Vascular endothelial growth factor in gingival tissues and crevicular fluids of diabetic and healthy periodontal patients

BACKGROUND: Periodontal disease is one of the major oral problems encountered in patients with diabetes mellitus (DM). Vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition observed in DM may contribute to periodontitis; and the vascular alterations observed in such patients may depend on vascular endothelial growth factor (VEGF) actions. Few reports are available about the mechanism of neovascularization and the angiogenic factors that contribute to the periodontal pathology and the role of VEGF in periodontal diseases. The aim of this study is to compare VEGF expression in healthy and periodontally diseased tissues with gingival crevice fluid (GCF) of healthy persons and diabetic patients. METHODS: Gingival tissue and GCF samples were collected from sites of periodontitis in 10 healthy subjects and in 10 type 2 diabetic patients, and from the sites of healthy gingiva within the same groups. Therefore, each patient became his/her own control. Additionally, 10 people without any systemic or periodontal diseases were enrolled, forming a negative control group. Thus, a total of 50 tissue and 50 GCF samples were provided. RESULTS: No VEGF staining was observed in the negative control group or in the systemically healthy people's healthy tissue samples, whereas four samples of diabetic patients showed positive staining (P < 0.05). However, VEGF was revealed in two tissue samples of periodontal sites of systemically healthy people and in six samples of the diabetic patients (P > 0.05). In all test groups, GCF VEGF levels were higher in periodontal sites (P < 0.05) than in healthy sites. CONCLUSION: The results of this study showed that VEGF is increased in all periodontal tissues of both groups and in the healthy sites of diabetic patients. Additionally, GCF VEGF values increased in periodontal sites of all test groups.     

Ⅱ型糖尿病伴牙周炎病人牙龈组织中VEGF表达及MVD计数的研究

目的:探讨Ⅱ型糖尿病伴牙周炎病人牙龈组织中血管内皮生长因子(VEGF)的表达水平及其作用。方法:选取Ⅱ型糖尿病伴重度牙周炎(DP)病人、单纯重度慢性牙周炎(CP)病人、健康对照者(N)各15例,分别切取牙龈组织,用免疫组化染色方法检测牙龈组织中VEGF表达和MVD计数。结果:DP组牙龈组织中VEGF表达和MVD计数均显著高于CP组和N组(P<0.05);CP组高于N组(P<0.05)。结论:Ⅱ型糖尿病伴重度慢性牙周炎病人牙龈组织中VEGF表达水平和MVD计数明显升高。     

人牙周膜细胞及炎症龈组织中mCD14表达的免疫组化研究

目的研究人PDLC及炎症龈组织中mCD14表达情况。方法采用免疫组化SABC方法。结果正常人牙周膜细胞mCD14染色为阳性反应，但细胞经脂多糖刺激2天后，胞浆染色要比未刺激的细胞染色深。健康牙龈组织中mCD14表达阴性；边缘性龈炎和牙周炎龈组织上皮棘层细胞mCD14呈强阳性表达，增生性龈炎也呈阳性表达。结论mCD14可在非髓样细胞中表达；mCD14可能在局部作为脂多糖受体参与牙周组织的破坏。     

CD44和CD133在口腔潜在恶性病变和口腔鳞状细胞癌中的表达及其临床意义

目的 探究肿瘤干细胞标记物CD44和CD133在口腔正常黏膜、口腔潜在恶性病变（OPMD）及口腔鳞状细胞癌（OSCC）中的表达情况及相互关系,评估其作为OPMD恶性转化早期诊断指标的临床价值。方法 回顾性分析60例OPMD患者、60例OSCC患者及10例口腔黏膜正常者的临床资料,采用免疫组织化学双重染色技术检测各组病理组织中CD44与CD133的表达,分析CD44和CD133表达之间的关系及其与各临床因素之间的关系。结果 口腔正常黏膜、OPMD、OSCC三组中,CD44的阳性表达率分别为100.00%、96.67%、71.67%（P<0.05）,CD133的阳性表达率分别为0.00%、35.00%、63.33%（P<0.05）。CD44与CD133二者间的表达具有相关性（P<0.05）,且其表达又都与OSCC的临床分期和淋巴结转移相关（P<0.05）。结论 CD44和CD133作为评估OPMD恶性转化潜能的指标具有重要临床价值。     

CD9 and HLA-DR expression by crevicular epithelial cells and polymorphonuclear neutrophils in periodontal disease

BACKGROUND, AIMS: The composition of gingival crevicular fluid (GCF) is likely to reflect inflammatory modifications that take place in the gingiva during periodontal diseases. METHOD: In this study, GCF was collected at 3 different sites from 23 periodontal patients. The sites were assessed to be healthy, presenting gingivitis or periodontitis. 10 healthy individuals without any form of periodontal disease formed the control group and were sampled at one site each. The cell content of GCF was collected using Durapore Millipore strips, and 2 types of cells were studied: epithelial cells (EC) and polymorphonuclear neutrophils (PMN). The expression of CD9 and HLA-DR within or on the surface of these cells was studied in immunofluorescence on cytospin smears. RESULTS: Both CD9 and HLA-DR expression on EC differed significantly from control subjects, and the latter decreased according to the severity of the pathology. None of the PMN found in controls expressed CD9 or HLA-DR. However, in periodontal patients, the expression of HLA-DR within PMNs was detectable and increased according to the severity of lesions. CD9 expression on PMNs also increased with inflammation. CONCLUSION: This study shows that clinically healthy sites of periodontal patients already present signs of immunological activation characterised by a down modulation of HLA-DR expression on EC and an upregulation of these 2 molecules in PMN.     

Markers of bone destruction and formation and periodontitis in type 1 diabetes mellitus

Aim: To determine plasma concentrations of bone metabolism markers in type 1 diabetes mellitus patients and non-diabetic and to evaluate the influence of periodontitis on biomarkers of bone formation in these patient groups.
Methods: Plasma concentrations of receptor activator of nuclear factor- κ B ligand (RANKL), osteoprotegerin (OPG), C-terminal telopeptide of type 1 collagen and osteocalcin were measured in type 1 diabetes mellitus patients ( n =63) and non-diabetics ( n =38) who were also subdivided on the basis of their periodontal status.
Results: Diabetics had significantly lower osteocalcin concentrations, lower RANKL to OPG ratios and higher OPG concentrations (as shown by other researchers) than non-diabetics. The ratio of RANKL to OPG was altered by the periodontal status. Osteocalcin had a negative correlation and OPG a positive correlation with the percentage of glycated haemoglobin in the blood.
Conclusion: Because, osteocalcin, a biomarker of bone formation, is lower in patients with periodontitis and in patients with type 1 diabetes mellitus with and without periodontitis than in non-diabetics without periodontitis, this might indicate that diabetics are less able to replace bone lost during active bursts of periodontitis and explain the greater severity of disease seen in studies of patients with diabetes.     

实验性牙移动初期大鼠牙周组织CD133+血管内皮祖细胞的表达

目的研究实验性牙移动初期大鼠牙周组织中CD133+血管内皮祖细胞（EPC）的表达。方法40只Wistar大鼠,随机分为实验组和对照组,建立牙移动动物模型。于加力后1、3、5、7、14 d处死动物并制作以上颌第一磨牙为中心近远中向的组织切片,进行苏木精－伊红染色、CD133免疫组织化学染色。结果大鼠正常牙周组织中未见CD133表达的新生血管。实验性牙移动初期,实验组牙周组织新生微血管内皮细胞中可见CD133阳性染色,牙周组织CD133表达于加力后1 d达到高峰,此后逐渐下降,但与对照组相比差异无统计学意义（P>0.05）。结论CD133+EPC参与了实验性牙移动牙周组织早期的血管改建,但其直接参与牙周组织血管生成较少,可能大多通过旁分泌作用参与新血管的形成。     

Lymphangiogenesis is induced during development of periodontal disease

Lymphangiogenesis, the formation of new lymphatics, is associated with chronic inflammation and tissue injury, and its role is to enhance lymphatic flow, immune cell transport, and antigen clearance. It is unknown if lymphangiogenesis takes place during periodontal disease development, and we hypothesized that growth of lymphatic vessels occurs in gingiva during development of periodontitis in mice. Inflammation was induced in gingiva with Porphyromonas gingivalis gavage, and bone resorption was verified after 42 days. Growth of lymphatic and blood vessels was measured after immunofluorescent staining with LYVE-1 and CD31. Expression of vascular endothelial growth factors and 2 inflammatory cytokines was investigated 10 days post-infection. Gingival lymphangiogenesis was found 10 days and 42 days post-infection, but proliferation of vessels was observed only in the shortest observation period. Epithelial expression of vascular growth factors (VEGF) A, C, and D was observed in gingiva, and increased numbers of immune cells expressing VEGF-C were found after infection, along with up-regulation of IL-1β and TNF-α at protein levels. We conclude that lymphangiogenesis takes place in gingiva during periodontal disease development, and that up-regulation of vascular growth factor C in recruited immune cells is likely important for the growth of lymphatic vessels.     

Serum sCD14, polymorphism of CD14−260 and periodontal infection

Background and Aims:  CD14 is a co-receptor involved in the recognition of Gram-negative and positive bacteria. Infections are known to influence serum sCD14 levels, and CD14 gene promoter polymorphism (CD14 C−260T) has been reported to be associated with many infectious diseases. Our aim was to investigate whether serum sCD14 concentration is associated with periodontal infection and the CD14⁻²⁶⁰ genotype.
Subjects and Methods:  The periodontal status of 56 subjects with chronic periodontitis and 28 controls was clinically examined. Serum sCD14 concentration was analyzed using ELISA and CD14⁻²⁶⁰ genotype using polymerase chain reaction (PCR).
Results:  The mean concentration of sCD14 in serum was significantly higher in subjects with periodontitis than in control subjects (4.9  μ g ml⁻¹ vs 3.8  μ g ml⁻¹, P  < 0.001). Serum sCD14 concentration associated significantly with the extent of advanced periodontal disease. In a regression analysis including both subject groups, the CD14⁻²⁶⁰ genotype was a significant determinant for serum sCD14 concentration. After stratification by periodontal health status (periodontitis vs controls), the influence of the CD14⁻²⁶⁰ genotype on serum sCD14 concentration was seen only in the control group.
Conclusions:  Periodontal infection is associated with the serum concentration of sCD14. Moderate to severe periodontal infection overshadows the influence of the genotype on serum sCD14 concentration.     

Association between type 1 and type 2 diabetes with periodontal disease and tooth loss

Aim: The aim of this study was to determine whether both type 1 (T1DM) and type 2 diabetes mellitus (T2DM) are associated with increased prevalence and extent of periodontal disease and tooth loss compared with non-diabetic subjects within a homogeneous adult study population.
Material and Methods: T1DM, T2DM and non-diabetic subjects were recruited from the population-based Study of Health in Pomerania. Additionally, T1DM subjects were retrieved from a Diabetes Centre. The total study population comprised 145 T1DM and 2647 non-diabetic subjects aged 20–59 years, and 182 T2DM and 1314 non-diabetic subjects aged 50–81 years. Periodontal disease was assessed by attachment loss (AL) and the number of missing teeth.
Results: Multivariable regression revealed an association between T1DM ( p <0.001) and T2DM ( p <0.01) with mean AL after full adjustment. After age stratification ( p =0.04 for interaction), the effect of T2DM was only statistically significant in the 60–69-year-old subjects (B=0.90 (95% confidence intervals [95% CI]; 0.49, 1.31). T1DM was positively associated with tooth loss (adjusted, p <0.001). The association between T2DM and tooth loss was statistically significant only for females (odds ratios=1.60 [95% CI: 1.10, 2.33]).
Conclusions: Our study confirmed an association between both T1DM and T2DM with periodontitis and tooth loss. Therefore, oral health education should be promoted in diabetic subjects.     

Effects of diabetes mellitus on periodontal and peri-implant conditions: update on associations and risks

Objectives: To review the evidence for the association between diabetes and periodontal and peri-implant conditions and the impact of periodontal therapy in subjects with diabetes.
Material and Methods: A search of MEDLINE-PubMed was performed up to and including December 2007. The search was limited to clinical studies published in English. Publications on animal studies were excluded. The selection criteria included all levels of available evidence.
Results: Evidence on the association between diabetes and periodontitis supports the concept of increased severity but not extent of periodontitis in subjects with poorly controlled diabetes. Subjects with controlled diabetes do not show an increase in extent and severity of periodontitis. Periodontitis is associated with poor glycaemic control and diabetes-related complications. It is inconclusive that periodontal therapy with or without the use of antibiotics results in improvements of glycaemic control and of markers of systemic inflammation. Evidence is lacking to indicate that implant therapy in subjects with diabetes yields long-term outcomes comparable with those of non-diabetic subjects.
Conclusions: Poorly controlled diabetes may be considered a risk factor for increased severity of periodontitis. The effects of periodontal therapy on glycaemic control and systemic inflammation is not proven beyond doubt and need to be confirmed in large-scale randomized-controlled clinical trials.     

脂多糖信号受体CD14和 TLR4在人炎症根尖周组织中的表达

目的 研究慢性根尖周炎患者根尖周组织中脂多糖(LPS)信号受体CD14和TLR4的表达和分布,探讨CD14和TLR4在炎性根尖周组织中可能的作用.方法 收集需做根尖外科手术的12例慢性根尖周炎患者和10例外科手术拔出的无牙髓炎和根尖周病变的阻生齿的根尖周组织,术中取炎症和正常根尖周组织后常规切片,采用免疫组织化学方法检测CD14和TLR4的表达和分布.结果 CD14和TLR4在炎症根尖周组织中呈强阳性表达,主要分布于单核细胞、巨噬细胞等炎症细胞;而在正常根尖周组织中未见明显CD14和TLR4阳性细胞.结论 炎症根尖周组织中CD14和TLR4的阳性表达,提示LPS可能通过CD14和 TLR4信号受体在根尖周炎症组织中发挥作用.     

Expression of CD14, CD16 and CD45RA on monocytes from periodontitis patients

BACKGROUND AND OBJECTIVE: Peripheral blood monocytes are a heterogeneous population, with phenotypes that change on activation or differentiation. Most of the monocytes express lipopolysaccharide (LPS) receptor, CD14 intensely, and do not express Fc gamma receptor III, CD16 (CD14++CD16- monocytes). But monocytes expressing CD16 with reduced CD14 (CD14+CD16+ monocytes) increase in inflammatory diseases as well as sepsis and bacteremia in hemodialysis patients. CD45RA is expressed on activated monocytes, and is regarded as an activation marker of peripheral blood monocytes. The purpose of this study was to determine the phenotypic and functional alteration of monocytes in periodontitis patients. METHODS: Peripheral blood was collected from 33 aggressive periodontitis patients (22 females, 11 males), 55 chronic periodontitis patients (35 females, 20 males) and 30 healthy subjects (16 females, 14 males), and the expression of CD14, CD16 and CD45RA on monocytes was determined using flow cytometry. The production of interleukin-6 (IL-6) by CD16+ and CD16- monocytes stimulated with LPS from Escherichia coli and Actinobacillus actinomycetemcomitans was also examined using flow cytometry. RESULTS: The percentage of CD14+CD16+ monocytes was significantly increased in chronic periodontitis patients. Percentage of monocytes expressing CD45RA was significantly increased in aggressive periodontitis patients compared to healthy subjects. CD16+ and CD16- monocytes produced IL-6 in response to LPS from E. coli and A. actinomycetemcomitans, and the percentage of IL-6 producing cells was higher in CD16+ monocytes than CD16- monocytes, suggesting that CD14+CD16+ monocytes represent a hyper-reactive phenotype. CONCLUSIONS: The present study demonstrated that CD14+CD16+ monocytes and CD45RA+ monocytes were increased in chronic and aggressive periodontitis, respectively. These findings suggest that alteration of monocytes in periodontitis patients could be evaluated by monitoring the surface expression of CD14, CD16 and CD45RA on monocytes.     

2型糖尿病家系成员牙周状况调查

目的调查2型糖尿病家系成员的牙周状况。方法共收集43个家系[167人，男性71人，女性96人，平均年龄为（49.2±12.2）岁]，分别抽取静脉血，并选取每位受检者的6颗代表牙，记录每颗牙的6个位点的菌斑指数（plaque index，PLI）、牙龈出血指数（bleeding index，BI）、牙周探诊深度（probing depth，PD）、附着丧失（attachment loss，AL）并记录总牙数及失牙数。结果43个家系中共有糖尿病患者101例，其中4例全口牙缺失，经牙周病问卷调查显示，均为牙齿松动自行脱落或拔除。97例糖尿病患者均患牙周炎，其中轻度牙周炎50例，中度牙周炎24例，重度牙周炎23例。48例非糖尿病者中牙龈炎5例，轻度牙周炎30例，中度牙周炎11例，重度牙周炎2例。糖尿病患者和非糖尿病者龈炎和轻、中、重度牙周炎患病率差异有统计学意义（X^2=17.96，P〈0.005），糖尿病患者的PD、AL及缺失牙数均高于非糖尿病者，差异有统计学意义（P〈0.05）。血糖控制不良的糖尿病患者BI、AL均显著高于血糖控制良好的糖尿病患者（P〈0.05）。血糖控制良好的糖尿病患者PLI、BI、PD及AL略高于非糖尿病者，但差异无统计学意义。结论在糖尿病家系成员中糖尿病患者的牙周炎患病率明显高于非糖尿病者，牙周破坏程度亦明显重于非糖尿病者；血糖控制良好患者的牙周状况与非糖尿病者相似。     

Superoxide dismutase activity in gingiva in type-2 diabetes mellitus patients with chronic periodontitis

OBJECTIVE: Antioxidant defence reduces in diabetes mellitus (DM) and periodontitis. This study investigates antioxidant enzyme; superoxide dismutase (SOD) activity in gingiva and blood glucose and lipid levels in type-2 DM patients and systemically healthy individuals with chronic periodontitis (CP). MATERIALS AND METHODS: Periodontal parameters, blood glycated-haemoglobin (HbA1c), glucose and lipid levels, and gingival-SOD activities (spectrophotometric assay) were measured in 17 DM patients with CP (DMCP), 17 systemically healthy CP patients, 18 periodontally healthy DM patients (DMPH), and 17 healthy controls (PH). RESULTS: Periodontal parameters were higher in periodontitis groups than the controls (p<0.05), while there was no difference between the periodontitis groups and between the control groups. HbA1c, glucose, and triglyceride levels were higher in diabetic groups than the non-diabetic groups (p<0.05). Low-density lipoprotein (LDL), very-LDL and cholesterol values of the DMCP group did not significantly differ from the CP group. No differences existed between diabetic patients with and without periodontitis in HbA1c, glucose, and lipid levels and the same was true for non-diabetic patients with and without periodontitis. Gingival-SOD activity was lower in periodontitis groups than the matched control groups (p<0.05). DMPH group had the highest and CP group had the lowest SOD levels. There were correlations between periodontal parameters, gingival-SOD activity, HbA1c, glucose and high-density lipoprotein (HDL) levels. CONCLUSION: The results suggest that gingival-SOD activity increases in diabetes and decreases in periodontitis and relations may exist between gingival-SOD activity, periodontal status, HbA1c, glucose and HDL levels. The higher gingival-SOD activity in diabetes may be attributed to an adaptive mechanism in the tissue.     

Ⅱ型糖尿病合并牙周病患者龈沟液中细胞因子/趋化因子的表达水平

目的 探讨Ⅱ型糖尿病合并牙周病患者与单纯牙周病患者龈沟液（gingival crevicular fluid,GCF）中细胞因子/趋化因子的表达水平。 方法 选取伴Ⅱ型糖尿病的牙周病患者52例,单纯牙周病患者40例,用Luminex FLEXMAP3D仪和Human Cytokine/Chemokine试剂盒检测GCF中14种细胞因子/趋化因子的表达水平。 结果 牙周病部位：嗜酸性粒细胞趋化因子、巨噬细胞炎症蛋白-1α、粒细胞-巨噬细胞集落刺激、白介素-6、肿瘤坏死因子-α和白介素-12的浓度,糖尿病组受试者高于非糖尿病组受试者（P<0.0035）。 结论 糖尿病可影响牙周病部位细胞因子/趋化因子的表达,糖尿病可能是牙周病的促进因素。     

CD29 expression on CD4+ gingival lymphocytes supports migration of activated memory T lymphocytes to diseased periodontal tissue

The cell surface phenotypes of CD4⁺ cells extracted from inflammatory periodontal disease tissues were analyzed using two- and three-color immunofluorescence and flow cytometry. Cells extracted from both adult periodontal and localized juvenile periodontitis lesions showed a depressed CD4/CD8 ratio (1.0±0.1 adult periodontitis and 1.1 ±0.1 localized juvenile periodontitis) compared with cells recovered from normal/marginal gingivitis tissue (1.8 ±0.2) or with normal peripheral blood cells (2.1 ±0.1) or periodontal disease blood cells (2.1±0.1 and 1.7±0.1 for adult periodontitis and juvenile periodontitis, respectively). The monoclonal antibodies anti-2H4 and anti-4B4 were used to identify the CD45RA and CD29 antigens respectively on CD4⁺ T cells from the periodontal disease lesions. In peripheral blood, CD29⁺ cells accounted for 66–77% of the CD4⁺ population, and CD45RA⁺ cells accounted for 22–27% of the CD4⁺ subset. No differences in expression were found between peripheral blood lymphocytes from normal subjects and from periodontal disease patients. Two-color analyses of lymphocytes from periodontal diseased tissues showed that 87–89% of the CD4⁺ population were CD29⁺ and that 70–79% of the CD4⁺ cells were CD45RA⁺. Normal tissues contained significantly fewer CD4⁺CD29⁺ cells (56±4%) and CD4⁺CD45RA⁺ cells (40±4%) on average, and few, if any double-labelled cells could be accounted for. These data implied that a significant percentage of the CD4⁺ cells from the diseased tissues were both CD29⁺ and CD45RA⁺ and that these populations are found in quite different proportions in diseased periodontal tissue than in peripheral blood or nondiseased tissue. In further analyses using three-color cytometry the mean percentage of CD4⁺CD29⁺CD45RA⁺ lymphocytes extracted from periodontal disease lesions was 43±9% of the CD4⁺ population. These results suggest that CD4⁺ T lymphocytes in periodontal disease not only demonstrate varying levels of maturity but also that the accumulation of CD4⁺ T cells within the periodontal tissues maybe a result of increased adhesion and transendothelial migration.     

Detection of, and anti-collagen antibody produced by, CD5-positive B cells in inflamed gingival tissues

This study was performed to investigate the frequency and distribution of CD5-positive (CD5+) B cells in inflamed gingival tissues using flow cytometric and immunohistochemical analyses. The ability of CD5+ B cells to produce anti-type I collagen antibody was also examined. CD5+ B cells expressed "low" fluorescence intensity in the peripheral blood of both healthy subjects and patients with adult periodontitis. However, in inflamed gingival tissues the intensity of this surface marker was high. The percentage of B cells bearing CD5 surface marker was statistically higher in gingiva than in peripheral blood obtained from both the patients and healthy subjects. These CD5+ B cells were observed in gingival subepithelial connective tissues from the bottom to the middle of the periodontal pocket. This area showed destruction of collagen fibers and dense cell infiltrations. Anti-collagen IgG antibody level in patients' gingival crevicular fluids (GCF) was higher than that in sera from healthy subjects, and slightly higher than in autologous sera. IgM anti-collagen antibody in GCF was lower than in autologous sera and in sera from healthy subjects. EBV-transformed CD5+ B cells produced considerably more IgM and IgG antibody to collagen than CD5- B cells. Therefore CD5+ B cells may contribute to the pathogenesis of inflamed gingival tissues.