investigated using transmission electron microscopy. At baseline, patients with RPP revealed much higher numbers of inflammatory cells than patients with AP. During initial therapy of patients with RPP, the amount of T cells, alphabeta-T cells, and gammadelta-T cells was reduced significantly (P<0.05). Biopsies of patients with AP revealed a statistically significant reduction of all cell types, except alphabeta-T cells and gammadelta-T cells in the deep connective tissue. The transmission electron microscopy of biopsies from patients with RPP and AP with severe inflammation taken at baseline revealed that collagen types I and III were destroyed nearly completely in areas with leukocyte infiltration, whereas collagen types V and VI revealed a more pronounced labeling reaction. The results revealed that, during initial therapy, the amount of inflammatory cells was reduced significantly more in biopsies of patients with AP than in patients with RPP. At baseline, the inflamed gingival tissue consists mainly of collagen types V and VI in areas with infiltrates of inflammatory cells.     

The evaluation of healing effect of triclosan and flurbiprofen-loaded nanogels in experimental periodontitis in rats by morphometric analysis

PurposeTo evaluate therapeutic effectiveness of antibacterial triclosan (TCS) and anti-inflammatory flurbiprofen (FLB)-loaded nanogels system in ligature-induced experimental periodontitis in rats.MethodologyA total of 72 Sprague–Dawley rats were used in this study. Four groups (n = 18 each) were randomly created: Group 1 – neither subjected to experimental periodontitis nor to any treatment; Group 2 – subjected to experimental periodontitis but not treated; Group 3 – subjected to experimental periodontitis and then treated with the developed nanogels; Group 4 – subjected to experimental periodontitis and then placed on a mixture of pure TCS and FLB treatment. The experimental periodontitis was induced on the lower incisors by applying a ligature which was kept for 14 days. Treatment was done for 7 days, and sampling was done at 7, 14, and 28 day of the post-induction experimental period. Morphometric analysis was conducted to assess the clinical outcomes and healing effect.ResultsThe morphometric findings showed that the group treated with the developed TCS and FLB-loaded nanogels recovered better and faster than a mixture of pure TCS and FLB. At 28 day of the experimental period, there was no significant difference (p > 0.05) between the baseline control group and the nanogels treated group.ConclusionsThe developed TCS and FLB-loaded nanogels was found to be effective in the treatment of experimental periodontitis in rats. The used experimental periodontitis model was found to be simple and easily reproducible.     

Comparison of scanning and transmission electron microscopy of the epithelial pocket wall in juvenile and adult periodontitis

Using scanning (SEM) and transmission (TEM) electron microscopy, this study compared fine structural features of the pocket walls in both juvenile and adult periodontitis (JP and AP, respectively) in 40 cases. Gingiva was also obtained from a control group consisting of periodontally noninvolved teeth. Clinical parameters were assessed in both JP and AP patients as well as in controls. Clinical findings showed low plaque accumulation, marked periodontal tissue destruction and less gingival inflammation in JP. Bone destruction and attachment loss were more marked in JP than in AP. AP had a higher plaque index and more evident gingival inflammation. SEM observations of JP as compared to AP showed gross distortions in pocket walls, an increased beaded appearance of microridges, and separation between pocket epithelial cells. TEM showed partially desquamated and separated superficial epithelial cells, but only in JP were fine granular precipitates observed in the intercellular spaces. The observations demonstrated structural features indicative of more prominent degenerative changes in JP than in AP. Also, these features were coincidental with a higher plaque index in AP than in JP, where clinical features (including a low plaque index) were not proportional to the epithelial destructive changes present.     

Relative resistance of long junctional epithelial adhesions and connective tissue attachments to plaque-induced inflammation

This study compared the resistance to periodontal disease of the long junctional epithelial adhesion and the naturally occurring dentogingival junction. Two groups were used, each containing three young male beagle dogs with all permanent teeth erupted. Periodontitis was induced around maxillary and mandibular premolars in the experimental dogs over a 42-day period, using subgingival ligatures and a soft diet. Fourteen days after ligature removal, flaps were reflected, granulation tissue was removed and the roots were planed to the alveolar crest. Reference grooves were placed in the root surfaces at the level of the alveolar bone, the flaps were positioned over the alveolar crests, and sutures were placed. A 60-day period permitted healing with formation of long junctional epithelial adhesions. During this 116-day period control dogs were maintained in gingival health by daily brushing and by prophylaxis every 14 days. Both groups had a high level of health (GI scores of 0) at the beginning of the 20-day combined disease phase. Inflammation was induced in both groups by subgingival ligature placement and a plaque-promoting diet. Right and left sides of both groups represented separate time intervals within the 20-day period. Block sections were secured at time of killing and the tissues were prepared for light and fluorescent microscopic evaluation. Mean GI scores and mean probing depths increased similarly in both groups. Tagge index scores of gingival inflammation were higher at the longer time periods in the experimental animals. However, they displayed an intact long junctional epithelial adhesion throughout the study, while control animals frequently showed ulceration of the sulcular epithelium. Neither group showed significant changes in location of the apical cells of the attachment epithelium. Crestal osteoblastic activity, confirmed with Procion labeling, predominated in the experimental animals, while osteoclastic activity predominated in the control. Under the conditions of this study, there appeared to be no appreciable difference in resistance to disease between a long junctional epithelial adhesion and a true connective tissue attachment.     

Inflammatory cells and pocket epithelium in periodontitis cases. Transmission and scanning electron microscopic study

Epithelial pocket walls of 10 hopeless teeth were separated from root surfaces after extraction. The tissues were prepared for examination by transmission (TEM) and scanning electron microscopies (SEM). The study focuses on inflammatory cells and their possible pathologic effects on gingival tissues. TEM observations showed widening of intercellular spaces and interruption or even complete deterioration of basal lamina when macrophages and lymphocytes were recognized between epithelial cells. SEM findings showed scattered lymphocytes and polymorphonuclear leukocytes (PMNs) on the pocket wall surfaces with several perforations that corresponded to their size. These observations led to the conclusion that separation between epithelial cells and perforations on pocket wall surfaces by inflammatory cells may promote periodontal tissue destruction by passage of irritant and destructive substances into the underlying connective tissue.     

Preventive effects of thymoquinone in a rat periodontitis model: a morphometric and histopathological study

Ozdemir H, Kara MI, Erciyas K, Ozer H, Ay S. Preventive effects of thymoquinone in a rat periodontitis model: a morphometric and histopathological study. J Periodont Res 2012; 47: 74–80. © 2011 John Wiley & Sons A/S Background and Objective: Thymoquinone has a variety of pharmacologic properties, including antihistaminic, antibacterial, antihypertensive, hypoglycemic, anti‐inflammatory and anti‐oxidative activities. Through its anti‐inflammatory and antioxidant properties, thymoquinone may play an important role in preventing periodontal diseases. The aim of this study was to evaluate the effectiveness of thymoquinone in preventing the initiation and progression of periodontitis in a rat periodontitis model. Material and Methods: Twenty‐four rats were randomly divided into three experimental groups: a nonligated (NL) treatment group (n = 8), a ligature‐only (LO) treatment group (n = 8) and a ligature plus thymoquinone (10 mg/kg, daily for 11 d) (TQ) treatment group. In order to induce experimental periodontitis, a 4/0 silk suture was placed at the gingival margin of the right‐mandibular first molars of the rats. Thymoquinone was administered by gastric feeding until the animals were killed on day 11. Changes in the alveolar bone levels of rats in each group were measured clinically, and tissues of rats in each group were examined histopathologically to determine inflammatory cell infiltration (ICI), osteoblast and osteoclast activities, and osteoclast morphology. Results: Alveolar bone loss around the mandibular molar tooth was significantly higher in the LO group compared with NL and TQ groups (p < 0.05). The ratio of the presence of ICI and osteoclast numbers was significantly higher in the LO group than in the NL and TQ groups (p < 0.05). Osteoblastic activity was significantly lower in the LO group than in the NL and TQ groups (p < 0.05). Conclusion: The present study showed that the oral administration of thymoquinone diminishes alveolar bone resorption in a rat periodontitis model.     

A morphometric and histopathologic evaluation of the effects of propolis on alveolar bone loss in experimental periodontitis in rats

BACKGROUND: Propolis collected by honeybees from various plant sources is a resinous hive product possessing a broad spectrum of biologic activities. Propolis has been used extensively in the diet to improve health and prevent disease. The purpose of this study was to analyze the morphometric and histopathologic changes associated with experimental periodontitis in rats in response to the systemic administration of propolis. METHODS: Forty Wistar rats were divided into four experimental groups: non-ligated (NL; N = 10); ligature only (LO; N = 10); and systemic administration of ligature and propolis (100 mg/kg body weight per day [Pro100; N = 10] or 200 mg/kg body weight per day [Pro200; N = 10]). Silk ligatures were placed at the gingival margin of the lower first molars in both mandibular quadrants. The study duration was 11 days, and the animals were sacrificed at the end of this period. Changes in alveolar bone levels were clinically measured, and tissues were histopathologically examined to assess the differences among the study groups. RESULTS: At the end of 11 days, alveolar bone loss was significantly higher in the LO group compared to the NL, Pro100, and Pro200 groups (P <0.05). Osteoclast numbers in the LO group were significantly higher than those of the NL, Pro100, and Pro200 groups (P <0.05). Both dosages of propolis significantly reduced the periodontitis-related bone loss, but the differences between the two propolis groups were not statistically significant (P >0.05). CONCLUSION: The findings of this study provide morphologic and histologic evidence that propolis, when administered systemically, prevents alveolar bone loss in the rat model.     

Ultrastructural study of bone cells and matrix incident in experimental periodontitis

The purpose of this study was to examine the process of bone destruction and also to examine the ultrastructural features of the cells and the resorbed sites of bone matrix in experimental periodontitis. To induce the periodontitis, a defect was prepared with a endodontic reamer in the proximal surfaces of the upper 1st and 2nd molars of rats. The process of the bone resorption was examined histopathologically once a week for 3 weeks. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) were used to examine the cells and the bone matrix using the specimens taken 2 or 3 weeks after the start of the experiment. The alveolar bone resorptions of interdental areas were observed 2 weeks after starting the experiment and it continued to progress longitudinally. After 3 weeks, concave bone loss appeared on the buccal surfaces of the bone. The resorbed bone surface revealed by TEM typical osteoclasts, macrophages and mononuclear cells resorbing collagen fibrils. These cells resorbing collagen fibrils which worked with the osteoclasts appeared frequently in resorbed sites. Numerous osteoblasts appeared on the resorbed area. However, judging from their undeveloped organelles, their function seemed to remain inactive and unproductive. SEM showed many resorbed lacunae in the alveolar bone in interdental areas and the differences in the ultrastructural features of the resorption lacunae were distinctive. These findings suggest that the massive and rapid bone resorption in experimental periodontitis is the result of increased osteoclastic activity and depressed osteoblastic activity. The different ultrastructural features of each lacunae were results of the resorbing stage.     

大黄素治疗实验性牙周炎的骨计量学研究

目的:观察不同浓度大黄素对实验性牙周炎大鼠附着丧失和牙槽骨吸收的治疗作用.方法:选取纯种雌性SD大鼠随机分为4 组,各30 只: 正常对照组(N组)、牙周炎组(P组)、低浓度大黄素治疗组(PL组)及高浓度大黄素治疗组(PH组).建立牙周炎动物模型并按分组用药,分别于4、 6、 8 周时处死动物,取上颌骨标本进行骨计量学观察.结果:PL组和PH组结缔组织附着丧失量、牙槽骨嵴高度丧失量和破骨细胞个数均明显小于P组,成骨细胞个数均明显大于P组(P<0.05).结论:大黄素可抑制牙周附着丧失和牙槽骨吸收,并能促进牙槽骨形成.     

Apoptosis in chronic adult periodontitis analyzed by in situ DNA breaks, electron microscopy, and immunohistochemistry

BACKGROUND: Apoptosis is an evolutionary form of physiological cell death. Previous studies suggest that apoptosis is involved in the pathogenesis of periodontal diseases. Therefore, we studied the apoptotic events in the gingival tissue of chronic adult periodontitis patients. METHODS: Gingival tissue biopsies from 22 patients with chronic adult periodontitis and from 11 healthy controls were obtained. Criteria for patient inclusion in the periodontitis group were a minimum of 14 natural teeth, excluding third molars, with at least 10 posterior teeth; 5 to 6 sites with probing depth > or = 5 mm; attachment loss > or = 3 mm; and extensive radiographic bone loss. The control group included healthy subjects with no prior history of periodontal disease. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique; electron microscopic analysis; and expression of Caspase-3, Fas, FasL, Bcl-2, and p53 by immunohistochemistry. RESULTS: TUNEL-positive cells and cells exhibiting chromatin condensation by electron microscopy were observed in the inflammatory infiltrate of biopsies obtained from periodontitis patients. Most of the TUNEL-positive cells belonged to neutrophil cell populations as they were stained with anti-myeloperoxidase. Positive staining for active-caspase 3, Fas, FasL, and p53 was only observed in the inflammatory infiltrate from periodontitis biopsies, whereas Bcl-2 cells were present in both periodontitis patients and healthy controls. CONCLUSIONS: Our findings establish that apoptosis is induced in the periodontal tissue by host and microbial factors and support the hypothesis that apoptotic mechanisms could be implicated in the inflammatory process associated with gingival tissue destruction observed in adult periodontitis patients.     

Histopathological study on qualitative changes in gingival collagen fibers for experimental periodontitis in rats. Remodeling of type I and III collagens detected by the Picrosirius-polarization method

The objective of this study was to demonstrate the movement of type I and III collagens accompanying gingival inflammatory destruction. Experimental marginal periodontitis was induced by a calculogenic diet and a high-sucrose diet with feces in 3-week-old Wistar rats. We observed the changes in the interdental periodontium histopathologically by using the picrosirius-polarization method and an electron microscope. 1. After 5 weeks of eating the calculogenic diet, mild gingivitis was found. A small number of inflammatory cells consisting of neutrophils were seen. At the 2nd week, a variety of bone resorptions of alveolar crests began to appear. At the 10th week, an epithelial downgrowth was observed. At the 64th week, migration of epithelial attachment to half of the apex and a high degree of inflammatory cell infiltration of plasma cells could be seen. 2. Observations under the polarization microscope showed that interdantal horizontal fibers became coarse and type I collagen decreased but at the middle type III increased. However when the horizontal fasciculus were newly formed, type I was always dominant. Between interdental horizontal fibers and the alveolar crest, type III was increased. 3. Under the electron microscope microfibrils were found around adjacent degraded fibroblasts at the locations where collagen fibrils were destroyed and disappeared. In contrast, at the locations detached from inflammatory cell infiltration small bundles of microfibrils were seen. It is suggested that at the areas of destructive collagen structures the relative increase in type III and the appearance of microfibrils were caused by the reduction of type I. At the same time at the sites of detachment from the lesions, complete growth of type III and the appearance of microfibrils were found and were considered to be newly formed juvenile collagen structures. Moreover it is concluded that a balance proceeds with the qualitative changes in the types of collagen fibers involving breakdown and new formation.     

Bacterial penetration of pocket soft tissues in chronic adult and juvenile periodontitis cases

This study investigates bacterial invasion of the soft tissue walls of deep pockets from cases with adult (AP) and juvenile periodontitis (JP). Transmission electron microscopy was used to examine pocket soft tissue walls removed from extracted teeth from 5 patients with AP and 2 patients with JP. Bacteria were sparse throughout the epithelium and connective tissue, regardless of the level of tissue breakdown. However many inflammatory cells were seen, and these did appear to be located in regions of marked collagen loss. Accumulations of large numbers of bacteria were extremely rare and found only on the epithelial surface or in artefactual spaces within the deeper tissues. The findings indicate that the tissue destruction associated with periodontitis is not directly related to bacterial invasion. The sparse organisms within the pocket tissues probably result from passive entry rather than an invasive action. Under these circumstances, it would seem reasonable to suggest that bacterial metabolic products rather than the micro-organisms themselves penetrate the tissues in periodontitis.     

Histopathological morphometric evaluation of 2 different hydroxyapatite-bone derivatives in sinus augmentation procedures: a comparative study in humans

BACKGROUND: Xenografts to augment the maxillary sinus have been used extensively. The aim of the present study was to evaluate, qualitatively and quantitatively, two different HA derivatives of natural and synthetic sources on newly formed bone in the augmented sinus. METHODS: A bilateral sinus augmentation procedure with simultaneous (16 out of 20 sites) or subsequent implant placement was performed in 10 patients. The antrum was randomly filled with a deproteinized, bovine hydroxyapatite mineral (B-HA) on one side and a non-ceramic resorbable hydroxyapatite (NC-HA) on the other. Cylindrical specimens were harvested from the augmented core at 12 months. Decalcified specimens were sectioned at a cross-horizontal plane and stained with hematoxylin and eosin for histopathologic and histomorphometric examinations. Tissue area fractions of bone, marrow, and the grafted particles were calculated for each specimen from the lateral to the deep region, and changes in values were compared within each material and between them. RESULTS: New bone formation was evident. B-HA and NC-HA particles were observed in all specimens surrounded by newly formed bone in direct connection or by soft tissue marrow. Morphometrically in the B-HA sites, from the lateral to deeper area, bone area fraction increased from 29.8% to 54.2% (average 42.1%) and marrow area fraction decreased from 37.9% to 26.7% (average 33.3%). The mineral area fraction decreased from 32.3% to 19.1% (average 24.7%). All increasing/decreasing patterns were statistically significant (P < 0.001). In the NC-HA sites, from the lateral to deeper area, bone area fraction increased from 25% to 36.5% (average 32.3%) and marrow area fraction decreased from 51.6% to 41.9% (average 43.2%) (P <0.001). The mineral area fraction decreased from 29% to 21.7% (average 24.6%) (P = 0.038). Comparison between the two HA derivative groups showed a significant difference between the bone area fraction averages (P = 0.0053) and between the increasing patterns along the core depth (P = 0.0006). There was also a significant difference between the decreasing marrow patterns (P = 0.003), but not between their averages. Comparison between the mineral area fractions showed no differences. CONCLUSIONS: B-HA and NC-HA were proven to be biocompatible materials. Although the B-HA-augmented sites showed a higher percentage of bone formation at 12 months, both are suitable bone derivatives in sinus augmentation procedures and can accommodate osseointegrated implants.     

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目的    探讨大鼠牙周炎与高脂血症的关系。方法    本实验于2005年6月至2011年6月在福建省高校口腔医学重点实验室及中国人民解放军南京军区福州总医院比较医学科完成。40只雄性SD大鼠按体重随机分为A、B、C、D组,每组10只。分别进行如下处理：A组,正常饲养;B组,单纯牙周结扎;C组,单纯高脂饮食;D组,牙周结扎+高脂饮食。3个月后处死大鼠,检测血清胆固醇（CHO）、甘油三酯（TG）、高密度脂蛋白（HDL-C）和低密度脂蛋白（LDL-C）浓度;同时,取大鼠下颌第一磨牙做组织学观察分析。结果    （1）牙周结扎大鼠磨牙牙槽骨出现不同程度的骨吸收。（2）各组大鼠血清TG浓度差异无统计学意义（P > 0.05）;高脂饮食大鼠血清CHO、HDL-C和LDL-C浓度均明显高于正常饮食大鼠,且D组大鼠血清CHO浓度高于C组（均P < 0.05）;C、D组HDL-C和LDL-C浓度差异无统计学意义（P > 0.05）。结论    通过高脂喂养和磨牙牙周结扎可建立高脂血症大鼠实验性牙周炎动物模型,牙周炎可致高脂饮食大鼠血清CHO浓度升高。