Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue?

BACKGROUND: Evidence of the role of matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in periodontal destruction is now well established. The purpose of this study was to quantify, in healthy and diseased upper gingival connective tissue, the area fraction (AA%) occupied by collagen fibers, the cell number belonging to inflammatory cell subsets, and the amounts of MMPs and TIMPs (tissue inhibitors of MMPs) in order to investigate the possible correlations, if any, between such molecules, collagen loss, and inflammatory cell subsets. METHODS: Gingival tissue specimens from 6 healthy controls (C) and 6 patients with severe periodontitis (P) were divided into 2 groups. The first group of specimens was frozen and used for the staining of collagen fibers by sirius red F3Ba and for immunohistochemistry with antibodies against CD8, CD4, CD22, CD68, and TIA-1 molecules. The second group was used for organ culture, zymography, Western blotting, and dot blotting. Morphometric and automated image analysis was performed for the evaluation of the area fraction occupied by collagen fibers, the number of inflammatory cell subsets and for enzymatic activities developed by MMPs, and the amounts of TIMPs expressed during periodontal disease. RESULTS: In group P, the area fraction of collagen fibers (33 +/- 10%) was significantly decreased (P < 0.0002) when compared to group C (60 +/- 7%), and was correlated with the number of all inflammatory cells and amounts of MMPs and TIMPs. In group P, there were significant increases of CD8+, CD22+, CD68+, and TIA-1+ cells, as well as increases in the amounts of MMP-1, MMP-2, MMP-3, MMP-9, and the active form of MMP-9. The active form of MMP-9 and the amount of TIMP-1 were positively correlated with the number of CD22+, CD68+, and TIA-1+ cells. CONCLUSIONS: The present study showed an imbalance between MMPs and TIMPs associated with the pathologic breakdown of the extracellular matrix during periodontitis. The active form of MMP-9 could be a marker for the clinical severity of periodontal disease.     

Are cytokines linked to collagen breakdown during periodontal disease progression?

BACKGROUND: Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established. The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index. METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA). Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis. RESULTS: The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers. Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF. The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3. CONCLUSION: The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss. These 2 cytokines could be markers of clinical severity during active periodontitis.     

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients

BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.     

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.     

Collagen fibers and inflammatory cells in healthy and diseased human gingival tissues: a comparative and quantitative study by immunohistochemistry and automated image analysis

BACKGROUND: Periodontal disease is histologically characterized by the degradation of extracellular matrix components associated with a gingival infiltration of inflammatory cell populations. The purpose of this in situ study was to quantify inflammatory cell subsets and the area fraction (AA%) occupied by collagen fibers in healthy and diseased upper gingival connective tissue in order to investigate the association, if any, between collagen loss and inflammatory cell infiltrate. METHODS: Paraffin gingival tissue sections from 10 healthy controls (C), 9 patients with gingivitis (G), and 10 patients with severe chronic periodontitis (P) were immunohistochemically stained by antibodies against CD45, CD3, CD8, CD20, CD68, TIA-1, and GrB molecules, and the collagen fibers were stained using sirius red F3Ba. The quantitative evaluations of inflammatory cell numbers and the AA% occupied by collagen fibers were performed by morphometric and automated image analysis. RESULTS: In group P, CD45+, CD20+, CD68+, TIA-1+, and GrB+ cell numbers were significantly increased (P<0.05) when compared to both C and G groups. The present study revealed significant differences (P <0.01) between means of AA% observed in group C (63%), group G (46%), and group P (26%), and AA% of group G and group P was inversely correlated with the numbers of TIA-1+ cells (P<0.01) and GrB+ cells (P<0.01 and P<0.05, respectively). CONCLUSIONS: This study showed great differences in the number of the distinct inflammatory cell subsets according to the severity of the periodontal disease and suggested that activated cytotoxic cells could play a pivotal role in the loss of collagen fibers observed during these pathological states. During periodontitis, collagen loss was significantly correlated with all inflammatory cell subset numbers. Finally, the quantitative evaluation of the area fraction occupied by gingival collagen fibers may reflect the clinical severity of the periodontal disease.     

基质金属蛋白酶及其组织抑制剂在牙本质生成性影细胞瘤和牙源性影细胞癌中的表达

目的 对牙本质生成性影细胞瘤(dentinogenic ghost cell tumor,DGCT)和牙源性影细胞癌(ghost cell odontogenic carcinoma,GCOC)中基质金属蛋白酶(matrix metallopmteinases,MMP)和金属蛋白酶组织抑制剂(tissue inhibitors of metalloproteinase,TIMP)的表达特点进行研究.方法 免疫组织化学分析15例DGCT和9例GCOC中MMP-2、MMP-9、MMP-14、TIMP-1和TIMP-2的表达.对DGCT和GCOC各一例分析MMP-2、MMP-9、MMP-14、TIMP-1和TIMP-2 mRNA的表达;明胶酶谱分析MMP-2和MMP-9酶原和活化蛋白的表达.结果 MMP-9和TIMP-1在GCOC中高表达(7/9,8/9);TIMP-1在GCOC中的高表达,与在DGCT中相比差异具有统计学意义(P＜0.05).对一例DGCT和一例GCOC病例研究发现,MMP-9酶原和活酶在GCOC中高表达,并且MMP-9和TIMP-1 mRNA在GCOC中高表达.结论 MMP-9和TIMP-1可能与GCOC的性质和生物学行为有关.     

Hydrocortisone affects the expression of matrix metalloproteinases (MMP-1, -2, -3, -7, and -11) and tissue inhibitor of matrix metalloproteinases (TIMP-1) in human gingival fibroblasts

BACKGROUND: There is a positive correlation between the course of periodontal disease and psychosocial stress status. Stress leads to activation of the hypothalamic-pituitary-adrenal axis, resulting in increased cortisol release. The present study evaluates the effect of two different hydrocortisone concentrations on mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) in cultured, human gingival fibroblasts. METHODS: Gingival fibroblasts were stimulated with 10(-7) or 10(-9) M hydrocortisone for 24 hours; untreated cells served as controls. Alterations in the expression of MMP-1, -2, -3, -7, -11 and TIMP-1 and -2 were evaluated using real-time polymerase chain reaction and Western blotting. beta-actin mRNA expression was used as a reference to normalize gene expression. RESULTS: Although the higher hydrocortisone concentration upregulated MMP-1, -2, -7, -11, and TIMP-1 (P <0.05) expression, the lower concentration induced downregulation or diminished upregulation. The lower hydrocortisone concentration induced a 23-fold increase in MMP-3 gene expression, whereas the higher concentration induced less upregulation; however, protein expression was regulated similarly by both hydrocortisone concentrations. The effect of hydrocortisone on TIMP-2 expression was not significant (P >0.05). CONCLUSIONS: Hydrocortisone produced a dose-dependent regulation of MMP and TIMP expression. The higher hydrocortisone concentration significantly upregulated expression of MMP-1, -2, -7, and -11 and TIMP-1 in human gingival fibroblasts, which may constitute a mechanism underlying the increased periodontal breakdown associated with psychosocial stress status.     

The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile periodontitis

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.     

Matrix metalloproteinases in mild and severe temporomandibular joint internal derangement synovial fluid

OBJECTIVES: The first objective of this study was to verify the presence of and identify the molecular forms of matrix metalloproteinases (MMPs), including collagenases (MMP-1, MMP-8, and MMP-13) and gelatinases (MMP-2 and MMP-9), in the synovial fluid (SF) of mild and severe temporomandibular joint internal derangement (TMJ-ID). Another objective was to evaluate whether the SF MMPs are potential diagnostic markers that reflect the stage of intra-articular inflammation in the TMJ. STUDY DESIGN: The subjects were 44 patients with mild (n = 16) or severe (n = 28) TMJ-ID; they were classified on the basis of subjective symptoms, clinical and radiographic findings, and surgical observations. The patients were surgically treated, and SF samples were collected immediately before the operation. The collagenase activity of SF samples was analyzed by means of a type I collagen degradation assay. The levels and molecular forms of the SF MMPs as well as the tissue inhibitors of MMPs (TIMP-1 and TIMP-2) were analyzed with Western immunoblotting and gelatin zymography. RESULTS: The SF of both the mild and the severe TMJ-ID patients exhibited free collagenase activity and activity capable of further degrading the (3/4)(alphaA) fragments. Ninety-two-kilodalton proMMP-9 and its 121-kD complex form, as well as 72-kD proMMP-2 were significantly increased in the mild TMJ-ID group (P <.05 in all cases). Both 70- to 80-kD neutrophil type and 45- to 55-kD mesenchymal cell-type MMP-8 (corresponding to the latent and active forms) were observed in mild and severe TMJ-ID SF, but they predominated in mild TMJ-ID. Both MMP-1 and MMP-13 were observed in both groups, and in mild TMJ-ID SF the low-molecular weight forms of MMP-1 indicated activation of the enzyme. CONCLUSIONS: The degradation of type I collagen in the TMJ is evidently due to the collective action of many collagenolytic MMPs present in the SF of patients with mild and severe TMJ-ID. The elevated levels of MMP-2, MMP-9, and MMP-8 in the SF of patients with mild TMJ-ID eventually reflect the active phase of TMJ destruction. These observations may have considerable diagnostic and therapeutic significance in the management of TMJ disorders.     

Matrix metalloproteinases and their inhibitors in gingival mast cells in persons with and without human immunodeficiency virus infection

BACKGROUND: Mast cells are a prominent cell type in the gingival infiltrate in periodontitis. In this study we examined the expression by gingival mast cells of matrix metalloproteinases, MMP-1, MMP-2, MMP-8 and the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. METHODS: Gingival specimens from 12 human immunodeficiency virus-negative (HIV-) and 15 HIV-positive (HIV+) patients with chronic marginal periodontitis (CMP), and from 10 HIV- and four HIV+ controls with clinically healthy gingiva (HG) were examined after double immunofluorescence staining for mast cell tryptase, combined with antibodies for MMP-1, MMP-2, MMP-8 or their inhibitors TIMP-1 and TIMP-2. RESULTS: In the HIV+CMP, HIV+HG and HIV-CMP groups, all mast cells expressed MMP-1 and MMP-8, whereas a smaller proportion (40-60%) in the HIV-HG controls displayed such staining. The former groups also displayed a significantly higher proportion (39-64%) of mast cells expressing MMP-2 as compared with the HIV-HG group (21-31%). All groups displayed similar proportions of TIMP-1 expressing mast cells (86-100%), whereas significantly increased proportions of TIMP-2+ mast cells were seen in the HIV+CMP, HIV+HG and HIV-CMP groups (18-25%) as compared with the HIV-HG group (8-13%). Mast cells were the cell type that most prominently expressed MMP-1 and MMP-8. MMP-2 expression was also strong in mast cells, but was also similarly expressed in other cell types. CONCLUSION: The chronically inflamed periodontal lesions in the present study appeared with little evidence of mast cell degranulation. The results show, however, that mast cells in inflamed gingiva have the potential to degrade extracellular matrix if appropriately triggered.     

黄芩苷对大鼠实验性牙周炎的保护效应及作用机制研究

目的 观察黄芩苷对丝线结扎诱导的大鼠牙周组织炎性破坏的保护作用,并探讨这一作用是否与牙龈组织中基质金属蛋白酶(matrix metalloproteinases,MMP)表达水平的改变有关.方法 将SD大鼠按随机数字表法分为3组,每组9只.用丝线结扎方法建立牙周炎模型,建模第7天处死动物.用药组(B200组)每天灌胃给予黄芩苷200 mg/kg;阴性对照组(L组)每天灌胃药物媒介0.5%羧甲基纤维素钠;空白对照组(C组)不予牙周炎诱导.评价牙槽骨吸收和胶原纤维的面积分数.免疫组化方法检测牙龈组织中MMP-1、MMP-2和MMP-9的表达.结果 B200组的骨吸收值[(0.93±0.04)mm]显著低于L组[(1.03±0.07)mm,P=0.009)];B200组的胶原纤维面积分数[(48.13±18.69)%]显著高于L组[(31.08±14.85)%,P=0.047];与L组相比,应用黄芩苷显著下调了MMP-1(P=0.023)及MMP-9(P=0.042)的水平,并降低了MMP-2的表达(P=0.099).结论 黄芩苷能够减少丝线结扎诱导的大鼠牙周炎的组织破坏,这一作用可能与其抑制MMP-1、MMP-2和MMP-9的表达有关.     

Nicotine increases the collagen-degrading ability of human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.     

Identification and characterization of gelatinases/type IV collagenases in jaw cysts

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and α-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blottings revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (<60 kD) were detected by α-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl). This indicates MMP-specific sensitivity to oxidative agents, but more specifically to preferential oxidative activation of PMN 92 kD MMP-9 and oxidative inactivation of the fibroblast-type 72 kD MMP-2.     

Immunohistochemical detection of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ameloblastomas

BACKGROUND: To evaluate the roles of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in tumor progression, expression of MMP-1, -2 and -9 and TIMP-1 and -2 was analyzed in ameloblastomas as well as tooth germs. METHODS: Frozen tissue sections of seven tooth germs and 22 ameloblastomas were immunohistochemically examined using anti-MMP-1, -2 and -9 and anti-TIMP-1 and -2 antibodies. RESULTS: MMP-1, -2 and -9 and TIMP-1 and -2 were expressed strongly in mesenchymal components of tooth germs, and stromal cells of ameloblastomas. Immunoreactivity for MMP-9 in stromal cells of ameloblastomas was significantly stronger than in mesenchymal cells of dental follicles and dental papillae. Dental laminae showed weak MMP-2 expression in six tooth germs, MMP-9 expression in two tooth germs and TIMP-1 expression in six tooth germs. Some tumor cells showed weak MMP-2 expression in 19 ameloblastomas, MMP-9 expression in four ameloblastomas and TIMP-1 expression in all cases. TIMP-2 reactivity was prominently found in basement membrane zones of dental laminae in tooth germs, and tumor cell islands or nests in ameloblastomas. CONCLUSION: Expression of MMPs and TIMPs was considered to be associated with interactions between epithelial cells and mesenchymal components in normal and neoplastic odontogenic tissues; these molecules might play a role in regulation of tumor progression in ameloblastomas as well as regulation of developmental processes in tooth germs.