Diabetes alters the involvement of myofibroblasts during periodontal wound healing.

OBJECTIVES: Myofibroblasts constitute a specific cell phenotype involved in connective tissue healing. Diabetes alters the wound healing response. However, it is not clear whether diabetes modifies the involvement of myofibroblasts in periodontal wounds. MATERIALS AND METHODS: Type I diabetes was induced in rats through streptozotocin injection, and periodontal wounds were performed. Wound healing was evaluated histologically at 2, 5, 7, and 15 days by measuring epithelial migration, neutrophil infiltration, and collagen and biofilm formation. Distribution of myofibroblasts was evaluated through immunofluorescence for α-smooth muscle actin. Data analyses were performed using the Shapiro-Wilk, ANOVA, or Kruskal-Wallis tests. RESULTS: Diabetic wounds were characterized by delayed epithelial closure, increased neutrophil infiltration, biofilm formation, and reduced collagen formation. Quantification of the myofibroblasts showed a significant reduction at 5 and 7 days in wounds of diabetic rats and an increase at 15 days when compared to wounds of non-diabetic rats. CONCLUSIONS: Diabetic wound healing was associated with decreased epithelial and connective tissue healing, increased levels of inflammation, and biofilm formation. Myofibroblast differentiation was delayed in diabetic periodontal wounds at early time points. However, myofibroblasts persisted at later time points of healing. The present study suggests that diabetes alters the involvement of myofibroblasts during periodontal wound healing.

Aged blood factors decrease cellular responses associated with delayed gingival wound repair.

Aging is a gradual biological process characterized by a decrease in cell and organism functions. Gingival wound healing is one of the impaired processes found in old rats. Here, we studied the in vivo wound healing process using a gingival repair rat model and an in vitro model using human gingival fibroblast for cellular responses associated to wound healing. To do that, we evaluated cell proliferation of both epithelial and connective tissue cells in gingival wounds and found decreased of Ki67 nuclear staining in old rats when compared to their young counterparts. We next evaluated cellular responses of primary gingival fibroblast obtained from young subjects in the presence human blood serum of individuals of different ages. Eighteen to sixty five years old masculine donors were classified into 3 groups: "young" from 18 to 22 years old, "middle-aged" from 30 to 48 years old and "aged" over 50 years old. Cell proliferation, measured through immunofluorescence for Ki67 and flow cytometry for DNA content, was decreased when middle-aged and aged serum was added to gingival fibroblast compared to young serum. Myofibroblastic differentiation, measured through alpha-smooth muscle actin (α-SMA), was stimulated with young but not middle-aged or aged serum both the protein levels and incorporation of α-SMA into actin stress fibers. High levels of PDGF, VEGF, IL-6R were detected in blood serum from young subjects when compared to middle-aged and aged donors. In addition, the pro-inflammatory cytokines MCP-1 and TNF were increased in the serum of aged donors. In old rat wound there is an increased of staining for TNF compared to young wound. Moreover, healthy gingiva (non injury) shows less staining compared to a wound site, suggesting a role in wound healing. Moreover, serum from middle-aged and aged donors was able to stimulate cellular senescence in young cells as determined by the expression of senescence associated beta-galactosidase and histone H2A.X phosphorylated at Ser139. Moreover, we detected an increased frequency of γ-H2A.X-positive cells in aged rat gingival tissues. The present study suggests that serum factors present in middle-aged and aged individuals may be responsible, at least in part, for the altered responses observed during wound healing in aging.

Tumor necrosis factor-α inhibits transforming growth factor-ß-stimulated myofibroblastic differentiation and extracellular matrix production in human gingival fibroblasts.

BACKGROUND: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components. METHODS: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-ß1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-ß1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-ß1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. RESULTS: TGF-ß1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-ß1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-ß1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. CONCLUSIONS: TNF-α inhibits several cell responses induced by TGF-ß1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.

Chemokine monocyte chemoattractant protein-3 in progressive periodontal lesions in patients with chronic periodontitis.

BACKGROUND: Chemokines are central in the activation and direction of leukocyte subsets to target tissues. However, the monocyte chemoattractant protein-3 (MCP-3) has not been associated with chronic periodontitis. Chronic periodontitis is an infection showing episodic supporting tissue destruction. The aim of this study is to determine the levels and expression of MCP-3 in periodontal sites characterized by active periodontal connective tissue destruction. METHODS: The study population consisted of 15 patients with a progression of periodontitis (15 of 56 patients), 18 patients with chronic periodontitis, and 10 healthy subjects without periodontal disease. As determined by the tolerance method, the 15 patients with moderate to advanced chronic periodontitis showed a progression of periodontitis over a 4-month period. Periodontitis was characterized by at least six sites with a probing depth >or=5 mm, clinical attachment level >or=3 mm, and radiographic bone loss. Gingival crevicular fluid was collected using a paper strip. The total protein concentration was determined. An enzyme-linked immunosorbent assay was performed to determine the total amount of MCP-3, and an immunoWestern blot was conducted to assess molecular MCP-3 forms. To determine the MCP-3 expression by immunohistochemistry, gingival biopsies were obtained from patients with chronic periodontitis and healthy subjects during third-molar extraction surgery. Statistical analyses were performed using statistical software. Data were expressed as subject means +/- SD, using the chi(2) and Student t tests. RESULTS: The total amount and concentration of chemokine MCP-3 were significantly higher in patients with chronic periodontitis than in healthy subjects (8.25 pg versus 0.53 pg, P = 0.006 and 2.95 pg/microl versus 0.45 pg/microl, P = 0.04, respectively). Active sites showed a significantly higher total amount and concentration of MCP-3 than inactive sites (11.12 versus 2.88 pg, P value = 0.005 and 3.95 versus 1.02, P value = 0.005, respectively). Western blot and immunohistochemical staining confirmed the presence of MCP-3 in periodontal disease, with observable differences between patients with chronic periodontitis and healthy subjects. CONCLUSIONS: MCP-3 was highly expressed in patients with chronic periodontitis, particularly in those with progressive periodontal lesions. MCP-3 could be involved in the recruitment of inflammatory cells toward periodontal tissues during the progression of the disease.

Efecto de cinco sistemas de pulido de resinas compuestas sobre superficie coronaria y radicular: Observación por medio de MEB y microscopía óptica / Effects of Five Composite Polishing Systems over Crown and Root Surface: SEM and Optical observations

El objetivo de este estudio in vitro fue describir la superficie coronaria y radicular alrededor de restauraciones de resina compuesta posterior a su pulido. Materiales y método: se realizaron 50 restauraciones estandarizadas sobre la superficie coronaria y 50 sobre la superficie radicular de dientes extraídos por indicación ortodóncica. Las cavidades se prepararon utilizando fresas cilíndricas de carbide (Meissinger®) con alta velocidad (Champion Dental Products Inc) bajo irrigación y fueron restauradas con resina compuesta híbrida (Filtek® Z 250, 3M®). Fueron separadas aleatoriamente en 5 grupos de 10 restauraciones coronarias y 10 radiculares. Cada grupo fue pulido utilizando un sistema diferente, siguiendo las indicaciones del fabricante. Grupo 1 (PB): Piedras de Arkansas (Dedeco). Grupo 2 (PD): piedras de diamante finas, extrafinas y ultrafinas (Diatech®). Grupo 3 (DOA): discos de óxido de aluminio (Sof lex®, 3M®). Grupo 4 (PG): puntas Enhance® (Dentsply®). Grupo 5 (FCT): fresas de carburo tungsteno de 16 y 30 cuchillo (Komet®). 10 dientes sin tratamiento se dejaron como grupo control (Grupo 6). Todas las muestras fueron observadas y fotografiadas mediante microscopía electrónica de barrido (TEAC Siemens®) y microscopía óptica (Carl Zeiss®). Las imágenes se compararon con las del grupo control. Resultados: la comparación de las microfotografías de los grupos tratados con las del grupo control mostró cambios en la corona y en la raíz en los grupos 1, 2, 3 y 4. En la corona se observó una pérdida de las características superficiales del esmalte y en la raíz una pérdida del cemento y exposición de dentina. En el grupo 5 algunas muestras presentaron modificación de la estructura superficial normal y otras no. Conclusión: los 5 sistemas de pulido de resinas compuestas analizados en este estudio modificaron la superficie del esmalte y el cemento.

The aim of this IN VITRO study was to describe the crown and root surface surrounding composite restorations, after polishing procedures. Materials and Methods: 50 standardized restorations were made over the crown surface and 50 over the root surface of teeth extracted by orthodontics indications. The cavities were prepared using cylindrical carbide burs (Meissinger) under water irrigation with a high speed hand piece (Champion Dental Products Inc) and restored with hybrid composite (Filtek Z250®, 3M®). They were randomized and separated into five groups of 10 restorations either in crown and root. Each group was polished using different systems, according to manufacturer’s instructions. Group 1(PB): Arkansas Stone (Dedeco). Group 2 (PD): Fine, extrafine and ultrafine Diamond Burs (Diatech®). Group 3 (DOA): Aluminum Oxide Discs (Sof lex® 3M®). Group 4 (PG): Enhance® Points (Dentsply®). Group 5 (FCT): 16 and 30 blades Tungsten Carbide Burs (Komet®). 10 teeth without treatment were used as control group (Group 6). All samples were analyzed and photographed by SEM (TEAC Siemens®), and Optical microscope (Carl Zeiss®). The images were compared with control group. Results: The comparison of the microphotography with control group showed changes in both crown and root surfaces in groups 1, 2, 3 and 4. It was seen a loss of the enamel superficial characteristics, in crown surfaces and a loss of the cementum and exposure of dentin in root. In group 5 some specimens resulted in a modification of normal tooth surface characteristics and in others there were no changes. Conclusions: The five composites polishing systems analyzed in this study resulted in enamel and cementum modification.

In situ detection of matrix metalloproteinase-9 (MMP-9) in gingival epithelium in human periodontal disease.

BACKGROUND AND OBJECTIVE: As the periodontal lesion develops, the junctional epithelium migrates apically in conjunction with the dissolution of the most coronal Sharpey's fibers. Because matrix metalloproteinase-9 (MMP-9) has been identified in migrating epithelial cells and invading tumors, we propose that this enzyme is produced by gingival keratinocytes in advanced periodontal lesions. METHODS: To test this idea, biopsies of inflamed gingival tissues were obtained from patients with advanced periodontitis. Healthy gingival tissue samples were utilized as controls. The presence and activity of MMP-9 was evaluated by combining indirect immunofluorescence of gingival tissue samples and gelatin zymography of gingival epithelium separated from connective tissue. RESULTS AND CONCLUSIONS: The staining pattern showed the presence of MMP-9 in junctional and pocket gingival epithelial cells, polymorphonuclear neutrophils (PMNs) and as a scattered deposit along connective tissues of periodontitis-affected gingival tissues. Gelatin zymography permitted the identification of pro-MMP-9 in surcular/pocket epithelium derived from inflamed gingival tissues. Lower levels of MMP-9 were detected in epithelium not exposed to inflammation. These observations suggest a role for MMP-9 in gingival epithelial response to periodontal infection.

Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans.

To test the hypothesis that a commercial solution of 5% NaOCl produces structural and molecular alterations in the collagen and glycosaminoglycans of mineralized and demineralized dentin, radicular segments from human teeth were treated with 5% NaOCl or 2 min at room temperature. As a control, distilled water replaced NaOCl. The experimental and control specimens were processed for indirect immunofluorescence by using antitype I collagen and antichondroitin sulfate antibodies. Tissue sections were morphometrically analyzed. A single exposure for 2 min to a 5% NaOCl solution produced a drastic loss of immunoreactivity in the dentin surface for both antibodies that were used in demineralized specimens. A narrow and irregular band of fluorescence loss was detected in mineralized-dentin segments when anticollagen antibodies were used. The results of this study suggest that 5% NaOCl induces alterations in dentin collagen and glycosaminoglycans and show the protective role of hydroxyapatite on organic matrix stability.

Presencia de microorganismos en cepillos dentales utilizados en higiene oral habitual / Presence of microorganisms in toothbrushes used in habitual oral hygiene

Los cepillos dentales se han utilizado ampliamente para el control mecánico de la placa bacteriana; sin embargo, las interacciones entre éstos y las distintas poblaciones celulares de la placa bacteriana son motivo de estudio. Para demostrar la hipótesis de que cepillos dentales utilizados en higiene bucal habitual, son posibles reservorios de poblaciones bacterianas, se realizó un estudio en 15 pacientes portadores de enfermedad periodontal moderada, los cuales realizaron su higiene bucal habitual durante 15 días utilizando cepillos dentales Oral-B I. Aleatoriamente se seleccionaron filamentos de cada muestra experimental y control, las que fueron procesadas para microscopía electrónica de barrido. Nuestras observaciones preliminares demuestran la presencia de poblaciones bacterianas cocáceas y bacilares en todas las muestras analizadas. Las bacterias se visualizaron formando placa bacteriana adherida a los extremos de los filamentos. Estos resultados sugieren claramente que los cepillos dentarios podrían constituir un vector de transmisión de bacterias. La supervivencia de dichas poblaciones bacterianas en los filamentos de los cepillos utilizados es desconocida