Produção, caracterização, degradação e citotoxicidade de arcabouços de PLGA+HA/?TCP com sinvastatina incorporada

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Odontologia, Florianópolis, 2017.As perdas ósseas decorrentes de perdas dentárias ou doenças degenerativas fizeram com que houvesse interesse na busca por estratégias de regeneração óssea, a fim de repor o osso que foi perdido. Devido às suas características biológicas e mecânicas, o osso exige estratégias complexas para permitir a reconstituição da sua estrutura e função. Os principais quesitos para uma estratégia eficaz de engenharia óssea incluem um número suficiente de células formadoras de osso, um arcabouço capaz de conduzir estas células e oferecer suprimento sanguíneo adequado, e fatores de crescimento capazes de induzir a diferenciação celular. Assim, o objetivo deste estudo foi produzir arcabouços de ácido polilático co-glicólico com hidroxiapatita e ß-tricálcio fosfato (PLGA+HA/ßTCP), adicionados de sinvastatina a 5%, bem como caracterizar e avaliar a degradação e a citotoxicidade destes arcabouços, de forma a verificar se os mesmos poderiam ser promissores para aplicação clínica. Os arcabouços foram obtidos pela técnica de evaporação de solvente. Após a dissolução completa do polímero em clorofórmio, foram adicionadas partículas de sacarose e de cerâmica bifásica (hidroxiapatita (70%) e ß-TCP (30%)), na proporção 1:1 entre polímero e cerâmica. Após a evaporação do solvente, a sacarose foi removida utilizando-se álcool polivinílico. Para os arcabouços com sinvastatina incorporada, o fármaco a 5% foi diluído em clorofórmio, ao qual foram adicionados sacarose, hidroxiapatita e ß-TCP ao polímero já diluído. Os arcabouços foram seccionados em discos de 6 mm por 1 mm e esterilizados por radiação gama. Após a síntese dos arcabouços, os mesmos sofreram testes de caracterização, degradação e citotoxicidade. Para tanto, as amostras foram imersas em PBS a 37 °C, sob agitação constante, durante 1, 7, 14, 21 e 28 dias. Amostras não degradadas foram tomadas como referência. Foram realizados microscopia eletrônica de varredura, para avaliar a estrutura física; espectroscopia no infravermelho transformada por Fourier, para analisar a composição química; calorimetria exploratória diferencial, para testar as propriedades térmicas; teste da gota, para verificar a hidrofobia; e citotoxicidade, para avaliar se as amostras afetam o crescimento celular. As imagens obtidas por microscopia revelam a presença de macro, meso e microporos na estrutura do polímero, onde partículas de HA e ßTCP encontram-se bem inseridas e dispersas. Para análises químicas, observa-se um padrão bastante semelhante entre os arcabouços sem sinvastatina e com sinvastatina a 5%, com prevalência de bandas de absorção de cerca de 1750 cm-1 (C = O), e de bandas entre 350 ? 1500 cm-1 (ésteres (C ? O) ehidrocarbonetos (CH2 e CH)), assim como traços de hidrocarbonetos (CH, CH3 e CH2) por volta de 3000 cm-1. Sobre as análises térmicas, observa-se que as curvas do termograma seguem um padrão semelhante entre os arcabouços sem sinvastatina e com sinvastatina a 5%, com valores numéricos muito próximos, onde os picos endotérmicos ocorrem em cerca de 150 - 160 °C. Os arcabouços com sinvastatina mostraram-se mais hidrofílicos e mais citotóxicos que os arcabouços sem sinvastatina, chegando a uma redução de até 20% na viabilidade celular ao terceiro dia de experimento em comparação com o controle positivo. Arcabouços de PLGA+HA/ßTCP com sinvastatina a 5% apresentaram boas características físico-estruturais, químicas e térmicas, demonstrando ser um biomaterial promissor para a regeneração de tecido ósseo juntamente com células-tronco. Entretanto, quando testado sobre fibroblastos, os mesmos arcabouços mostraram-se citotóxicos na concentração de testada de sinvastatina.Abstract : Bone loss due to tooth extraction or degenerative diseases cause interest in the search for bone regeneration strategies. Due to its biological and mechanical characteristics, bone requires complex strategies to allow the reconstitution of its structure and function. The main requirements for an effective bone engineering strategy include sufficient number of bone-forming cells, scaffolds capable of conducting these cells and providing adequate blood supply, and growth factors able to induce cell differentiation. Thus, the aim of this study was to develop scaffolds of polylactic coglycolic acid, hydroxyapatite and ß-tricalcium phosphate (PLGA+HA/ß-TCP), added with 5% simvastatin, as well as to characterize and evaluate the degradation and cytotoxicity of the scaffolds in order to assess if these biomaterials could be used for further clinical proposes. The samples were obtained by the solvent evaporation technique. After complete dissolution of the polymer in chloroform, sucrose and biphasic ceramic particles (HA (70%) and ß-TCP (30%)) were added in a ratio of 1:1 between polymer and ceramic. After evaporation of the solvent, sucrose was removed using polyvinyl alcohol. For the samples with simvastatin, 5% of the medication were diluted in chloroform to which sucrose, HA and ß-TCP were added to the diluted polymer. Then, the samples were sectioned on 6 mm x 1 mm discs and sterilized by gamma radiation. After the synthesis of the scaffolds, characterization and degradation tests were conducted. For degradation, the samples were immersed in PBS at 37 °C under constant stirring for 1, 7, 14, 21, and 28 days. Non-degraded samples were taken as reference. Scanning electron microscopy was performed to evaluate the physical structure; Fourier transform infrared spectroscopy to analyze the chemical composition; differential scanning calorimetry to test the thermal properties; drop test to check for hydrophobicity; and cytotoxicity to evaluate whether the samples could affect cell growth. Microscopy images revealed the presence of macro, meso and micropores in the polymer structure, where particles of HA and ßTCP were well inserted and dispersed. On chemical analyzes, a very similar pattern is observed between the scaffolds without simvastatin and with 5% simvastatin, and a prevalence of absorption bands of about 1750 cm-1 (C = O), and bands between 350 - 1500 cm-1, characteristic of esters (C ? O) and hydrocarbons (CH2 and CH), as well as traces of CH, CH3 and CH2 hydrocarbons at about 3000 cm-1. On thermal analyzes, it was observed that the thermogram curves follow a similar pattern between the scaffolds without simvastatin and with 5% simvastatin, with very close numericalvalues, where the endothermic peaks occur at about 150 - 160 °C. Scaffolds with 5% simvastatin were more hydrophilic and cytotoxic than scaffolds without simvastatin, reaching a reduction of up to 20% in cell viability on the third day of experiment compared to the positive control. Scaffolds of PLGA+HA/ßTCP with 5% simvastatin presented good structural, chemical and thermal characteristics, revealing to be a promising biomaterial for regeneration of bone tissue along with stem cells. However, when tested on fibroblasts, the scaffolds were cytotoxic at the concentration of 5% simvastatin

Avaliação de lesões bucais em pacientes usuários de substâncias químicas ilícitas

TCC (graduação) - Universidade Federal de Santa Catarina. Centro de Ciências da Saúde. Odontologia.O consumo de drogas ilícitas aumentou muito nos últimos anos. Dentre elas, a maconha e a cocaína/crack são as mais comumente utilizadas. Na cavidade bucal, essas substâncias produzem diversas alterações celulares e teciduais que indicam possibilidade de indução ao câncer de boca. Esse trabalho teve como objetivo avaliar a presença de lesões na mucosa oral, dados demográficos e saúde geral de pacientes usuários de drogas ilícitas. O estudo foi realizado com 35 dependentes químicos de ambos os sexos acima de 18 anos de idade e que se encontravam em tratamento no Núcleo de Psiquitria do Hospital Universitário (HU/UFSC) e no Instituto de Psiquitra de Santa Catarina (IPq/SC).Um número equivalente de pacientes não usuários foi usado como grupo controle. A análise estatística foi realizada pelo teste qui-quadrado com intervalo de confiança de 95% (p < 0,05). Foi encontrado que 91,4% do grupo experimental eram do sexo masculino, com média de idade de 36 anos. Para variáveis de saúde geral (comorbidades, internações, cirurgias, alergias e uso de medicamentos), não houve diferença estatisticamente significante entre os grupos. Entretanto, as variáveis tabagismo, etilismo e presença de lesões apresentaram diferença estatística (p < 0,05). Dos 35 participantes da pesquisa, nenhuma lesão que tivesse hipótese de diagnóstico de câncer de boca ou de lesão potencialmente maligna foi observada, porém alterações diversas como aftas, ceratoses friccionais provocadas por bordos cortantes ou morsicátio, candidíase, cicatriz de extração dentária e despapilação da língua foram relatadas no grupo experimental. Dentre o grupo controle, apenas duas lesões foram observadas, sem, entretanto,hipótese de diagnóstico de câncer de boca. Embora esta pesquisa não tenha comprovado a relação entre câncer de boca e a dependência de substâncias químicas ilícitas, os fatores de risco são conhecidos e comprovados e precisam ser levados em conta para a prevenção deste tipo câncer.The use of illicit drugs has increased greatly in recent years. Among them, marijuana and cocaine/crack are the most commonly used drugs. In the oral cavity, these substances produce several changes on cell and tissues that indicate the possibility of induction of oral cancer. The aim of this study was to evaluate the presence of lesions in oral mucosa, besides demographic and general health of patients using illicit drugs. The study was conducted with 35 individuals chemically dependents of both genders above 18 years old and who were undertreatmentment at Núcleo de Psiquitria do Hospital Universitário (HU/UFSC) and Instituto de Psiquitra de Santa Catarina (IPq/SC). An equivalent number of patients nonusers of drugs was used as a control group. Statistical analysis was performed using Chi-square test with a confidence interval of 95% (p <0.05). It was found that 91.4% of the experimental group was male, with a mean range of 36 years of age. For general health variables (comorbidities, hospitalizations, surgeries, allergies and medications), there were no statistically significant difference between both groups. However, variables such as smoking, alcoholism and presence of oral lesions differ significantly (p <0.05). Of the 35 participants, any lesion withhypothesis of diagnosis of oral cancer or potentially malignant lesion was observed, but several changes as aphthae, frictional keratoses caused by sharp edges or morsicatio, candidiasis, tooth extraction scar and lingual despapilation have been reported in the experimental group. Only two lesions without diagnosis of oral cancer were observed in the control group. Although this study had not proven the relationship between oral cancer and the dependence of illicit drugs, the risk factors are known and must be considered to prevent this kind of cancer

Oral squamous carcinoma cell lysates provoke exacerbated inflammatory response in gingival fibroblasts.

OBJECTIVES To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells. MATERIALS AND METHODS Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR. RESULTS Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials. CONCLUSIONS Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory. CLINICAL RELEVANCE Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling

TGF-β Signalling Mediates the Anti-Inflammatory Activity of Enamel Matrix Derivative In Vitro.

Enamel matrix derivative (EMD) prepared from extracted porcine fetal tooth material can support the regrow of periodontal tissues. Previous findings suggest that EMD has anti-inflammatory properties and TGF-β activity in vitro. However, the anti-inflammatory activity of EMD is mediated via TGF-β has not been considered. To this aim, we first established a bioassay to confirm the anti-inflammatory activity of EMD. The bioassay was based on the RAW 264.7 macrophage cell line and proven with primary macrophages where EMD significantly reduced the forced expression of IL-6. We then confirmed the presence of TGF-β1 in EMD by immunoassay and by provoking the Smad2/3 nuclear translocation in RAW 264.7 macrophages. Next, we took advantage of the TGF-β receptor type I kinase-inhibitor SB431542 to block the respective signalling pathway. SB431542 reversed the anti-inflammatory activity of EMD and TGF-β in a bioassay when IL-6 and CXCL2 expression was driven by the LPS stimulation of RAW 264.7 macrophages. This central observation was supported by showing that SB431542 reversed the anti-inflammatory activity of EMD using IL-1β and TNF-α-stimulated ST2 bone marrow stromal cells. Together, these findings implicate that the TGF-β activity mediates at least part of the anti-inflammatory activity of EMD in vitro

Platelet-Rich Fibrin Reduces IL-1β Release from Macrophages Undergoing Pyroptosis.

BACKGROUND Pyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1β (IL-1β) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1β, remains unknown. The question arises whether PRF could regulate IL-1β release from macrophages in vitro. METHODS To answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1β secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected. RESULTS PRF lowered the LPS-induced expression of IL-1β and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1β and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1β at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1β release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1β release in RAW 264.7 cells and a trend to diminish IL-1β release in primary macrophages. CONCLUSION These findings suggest that PRF can reduce IL-1β release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages

Enamel Matrix Derivative Decreases Pyroptosis-Related Genes in Macrophages.

Background: Pyroptosis is a caspase-dependent catabolic process relevant to periodontal disorders for which inflammation is central to the pathophysiology of the disease. Although enamel matrix derivative (EMD) has been applied to support periodontal regeneration, its capacity to modulate the expression of pyroptosis-related genes remains unknown. Considering EMD has anti-inflammatory properties and pyroptosis is linked to the activation of the inflammasome in chronic periodontitis, the question arises whether EMD could reduce pyroptosis signalling. Methods: To answer this question, primary macrophages obtained from murine bone marrow and RAW 264.7 macrophages were primed with EMD before being challenged by lipopolysaccharide (LPS). Cells were then analysed for pyroptosis-signalling components by gene expression analyses, interleukin-1β (IL-1β) immunoassay, and the detection of caspase-1 (CAS1). The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: We report here that EMD, like the inflammasome (NLRP3) and CAS1 specific inhibitors-MCC950 and Ac-YVAD-cmk, respectively-lowered the LPS-induced expression of NLRP3 in primary macrophages (EMD: p = 0.0232; MCC950: p = 0.0426; Ac-YVAD-cmk: p = 0.0317). EMD further reduced the LPS-induced expression of NLRP3 in RAW 264.7 cells (p = 0.0043). There was also a reduction in CAS1 and IL-1β in RAW 264.7 macrophages on the transcriptional level (p = 0.0598; p = 0.0283; respectively), in IL-1β protein release (p = 0.0313), and CAS1 activity. Consistently, EMD, like MCC950 and Ac-YVAD-cmk, diminished the ROS release in activated RAW 264.7 cells. In ST2 murine mesenchymal cells, EMD could not be tested because LPS, saliva, and IL-1β + TNF-α failed to provoke pyroptosis signalling. Conclusion: These findings suggest that EMD is capable of dampening the expression of pyroptosis-related genes in macrophages

Heartwood of Dalbergia cochinchinensis: 4,7,2'-Trihydroxy-4'-methoxyisoflavanol and 6,4'-Dihydroxy-7-methoxyflavane Reduce Cytokine and Chemokine Expression In Vitro.

Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2'-trihydroxy-4'-methoxyisoflavanol (472T4MIF) and 6,4'-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1β and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells

Damaged Mesenchymal Cells Dampen the Inflammatory Response of Macrophages and the Formation of Osteoclasts.

Damage to mesenchymal cells occurs by dental implant drills as a consequence of shear forces and heat generation. However, how the damaged mesenchymal cells can affect the polarization of macrophages and their differentiation into osteoclastogenesis is not fully understood. To simulate cell damage, we exposed suspended ST2 murine bone marrow stromal cells to freeze/thawing or sonication cycles, followed by centrifugation. We then evaluated the lysates for their capacity to modulate lipopolysaccharide-induced macrophage polarization and RANKL-MCSF-TGF-β-induced osteoclastogenesis. We report that lysates of ST2, particularly when sonicated, greatly diminished the expression of inflammatory IL6 and COX2 as well as moderately increased arginase 1 in primary macrophages. That was confirmed by lysates obtained from the osteocytic cell line IDG-SW3. Moreover, the ST2 lysate lowered the phosphorylation of p65 and p38 as well as the nuclear translocation of p65. We further show herein that lysates of damaged ST2 reduced the formation of osteoclast-like cells characterized by their multinuclearity and the expression of tartrate-resistant phosphatase and cathepsin K. Taken together, our data suggest that thermal and mechanical damage of mesenchymal cells causes the release of as-yet-to-be-defined molecules that dampen an inflammatory response and the formation of osteoclasts in vitro

Blocking of Caspases Exerts Anti-Inflammatory Effects on Periodontal Cells.

Periodontitis is an inflammatory process that is associated with caspase activity. Caspases could thus become molecular targets for the modulation of the inflammatory response to harmful factors, such as lipopolysaccharides (LPS) and TNFα. Here, the impact of the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methyl ketone) on the modulation of the LPS-induced inflammatory response of murine RAW 264.7 cells and primary macrophages was examined. Moreover, the inflammatory responses of human gingival fibroblasts, HSC2 oral squamous carcinoma cells and murine ST2 mesenchymal fibroblasts when exposed to TNFα were studied. Data showed that Z-VAD-FMK significantly lowered the inflammatory response of RAW 264.7 cells and primary macrophages, as indicated by the expression of IL1 and IL6. In murine ST2 mesenchymal fibroblasts, the TNFα-induced expression of CCL2 and CCL5 was significantly reduced. In human gingival fibroblasts and HSC2 cells, Z-VAD-FMK considerably reduced the TNFα-induced expression of CXCL8 and CXCL10. These findings suggest that pharmacological blocking of caspases in an inflammatory environment lowers the expression of cytokines and chemokines in periodontal cells

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms

Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. Objective To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. Material and methods We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 μg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. Results Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. Conclusions Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect