Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System

Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Resumo Este estudo avaliou marcadores de perda óssea e da resposta imune presentes na evolução da doença periodontal. Cento e dois ratos Wistar foram divididos em três grupos de animais: PD0, sem ligadura e PD15 dias e PD60 dias, submetidos a colocação de ligadura com um fio de seda estéril 3-0 na região cervical do primeiro molar superior em ambos os lados. Foram obtidas amostras de tecido gengival para análise histomorfométrica, análises imunohistoquímicas de RANK, RANKL, OPG, caracterização do infiltrado inflamatório, quantificação de óxido nítrico, expressão de quimiocinas MCP-1, RANTES, IP10 e do TGF-b1, VEGF e bFGF . O número de células inflamatórias no tecido gengival foi maior nas amostras PD60. O teor de colágeno na área ocupada pelas fibras de colágeno birrefringentes foram menores para PD60. A contagem diferencial de leucócitos mostrou que houve um influxo polimorfonuclear significativamente maior no grupo PD15, enquanto que PD60 mostrou número maior de linfócitos. PD60 apresentou transcritos de genes RANTES, IP-10, MCP-1 mais elevados, bem como uma maior concentração de óxido nítrico. A avaliação clínica revelou que o grupo PD60 apresentou aumento da área óssea exposta na região da furca. Em conclusão, neste modelo animal o aumento dos marcadores RANK/RANKL e HGF está relacionado a uma resposta imunológica específica e provavelmente contribuiu para a evolução da doença periodontal. Investigar o efeito destes biomarcadores pode ajudar na terapia dirigida para a reabsorção óssea, uma vez que bloquear estes pode inibir a perda óssea.

Prolonged viability of human organotypic skin explant in culture method (hOSEC)

Abstract BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. .

Phototherapy promotes healing of cutaneous wounds in undernourished rats

BACKGROUND: Various studies have shown that phototherapy promotes the healing of cutaneous wounds. OBJECTIVE: To investigate the effect of phototherapy on healing of cutaneous wounds in nourished and undernourished rats. METHODS: Forty rats, 20 nourished plus 20 others rendered marasmus with undernourishment, were assigned to four equal groups: nourished sham, nourished Light Emitting Diode treated, undernourished sham and undernourished Light Emitting Diode treated. In the two treated groups, two 8-mm punch wounds made on the dorsum of each rat were irradiated three times per week with 3 J/cm2 sq cm of combined 660 and 890nm light; wounds in the other groups were not irradiated. Wounds were evaluated with digital photography and image analysis, either on day 7 or day 14, with biopsies obtained on day 14 for histological studies. RESULTS: Undernourishment retarded the mean healing rate of the undernourished sham wounds (p < 0.01), but not the undernourished Light emission diode treated wounds, which healed significantly faster (p < 0.001) and as fast as the two nourished groups. Histological analysis showed a smaller percentage of collagen in the undernourished sham group compared with the three other groups, thus confirming our photographic image analysis data. CONCLUSION: Phototherapy reverses the adverse healing effects of undernourishment. Similar beneficial effects may be achieved in patients with poor nutritional status. .

The vegetal biomembrane in the healing of chronic venous ulcers / Biomembrana vegetal na cicatrização de úlceras venosas crônicas

BACKGROUND: The vegetal biomembrane has been used to treat cutaneous ulcers. OBJECTIVES: To assess the role of the vegetal biomembrane on the chronic venous ulcers treatment compared to treatment with collagenase cream. METHODS: Fourteen patients were selected to be treated with vegetal biomembrane and 7 with Fibrase®(CONTROL), followed clinically and photographically by the Wound Healing Index by ImageJ during 120 days and biopsied on the 1st and 30th days for histological examination. RESULTS: The vegetal biomembrane was better in promoting healing of the ulcers, especially on the inflammatory phase, confirmed by abundant exudation and wound debridement than the CONTROL group, on the 30th day. There was a greater tendency to angiogenesis followed by re-epithelialization with highest wound healing index on the 90th and 120th days. CONCLUSION: A combined analysis of clinical and histopathological findings suggests that the vegetal biomembrane acted as a factor inducing wound healing, especially on the inflammatory phase, confirmed by abundant exudation of the lesions promoting the transformation of the microenvironment of the chronic venous ulcers, and also stimulating angiogenesis and subsequent re-epithelialization.

FUNDAMENTOS: A biomembrana vegetal tem sido usada para tratamento de úlceras cutâneas. OBJETIVOS: Avaliar a ação da biomembrana vegetal no tratamento de úlceras venosas crônicas, comparando-a ao tratamento à base de colagenase. MÉTODOS: Foram selecionados 14 pacientes tratados com biomembrana vegetal e sete com Fibrase® (grupo controle), acompanhados clínico-fotograficamente pelo índice de cicatrização das úlceras (ICU) por 120 dias, por meio do software ImageJ, e biopsiados no primeiro e 30º dias para estudo histopatológico. RESULTADOS: A biomembrana vegetal foi superior em relação ao controle na cicatrização das úlceras no 30º dia, especialmente na fase inflamatória, confirmada pela exsudação abundante e pelo desbridamento. Houve tendência superior à angiogênese seguida de reepitelização com maiores ICUs no 90º e 120º dias. CONCLUSÃO: A análise conjunta dos achados clínicos e histopatológicos sugere que a biomembrana vegetal atuou como um fator indutor da cicatrização, especialmente na fase inflamatória, confirmada pela exsudação abundante das lesões, promovendo a transformação do microambiente das úlceras venosas crônicas e estimulando a angiogênese e a posterior reepitelização.

Curativo de biomembrana vegetal e hipersensibilidade / Natural-biomembrane dressing and hypersensitivity

FUNDAMENTOS: A biomembrana vegetal do látex da seringueira Hevea brasiliensis tem sido usada como curativo para úlceras cutâneas. OBJETIVOS: Avaliar a segurança da biomembrana vegetal como curativo em relação à hipersensibilidade ao látex. MÉTODOS: Foram selecionados pacientes com úlceras cutâneas constituindo-se os grupos: controle - baixa exposição profissional ao látex (n=17); alta exposição profissional (n=14); ulcerados em uso da biomembrana vegetal (n=13); ulcerados-controle sem uso da biomembrana vegetal (n=14) e casos novos (n=9), submetidos à avaliação pré e após 3 meses de uso da biomembrana vegetal. Todos foram submetidos à avaliação clínico-epidemiológica quanto à hipersensibilidade ao látex e IgE específica (UniCap®), e os grupos controle e controle exposto ao látex ao "patch test". RESULTADOS: A história de hipersensibilidade foi positiva em 64,7 por cento dos pacientes do grupo-controle, 71,4 por cento do controle exposto ao látex, 61,5 por cento dos ulcerados em uso da biomembrana vegetal, 35,7 por cento dos ulcerados-controle, e apenas 22,2 por cento no grupo casos novos. Ao teste de contato dos grupos controle e controle exposto ao látex, apenas um indivíduo do grupo C (baixo contato) apresentou eritema na primeira leitura, negativando-se na segunda. A média de contato com látex no grupo-controle exposto ao látex foi de 3,42 horas/dia. No ensaio fluoroimunoenzimático, a grande maioria dos soros foi classificada como zero (variação 0 a 6). Nenhum soro recebeu classificação acima de 2, não sendo considerada classificação significante para hipersensibilidade (classificação > 4). CONCLUSÃO: A biomembrana vegetal mostrou-se segura como curativo, pois não induziu reações de hipersensibilidade entre os voluntários submetidos ao "patch test", nem entre os usuários da biomembrana vegetal, como demonstrado clinica e imunologicamente pela dosagem de IgE.

BACKGROUND: The natural biomembrane of latex extracted from Hevea brasiliensis has been used as a dressing for skin ulcers. OBJECTIVES: To evaluate how safe the natural biomembrane is in relation to hypersensitivity to latex when used as a dressing. METHODS: We selected patients with skin ulcers, forming the following groups: control - low occupational exposure to latex (n = 17); latex-exposed control - high occupational exposure (n = 14); ulcerated, using the natural biomembrane (n = 13); ulcerated control, not using the natural biomembrane (n = 14); and new cases (n = 9), assessed before and after 3 months of using the natural biomembrane. All patients underwent clinical and epidemiological evaluation for latex hypersensitivity and specific IgE (UniCap®), and the control and latex-exposed control groups underwent the patch test. RESULTS: Hypersensitivity was positive in 64.7 percent of the patients in the control group, 71.4 percent of the patients in the latex-exposed control group, 61.5 percent of the ulcerated using the natural biomembrane, 35.7 percent of the ulcerated control, and only 22 , 2 percent of the new cases. In the patch test of the control and latex-exposed control groups, only one individual in the control group (low contact) showed erythema in the first reading, which became negative in the second. The mean contact with latex in the latex-exposed control group was 3.42 hours / day. In the fluoroimmunoenzymatic assay, most of the sera was classified as zero (range 0-6). No serum was rated above 2, which is not considered significant for hypersensitivity (classification > 4). CONCLUSION: The natural biomembrane proved to be safe as a dressing, for it did not induce hypersensitivity reactions among the volunteers who underwent the patch test or among users of the natural biomembrane, as it was clinically and immunologically demonstrated by IgE levels.

Modelos experimentais de desnutrição e sua influência no trofismo cutâneo / Experimental models of malnutrition and its effect on skin trophism

FUNDAMENTOS: A pele, para exercer suas funções, necessita de níveis adequados de nutrientes. OBJETIVO: Analisar o trofismo cutâneo de ratos nutridos e desnutridos por meio de dois modelos de desnutrição. MÉTODOS: No Modelo Marasmo, utilizaram-se 60 ratos Wistar em controle dietético, dos quais 30 foram selecionados aleatoriamente para receber metade da dieta diária durante 60 dias. No Modelo Gelatina, empregaram-se 60 ratos, dos quais 30 receberam dieta associada a proteína de baixa qualidade (gelatina) durante 30 dias. Avaliou-se o estado nutricional dos animais por meio da massa corporal, dos sinais clínicos e da dosagem de albumina sérica. Após o período de desnutrição, fez-se a histologia da pele dos animais para análise da espessura da derme e epiderme com o software Leica Application Suite; nas lâminas coradas com tricrômio de Gomori, analisou-se a colagênese com o software ImageJ. RESULTADOS: A massa corporal dos animais desnutridos pelo marasmo e gelatina foi significativamente menor (p<0,0001 e p<0,0001) do que a dos grupos nutridos. Quanto à albumina sérica, não houve diferença entre os grupos nos dois modelos. Em relação à análise histológica da espessura da pele, os desnutridos apresentaram a derme significativamente menos espessa em comparação aos nutridos (p<0,0001 e p<0,0001). No que respeita à colagênese, os grupos desnutridos apresentaram menores percentuais de colágeno em relação aos nutridos (p<0,0005 e p<0,003). CONCLUSÕES: Os animais desnutridos pelos dois modelos apresentaram diminuição na espessura dérmica, confirmada histologicamente pelo menor percentual de colágeno, mostrando a influência negativa da desnutrição no trofismo cutâneo.

BACKGROUND: The skin requires adequate levels of nutrients to function properly. OBJECTIVE: To analyze skin trophism in well-nourished and undernourished rats using two models of malnutrition. METHODS: In the marasmus model, 60 Wistar rats were kept on a controlled diet, 30 being randomly selected to receive half the established diet for 60 days. In the gelatin model, 60 rats were used, 30 of which received a diet consisting of poor quality protein (gelatin) for 30 days. The nutritional status of the animals was evaluated according to body mass index, clinical signs and serum albumin measurement. After the period of malnutrition, histology was performed on the animals' skin to analyze the thickness of the dermis and epidermis using the Leica Application Suite software. Collagen was analyzed on slides stained with Gömöri trichrome using the ImageJ software program. RESULTS: The body mass index of the malnourished animals in the marasmus and gelatin groups was significantly lower than that of the well-nourished animals in the two groups (p<0.0001 in both models). With respect to serum albumin, there was no difference between the groups in either of the two models. In relation to the histological analysis of skin thickness, the dermis of the malnourished animals was significantly thinner compared to that of the well-nourished animals (p<0.0001 in both models). The percentage of collagen was lower in the malnourished animals compared to the well-nourished animals (p<0.0005 and p<0.003 in the marasmus and gelatin model, respectively). CONCLUSIONS: Skin thickness measurements were lower in the malnourished animals in both models, and this finding was histologically confirmed by the lower percentage of collagen, showing the negative effect of malnutrition on skin trophism.