Photoelastic analysis of polymerization shrinkage stress of composites under the influence of restorative techniques

Orientador: Simonides ConsaniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A tensão de contração da polimerização é uma característica indesejável e inevitável das restaurações adesivas. É importante entender a distribuição da tensão para melhorar a efetividade clínica das restaurações de resina composta. Capítulo 1: o objetivo foi avaliar a tensão de contração da polimerização e o grau de conversão de um compósito resinoso com diferentes modos de ativação e densidades de energia. O compósito Z350 foi inserido no orifício central do disco de resina fotoelástica e fotoativado com LED (Bluephase-2) com densidade de energia de 12, 24 ou 36 J/cm2 usando os seguintes protocolos de fotoativação: contínuo de alta intensidade (HI: 1200 mW/cm2 por 10, 20 ou 30 s), contínuo de baixa intensidade (LI: 650 mW/cm2 por 18, 36 ou 54 s) e soft-start (SS: 150 mW/cm2 por 5 s + 1200 mW/cm2 por 9, 19 ou 29 s) (n=5). A tensão de contração de polimerização foi avaliada por meio de análise fotoelástica e o FTIR foi usado para análise do grau de conversão imediatamente e 24 horas após a ativação. ANOVA 3-fatores foi usado para avaliar os principais efeitos e as interações correspondentes, depois ANOVA 2-fatores foi realizado para cada tempo (p<0,05). As imagens mostraram que a tensão aumentou quando maior densidade de energia foi usada. A tensão não foi estatisticamente significante entre os métodos de ativação de alta e baixa intensidade. O método soft-start gerou tensões menores do que os modos contínuos quando 12 J/cm2 usado. O grau de conversão do modo soft-start foi estatisticamente menor que os modos de alta e baixa intensidade. Não foram encontradas diferenças no grau de conversão entre as doses de energia testadas. Pode-se concluir que a tensão de contração foi influenciada pela dose de energia e pelos protocolos de polimerização. O grau de conversão não foi influenciado pelo aumento das densidades de energia. Capítulo 2: O objetivo foi avaliar a tensão de contração criada em modelos fotoelásticos usando diferentes técnicas de inserção do compósito. Vinte réplicas de dentes em resina fotoelástica com cavidade classe II foram obtidos e distribuídos em quatro grupos (n=5) de acordo com as seguintes técnicas restauradoras: inserção horizontal (H), inserção oblíqua (O), compósito fluido + inserção horizontal (F+H), compósito fluido + inserção oblíquoa (F+O). Os modelos restaurados foram analisados por meio de polariscópio plano. A tensão ao longo de 7 áreas da interface adesiva foi analisada a partir das imagens obtidas após inserção de cada incremento e 24h após a fotoativação. Os valores de tensão máxima foram submetidos a ANOVA 2-fatores, seguido pelo teste de Tukey (p<0.05). As técnicas de inserção horizontal e oblíqua apresentaram diferenças no padrão de tensão. Apesar disso, nenhuma diferença no valor de tensão entre as técnicas horizontal e oblíqua foi encontrada. Os grupos com resina fluida mostraram maior tensão que aqueles restaurados apenas com compósito convencionalAbstract: Polymerization shrinkage stress is an undesirable and inevitable characteristic of adhesive restorations. It is important to understand the stress distribution to improve the clinical effectiveness of resin composite restoration. Chapter 1: The aim was to evaluate the polymerization shrinkage stress and degree of conversion of a resin composite material submitted to different photoactivation protocols. A bulk increment of composite Filtek Z350 was placed in the central perforation of photoelastic disc and photoactivated using LED-based curing unit (BluePhase II) with energy density of 12, 24 or 36 J/cm2 using the following photopolymerization protocols: continuous high intensity (HI: 1200 mW/cm2 during 10, 20 or 30 s), continuous low intensity (LI: 650 mW/cm2 during 18, 36 or 54 s) and soft-start (SS: 150 mW/cm2 during 5 s + 1200 mW/cm2 during 9, 19 or 29 s) (n=5). Photoelastic analysis was used to evaluate polymerization shrinkage stress of composite and FTIR was performed to access the degree of conversion of composite according to the curing modes. Statistical test ANOVA 3-way was used to determine the main effects and the corresponding interactions, then ANOVA 2 way was performed for each time (p<0.05). The images showed that polymerization shrinkage stress increases with higher values of power density. No significant differences should be noticed on polymerization shrinkage stress between high and low intensity modes of activation. Soft-start method generates stresses that were statistically lower than continuous modes except when 12 J/cm2 was applied. Similar degree of conversion was observed for photoactivation modes used, except for soft-start mode with 12, 24 and 36 J/cm2 that showed lowest levels of conversion. It could be concluded that polymerization shrinkage stress was significantly influenced by energy density and the curing protocol. No benefits on degree of conversion were observed by increasing the energy density. Chapter 2: The aim was to evaluate the polymerization shrinkage stress created in tooth photoelastic models using different filling techniques. Twenty class II tooth models were obtained. They were distributed into four experimental groups (n=5) according to the following restorative techniques: resin composite with horizontal insertion (H), resin composite with oblique insertion (O), flowable composite + resin composite with horizontal insertion (F + H) and flowable composite + resin composite with oblique insertion (F + O). Restored photoelastic models were analyzed using a plane polariscope. The stress along seven points of adhesive interface was analyzed from images of each insertion, at the baseline (immediately) and 24 h after polymerization. Maximum shear stress data was obtained and submitted to ANOVA 2-way, followed by Tukey's post hoc test at p = 0.05. Horizontal and oblique techniques have shown differences in stress patterns. No difference between horizontal and oblique techniques was found. Groups restored with flow composite showed significant higher stress levels than those restored only with regular composite. In conclusion, the use of flow composite created higher polymerization stress effects on the class II restoration than does the conventional restoration techniqueDoutoradoMateriais DentariosDoutor em Materiais Dentário

Photoelastic analysis of polymerization shrinkage stress in resin composites

Orientador: Simonides ConsaniDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O objetivo deste trabalho foi comparar a tensão de contração de polimerização gerada por compósitos resinosos em diferentes tempos após a fotoativação, por meio de análise fotoelástica. Três compósitos experimentais com diferentes tipos de fotoiniciadores, e dois compósitos comerciais, sendo um à base de monômeros metacrilatos e o outro à base de siloranos foram comparados. Compósitos experimentais foram fabricados com 40% em massa de matriz orgânica e 60% de partículas de carga inorgânica, canforoquinona (CQ) ou fenil propanodiona (PPD) foram adicionados como sistemas fotoiniciadores. Discos deresina fotoelástica (base: Araldite GY 279/ endurecedor: Aradur 2963) foram confeccionados com perfurações de 6mm de diâmetro e 2mm de espessura no centro de cada disco. A superfície interna da resina fotoelástica foi condicionada com agente adesivo. Os compósitos foram separados em 5 grupos, sendo que os grupos 1 e 2 representaram os compósitos comerciais Filtek Silorane e Filtek Z250, respectivamente. Enquanto que os grupos 3, 4 e 5 constituíam os compósitos experimentais com os sistemas iniciadores CQ/Amina, CQ/PPD/Amina e PPD/Amina, respectivamente. O compósito foi inserido em incremento único no preparo da resina fotoelástica e fotoativado com luz LED (LED Ultralume 5) por 60s. As tensões geradas pelos compósitos foram avaliadas logo após a fotoativação, em 24 horas e 7 dias, por meio de análise fotoelástica com polariscópio plano, diretamente sobre luz branca ou com filtro vermelho resultando em imagens coloridas e monocromáticas, respectivamente. A análise qualitativa das imagens fotoelásticas revelaram que o grupo 1 apresentou a menor tensão de contração de polimerização em todos os tempos avaliados. Comparando os grupos das resinas experimentais, notou-se que o grupo 3 gerou maior tensão na resina fotoelástica em relação aos grupos 4 e 5 imediatamente após a fotoativação. Porém, 24 horas e 7 dias após a fotoativação as tensões geradas se equipararam para os grupos das resinas experimentais. A análise quantitativa realizada com avaliação das imagens monocromáticas mostrou que a tensão gerada pelo compósito Filtek Silorane foi similar à do compósito Filtek Z250 imediatamente após a fotoativação; porém, em 24 horas e 7dias o Filtek Z250 apresentou maiores tensões quando comparado ao Filtek Silorane. Os compósitos experimentais não apresentaram diferença estatisticamente significativa entre eles em nenhum dos tempos avaliados. Em geral, as tensões geradas aumentaram com o passar do tempo. Concluiu-se que o PPD não foi efetivo para minimizar as tensões geradas durante o processo de polimerização dos compósitos. Compósitos à base de silorano apresentaram os menores valores de tensão de contração de polimerização.Abstract: The aim of this study was to compare polymerization shrinkage stresses of dental composites in different moments after light-activation. Three experimental composites with different photoinitiator systems, and two commercial composites, one methacrilate based and the other silorane based were compared. Experimental composites were fabricated with 40% of organic matrix and 60% of inorganic filler, canforoquinone (CQ) or phenyl propanodione (PPD) were added to the resin as photoinitiator systems. Discs of the photoelastic resin (Araltide GY279/Aralur 2963) were prepared with cylindrical cavities (6mm diameter, 2mm thick) in the center of each disc. Internal surfaces of the photoelastic resin were roughened and prepared with adhesive system application. The composites were divided into five groups: groups 1 and 2 represented commercial composites Filtek Silorane and Filtek Z250, respectively; groups 3, 4 and 5 represented experimental composites with CQ/Amine, CQ/PPD/Amine and PPD/Amine as initiator system, respectively. A bulk increment of the composite was inserted into the cavity of photoelastic resin and photo-polymerized with a LED light-cure unit (LED Ultralume 5) for 60s. The stresses created by polymerization shrinkage were analyzed immediately after photopolymerization, in 24 hours and 7 days, by photoelastic analysis with a plane polariscope, with white light, with or without a red filter resulting in color and black and white images, respectively. Qualitative analysis of photoelastic images showed that group 1 had the lowest polymerization shrinkage stress in all times evaluated. Comparing experimental composites, it was observed that group 3 created more stress in photoelastic resin than groups 4 and 5 immediately after light-activation. However, 24 hours and 7 days after photopolymerization the stresses had equalized in resin experimental groups. Quantitative analysis showed that shrinkage strain of Filtek Silorane was similar to that of Filtek Z250 immediately after photopolymerization. In 24 hours and 7 days, the Filtek Z250 showed higher strain than Filtek Silorane. Experimental composites didn't differ statistically between each other in all times evaluated. Generally, the tension increased with the time. It was concluded that PPD wasn't capable of decrease the stresses created during polymerization process of composites. Silorane based composites had the lowest stresses during polymerization shrinkage process.MestradoMestre em Materiais Dentário

Membrane potential (Vmem) measurements during mesenchymal stem cell (MSC) proliferation and osteogenic differentiation

Background Electrochemical signals play an important role in cell communication and behavior. Electrically charged ions transported across cell membranes maintain an electrochemical imbalance that gives rise to bioelectric signaling, called membrane potential or Vmem. Vmem plays a key role in numerous inter- and intracellular functions that regulate cell behaviors like proliferation, differentiation and migration, all playing a critical role in embryonic development, healing, and regeneration. Methods With the goal of analyzing the changes in Vmem during cell proliferation and differentiation, here we used direct current electrical stimulation (EStim) to promote cell proliferation and differentiation and simultaneously tracked the corresponding changes in Vmem in adipose derived mesenchymal stem cells (AT-MSC). Results We found that EStim caused increased AT-MSC proliferation that corresponded to Vmem depolarization and increased osteogenic differentiation that corresponded to Vmem hyperpolarization. Taken together, this shows that Vmem changes associated with EStim induced cell proliferation and differentiation can be accurately tracked during these important cell functions. Using this tool to monitor Vmem changes associated with these important cell behaviors we hope to learn more about how these electrochemical cues regulate cell function with the ultimate goal of developing new EStim based treatments capable of controlling healing and regeneration

Electrical stimulation in bone tissue engineering treatments

Electrical stimulation (EStim) has been shown to promote bone healing and regeneration both in animal experiments and clinical treatments. Therefore, incorporating EStim into promising new bone tissue engineering (BTE) therapies is a logical next step. The goal of current BTE research is to develop combinations of cells, scaffolds, and chemical and physical stimuli that optimize treatment outcomes. Recent studies demonstrating EStim’s positive osteogenic effects at the cellular and molecular level provide intriguing clues to the underlying mechanisms by which it promotes bone healing. In this review, we discuss results of recent in vitro and in vivo research focused on using EStim to promote bone healing and regeneration and consider possible strategies for its application to improve outcomes in BTE treatments. Technical aspects of exposing cells and tissues to EStim in in vitro and in vivo model systems are also discussed

Role of bioelectricity during cell proliferation in different cell types

Most living organisms possess varying degrees of regenerative capabilities but how these regenerative processes are controlled is still poorly understood. Naturally occurring bioelectric voltages (like Vmem) are thought to be playing instructive role in tissue regeneration, as well as embryonic development. The different distribution of ions on the either side of the cell membrane results in intra- and extra-cellular voltage differences, known as membrane potential or Vmem. The relationship between Vmem and cell physiology is conserved in a wide range of cell types and suggests that Vmem regulation is a fundamental control mechanism for regeneration related processes e.g., proliferation and differentiation. In the present study we measured Vmem in three different cell types (human osteogenic sarcoma cell line (OSC), rat bone marrow derived mesenchymal stem cells (BM-MSC), and rat dermal fibroblasts) and characterized the relationship between their Vmem and proliferation. In order to find out if Vmem controls proliferation, or visa-versa, we blocked and then unblocked Na+/K+-exchanging ATPase using ouabain and measured the proliferation. Our results demonstrate that Vmem can be pharmacologically manipulated to control proliferation in certain cell types like BM-MSC. Taken together, it is clear that control of bioelectrical properties in non-excitable cells could prove to be potentially a useful tool in regenerative medicine efforts

Photoelastic evaluation of the effect of composite formulation on polymerization shrinkage stress

We compared polymerization stress in two commercial composites and three experimental composites made using camphorquinone (CQ) and/or phenylpropanedione (PPD) as photoinitiators. The internal surfaces of photoelastic resin discs with cylindrical cavities were roughened and treated with adhesive. Composites were divided into five groups: two commercial composites (Filtek Silorane and Filtek Z250) and three experimental composites with CQ/amine, CQ/PPD/amine, and PPD/amine. Composites were photopolymerized inside cavities, and subjected to photoelastic analysis immediately and at 24 hours and 7 days later using a plane polariscope. Stress created by Silorane (3.08 ± 0.09 MPa) was similar to that of Z250 (3.19 ± 0.13 MPa) immediately after photopolymerization (p > 0.05). After 24 hours and 7 days, Z250 (3.53 ± 0.15 and 3.69 ± 0.10 MPa, respectively) showed higher stress than Silorane (3.19 ± 0.10 and 3.16 ± 0.10 MPa, respectively). Qualitative analysis immediately after photopolymerization showed composite/CQ promoted higher stress than PPD, but stress levels at other evaluated times were statistically similar, varying between 3.45 ± 0.11 MPa and 3.92 ± 0.13 MPa. At 24 hours and 7 days, Silorane created the lowest stress. All photoinitiators created comparable tensions during polymerization

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects

Background: Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES’s demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Methods: Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. Results: We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure. Discussion: This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential

Time course of traumatic neuroma development

This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA