Evaluation of tissue in repair with natural latex and / or hyaluronic acid in surgical bone defects

Abstract This study evaluated the bone repair in surgical defects of rats treated with hyaluronic acid (HA) associated or not with Hevea brasiliensis fraction protein (F-1). Bone defect were created in 15 albino Wistar rats divided into 3 groups (n=5): Control group (1) - blood clot; HA group (2) - 0.5% hyaluronic acid; HAF1 group (3) - 0.1% F-1 protein fraction dissolved in 0.5% hyaluronic acid. After 4 weeks, the animals were euthanized and the bone repair was evaluated through histomorphometric analysis, zymography and immunohistochemistry. The neoformed bone area did not show a significant difference (p = 0.757), but there was a tendency for bone trabeculation to increase in the groups HA and HAF1. For immunohistochemically analysis, there was a difference in vascular endothelial growth factor (VEGF) labeling (p = 0.023), being higher in the groups HA and HAF1 than the control group. No significant difference in bone sialoprotein (BSP) (p = 0.681), osteocalcin (p = 0.954), however, significant difference in platelet endothelial cell adhesion molecule-1 (CD-31) (p = 0.040), with HAF1 group being significantly lower than the control. For zymographic analysis, there was no significant difference for metalloproteinase-2 (MMP-2) (p = 0.068), but there was a tendency to increase MMP-2 in the HA group. Despite the influence on angiogenic factors and the apparent tendency for greater trabeculation in the HA and HAF1 groups, there was no significant difference in the area of ​​newly formed bone tissue in the analyzed period.

Resumo Este estudo avaliou o reparo ósseo em defeitos cirúrgicos de ratos tratados com ácido hialurônico (AH) associado ou não à fração proteica de Hevea brasiliensis (F-1). Foram criados defeitos ósseos em 15 ratos albinos Wistar divididos em 3 grupos (n = 5): Grupo controle (1) - coágulo sanguíneo; Grupo HA (2) - ácido hialurônico 0,5%; Grupo HAF1 (3) - fração proteica F-1 0,1% dissolvida em ácido hialurônico a 0,5%. Após 4 semanas, os animais foram submetidos à eutanásia e o reparo ósseo avaliado por meio de análise histomorfométrica, zimografia e imunohistoquímica. A área óssea neoformada não apresentou diferença significativa (p = 0,757), mas houve tendência de aumento da trabeculação óssea nos grupos HA e HAF1. Para a análise imunoistoquímica, houve diferença na marcação do fator de crescimento endotelial vascular (VEGF) (p = 0,023), sendo maior nos grupos HA e HAF1 do que no grupo controle. Nenhuma diferença significativa na sialoproteína óssea (BSP) (p = 0,681), osteocalcina (p = 0,954), no entanto, diferenças significativas foram encontradas para a molécula de adesão de células endoteliais plaquetárias-1 (CD-31) (p = 0,040), com o grupo HAF1 sendo significativamente inferior ao controle. Para a análise zimográfica, não houve diferença significativa para metaloproteinase-2 (MMP-2) (p = 0,068), mas houve tendência de aumento da MMP-2 no grupo HA. Apesar da influência sobre os fatores angiogênicos e da aparente tendência de maior trabeculação nos grupos HA e HAF1, não houve diferença significativa na área de tecido ósseo neoformado no período analisado.

Análise in vitro da capacidade de soluções contendo fluoreto e/ou trimetafosfato na remineralização da dentina e na inibição da atividade de metaloproteinases da matriz dentinária / In vitro analysis of the capacity of solutions containing fluoride and/or trimetaphosphate in the remineralization of dentin and in the inhibition of the activity of dentin matrix metalloproteinases

Objetivo: O objetivo deste estudo foi investigar o potencial anti-proteolítico contra MMP-2 e MMP-9 e a capacidade de induzir a remineralização da dentina, através de soluções contendo diferentes concentrações de TMP (na sua forma cíclica), diferentes concentrações de F e associação TMP/F. Métodos: Blocos de dentina radicular (6mmx4mmx2mm, n = 130) foram selecionados e divididos aleatoriamente em 13 grupos/soluções experimentais (n = 10): 1) Placebo (sem F/TMP); 2) 0,3% TMP hidrolisado; 3) 1% TMP hidrolisado; 4) 3% TMP hidrolisado; 5) 0,3% TMP; 6) 1% TMP; 7) 3% TMP; 8) 250 ppm F; 9 500 ppm F; 10) 1100 ppm F; 11) 250 ppm F associado a 0,3% TMP; 12) 500 ppm F associado a 1% TMP e 13) 1100 ppm F associado a 3% TMP. A avaliação do potencial anti-proteolítico das soluções experimentais contra as metaloproteinases da matriz dentinária (- 2 e -9) foi realizado por meio da análise zimográfica. Para as análises mecânicas, três áreas foram determinadas para cada espécime: 1- controle (sem tratamento); 2- desmineralizado (cárie artificial); 3- tratado (desmineralizado e submetido a ciclagem de pH por 7 dias, e tratado por 1 min com as soluções experimentais). Os espécimes de dentina foram analisados quanto à porcentagem de recuperação de dureza superficial (%SHR), em dureza transversal (%CSHR) e por microtomografia computadorizada (IMC). Para os dados de dureza e Micro-CT, os resultados foram analisados por ANOVA de medidas repetidas seguida do teste de Student-Newman-Keuls (p <0,05). Resultados: A análise zimográfica mostrou que 1100 ppm F + 3% de TMP promoveu inibição completa da atividade gelatinolítica (MMP-2; MMP-9). Os grupos com TMP não hidrolisados apresentaram efeito remineralizador (% SHR e % CSHR) superior aos grupos hidrolisados. O grupo 1100F + TMP promoveu a maior %SHR e %CSHR entre todos os grupos (p <0,001), sendo respectivamente 15,4 e 10,5%, superior ao grupo 1100F. Os grupos contendo 1100F e 1100F + 3%TMP apresentaram maior concentração mineral. Conclusão: Com base nos resultados, 3% TMP atua como um agente antiproteolítico contra metaloproteinases da matriz dentinária. Além disso, quando suplementado com 1100F, 3% TMP potencializa a remineralização, aumentando significativamente as propriedades mecânicas da dentina tratada. Os tratamentos com TMP não hidrolisado e associado ao F maior que as soluções fluoretadas sem o TMP. Significância clínica: Dessa forma, uma potencial estratégia preventiva e terapêutica pode ser considerada na clínica odontológica, principalmente na terapia da cárie radicular, como também para método preventivo de lesões iniciais de cárie dentinária ou no pré-tratamento da dentina, utilizando-os como agentes cross-linker em procedimentos restauradores(AU)

Objective: The aim of this study was to investigate the anti-proteolytic potential against MMP-2 and MMP-9 and the ability to induce dentin remineralization, through solutions containing different concentrations of TMP (in its cyclic form), different concentrations of F and TMP / F association. Methods: Root dentin blocks (6mmx4mmx2mm, n = 130) were selected and randomly divided into 13 groups / experimental solutions (n = 10): 1) Placebo (without F / TMP); 2) 0.3% hydrolyzed TMP; 3) 1% hydrolyzed TMP; 4) 3% hydrolyzed TMP; 5) 0.3% TMP; 6) 1% TMP; 7) 3% TMP; 8) 250 ppm F; 9,500 ppm F; 10) 1100 ppm F; 11) 250 ppm F associated with 0.3% TMP; 12) 500 ppm F associated with 1% TMP and 13) 1100 ppm F associated with 3% TMP. The evaluation of the antiproteolytic potential of experimental solutions against dentin matrix metalloproteinases (- 2 and -9) was carried out by means of zymographic analysis. For mechanical analysis, three areas were determined for each specimen: 1- control (without treatment); 2- demineralized (artificial caries); 3- treated (demineralized and subjected to pH cycling for 7 days, and treated for 1 min with the experimental solutions). The dentin specimens were analyzed for the percentage of recovery of superficial hardness (% SHR), in crosssectional hardness (% CSHR) and by computed microtomography (∆IMC). For the hardness and Micro-CT data, the results were analyzed by repeated measures ANOVA followed by the Student-Newman-Keuls test (p <0.05). Results: The zymographic analysis showed that 1100 ppm F + 3% TMP promoted complete inhibition of gelatinolytic activity (MMP-2; MMP-9). The groups with non-hydrolyzed TMP showed a remineralizing effect (% SHR and % CSHR) superior to the hydrolyzed groups. The 1100F + TMP group promoted the highest % SHR and % CSHR among all groups (p <0.001), being 15.4 and 10.5%, respectively, higher than the 1100F group. The groups containing 1100F and 1100F + 3% TMP showed higher mineral concentration. Conclusion: Based on the results, 3% TMP acts as an antiproteolytic agent against dentinal matrix metalloproteinases. In addition, when supplemented with 1100F, 3% TMP enhances remineralization, significantly increasing the mechanical properties of the treated dentin. Treatments with non-hydrolyzed TMP and associated with F greater than fluoridated solutions without TMP. Clinical significance: Thus, a potential preventive and therapeutic strategy can be considered in the dental clinic, mainly in the treatment of root caries, as well as for the preventive method of initial lesions of dental caries or in the pretreatment of dentin, using them as agents cross-linker in restorative procedures(AU)

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model

Abstract Objective The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. Material and Methods Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. Results Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1β, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. Conclusions This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Avaliação do efeito antiproliferativo da apocinina e diapocinina em células de osteossarcoma humano / Evaluation of the antiproliferative effect of apocynin and diapocynin on human osteosarcoma cells

O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)

Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice

Abstract Objective This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. Material and Methods Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. Results We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). Conclusions This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

Periodontitis and type 2 diabetes among women with previous gestational diabetes: epidemiological and immunological aspects in a follow-up of three years

Abstract Periodontitis can contribute to the development of insulin resistance. Gestational diabetes is a risk factor for type 2 diabetes. Therefore, periodontitis, when associated with gestational diabetes, could increase the risk for the development of type 2 diabetes after pregnancy. Objective The aim of this study was to verify the incidence on the development of type 2 diabetes in women with previous gestational diabetes with and without periodontitis after a three-year time interval. Material and Methods Initial sample of this follow-up study consisted of 90 women diagnosed with gestational diabetes who underwent periodontal examination. After three years, 49 women were subjected to new periodontal examination and biological, behavioral, and social data of interest were collected. Additionally, the quantification of the C-reactive protein in blood samples was performed. Fasting glucose and glycated hemoglobin levels were requested. Saliva samples were collected for quantification of interleukin 6 and 10, tumor necrosis factor α, matrix metalloproteinase 2 and 9. Results The incidence of type 2 diabetes mellitus was 18.4% and of periodontitis was 10.2%. There was no significant difference in the incidence of type 2 diabetes mellitus among women with and without periodontitis. It was observed impact of C-reactive protein in the development of type 2 diabetes mellitus. However, it was not observed impact of periodontitis on the development of type 2 diabetes mellitus among women with previous gestational diabetes. Conclusions It was not observed impact of periodontitis on the development of type 2 diabetes among women with previous gestational diabetes. The impact of C-reactive protein in the development of type 2 diabetes mellitus highlights the importance of an inflammatory process in the diabetes pathogenesis.

Associação entre periodontite e disfunção endotelial: parâmetros clínicos periodontais e níveis dos marcadores inflamatórios IL-1ß, TNF-α, MMP2 e óxido nítrico / Association between periodontitis and Endothelial dysfunction: periodontal clinical parameters and levels of inflammatory markers IL-1ß, TNF-α, MMP2 and nitric oxide

Objetivos: A periodontite aumenta o risco das doenças cardiovasculares ateroscleróticas (DCA) e ambas doenças apresentam mecanismos fisiopatológicos semelhantes. A disfunção endotelial (DE) é um parâmetro precoce das DCA e sua associação com a periodontite foi pouco investigada até o presente momento. Neste contexto o objetivo geral deste estudo foi avaliar a associação entre a periodontite e a disfunção endotelial por meio de parâmetros clínicos periodontais e a presença dos marcadores inflamatórios IL-1ß, TNF-α, MMP2/TIMP2 e óxido nítrico. Métodos: A amostra foi constituída por 47 indivíduos de ambos os gêneros, sem alterações sistêmicas conhecidas, divididos em dois grupos: um grupo com 24 indivíduos com periodontite crônica e outro com 23 indivíduos sem periodontite crônica. Foram realizados exames periodontais de sangramento a sondagem (SS), profundidade de sondagem (PS), nível de inserção clínica (NIC) e avaliada a DE por meio da dilatação mediada por fluxo (DMF) da artéria braquial. A concentração dos mediadores inflamatórios IL-1ß, TNF-α, MMP2/TIMP2 foi avaliada pelo método ELISA e o óxido nítrico (ON) pela reação de Griess na saliva. Resultados: Os indivíduos com periodontite apresentaram significativamente maior DE quando comparados com indivíduos sem periodontite (p= 0,034 após hiperemia reativa e p= 0,049 após nitrato sublingual). Em relação aos mediadores inflamatórios avaliados, houve uma associação significativa entre a produção de MMP2/TIMP2 com a presença de periodontite (p=0,008) além de uma correlação positiva com todos parâmetros clínicos de gravidade da inflamação periodontal avaliados (PS, NIC, SS). Para os demais marcadores (IL-1ß, TNF-α e ON) os grupos com e sem periodontite apresentaram resultados similares. Foi identificada também uma correlação positiva significativa entre a produção de ON e uma menor DE (após hiperemia reativa p= 0,027 e após nitrato sublingual p=0,016). . Conclusão: Indivíduos com periodontite apresentaram maior DE, expressa por menor % de DMF da artéria braquial e maiores níveis de MMP2/TIMP2 que indivíduos sem periodontite. O ON foi significativamente associado com uma menor DE

Background: The periodontitis and atherosclerotic cardiovascular diseases (ACD) are chronic diseases which have a large number of risk factors and inflammatory mediators in common. The ACD has endothelial dysfunction (ED) with an important role in the initiation and progression of atherosclerosis. However, few investigations were conducted on a possible association between periodontitis and higher ED. In this context the general aim of this study is to evaluate the association between the endothelial function and the periodontitis using periodontal clinical parameters and the presence of inflammatory markers IL-1ß, TNF-α, MMP2 and nitric oxide. Methods: This study consisted of 47 subjects of both genders, systemically healthy divided into two groups: a group with 24 subjects with chronic periodontitis and another with 23 subjects without chronic periodontitis. Were done complete periodontal examination and evaluated ED by the flow-mediated dilatation (FMD) of the brachial artery and measured the levels of systemic inflammation mediators IL1ß, TNF-α, MMP2 and the nitric oxide (NO) in the saliva. Results: The subjects with periodontitis showed % DMF significantly worse when compared with subjects without periodontitis (p= 0.034 after reactive hyperemia and p= 0.049 after sublingual nitrate), thus subjects with periodontitis showed significantly higher ED. In relation to inflammatory mediators evaluated, there was a significant association between MMP2 with the presence of periodontitis (p=0.008) and a positive correlation with all clinical parameters of periodontal variation. For the other markers (IL-1ß, TNF-α, and nitric oxide) the groups with and without periodontitis showed similar results. It was also identified a significant positive correlation between the nitric oxide and a better endothelial function. Conclusion: subjects with periodontitis showed higher ED, expressed by smaller % of DMF brachial artery and higher levels of MMP2 that subjects without periodontitis