The effect of colchicine on alveolar bone loss in ligature-induced periodontitis

Abstract Colchicine is widely used in the treatment of several inflammatory diseases due to its anti-inflammatory effect, but effects on bone metabolism are unclear. The aim of this study was to evaluate the effects of systemically-administered colchicine on healthy periodontium and experimentally-induced periodontitis. In total, 42 male Wistar rats were included in this study. A non-ligated group constituting the negative control group (Control, C, n = 6) and a ligature-only group forming the positive control group (LO, n = 12) were created separately. Twelve rats were treated with 0.4 mg/kg colchicine and another 12 with 1 mg/kg colchicine. In the colchicine-administered groups, right mandibles constituted the ligated groups (1 mgC-L or 0.4 mgC-L) and left mandibles formed the corresponding non-ligated controls (1mgC or 0.4mgC). Silk ligatures were placed at the gingival margin of the lower first molars. The animals were euthanized at different time-points of healing (11 or 30 days). Alveolar bone loss was clinically measured and TRAP+ osteoclasts, osteoblastic activity, and MMP-1 expression were examined histologically. There was no increase in alveolar bone loss with either colchicine dose in healthy periodontium (p > 0.05) and the highest level of alveolar bone loss, TRAP+ osteoclast number, and MMP-1 expression were measured in the LO group (p < 0.05). The 0.4 mgC-L group showed less alveolar bone loss at 11 days (p < 0.05), but greater loss at 30 days. The 1 mgC-L group showed higher osteoblast number than the other ligated groups (p < 0.05) at both time-points. In summary, colchicine did not increase alveolar bone loss in healthy periodontium and also may tend to reduce periodontitis progression. However, further extensive study is necessary to understand the mechanism of colchicine action on alveolar bone loss in periodontitis.

MMP1-1607 polymorphism increases the risk for periapical lesion development through the upregulation MMP-1 expression in association with pro-inflammatory milieu elements

ABSTRACT Increased matrix metalloproteinases (MMPs) activity is a hallmark of periapical granulomas. However, the factors underlying the MMPs expression modulation in healthy and diseased periapical tissues remains to be determined. Objective In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. Material and Methods MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). Results The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. Conclusion The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.

Effects of bromopride on expression of metalloproteinases and interleukins in left colonic anastomoses: an experimental study

Anastomotic dehiscence is the most severe complication of colorectal surgery. Metalloproteinases (MMPs) and interleukins (ILs) can be used to analyze the healing process of anastomosis. To evaluate the effects of bromopride on MMP and cytokine gene expression in left colonic anastomoses in rats with or without induced abdominal sepsis, 80 rats were divided into two groups for euthanasia on the third or seventh postoperative day (POD). They were then divided into subgroups of 20 rats for sepsis induction or not, and then into subgroups of 10 rats for administration of bromopride or saline. Left colonic anastomosis was performed and abdominal sepsis was induced by cecal ligation and puncture. A colonic segment containing the anastomosis was removed for analysis of gene expression of MMP-1α, MMP-8, MMP-13, IL-β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). On the third POD, bromopride was associated with increased MMP-1α, MMP-13, IL-6, IFN-γ, and IL-10 gene expression. On the seventh POD, all MMP transcripts became negatively modulated and all IL transcripts became positively modulated. In the presence of sepsis, bromopride administration increased MMP-8 and IFN-γ gene expression and decreased MMP-1, TNF-α, IL-6, and IL-10 gene expression on the third POD. On the seventh POD, we observed increased expression of MMP-13 and all cytokines, except for TNF-α. In conclusion, bromopride interferes with MMP and IL gene expression during anastomotic healing. Further studies are needed to correlate these changes with the healing process.

Liver cirrhosis on the colonic anastomotic healing in rats / Cirrose hepática na cicatrização de anastomose intestinal em ratos

PURPOSE: To investigate the effects of cirrhosis on colonic anastomosis healing in rats. METHODS: Fifty five Wistar male rats were used (23 in the control group and 32 in the cirrhosis group). On the first day of the procedure, the rats in the cirrhosis group underwent double ligation and folding of the common bile duct to induce liver cirrhosis, and the control rats underwent a laparotomy and intestinal manipulation. On the fourteenth and thirty-fifth days, all of the animals were biochemically assessed for serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, bilirubin, total protein, and albumin and for liver histopathology. On the thirty-fifth day, cirrhosis was confirmed. On the twenty-eighth day, all of the animals were subjected to left colon transection and anastomosis. On the seventh day after the colonic anastomosis, the rats were sacrificed and macroscopically evaluated for dehiscence. The region of the colonic anastomosis was removed and subjected to hydroxyproline content measurement, conventional histology, and the immunohistochemical determination of vascular endothelial growth factor (VEGF) and matrix metalloproteinase type 1 (MMP 1). RESULTS: The biochemical and histopathological examinations confirmed cirrhosis in all of the animals in the cirrhosis group. More deaths occurred after anastomosis in the cirrhosis group (5/25) than in the control group (0/21), and anastomotic dehiscence was more frequent in the cirrhosis group (8/25) than in the control group (0/21). The average hydroxyproline concentration was lower in the cirrhosis group than in the control group. The immunohistochemical studies showed that the average VEGF expression in the cirrhosis group was lower than in the control group, and the average MMP1 expression was higher in the cirrhosis group. CONCLUSION: Hepatic cirrhosis leads to increased mortality and colonic anastomotic dehiscence, an increased distance between the mucosal edges of the anastomosis area, and a lower hydroxyproline concentration in the colonic anastomosis; therefore, these conditions are deleterious to healing.

OBJETIVO: Investigar os efeitos da cirrose hepática na cicatrização de anastomose intestinal em ratos. MÉTODOS: Este estudo avaliou o efeito da cirrose hepática na cicatrização de anastomoses em ratos. 55 ratos Wistar machos foram utilizados (23 controles e 32 no grupo cirrose). No primeiro dia os ratos do grupo cirrose for submetidos à dupla ligadura e enovelamento do ducto hepático comum para indução de cirrose e os ratos controles foram submetidos à laparotomia e manipulação das alças intestinais. No dia 14 e 35, todos os animais foram avaliados bioquimicamente para dosagem sérica da alanina aminotransferase (AST), alanina aminotransferase (ALT), fosfatase alcalina, bilurrubinas, proteínas totais, albumina e histologia do fígado. No dia 35 a cirrose foi confirmada. No dia 28 todos os animais foram submetidos à colectomia esquerda e anastomose. 70 dias após anastomose os ratos foram submetidos à eutanásia e foram avaliados macroscopicamente a procura de deiscência. A região da anastomose colônica foi removida para dosagem de hidroxiprolina, histologia convencional e imunohistoquímica para determinação do fator de crescimento endotelial vascular (VEGF) e metaloproteinase tipo 1 (MMP 1). RESULTADOS: A análise bioquímica e histológica confirmou a cirrose em todos os animais do grupo cirrose. Óbito ocorreu em maior freqüência após a anastomose no grupo cirrose (5/25) se comparado com grupo controle (0/21), e a deiscência da anastomose foi mais freqüente no grupo cirrose (8/25) se comparado com controle (0/21). A concentração média de hidroxiprolina foi menor no grupo cirrose se comparado com grupo controle. A análise imonuhistoquímica mostrou que a expressão VEGF no grupo cirrose foi menor que no grupo controle e a expressão média da MMP1 foi maior no grupo cirrose. CONCLUSÃO: A cirrose hepática leva a aumento de mortalidade e aumento na deiscência de anastomose, aumento na distância entre as pontes mucosas na area da anastomose, menor nível de hidroxiprolina na anastomose colônica e assim pior condição de cicatrização.

Matrix metalloproteinase-1 expression in oral submucous fibrosis: An immunohistochemical study.

Context: Oral submucous fibrosis (OSF) is a form of pathological fibrosis affecting the oral mucosa. There is compelling evidence to implicate the habitual chewing of areca nut with the development of OSF. Because collagens are the major structural components of connective tissues, including oral submucosa, the composition of collagen within each tissue needs to be precisely regulated to maintain tissue integrity. Arecoline stimulates fibroblasts to increase the production of collagen by 150%. Aim: As the role of collagenase is implicated in cleaving the collagen under physical conditions, this study was carried out to evaluate the role of collagenase-1 (matrix metalloproteinase [MMP]-1) in a pathologic condition like OSF. Settings and Design: A total of 40 patients were included in the study, comprising of 30 OSF as Group 1 and 10 normal buccal mucosa tissue as Group 2. Materials and Methods: Both the groups were stained for MMP-1 by the immunohistochemical method using the streptavidin HRP-biotin labeling technique. MMP-1 expression intensity in the epithelium and connective tissue was decreased in Group 1 when compared to Group 2. Statistical Analysis Used: Chi-square test of association was used to determine the difference in the expression of MMP-1 between OSF and normal buccal mucosa and among different histological gradings of OSF. Results: The results were statistically significant. However, there was no statistically significant difference between the expression of MMP-1 among different histological grades of OSF in Group 1.

Effect of thalidomide on the healing of colonic anastomosis, in rats / Efeito da talidomida na cicatrização de anastomoses colônicas em ratos

PURPOSE: Thalidomide, because of its anti-inflammatory properties, as re-emerged as an option for the treatment of Crohn's disease refractory to standard therapy. We studied the effect of thalidomide on the healing of colonic anastomosis. METHODS: Sixty male rats (Rattus norvegicus), were divided into 3 groups of 20 animals each, respectively receiving 0.5 or 1.0 mg/kg thalidomide by the oral route for 7 days, or saline solution (control). All animals were submitted to continuous end-to-end anastomosis with 6-0 Prolene sutures. After sacrifice the anastomoses were analyzed macroscopically and submitted to determination of hydroxyproline, to histology and to immunohistochemistry for metalloproteinase 1, metalloproteinase 1 inhibitor and vascular endothelial growth factor (VEGF). RESULTS: Statistical analysis of the data showed no significant difference in macroscopic aspect or hydroxyproline determination (p= 0.5403). In the immunohistochemical analysis, the following p values were obtained: p = 0.5817 for VEGF, p = 0.1854 for metalloproteinase 1, and p = 0.0023 for metalloproteinase 1 inhibitor, with this last value being considered statistically significant. CONCLUSION: We conclude that thalidomide influenced collagen maturation. There was a stronger action of metalloproteinases, possibly indicating a negative tendency for the healing process.

OBJETIVO: Sabe-se que agentes farmacológicos podem influenciar no processo de cicatrização. A talidomida, devido às suas propriedades antiinflamatórias, tem ressurgido como uma opção no tratamento da doença de Crõhn refratária à terapêutica convencional. Neste trabalho, estudamos o efeito da talidomida na cicatrização de anastomoses colônicas no rato. MÉTODOS: Foram utilizados 60 animais Rattus norvegius, com peso médio de 300g. Organizou-se 3 grupos de 20 animais, sendo um grupo controle (AC), um grupo (BD), com administração de talidomida 0,5 mg/kg por 7 dias e um grupo (AD) com administração de talidomida 1,0 mg/kg por 7 dias. Foi realizada anastomose término-terminal contínua de prolene 6-0. O sacrifício foi no 7º. dia pós operatório e as anastomoses foram analisadas quanto a aspecto macroscópico, dosagem de hidroxiprolina, histologia em hematoxilina-eosina e imuno-histoquímica para metaloproteinase 1, inibidor de metaloproteinase 1 e VEGF. RESULTADOS: Não houve diferença estatisticamente significante para a observação macroscópica e para dosagem de hidroxiprolina (p=0,5403). Na análise imunohistoquímica, para VEGF houve p=0,5817, para metaloproteinase 1, p=0,1854 e para inibidor de metaloproteinase, p=0,0023, considerado estatisticamente significante. CONCLUSÃO: Concluímos que a talidomida influenciou na maturação do colágeno. Notou-se maior ação das metaloproteinases, que pode significar uma tendência negativa para o processo cicatricial.

Effects of preoperative pelvic irradiation on colonic anastomosis healing: an experimental study in rats / Efeito da radioterapia pélvica pré-operatória na cicatrização de anastomoses colônicas: estudo experimental em ratos

PURPOSE: Colorectal anastomosis is a constant worry-issue among surgeons because of high rates of complications, specially the dehiscence. The preoperative irradiation on cancer surgeries might interfere in the healing process, leading to an unfavorable outcome. METHODS: In the present study, two groups of rats were irradiated previously to a colorectal anastomosis surgery, with intervals of 4 and 8 weeks between the procedures. Seven days after the surgery, healing process was evaluated for dehiscence presence and histologic inflammatory characteristics. Also, levels of hydroxyproline, metalloproteinases and vascular endothelial growth factor were measured. RESULTS: Our results showed a higher incidence of dehiscences on the animals submitted to irradiation, compared to controls, with a reduced inflammatory activity in the healing tissue. DISCUSSION: Comparing both irradiated groups, those irradiated 8 weeks before surgery showed higher levels of hydroxyproline and metalloproteinases, indicating higher efficiency of the healing process. In conclusion, preoperative irradiation interferes with intestinal anastomosis healing and a larger time interval between both procedures is safer in terms of the healing quality.

INTRODUÇÃO: As anastomoses colorretais são motivos constante de preocupação por parte dos cirurgiões, em virtude do alto índice de complicações, principalmente as deiscências. O uso da radioterapia previamente à cirurgia, nos casos de doença neoplásica, pode interferir no processo cicatricial das anastomoses, e levar a uma evolução desfavorável. MÉTODOS: Os autores estudaram dois grupos de ratos, submetidos a radioterapia e à confecção de uma anastomose no cólon, com intervalo de 04 e de 08 semanas entre os dois procedimentos, comparando com um grupo controle. Após 07 dias da cirurgia, estudaram-se vários aspectos do processo cicatricial: presença de deiscência, características inflamatórias do tecido, dosagem de hidroxiprolina, de mateloproteinase e de VEGF. RESULTADOS: Os autores detectaram maior índice de deiscência nos animais submetidos à radioterapia, com prejuízo da atividade inflamatória característica de um tecido em cicatrização. DISCUSSÃO: Dentre os dois grupos irradiados, aquele com intervalo de oito semanas entre a radioterapia e a confecção da anastomose teve dosagem mais alta de hidroxiprolina e metaloproteinase, demonstrando maior eficiência do processo cicatricial. CONCLUSÃO: A radioterapia prévia interfere no processo de cicatrização das anastomoses intestinais, e que um maior intervalo de tempo entre os dois procedimentos é melhor para garantia de uma cicatrização satisfatória.

Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in oral submucous fibrosis.

Immunohistochemical staining of formalin fixed, paraffin embedded tissue sections of OSF for MMPs-1,2,9 and their tissue inhibitors TIMP-1and 2 was performed using monospecific antibodies coupled with gelatin zymography (MMP-2 and 9) for measuring enzymatic activity quantitatively and for distinguishing the active from the inactive variants of enzymes. The present study, contrary to earlier reports, recorded statistically significant increase in the levels of stromal expression of MMP-1, MMP-2 and MMP-9 and TIMP-1 and TIMP-2 using monospecific antibodies reacting against tissue antigens.The simultaneous increase in reactivity of MMPs and TIMPs poise difficulty in interpretingthe results of this study. The possible reasons for this result, against the backdrop of existing knowledge, were attempted in this study.