Regulation of matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase-1, and extracellular metalloproteinase inducer by interleukin-17 in human periodontal ligament fibroblasts.

INTRODUCTION: Under physiological conditions, matrix metalloproteinases (MMPs) are involved in the turnover of periapical tissue, and their activity is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs). Disturbances in the balance between MMPs and TIMPs may result in excessive tissue destruction. In addition, the extracellular metalloproteinase inducer (EMMPRIN) capable of inducing MMPs may also play a role in the pathologic processes. This study aimed to investigate the effects of interleukin (IL)-17 on the mRNA expression and protein production of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN through human periodontal ligament cells. METHODS: The cells were stimulated with IL-17 (1, 10, and 50 ng/mL) for different time periods. The mRNA levels of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN were evaluated via quantitative real-time polymerase chain reaction analysis, whereas the protein secretion into the culture medium was assessed via enzyme-linked immunosorbent assay and zymography analysis. RESULTS: IL-17 significantly up-regulated MMP-1 and MMP-13 mRNA expression but down-regulated MMP-2, MMP-9, and TIMP-1 mRNA expression. Furthermore, IL-17 (50 ng/mL) increased the secreted protein level of MMP-1 and MMP-13 and conversely reduced the level of MMP-2, MMP-9, and TIMP-1. However, IL-17 exerted no effect on EMMPRIN mRNA or protein secretion. CONCLUSIONS: This study first reported the ability of IL-17 to regulate MMP and TIMP-1 production through human periodontal ligament cells, a phenomenon that may contribute to periapical tissue destruction.

Inmunodetección de metaloproteinasas de matriz extracelular (MMPs)-2, -9, -13 y -14 en lesiones apicales asociadas con periodontitis apical asintomática / Immunodetection of matrix metalloproteinases (MMPs)-2, -9, -13 and -14 in periapical lesions associated with asymptomatic apical periodontitis

La periodontitis apical asintomática (PAa) es una patología infecciosa caracterizada por destrucción ósea perirradicular asociada a un proceso inflamatorio crónico y producción de mediadores inflamatorios, entre los cuales se encuentran las metaloproteinasas de matriz extracelular (MMPs). Entre éstas, las MMPs-13, -14, -2 y -9, son producidas por el tejido óseo y degradan sinérgicamente el colágeno tipo I, principal componente de los tejidos periodontales, y gelatina, producto de la degradación y desnaturación del colágeno. El objetivo de este estudio fue determinar el patrón de expresión de las MMPs-2, -9, -13 y -14 en granulomas periapicales (GPAs), quistes radiculares inflamatorios (QRIs) y ligamento periodontal sano (LS). Materiales y Métodos: Se seleccionaron 12 pacientes con diagnóstico clínico de PAa e indicación de exodoncia a partir de los cuales se obtuvieron biopsias de lesiones periapicales (LPAs). Como controles, se seleccionaron 7 individuos con indicación de exodoncia de premolares por ortodoncia, obteniéndose biopsias de LS. Se efectuó el diagnóstico anátomo-patológico de los especímenes y se caracterizó la expresión de las MMPs en estudio mediante inmunohistoquímica. Resultados: Las MMPs en estudio sólo se detectaron en GPAs y QRIs, y se inmunolocalizaron principalmente en el infiltrado inflamatorio de éstos. Adicionalmente, la MMP-2 se identificó en fibroblastos del tejido conectivo. Conclusiones: MMPs-2, -9, -13 y -14 se expresan predominantemente en el infiltrado inflamatorio de las LPAs y no en LS, y por tanto se sugiere la participación de estos mediadores en la patogénesis de la PAa.

Asymptomatic apical periodontitis (aAP) is an infectious disease characterized by perirradicular bone destruction associated with chronic inflammation and release of inflammatory mediators, such as matrix metalloproteinases (MMPs). MMPs-13, -14 and -2, -9 are bone-expressed enzymes that can synergistically degrade collagen I, the main component of periodontal extracellular matrix, and gelatin, the product of degradation and denaturation of collagen. The aim of this study was to characterize the expression pattern of MMPs-2, -9, -13, and -14 in periapical granulomas (PGs), radicular cysts (RCs) and healthy periodontal ligament (PDL). Materials and Methods: Individuals with clinical diagnosis of aAP and indication of extraction were selected (N=12), and biopsies of periapical lesions (PLs) were obtained. For controls, 7 subjects with indication of premolar extraction for orthodontic reasons were selected, and PDL biopsies were obtained. Samples were diagnosed by anatomopathological examination and immunohistochemical staining was carried out to characterize MMPs expression. Results: MMPs-2, -9, -13 and -14 detection was limited to PLs and were localized mainly to inflammatory infiltrate on both, PGs and RCs. Additionally, MMP-2 was immunolocalized to fibroblasts from the connective tissue. Conclusions: Whereas MMPs-2, -9, -13 and -14 were not detected in healthy periodontal ligament, they were highly expressed on inflammatory infiltrate from PGs and RCs, suggesting a role of these mediators in aAP pathogenesis.

Rat model for studying tissue changes induced by the mechanical environment surrounding loaded titanium implants.

PURPOSE: This study presents a new rat oral implant model for assessing histologic changes in the mechanical environment surrounding loaded and unloaded dental implants. MATERIALS AND METHODS: The maxillary left first molar from retired breeder rats was extracted, and the site was allowed to heal for 1 month. A titanium miniscrew implant was then placed into the site and allowed to heal for 21 days. The mandibular left first molars in one group of rats were extracted to create an unloaded condition; in a second group of rats the mandibular left first molars were left in occlusion with the opposing screw head to simulate loading. Radiographs were taken on the day of placement and again at 7 days, 14 days, and 21 days after placement and were used to estimate the bone-implant contact ratio. The rats were sacrificed after 21 days. Peri-implant tissue samples from day 21 were processed for histology and immunohistochemistry with antibodies to osteocalcin and matrix metalloproteinase 13 (MMP-13). Two-dimensional finite element models were created from images of the histologic sections and immunohistochemical samples to observe tissue changes. RESULTS: Areas of high shear stress adjacent to the helical threads of loaded implants were associated with osteocalcin localization and bone formation but only minimal localization of MMP-13. Bone adjacent to unloaded implants showed fibrous tissue and extensive MMP-13 localization surrounding the apical two-thirds of each implant. These results agree with estimated bone-implant contact ratios, which showed a steady decrease in contact ratio for the unloaded implant group but a significantly higher contact ratio in the loaded group between 14 and 21 days. CONCLUSION: The rat oral implant model is useful for studies of the mechanical and physiologic environment affecting osseointegration in loaded and unloaded implants.

Immunolocalization of matrix metalloproteinases-2 and -9 during apical periodontitis development.

OBJECTIVE: The objective of this study was to determine the expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) during apical periodontitis development. METHODS: Using an experimental design of induced periapical lesions in rats and immunohistochemistry assay as investigative tool, the MMP-2 and MMP-9 expression and distribution were evaluated at 3, 7, 14, 21, 30, 60 and 90 days after coronary access and pulp exposure of the first left mandibular molar to the oral environment. Two blind observers scored the immunoreactivity. A semi-quantitative analysis was performed. RESULTS: Except at day 3, MMP-2 and MMP-9 immunostaining was observed in all experimental periods. The MMP-2 (p=0.004) and MMP-9 (p=0.005) immunostaining was higher in the period between 7 and 21 days. They were mainly observed in cells surrounding the apical foramen and adjacent periapical areas. Cells into the hypercementosis areas were strongly stained while both osteoblasts and osteoclasts presented discrete staining along of this study. No staining was observed on epithelial walls. At 30, 60 and 90 days, the subjacent connective tissue presented intense MMP-2 and MMP-9 immunostaining in mononuclear cells (suggestive of fibroblasts, macrophages, infiltrating neutrophils and lymphocytes). CONCLUSION: The results observed in this study suggest that MMP-2 and MMP-9 play a critical role in the development of inflammatory periapical lesions, probably involved in the extracellular matrix (ECM) degradation during the initial phase of the lesion development.

The effect of endodontic therapy on periapical exudate neutrophil elastase and prostaglandin-E2 levels.

This study aimed to investigate periapical exudate neutrophil elastase (NE) and prostaglandin E2 (PGE2) levels and their relationships with clinical symptoms, and to determine the changes of their levels following first treatment visit. Periapical exudate samples were collected from the canals of 34 nonvital single-rooted teeth at two sequential treatment visits. Periapical exudate NE and PGE2 levels were found to be higher in the presence of clinical symptoms (pus discharge, swelling) (p < 0.05). The canals of teeth with larger periapical radiolucent area (>or=1 cm) contained more PGE2 levels than with smaller ones (<1 cm) (p < 0.05). Periapical exudate NE levels were significantly correlated with PGE2 levels (p < 0.05), and their levels at first treatment visit did not change following root canal therapy (p > 0.05). The periapical exudate NE and PGE2 levels may regulate periapical disease expression, but the results of this study were unable to reveal this association.

Collagenase-3 (MMP-13) is expressed in periapical lesions: an immunohistochemical study.

AIM: To determine whether or not matrix metalloproteinase 13 (MMP-13) is present in periapical granulomas with and without epithelium. METHODOLOGY: Seventeen open periapical granulomas of pulpal origin (seven lesions without epithelium and 10 with proliferating epithelium) were fixed in formalin and then embedded in paraffin prior to being processed for immunohistochemical analysis. A monoclonal antibody against human MMP-13 was used to evaluate MMP-13 expression. Immunocomplexes were subsequently treated with the secondary antibody and then detected by means of streptavidin peroxidase. Immunoreactivity was visualized by development with 3,3'-diaminobenzidine. RESULTS: An immunopositive cytoplasmatic reaction for MMP-13 was observed in all the specimens, although the immunostaining by anti-MMP-13 antibody was heterogeneous and its levels varied according to histopathological findings. In periapical lesions without epithelium MMP-13 immunolabelling was detected in a few fibroblast-like cells, and in some plasma cells within the granulomatous tissue. A clear upregulation of MMP-13 expression was detected in periapical lesions with epithelium, especially in small island and thin strands of epithelium. CONCLUSIONS: The expression pattern of MMP-13 demonstrates that it is involved in the conversion of a periapical granuloma with epithelium into a radicular cyst. This property is related to the ability of MMP-13 to influence not only the migration of epithelial cell but also the invasion of granulomatous tissue.

Regulation of tissue inhibitors of metalloproteinase-1 gene expression by cytokines in human gingival fibroblasts.

Tissue inhibitors of metalloproteinase (TIMP) are important participants in various physiological processes that involve tissues remodeling. They help maintain a delicate balance between physiological degradation and synthesis of the extracellular matrix. A better understanding of TIMP activity will be helpful in understanding the etiology of periapical lesions and their means of treatment. The fibroblast is a prominent cellular component of the periapical tissues. The potential implications of cytokine-mediated tissue destruction still remain to be elucidated. The purpose of this study was to determine the effects of interleukin (IL)-1alpha and transforming growth factor (TGF)-beta on the expressing of TIMP-1 by primary gingival fibroblast cultures. After exposure to cytokines for 8 h, total RNA in gingival fibroblasts was isolated and evaluated by reverse-transcriptase polymerase chain reaction. Densitometric analysis of the TIMP-1 mRNA gene expression, after normalization by beta-actin, demonstrated that exposure to IL-1alpha resulted in a decreased level of TIMP-1 mRNA compared with the control groups. However, the TIMP-1 mRNA was up-regulated by TGF-beta. In addition, when the cells were cultured in combination with TGF-beta (1 ng/ml) and IL-1alpha for 8 h, the level of TIMP-1 mRNA was dramatically reduced. These results demonstrated that in human periapical tissue cytokines differentially and specifically regulate expression of TIMP-1 mRNA. An understanding of the actions of cytokines on gingival fibroblasts may result in new therapies to augment current treatment of periapical lesions.

Analysis of arylsulfatases A and B, acid phosphatase, lactate dehydrogenase, and aspartate transaminase in chronic periapical lesions of endodontic origin.

Attempts were made to detect and measure the activities of arylsulfatases. A&B acid phosphatase, lactate dehydrogenase, and glutamate oxaloacetate transaminase (aspartate transaminase) enzymes in human chronic lesions of endodontic origin. Thirteen periapical lesions of endodontic origin and 11 noninflamed control periapical tissues were obtained. The specimens were carried to the laboratory on liquid nitrogen and kept at -70 degrees C. Samples were thawed, homogenized, and then assayed for enzyme activities. The specific activities of arylsulfatase A (nmol/hr/mg protein) were 55.0+/-10.7 (chronic lesions) vs. 3.4+/-2.2 (controls) (p < 0.01). Arylsulfatase B specific activities (nmol/hr/mg protein) were 50.3+/-6.4 (chronic lesions) vs 91.8+/-18.4 (controls). Total acid phosphatase activities (mU/mg protein) were 45.8+/-6.6 (chronic lesions) vs. 26.8+/-3.1 (controls). Lactate dehydrogenase activities (Berger-Broida units/mg protein) of the chronic periapical lesions were significantly higher than the control group (362+/-63.2) vs. (140+/-46.0) (p < 0.05). There was no significant difference between the specific activities of aspartate transaminase in chronic lesions and the control group (68.0+/-14.5) vs. (53.0+/-10.4) mU/mg protein).

Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1.

Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 microm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation.

Production of human-inducible nitric oxide synthase in radicular cysts.

To examine if nitric oxide (NO) is produced in radicular cysts, NO synthase (NOS) production was analyzed. Periapical tissues were removed from patients at the time of endodontic surgery. Frozen tissue sections were histologically evaluated with hematoxylin-eosin staining. Production of human-inducible NOS (iNOS) in apical cysts was then immunohistochemically examined. Immunoreactive human iNOS was widely distributed in epithelial cells, endothelial cells, fibroblasts, macrophages, or polymorphonuclear leukocytes. Remarkably, iNOS-positive cells were significantly present around blood vessels, and cells residing apart from the blood vessels showed weak or no iNOS production, suggesting that only cells around blood vessels could be stimulated for iNOS synthesis. These data demonstrated the possibility that several, but not all, cells could be stimulated to synthesize iNOS in inflamed tissues. In the presence of iNOS, NO can be produced spontaneously in periapical lesions and may play a crucial role in the regulation of chronic infection.

Non specific acid esterase activity in human periapical inflammatory cells.

The fine structural localization of non specific acid alpha-naphthyl acetate esterase activity (ANAE) in human periapical inflammatory cells was studied in sections of paraffin embedded tissue of 20 human periapical lesions (granulomas). Examination of specimens incubated with ANAE resulted in ANAE+ cells interpreted as T-lymphocytes, monocytes, macrophages, giant cells and plasma cells. ANAE- lymphocytes were interpreted as B cells. Our findings do not seem to confirm the presence among human periapical inflammatory cells of NK (natural killer) cells. T-lymphocytes were the most represented cellular type. The macrophages with ANAE+ reaction were numerous in all specimens observed and the variation in staining intensity could reflect a varying stage of activation. These findings allow conclusions about the role of T-lymphocyte mediated immune reaction in the pathogenesis of periapical lesions. The possibility that the activated T-lymphocytes within the periapical lesions may have a critical role in establishing and maintaining granuloma formation is also discussed.