A biomineralização óssea é um processo complexo e multifatorial sendo um grande desafio para a ciência à compreensão dos seus mecanismos regulatórios. Este processo é mediado pela liberação de vesículas da matriz (MVs), as quais surgem das superfícies de osteoblastos e são secretadas no local específico do início da biomineralização. MVs têm a capacidade de acumular altas concentrações de íons Ca2+ e fosfato (Pi), proporcionando um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita. Especial atenção deve ser dada a duas proteínas: Anexina V (AnxA5) e Fosfatase Alcalina (TNAP). As anexinas são as proteínas mais abundantes detectadas nas MVs e responsáveis pela formação de canais de cálcio. TNAP apresenta atividade fosfomonohidrolítica, produzindo Pi a partir, principalmente, de pirofosfato (PPi) e ATP. O enfoque deste projeto foi produzir e caracterizar proteolipossomos com diferentes composições lipídicas de dipalmitoil fosfatidilcolina (DPPC) e dipalmitoil fosfatidilserina (DPPS) contendo TNAP e AnxA5, e manter a funcionalidade das proteínas após incorporação nos sistemas miméticos. Foi possível incorporar AnxA5 em DPPC-proteolipossomos (11,64 µg/mL), mas na presença de DPPS houve um aumento significativo de AnxA5 incorporada (25,79 µg/mL) a DPPC:DPPS 10%-proteolipossomos (razão molar). A presença das proteínas nos proteolipossomos compostos por DPPC e DPPC:DPPS 5, 10 e 15% (razão molar) foi confirmada por SDS-PAGE e Immunoblotting. Melhores rendimentos de incorporação das duas proteínas foram obtidos quando ambas foram incorporadas concomitantemente. DPPC-proteolipossomos e DPPC:DPPS 10%-proteoliposomos revelaram conter 75% de AnxA5 e 25% de TNAP em concentração de proteína. A presença de DPPS não afetou significativamente as porcentagens de proteínas incorporadas. Os parâmetros cinéticos da TNAP na hidrólise de diferentes substratos fisiológicos (ATP, ADP e PPi) foram determinados na presença e ausência de AnxA5, em pH fisiológico, e para os diversos sistemas lipídicos. A melhor eficiência catalítica da enzima foi obtida para sistemas contendo 10% de DPPS (razão molar) (kcat/K0.5= 183,02; 776,06 e 657,08 M-1.s-1, respectivamente). A TNAP apresentou maior especificidade para a hidrólise de PPi quando comparado com ATP e ADP. Estudos utilizando Calorimetria Diferencial de Varredura (DSC) mostraram que o aumento da concentração de DPPS em DPPC-lipossomos proporcionou um progressivo alargamento no pico de transição de fase, diminuição na t1/2 e H. A pré-transição de fase só foi detectada até a concentração de 15% de DPPS em DPPC. Para 20% de DPPS e acima, observou-se uma segregação lateral de fase com a formação de possíveis microdomínios ricos em DPPS. A interação da AnxA5 com DPPC-lipossomos e DPPC:DPPS 10%-lipossomos resultou em uma redução nos valores de H (de 8,73 para 5,68 e 8,43 para 5,37 Kcal.mol-1, respectivamente). Quando a TNAP está presente nos proteolipossomos, este efeito é ainda maior. A AnxA5 incorporada em DPPC-proteolipossomos e DPPC:DPPS 10%-proteolipossomos (razão molar) foi capazes de mediar o influxo de 45Ca2+ para dentro das vesículas (~ 800 nmol Ca2+) quando utilizados faixas de concentração de cálcio em níveis fisiológicos (~2 mM). A presença da TNAP nos proteolipossomos não afetou o influxo de Ca2+ mediado pela AnxA5. Entretanto, a presença da AnxA5 afetou significativamente os parâmetros cinéticos da TNAP para os diferentes substratos. Estudos com vesículas unilamelares gigantes (GUVs) também confirmaram a inserção funcional da AnxA5 em vesículas constituídos de dioleoil fosfatidilcolina (DOPC) e DOPC:DPPS 10% (razão molar). O principal efeito causado pela AnxA5 na morfologia das GUVs foi a perda de contraste óptico devido a formação de poros nas membranas das vesículas. Neste caso, a presença de DPPS não proporcionou mudanças significativas para a incorporação da AnxA5. TNAP quando inserida em GUVs provocou intensa flutuação e excesso de área das vesículas com formação de filamentos. A presença do DPPS provavelmente dificulta a inserção da TNAP à membrana das GUVs. Quando há microdomínios lipídicos heterogêneos na composição de GUVs compostas por DOPC:Colesterol:Esfingomielina (8:1:1) e DOPC:Colesterol:Esfingomielina:Gangliosídeo (7:1:1:1) (razão molar), a inserção da TNAP provocou uma maior segregação lateral de fase evidenciada por imagens com fluorescência. A presença da TNAP e AnxA5 em DPPC:DPPS 10%-proteolipossomos proporcionou mudanças significativas nas propriedades mecânicas visco-elásticas dos proteolipossomos detectadas por imagens de Microscopia de Força Atômica. Assim, no presente trabalho foi possível obter uma inédita metodologia para a formação de proteolipossomos contendo TNAP e AnxA5 concomitantemente, os quais apresentaram uma reconstituição funcional das proteínas, apresentando capacidade de captar Ca2+ para dentro das vesículas e habilidade de hidrolisar fosfosubstratos em sua superfície. / Bone biomineralization is a multifactorial and complex process, being a challenge for the science the understanding of their regulatory mechanisms. This process is mediated by the release of matrix vesicles (MVs), structures which arise by budding from osteoblast and chondroblast surface and are secreted in the specific site where biomineralization begins. MVs have the ability of accumulating high concentrations of Ca2+ and Pi ions, providing an adequate microenvironment for the initial formation and propagation of hydroxyapatite crystals. Two protein families present in MVs merit special attention: Annexins and Phosphatases. The annexins were the most abundant proteins detected in MVs and are responsible for the Ca2+-channels formation (especially AnxA5). Tissue-nonspecific alkaline phosphatase (TNAP) exhibits phosphomonohydrolytic activity, producing Pi mainly from PPi and ATP. Such proteins regulate the formation of calcium phosphate crystals, acting directly in the bone mineralization process. The goal of this project was to produce and characterize proteoliposomes with different lipid compositions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) harboring TNAP and AnxA5, keeping the functions of both proteins after their incorporation into the mimetic systems. AnxA5 was able to incorporate into DPPC-proteoliposomes (11.64 µg/mL), but the presence of DPPS increased significantly the AnxA5 incorporation (25.79 µg/mL) into DPPC:DPPS 10%-proteoliposomes. The presence of both proteins into DPPC and DPPC:DPPS 5, 10 and 15% (molar ratios) proteoliposomes was confirmed by SDS-PAGE and Immunoblotting analysis. Better yield of TNAP and AnxA5 incorporation was observed when both proteins were reconstituted simultaneously. DPPC-proteoliposomes and DPPC:DPPS 10%-proteoliposomes (molar ratio) incorporated about 75% of AnxA5 and 25% of TNAP (protein concentration). DPPS presence did not affect significantly the yield of incorporation of both proteins. The kinetic parameters for the hydrolysis of different physiological substrates (ATP, ADP and PPi) by TNAP were determined in the presence and absence of AnxA5, at physiological pH, for the different systems. The best catalytic efficiencies were achieved with proteoliposomes containing DPPS 10% (molar ratio) (kcat/K0.5= 183.02; 776.06 and 657.08 M-1.s-1 for ATP, ADP and PPi, respectively), condition that also favored PPi hydrolysis by TNAP when compared to ATP and ADP hydrolysis. Studies by Differential Scanning Calorimetry (DSC) showed that the increasing DPPS concentrations in the DPPC-liposomes resulted in a progressive broadening of the phase transition peaks and decreased t1/2 and H values. The pre-transition was detected only in concentrations up to DPPS 15% in DPPC. Phase lateral segregation can be observed for DPPS 20% and above, suggesting the formation of DPPS-rich microdomains. The interaction of AnxA5 with DPPC and DPPC:DPPS 10%-liposomes resulted in a decrease of H values (from 8.73 to 5.68 and from 8.43 to 5.37 Kcal.mol-1, respectively). When TNAP was present in the proteoliposomes, this effect was even greater. AnxA5 incorporated into DPPC and DPPC:DPPS 10%-proteoliposomes (molar ratio) was able to mediate 45Ca2+-influx (~ 800 nmol Ca2+) into the vesicles at physiological Ca2+-concentrations (~ 2 mM), and this process was not affected by the presence of TNAP in the systems. However, AnxA5 affected significantly the hydrolysis of substrates by TNAP. Studies with Giant Unilamellar Vesicles (GUVs) also confirmed the functional reconstitution of AnxA5 in dioleoylphosphocholine (DOPC) and DOPC:DPPS 10% (molar ratio) vesicles. The main effect caused by AnxA5 in the GUVs morphology was the formation of pores in the vesicles membrane. In this case, DPPS presence did not affect the AnxA5 incorporation. The presence of TNAP in GUVs caused a several fluctuation, indicating that the vesicles acquired an excess of area and undergoes sequential budding transitions. It is suggested that the presence of DPPS makes the TNAP insertion into the GUVs membrane difficult. With the presence of heterogeneous lipid microdomains in GUVs composed of DOPC, Cholesterol (Chol), Sphingomyelin (SM) and Ganglioside (GM1) in the proportions DOPC:Chol:SM 8:1:1 and DOPC:Chol:SM:GM1 7:1:1:1 (molar ratios), the TNAP insertion caused a greater phase lateral segregation, evidenced by fluorescence analysis. Atomic Force Microscopy (AFM) analysis indicated that the presence of both proteins into DPPC:DPPS 10%-proteoliposomes (molar ratio) caused significant changes in the visco-elastic mechanical properties of the vesicles. In conclusion, the present work describes the synthesis of proteoliposomes harboring TNAP and AnxA5 concomitantly, with the functional reconstitution of both proteins, with the ability to transport Ca2+ into the vesicles and hydrolyze phosphosubstrates on their surface.

Background: Diabetic nephropathy (DN) is defined as a rise in urinary albumin excretion rate, often associated with an increase in blood pressure. It is the leading cause of end-stage renal disease and carries an increased risk for cardiovascular mortality. Microalbuminuria is the first sign of diabetic renal impairment or incipient nephropathy and is generally considered the best noninvasive predictor for the development of DN. Urinary markers of tubular damage are mainly composed of enzymes or plasma proteins of low molecular weight that are normally freely filtered by the glomerulus, and these biomarkers can be useful for diagnosis of DN. Thus, the aim of this study was to test the diagnostic accuracy of the urinary excretion of gama-glutamyltransferase (GGT) and alkaline phosphatase (ALP) for diagnosis of diabetic nephropathy (DN). Methods: Fasting glucose, fructosamine, serum creatinine, glomerular filtration rate (GFR), serum uric acid, serum albumin, and urinary albumin, creatinine, GGT and ALP were assessed in 74 type 2 diabetic patients without nephropathy and 38 type 2 diabetic patients with nephropathy. Results: Urinary GGT and ALP were threefold higher in type 2 diabetic patients with nephropathy. Significant correlations were observed between urinary albumin and GGT (r=0.439, P<0.001) and urinary albumin and ALP (r=0.305, P<0.01). Areas under the curve for GGT and ALP were 0.7696 (P<0.001) and 0.7233 (P<0.001), respectively. At a cut-off value of 72 U/g creatinine, GGT demonstrated a sensitivity of 96.0% and a specificity of 52.6%. At a cut-off value of 20 U/g creatinine, ALP demonstrated a sensitivity and specificity of 83.8% and 36.8%, respectively. Conclusions: Urinary GGT and ALP have potential value in the diagnosis of nephropathy in type 2 diabetic patients, but GGT has a slightly higher ability to discriminate nephropathy than ALP. / Introdução: A nefropatia diabética (ND) é definida como um aumento na taxa de excreção urinária de albumina, sendo esta frequentemente associada ao aumento da pressão sanguínea. A ND é a principal causa de doença renal em estágio final, estando também associada ao aumento do risco de mortalidade cardiovascular. A microalbuminúria é o primeiro sinal de dano renal ou nefropatia incipiente, sendo geralmente considerado o melhor preditor não-invasivo para o desenvolvimento de ND. Os marcadores urinários de dano tubular são principalmente compostos por enzimas ou proteínas plasmáticas de baixo peso molecular que são normalmente filtradas pelo glomérulo, sendo que estes biomarcadores podem ser úteis para o diagnóstico da ND. Assim, o objetivo deste estudo foi avaliar as características diagnósticas dos níveis urinários de gama-glutamiltransferase (GGT) e fosfatase alcalina (FAL) para o diagnóstico da ND. Métodos: Glicemia de jejum, frutosamina, creatinina sérica, taxa de filtração glomerular (TFG), ácido úrico, albumina, além dos níveis urinários de albumina, GGT e FAL foram mensurados em 74 pacientes diabéticos tipo 2 sem nefropatia e 38 pacientes diabéticos tipo 2 com nefropatia. Resultados: As enzimas GGT e FAL, mensuradas em amostras de urina, foram três vezes mais elevadas nos pacientes diabéticos tipo 2 com nefropatia. Foram observadas correlações significativas entre a albumina urinária e GGT (r=0,439, P<0,001) e albumina urinária e FAL (r=0,305, P<0,01). As áreas sob a curva para GGT e FAL foram 0,7696 (P<0,001) e 0,7233 (P<0,001), respectivamente. Ao considerar o ponto de corte de 72 U/g de creatinina, a GGT demonstrou uma sensibilidade de 96,0% e especificidade de 52,6%. Considerando o ponto de corte de 20 U/g de creatinina, a FAL demonstrou uma sensibilidade e especificidade de 83,8% e 36,8%, respectivamente. Conclusões: As enzimas GGT e FAL mensuradas em amostras de urina apresentaram potencial valor para o diagnóstico de nefropatia em pacientes com diabetes tipo 2, mas a GGT apresentou uma habilidade discretamente superior à FAL nesta diferenciação.

Este trabalho teve como objetivo avaliar o efeito da aplicação do ultra-som (US) de 1 MHz comparando as freqüências de repetição de pulso de 100 Hz e 16 Hz na recuperação da fratura de fíbula, por análise morfológica e bioquímica, entre animais tratados e não-tratados. Foram utilizados 60 ratos machos albinos Wistar divididos em 4 grupos experimentais: referência, controle, tratados com US com freqüência de repetição de pulso de 16 Hz ou 100 Hz. Os animais dos grupos tratado e controle foram submetidos a uma fratura com perda óssea da fíbula direita. O tratamento teve início 24 h após a fratura, durante 5 dias por semana, com intensidade de 0,5 W/'CM POT.2', modo pulsado 1/5, freqüência de repetição de pulso a 100 Hz ou 16 Hz, por 3 min/dia. No 7°, 14º e 21º dia após a indução da fratura foi realizada coleta do sangue através de punção cardíaca para quantificação dos níveis de fosfatase alcalina e cálcio sérico e em seguida os animais foram submetidos à eutanásia e a fíbula foi removida para análise histológica. Foram determinadas a densidade de matriz óssea, condrócitos e fibroblastos. Os níveis de fosfatase alcalina e cálcio foram significativamente (P < '10 POT.-7') diferentes nos grupos experimentais. A densidade de matriz óssea, condrócitos e fibroblastos também foram significativamente diferentes entre os grupos experimentais. O tratamento com US acelerou a regeneração óssea e modulou os níveis sanguíneos de fosfatase alcalina e cálcio, sendo que, o US pulsado com freqüência de repetição de pulso a 100 Hz e freqüência de base da 1 MHz demonstrou ser mais eficaz / The aim of this work was to evaluate the effect of 1 MHz ultrasound application, comparing the 100 Hz and 16 Hz pulse repetition frequency in the recovering of fibula fracture, using morphological and biochemical analysis, between treated and non-treated animals. We used 60 male albino Wistar rats divided into 4 experimental groups: reference; control; treated with 100 Hz or 16 Hz pulse repetition frequency ultrasound. The treatment began 24 h after fracturing, lasted for 5 days per week, with 0.5 W/'CM POT. 2', pulsed mode 1/5, pulse repetition frequency of 100 Hz or 16 Hz, for 3 min a day. In the 7th, 14th and 21st after fracture induction, blood was collected through cardiac punction to alkaline phosphatase and calcium levels quantification and following that, the animals were euthanised and the fibula was removed to histological analysis. The bone matrix, condrocytes and fibroblasts densityes were determined. The levels of alkaline phosphatase and calcium were significantly (P < '10 POT.-7') different among the experimental group. The bone matrix, condrocytes and fibroblasts densities were significantly different among experimental groups. The US treatment accelerated the bone regeneration and modulated the alkaline phosphatase and calcium blood levels, being the pulsed US with 100 Hz pulse repetition frequency and base current 1 MHz the most efficient

V této diplomové práci je popsána přechodná transfekce buněk 293 HEK adaptovaných na růst při suspenzní kultivaci bez přítomnosti séra za použití polyethyleniminů (PEI). Buňky byly transfekovány plasmidem pcDNA5/SEAP, který exprimuje sekretovanou formu lidské placentální alkalické fosfatázy. K porovnání účinnosti jednotlivých transfekcí byla měřena koncentrace exprimované fosfatázy v buněčném supernatantu. Cílem této práce bylo optimalizovat různé faktory ovlivňující účinnost transfekcí s důrazem na nalezení optimálního poměru DNA:PEI.

Objective: To determine the ameliorative effect of the ethanolic extract of Chuquiraga spinosa (ChS) on 7,12-Dimethylbenz[a]anthracene (DMBA)-induced breast cancer in rats. Methods: 36 female Holztman rats were divided into 6 groups. I) The negative control group received physiological saline (PS). II) ChS-200 group received 200 mg/kg of ChS. III) DMBA group was induced with DMBA (20 mg/Kg) dissolved in PS and administrated orally for 15 weeks. IV) DMBA + ChS-50 group, V) DMBA + ChS-250 group, and VI) DMBA + ChS-500 group, which received the extract orally for 15 weeks after DMBA induction. All data were expressed as mean and standard deviation. One-way analysis of variance (ANOVA) followed by Dunnet test was carried out to compare the mean value of different groups Histopathological analysis was evaluated by using Image J software. Results: Hematology showed that the triglyceride level was significantly lowered (P< 0.01) and high-density lipoprotein (HDL) level was significantly increased (P <0.01) in groups III, IV and V. Also, ChS extract significantly lowered the C reactive protein (CRP) level (P <0.01) and malondialdehyde level (P<0.05). There was a significant decrease in the frequency of DMBA-induced micronucleated polychromatic erythrocyte (P<0.01). Conclusions: Chuquiraga spinosa showed an ameliorative effect on DMBA-induced breast cancer in rats as well as antioxidant, antitumor and antigenotoxic properties. / Revisión por pares

Silver nanoparticles (AgNPs) are expected to enter aquatic systems, but there are limited data on how they might affect microbial communities in pathogen impaired streams. We examined microbial community responses to citrate-AgNP (10.9 ± 0.7 nm) and polyvinylpyrrolidone (PVP)-AgNP (11.0 ± 0.7 nm) based on microbial concentration and enzyme activity in sediment from a pathogen impaired stream. Addition of each nanoparticle to sediment caused at least a 69% decrease in microbial concentration (1,264 ± 93.6 to 127 ± 29.5 CFU/g) and a 62% decrease in β-glucosidase activity (11.7 ± 2.1 to 1.3 ± 0.3 μg/g/h). Each AgNP reduced alkaline phosphatase activity but their effects were not statistically significant. Sediment exposed to 0.108 mg Ag/kg of AgNO3 resulted in a 92% decrease in microbial concentration and a reduced enzyme activity which was not statistically significant. Measured total silver in sediments treated with AgNPs which exhibited significant inhibition effects on the microbial community ranged from 0.19 ± 0.02 to 0.39 ± 0.13 mg Ag/kg. These concentrations tested in this study are much lower than the expected concentrations (2-14 mg Ag/kg) in freshwater sediments. The results of this study demonstrate that AgNPs can alter microbial community activity and population size, which may lead to false negative fecal indicator bacteria detection and enumeration using methods that rely on β-glucosidase activity. We conclude that the presence of AgNPs in impaired streams and recreational waters can influence pathogen detection methods, potentially affecting public health risk estimates.

Indiana University School of Dentistry / Introduction: The goal of nonsurgical root canal therapy is to reduce the bacterial load within an infected root canal system, and the subsequent objective is to prevent or treat apical periodontitis. Clinical studies have shown more expedient healing of apical periodontitis treated with electromagnetic wave stimulation (EMS) as compared to apical periodontitis not treated with EMS. Stimulation of osteoblasts and growth factors has been shown when EMS was applied to rat calvaria, resulting in increased bone healing. Objective: The purpose of this vitro study was to evaluate the effects of EMS on the proliferation and differentiation of osteoblasts. Using primary neonatal calvaria osteoblast-lineage cells, the effects of different EMS regimens on proliferation, alkaline phosphatase (ALP) activity, and mineral deposition were determined. Materials and Methods: EMS regimen included currents of 0mA, 0.1mA, 1mA, and 10mA delivered for five consecutive 1s pulses per day for one, two, and three days. Cell proliferation was assayed after 1 or 2 days using an MTS assay. Alkaline phosphatase activity and mineral deposition were assayed after culturing the cells in osteogenic media containing ascorbic acid and -glycerol phosphate for 7 days. Comparisons were performed using analysis of variance, with a 5% significance level. Results: There was no statistically significant differences noted in MTS proliferation and mineral deposition between the experiment EMS treatment groups of 0.1, 1.0, and 10.0 mA compared to the control group of 0 mA current on calvaria-derived osteoblast. While there were no statistically significant differences noted in ALP activity in the 0.1, and 1.0 mA EMS groups, compared to 0 mA control, alkaline phosphatase activity was significantly increased in the 10 mA EMS group. Conclusion: There was no significant differences in MTS proliferation and mineral deposition of the EMS group compared to the control group. However, 10 mA EMS favored increased ALP activity suggesting EMS can promote matrix maturation by osteoblasts. Additional in vitro experimental studies, including different stem cell populations, culture duration and EMS treatment regimens are needed to understand the mechanism of action of EMS for future applications in regenerative endodontics.

Thesis (M.S.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), February 2001. / Includes bibliographical references (leaves 144-149). / A bi-weekly seasonal study was conducted in Ashumet Pond (Cape Cod, Massachusetts). The Redfield Ratio (106C:16N:1P) and alkaline phosphatase activity (APA) were utilized in tandem as nutrient deficiency indicators (NDIs) for phytoplankton. The study objective was to evaluate the limiting nutrient status of the pond throughout the growing season. The development of a high throughput method for fluorometrically measuring APA allowed for a large quantity of pond-water samples to be analyzed. The new method utilized a cytofluor, a fluorescence multi-well plate reader, which increased sample throughput by 75% compared to a standard filter fluorometer method. The detection limit, capability to measure APA at different time intervals, and performance at sea were tested. APA measurements made using the cytofluor were comparable to those made using a standard filter fluorometer, thus indicating that the cytofluor is a suitable and preferred replacement to the fluorometer for APA measurements. The presence of alkaline phosphatase, an inducible phospho-hydrolytic enzyme, is commonly used as an NDI diagnostic for phosphate limitation. A nutrient enrichment incubation re-affirmed the use of APA as a robust indicator of phosphate limitation in phytoplankton. APA data indicate that the system experienced episodic periods of phosphate-deficiency, implying that the limiting nutrient regime was not static, but was changeable throughout the growing season. Seasonal trends in dissolved N:P and particulate C:P ratios often contradict the APA results, however, suggesting that the Redfield Ratio is an unreliable indicator of the overall nutrient limitation regime of the pond. The observed discrepancies between C:N:P and APA can be reconciled by taking into account seasonal changes in species composition, which played an important role in driving seasonal APA trends. / by Christie Lynn Haupert. / M.S.

Strontium (Sr2+) är det aktiva ämnet i läkemedel som används för att reducera frakturrisken hos patienter som lider av osteoporos. På senare tid har Sr2+ kombinerats med olika biomaterial i syfte att gynna benbildning, emellertid är ämnet vagt studerat avseende effekten på parodontala vävnader. Trots omfattande användning, är verkningsmekanismer av Sr2+ inte helt klarlagda. Denna studie syftar därmed till att utvärdera effekten av Sr2+ på humana parodontalligament-celler (PDL-celler) och osteoblaster avseende proliferation och osteogen aktivitet. Odlade humana PDL celler och osteoblastcellinjerna MG63 och hFOB 1.19 behandlades med SrCl2 (0,1-10 mM) eller vehikel under 72 h. Manuell cellräkning utfördes med en Bürker kammare. Det totala proteininnehållet fastställdes med en kolorimetrisk mätning genom användning av Bio-Rad proteinanalys. Aktiviteten av alkaliskt fosfatas bestämdes enzymatiskt och normaliserades till totalt proteininnehåll. SrCl2 hade ingen signifikant effekt på PDL celler (p> 0.05), däremot observerades en tendens till inducerade osteoblastiska egenskaper. I motsats till det, ökade 5 mM SrCl2 den totala proteinhalten i MG63 celler med 37% (p <0,01) jämfört med vehikel, medan en lägre koncentration (0,1 mM) inte hade någon påverkan. 5 mM SrCl2 ökade MG63 cellantalet med 38% (p <0,001), medan en högre koncentration (10 mM) inte uppvisade en signifikant ökad effekt jämfört med 5 mM (+54%, jämfört med vehikel, p<0.05). Resultaten visar att 72 h administrering av ≥ 5 mM SrCl2 ger en pro-proliferativ effekt på humana osteoblastliknande MG63 celler och uppvisar en tendens till att stimulera osteogena egenskaper hos primära humana PDL-celler. / Strontium (Sr2+) is the active substance of pharmaceuticals used for reducing fracture risk in osteoporotic patients. Lately, Sr2+ is combined with biomaterials to enhance osteogenesis, which has been vaguely studied considering periodontal tissue regeneration. Despite extensive use, the mechanisms of action of Sr2+ are not fully understood. The present study assesses the impact of Sr2+ on primary human periodontal ligament cells (PDL cells) and human osteoblasts in regard to proliferation and pro-osteogenic activity. Cultured human PDL cells and osteoblast cell lines MG63 and hFOB 1.19 were treated with SrCl2 (0.1-10 mM) or vehicle for 72 h. Cells were counted manually using a Bürker chamber. Total protein content was determined by colorimetric analysis using Bio-Rad protein assay. Alkaline phosphatase activity was determined enzymatically and normalized to total protein content. SrCl2 had no significant effect on PDL cells (p>0.05), but a tendency towards induced osteogenic characteristics was observed. In contrast, 5 mM SrCl2 enhanced total MG63 cell protein content by 37% (p<0.01), compared to vehicle, whereas a lower concentration (0.1 mM) did not. 5 mM SrCl2 increased MG63 cell number by 38% (p<0.001), while a higher concentration (10 mM) did not have a significant additional effect over the 5 mM (+54%, compared to vehicle, p<0.05). The results demonstrate that 72 h administration of ≥ 5 mM SrCl2 exerts a pro-proliferative effect on human osteoblast-like MG63 cells and display a tendency to induce osteogenic characteristics in primary human PDL cells.

La grossesse est une période de majoration du risque vasculaire, participant à une morbi-mortalité maternelle et fœtale pouvant justifier des mesures de prévention primaire et secondaire. Notre travail évalue l'impact de certains déterminants et l'apport de nouveaux acteurs moléculaires impliqués dans la dysfonction vasculo-placentaire. Le but ultime étant d'optimiser les prises en charge et de développer de nouvelles stratégies thérapeutiques. Nous avons étudié les complications vasculaires placentaires associées à des marqueurs biologiques connus : mutation du facteur V Leiden, mutation du gène de la prothrombine et marqueurs conventionnels du syndrome des anticorps anti-phospholipides (SAPL). Nos résultats montrent que les femmes à antécédents de fausses couches précoces répétitives et porteuses, soit du polymorphisme du facteur V, soit du polymorphisme du facteur II, soit d'un SAPL (traité par héparine et aspirine faible dose), ont un risque élevé de fausse couche tardive lors d'une nouvelle grossesse. Les femmes à antécédent de fausse couche tardive et porteuses des mêmes particularités biologiques, traitées pendant leur grossesse selon les recommandations (héparine pour l'anomalie du facteur V ou II, héparine plus aspirine faible dose pour le SAPL), ont un risque diminué de récidive de perte fœtale tardive mais demeurent, dans le groupe SAPL, fréquemment exposées aux complications tardives de la grossesse malgré la prophylaxie antithrombotique. Nous avons évalué l'apport de nouveaux marqueurs de la dysfonction vasculaire placentaire. Nous montrons que le polymorphisme Ile89Leu du gène de la phosphatase alcaline placentaire (PLAP), enzyme exprimée par les cellules du syncytiotrophoblaste -polymorphisme associé à une augmentation de l'activité PLAP-, exerce un effet protecteur sur l'échec d'implantation et la survenue d'une fausse couche primaire. Un facteur angiogénique (brevet en cours) a également été étudié (génétique, dosage plasmatique, fécondation in vitro) et nous montrons une association de ce marqueur avec les échecs d'implantation et les fausses couches idiopathiques. L'ensemble de ces travaux suggère que ces nouveaux marqueurs moléculaires pourraient contribuer au diagnostic des complications vasculaires de la grossesse et fournir des biomarqueurs d'implantation embryonnaire et/ou de développement placentaire. Ils pourraient suggérer de nouvelles cibles et stratégies thérapeutiques, répondant aux limites des traitements disponibles. / Vascular risk increases during pregnancy, contributing to maternal and foetal morbidity and mortality, and potentially justifying primary and secondary preventive measures. Our work evaluates the impact of some determinants and the contribution of new molecular actors implicated in placental vascular dysfunction. The ultimate aim is to optimize management and to develop new therapeutic strategies. We studied the placental vascular complications associated with known biological markers: the factor V Leiden or prothrombin polymorphisms, and conventional markers of the antiphospholipid antibody syndrome (APS). Women with previous recurrent abortions carrying polymorphisms of either factor V or factor II, or with APS (treated with heparin and low-dose aspirin), had an increased risk of foetal loss during subsequent pregnancies. Women with a previous foetal loss carrying these biological markers, treated according to recommendations during a new pregnancy (heparin for the polymorphisms, heparin plus low-dose aspirin for APS) had a lower risk of foetal loss, but an excess of late complications was observed in the APS group despite prophylaxis. We evaluated the contribution of new markers of placental vascular dysfunction. The placental alkaline phosphatase enzyme (PLAP) is synthesized and expressed by syncytiotrophoblastic cells. We found that the Ile89Leu polymorphism of the PLAP gene provides protection against implantation failure and primary miscarriage and induces increased PLAP activity. We also studied (genetics, plasma determinations, in vitro fertilisation) an angiogenic factor (patent application underway), which we showed to be associated with idiopathic implantation failure and miscarriage. These findings suggest that these molecular actors are potentially useful for the diagnosis of placenta-mediated pregnancy complications and may be relevant biomarkers of embryo implantation and/or placental development. They may indicate new targets for relevant therapeutic strategies, potentially overcoming the limitations of the currently available treatments.