Effect of diabetes on collagen metabolism and hypoxia in human gingival tissue: a stereological,histopathological, and immunohistochemical study

Diabetes mellitus and periodontitis are chronic inflammatory diseases that disrupt soft tissue metabolism. The diseases separately or together increase apoptosis in gingival fibroblast cells and reduce cell renewal. We investigated the effects of diabetes and periodontitis on the composition and structure of gingival connective tissue. We used gingival biopsies from 16 healthy individuals (control group, C), 16 type 2 diabetic patients with chronic periodontitis (diabetes + periodontitis group, D + P) and 16 healthy chronic periodontitis patients (periodontitis group, P). Biopsies were obtained under local anesthesia. Clinical attachment level (CAL), gingival index (GI) and plaque index (PI) were measured prior to gingival biopsies. Fibroblast cells were counted stereologically. Inflammatory cells were counted histomorphometrically. Hypoxia-inducible factor (HIF)-1α, lysyl hydroxylase (PLOD-2), neutrophil collagenase (MMP-8), and vascular endothelial growth factor (VEGF) levels were evaluated immunohistochemically. CAL, GI and PI for the C group were lower than for the other groups (p < 0.05). Fibroblast cell counts were lower for the D + P group than for the other groups (p < 0.05). Diabetes increased inflammatory cell numbers in the D and D + P groups compared to the C and P groups. MMP-8 levels were higher for the D + P group than for the other groups. VEGF was elevated in both the P and D + P groups compared to the C group, while HIF-1α and PLOD-2 levels were comparable. Diabetes increased tissue destruction and inflammation, and decreased fibroblast cell numbers without affecting collagen crosslinking and HIF-1α levels.     

纤粘连蛋白在实验性牙周炎鼠牙龈组织中的表达

目的:观察分析大鼠实验性牙周炎牙龈组织中纤粘连蛋白(fibronectin,FN)的表达及意义。方法:26只8周龄雄性Wistar大鼠,随机分为局部丝线结扎高糖软食4周和8周两组,每组实验动物10只,空白对照组3只。运用免疫组化方法,观察分析FN在健康牙龈组织中、牙周炎牙龈组织中的表达及意义。结果:健康牙龈组织中,FN相对均匀弥漫表达于整个牙龈结缔组织基质内;牙周炎牙龈组织中,FN表达具有部位特异性,即上皮下结缔组织最上部基质内FN表达明显下调;结合上皮根端基底细胞基底面下FN表达明显上调,表达强度和范围随结合上皮根向增生程度的增加而增强。结论:炎性刺激下调炎症中心区牙龈结缔组织基质内FN表达;炎症刺激上调结合上皮根端基底细胞基底面下FN表达,其表达强度和范围随结合上皮根向增生程度的增加而增强扩大,暗示FN参与牙龈结合上皮根向增生迁移活动。     

A new paradigm in the periodontal disease progression: gingival connective tissue remodeling with simultaneous collagen degradation and fibers thickening

Bacterial dental plaque is considered to be the main cause of periodontal diseases, but progression of the disease is also related to the host inflammatory response. The earliest affected tissue is the gingiva, but the specific mechanisms involved in the onset of this condition remain unclear. Frequently, collagen degradation is pointed as the main marker of periodontal disease progression, but the organization of the fibers in the gingival tissue is still unknown. The aim of the present study was to investigate the gingival extracellular matrix in a model of ligature-induced periodontal disease. Analysis of the microbiota indicated a progressive increase in the ratio of Gram-negative/Gram-positive microorganisms. There was no difference in the organization of reticulin fibers next to the epithelial basement membrane, whereas the arrangement of collagen fibers in the gingival connective tissue was significantly affected. Animals with inflammation presented a reduction of 35% in the total area occupied by collagen fibers. However, these fibers were thicker and more densely packed. These alterations involve type I, type III and type VI collagens as determined by immunohistochemistry. The results demonstrated the occurrence of marked reorganization of the gingival extracellular matrix in response to the inflammatory process, indicating a new paradigm in the periodontal disease progression: collagen degradation and fibers thickening, simultaneously.     

Interleukin 1beta-secreting cells in inflamed gingival tissue of adult periodontitis patients.

Interleukin 1beta (IL-1beta) is a cytokine with a wide range of biological activities. It is produced by various cell types including macrophages, fibroblasts, and neutrophils. The inflammatory responses mediated by IL-1beta play an important role in periodontal tissue destruction. The purposes of this study were: (1) to determine the location of IL-1beta in inflamed human gingival tissues by the immunofluorescence method; and (2) to correlate this location to the concomitant presence of macrophage or neutrophils by immunohistochemistry. Five patients with moderate to advanced adult periodontitis receiving periodontal phase I therapy were included in this study. One month after phase I therapy, 15 sites with a probing pocket depth >/=5 mm and gingivitis index >/=1 were arranged for modified Widman flap operation. Another three sites with a probing pocket depth 相似文献   

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Protective effects of crocin on biochemistry and histopathology of experimental periodontitis in rats

ABSTRACT

We investigated the effectiveness of crocin for preventing oxidative damage in experimentally produced periodontitis. We used three groups of 10 female Wistar rats divided into: control (C); experimental periodontitis (EP), experimental periodontitis + crocin (Cr-EP). Malondialdehyde (MDA), glutathione (GSH), total antioxidant status (TAS), total oxidant status (TOS) and superoxide dismutase (SOD) and catalase (CAT) enzyme activities were measured. We examined histopathology and inflammatory cell infiltration in gingiva and periodontal ligament. MDA and TOS levels, and SOD and CAT activities increased significantly in rats with induced periodontitis compared to the control group, while GSH and TAS levels were decreased significantly compared to the control group. Histopathologic examination revealed inflammatory cell infiltration in gingiva epithelium and subepithelial connective tissue in the EP group. Histological damage was reduced significantly after crocin treatment compared to the EP group. Crocin supplementation may help reduce oxidative damage to periodontal tissues.     

Fractionation of T cell subsets on Ig anti-Ig columns: isolation of helper T cells from nonresponder mice, demonstration of antigen-specific T suppressor cells, and selection of CD-3 negative variants of Jurkat T cells

In the present experiments we have explored the possibilities of a modified immunoadsorbent technique to select for (1) mutagenized T cell receptor (Tcr) negative variants of Jurkat T lymphoma cells and (2) purified CD-4+ or CD-8+ T lymphocytes. The basic principle was to make large numbers of immunoglobulin (Ig) negative T cells Ig+ by T cell subset-specific monoclonal antibodies (mAb), and to select such cells on Ig anti-Ig columns. Our results demonstrated that Thy-1+, Fc receptor positive, antigen-specific T cells regulate the immune response in mice nonresponders to pork insulin, and the "autologous" mixed lymphocyte reaction. In addition, the immunoadsorbent method very efficiently selects Tcr/CD-3- variants from mutagenized Jurkat cell populations incubated with anti-CD3 mAb. The described method is easy and quick and can fractionate large numbers of cells; it is the "poor-man's cell sorter." The most important finding is the demonstration of antigen-specific Thy-1+, CD-8+, and Fc receptor+ T suppressor cell that apparently react with antigen in a non-major histocompatibility complex-restricted manner.     

Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue

Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet-activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In this study, hydroxyl-PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of hydroxyl-PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.     

Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-κB (p65) DNA binding and promoted periodontal repair in rats

We wished to evaluate whether epigenetic modifiers have a beneficial effect on treating experimental periodontitis and mechanisms for regulating the cell fate of mesenchymal stem cells (MSCs) in inflammatory microenvironments. We isolated MSCs from healthy and inflamed gingival tissues to investigate whether trichostatin A (TSA) could improve osteogenic differentiation and resolve inflammation in vitro. The tissue regenerative potentials were evaluated when treated with a temperature-dependent, chitosan-scaffold-encapsulated TSA, in a rat model of periodontitis. After induction with the conditioned medium, TSA treatment increased the osteogenic differentiation potential of inflamed MSCs and healthy MSCs. In addition, interleukin-6 and interleukin-8 levels in supernatants were significantly decreased after TSA treatment. Moreover, TSA promoted osteogenic differentiation by inhibiting nuclear factor-κB (p65) DNA binding in MSCs. In rats with experimental periodontitis, 7 weeks after local injections of chitosan-scaffold-encapsulated TSA, histology and microcomputed tomography showed a significant increase in alveolar bone volume and less inflammatory infiltration compared with vehicle-treated rats. The concentrations of interferon-γ and interleukin-6 were significantly decreased in the gingival crevicular fluid after TSA treatment. This study demonstrated that TSA had anti-inflammatory properties and could promote periodontal tissue repair, which indicated that epigenetic modifiers hold promise as a potential therapeutic option for periodontal tissue repair.     

牙周组织再生术联合正畸治疗对牙周炎患者牙周状况及满意度的影响

目的:探讨牙周组织再生术联合正畸治疗对牙周炎患者牙周状况及满意度的影响。方法:选取我院于2016年3月-2017年4月期间收治的牙周炎患者76例为研究对象。按照随机数字表法将患者分为研究组(n=38)与对照组(n=38),对照组行牙周组织再生术,研究组行牙周组织再生术联合正畸治疗。于治疗前、治疗后3个月检查患者牙周指标情况和X线头影测量情况,对比两组患者治疗前后视觉模拟疼痛评分(VAS),随访一年,于治疗1年后采用医院自制调查问卷评价患者满意度,观察并比较两组患者治疗后并发症发生情况。结果:治疗后3个月,两组患者的牙龈指数(GI)、菌斑指数(PLI)、龈沟出血指数(SBI)、牙周探诊深度(PD)以及临床牙周平均附着丧失(CAL)均显著降低,且研究组低于对照组(P0.05)。治疗后3个月,两组患者的SNA角、SNB角均显著降低,且研究组低于对照组,两组患者的ANB角显著升高,且研究组高于对照组(P0.05)。两组患者治疗后VAS评分较治疗前显著降低,且研究组低于对照组(P0.05)。研究组患者牙龈健康状况评分、口腔清洁能力评分、美观评分、满意度均高于对照组(P0.05),而两组患者咀嚼功能评分相比无统计学差异(P0.05)。两组患者治疗后并发症发生率比较无统计学差异(P0.05)。结论:牙周组织再生术与正畸治疗联合运用,对牙周组织健康状况有显著改善作用,且安全有效,提升患者满意度。     

The influence of type I diabetes mellitus in periodontal disease induced changes of the gingival epithelium and connective tissue

Periodontal disease constitutes the most frequent chronic diseases in human dentition. Bacterial plaque is the main etiologic agent, although it is the host immune response that causes periodontal tissue destruction. Diabetes is considered an important risk factor, not only for the onset but also for progression of the disease. The aim of this study was to analyze structural changes in the rat gingival epithelium and connective tissue in response to the experimental periodontal disease induced by the ligature technique, under the influence of diabetes. The results showed that experimental periodontal disease is characterized by marked inflammation, affecting both the epithelial and connective tissues, causing degeneration of the dermal papilla, increase in the number of inflammatory cells, destruction of reticulin fibers, and accumulation of dense collagen fibers (fibrosis). These changes were worsened by diabetes, apparently by hampering the inflammatory response and affecting tissue repair of the affected tissues.     

Cigarette smoking enhances T cell activation and a Th2 immune response; an aspect of the pathophysiology in periodontal disease

Smoking is a strong risk factor for periodontitis. Treated patients who smoke show increased risk for further periodontal breakdown, despite receiving maintenance care. Previous work indicated that such patients have a monocytic cytokine response favoring Th2 activity. The purpose of this study was to investigate the T lymphocytic cytokine production representing Th1 and Th2 subpopulations in smokers and non-smokers. Venous blood was collected from 30 treated periodontitis patients (12 smokers) and 24 healthy subjects (12 smokers). Whole blood cell cultures were stimulated and interferon (IFN)-γ and interleukin (IL)-13 were measured in the culture supernatants, representing types 1 and 2 Th subpopulations, respectively. Unadjusted data showed that smokers had more lymphocytes, and higher levels of IFN-γ and IL-13, irrespective of being periodontal patient. However in a multivariate analysis, increased IFN-γ production was not significantly explained by smoking, while higher IL-13 was strongly explained by smoking (21%, p < 0.001). We suggest that the increased Th activity and specifically an elevated Th2 profile in smokers may constitute a risk for smoking patients which may induce conversion of periodontal stability into progressive disease. This phenomenon may be equally important in other conditions, where connective tissue and bone loss are hallmarks of disease pathophysiology.     

Role of PTPα in the Destruction of Periodontal Connective Tissues

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα^+/+ and PTPα^−/− mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5–3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα^−/− mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα^KO and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.     

Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss.     

Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases

Anaerobic gram-negative oral bacteria such as Treponema denticola, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum are closely associated with periodontal diseases. We measured the relative population (bacterial levels) of these oral pathogens in subgingival tissues of patients at different stages of Korean chronic periodontal diseases. We divided the individuals into those with chronic gingivitis (G), moderate periodontitis (P1), severe periodontitis (P2), and normal individuals (N) (n?=?20 for each group) and subgingival tissue samples were collected. We used real-time PCR with TaqMan probes to evaluate the change of periodontal pathogens among different stages of periodontitis. Bacterial levels of A. actinomycetemcomitans and C. rectus are significantly increased in individuals with chronic gingivitis and moderate periodontitis, but unchanged in severe periodontitis patients. These results suggest that analyzing certain bacterial levels among total oral pathogens may facilitate understanding of the role of periodontal bacteria in the early stages of periodontitis.     

The cytology of submandibular glands of the opossum

Tooth attachment in the majority of the bony fish is by ankylosis or fibrous membrane. However, in one group of the osteichthys, the trigger-fish or balistids, tooth attachment is by means of a periodontium composed of a shallow alveolar socket, a periodontal ligament and acellular cementum. Histologically, the balistid periodontal ligament is composed of a dense fibro-cellular connective tissue possessing an abundance of typical fibrocytes, collagen fiber bundles, and oxytalan fibers. The collagen fiber bundles which resemble the principal fiber bundles of the mammalian periodontal ligament are inserted into the bone of the shallow alveolar sockets and are anchored to the teeth by means of a layer of amorphous acellular cementum that covers the radicular dentin. No cementoblasts were found in functional teeth, and epithelial rests are lacking. The mid-central zone of the balistid periodontal ligament is occupied by small blood vessels.     

牙周夹板联合正畸治疗对牙周炎所致前牙扇形移位患者咀嚼功能和龈沟液PGE2、sICAM-1、PAK5的影响

目的:探讨牙周夹板联合正畸治疗对牙周炎所致前牙扇形移位患者咀嚼功能和龈沟液中前列素E2(PGE2)、可溶性细胞间黏附分子-1(s ICAM-1)、p2l活化激酶5(PAK5)的影响。方法:采用随机数字表法,将我院2018年2月~2020年2月间接收的93例牙周炎所致前牙扇形移位患者分为对照组(46例,牙周基础治疗、牙周夹板治疗)和研究组(47例,牙周基础治疗、牙周夹板治疗联合正畸治疗),观察两组疗效,对比两组治疗前后的牙周情况,咀嚼功能、美观度,龈沟液PGE2、s ICAM-1、PAK5水平。结果:研究组的临床总有效率高于对照组(P<0.05)。研究组治疗后牙周菌斑指数(PLI)、探诊深度(PD)、牙龈指数(GI)、附着丧失(AL)、龈沟出血指数(SBI)低于对照组(P<0.05)。研究组治疗后探诊出血率、前牙咬合低于对照组,咀嚼功能评分高于对照组(P<0.05)。研究组治疗后牙周袋深度、前牙覆盖度、牙槽骨高度低于对照组(P<0.05)。研究组治疗后龈沟液PGE2、s ICAM-1、PAK5水平低于对照组(P<0.05)。结论:牙周夹板联合正畸治疗可有效恢复牙周炎所致前牙扇形移位患者牙周功能和咀嚼功能,且美观效果好,还可减轻对龈沟液PGE2、s ICAM-1、PAK5水平的影响。