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The mechanisms that mediate acute exacerbations in chronic inflammatory diseases such as periodontitis are not understood. IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions. We investigated the role of fibroblast-mast cell interactions on short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC-1). After co-culture, the concentration of IL-8 was measured by ELISA. HMC co-cultured with fibroblasts increased IL-8 secretion by >6-fold, which required intercellular contact and was blocked by the gap junction inhibitor BGA. Thapsigargin-induced elevations of intracellular calcium increased IL-8 levels by 15-fold. Chemotaxis of human neutrophils was significantly enhanced in response to conditioned medium from co-cultures. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on β1 integrins. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, thereby enhancing neutrophil chemotaxis and possibly the perpetuation of the inflammatory response.

The mechanisms that mediate acute exacerbations in chronic inflammatory diseases such as periodontitis are not understood. IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions. We investigated the role of fibroblast-mast cell interactions on short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC-1). After co-culture, the concentration of IL-8 was measured by ELISA. HMC co-cultured with fibroblasts increased IL-8 secretion by >6-fold, which required intercellular contact and was blocked by the gap junction inhibitor BGA. Thapsigargin-induced elevations of intracellular calcium increased IL-8 levels by 15-fold. Chemotaxis of human neutrophils was significantly enhanced in response to conditioned medium from co-cultures. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on β1 integrins. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, thereby enhancing neutrophil chemotaxis and possibly the perpetuation of the inflammatory response.

Os componentes protéticos que fazem a união entre implante e prótese são importantes para a manutenção da saúde peri-implantar sendo responsáveis pela vedação permucosa e impedindo a penetração de agentes químicos ou bacterianos desencadeadores de processos destrutivos nas estruturas ao redor do implante. Assim faz-se importante o desenvolvimento de técnicas que preservando as características de citocompatibilidade dos componentes protéticos que incorporem a sua estrutura, agentes antibacterianos que assegurem a saúde peri-implantar e que não permitam o desenvolvimento de resistência dos micro-organismos sendo ainda inócuos aos tecidos circunjacentes e ao organismo como um todo. Este trabalho teve o objetivo de testar a citocompatibilidade de discos de titânio com uma cobertura revestida usando nanotecnologia de prata em cultura de fibroblastos gengivais humanos. Discos de titânio de 5,9 mm foram preparados para o ensaio, sendo compostos por disco controle ST (N=1) sem nenhuma espécie de tratamento e disco com tratamento CT (N=3) com 3 camadas de cobertura com filme carregado com partículas de prata. O aditivo antimicrobiano à base de prata foi obtido pelo método wet chemistry por impregnação de metal homogeneamente distribuída sobre a superfície do disco. Fragmentos de gengiva foram cortados e colocados em placas de petri com meio de cultura Eagle modificado por Dulbecco. Com aproximadamente 15 dias foram retirados os fragmentos de gengiva, seguido da tripsinização das células para sua expansão. O meio de cultura foi trocado a cada 2-3 dias e a progressão da cultura foi avaliada por microscópio de fase. Os FGHs (fibroblastos de gengiva humana) na terceira passagem (p3) foram contados em um hemocitômetro e plaqueados em placas de 96 e 24 poços. As análises foram feitas com 24, 48 e 72 horas após o plaqueamento. Após a solubilização, as soluções foram transferidas para uma nova placa de 96 poços, e efetuada a leitura. A citotoxicidade dos materiais foi avaliada por espectrofotometria no aparelho Leitor de Elisa, com comprimento de onda de 590 nm. As amostras foram metalizadas e o MEV foi usado para avaliar a morfologia das células. O comportamento da cultura de fibroblastos sobre os discos de titânio em um período de 24 horas mostrou-se mais favorável na superfície ST mesmo após 48 horas quando CT apresentou valores inferiores ao ST, porém estatisticamente não significantes ao tempo de armazenamento. A avaliação após 72 horas indica que CT apresentou queda no desenvolvimento da cultura de fibroblastos em relação a ST sendo o resultado estatisticamente significante o que sugere menor característica de citocompatibilidade do material utilizado no tratamento de superfície que se potencializa com o aumento do tempo de exposição visto que quando comparado aos demais espécimes com meio de cultura com nutrientes (CC), controle sobre a superfície da placa em H2O sem nutrientes (H2O) e controle sobre a superfície da placa com meio de cultura com nutrientes acrescido de fenol a 1 % (CM) o espécime CT somente teve valores absolutos superiores para presença das células quando comparado ao espécime CM. Nossos dados indicaram a necessidade de se promover aperfeiçoamento no complexo estrutural do material de cobertura dos discos de forma que se tornem mais citocompatíveis. / The prosthetic components that make the bond between implant and prosthesis are important for maintaining healthy peri-implant permucosal being responsible for sealing and preventing penetratíon of chemicals or bacterial triggers of destructive processes in the structures around the implant. So it is important to develop techniques that preserve the characteristics of cell compatibility of prosthetic components that incorporate its structure, antibacterial agents to ensure the peri-implant health and not allow the development of resistance of micro-organisms are harmless to the tissues even and surrounding the body as a whole. This study aimed to test the cell compatibility of titanium disks coated with a covering of silver using nanotechnology in cultured human gingival fibroblasts. Titanium disks of 5.9 mm were prepared for trial, being composed of hard control ST (N = 1) without any kind of treatment and treatment disc with CT (n = 3) with 3 layers of film cover loaded with silver particles. The additive antimicrobial silver-based was obtained by wet chemistry by impregnatíon of metal homogeneously distributed over the disk surface. Gingival fragments were distributed in petri dishes with culture medium Dulbecco\'s modified Eagle. With approximately 15 days they were removed, followed by trypsinizatíon of the cells for their expansíon. The culture medium were changed every 2-3 days of culture and progressíon was evaluated by phase microscopy. The FGHs (human gingival fibroblasts) in the third passage (p3) were counted in a hemocytometer and plated in a 96 and 24 wells. Analyses from 24, 48 and 72 hours after plating were made. After solubilizatíon, the solutíons were transferred to a new 96-well plate, and made reading. The cytotoxicity of the materials were evaluated by spectrophotometric ELISA reader device with a wavelength of 590 nm. The samples were metallized and SEM was used to assess cell morphology. The behavior of cultured fibroblasts on titanium disks in a period of 24 hours was more favorable on the surface of ST even after 48 hours when the CT were lower than the ST but not statistically significant at the time of storage. The evaluatíon afetr 72 hours indicates that the CT fell in the development of cultured fibroblasts in relatíon to the ST and the result was statistically significant which suggests lower cell compatibility characteristic of the material used in surface treatment that enhances with increasing time exposure as compared to other disk with culture medium with nutrients (CC), control over the surface of the plate without nutrients H2O and control over the surface of the plate with culture medium with nutrients plus phenol 1 % (CM) the CT only had higher absolute values for the presence of cells when compared to CM. Our data indicated the need to promote improvement in the structural complex of the covering material so that the disks become more compatibility.

"Mechanical loads play a pivotal role in the growth, maintenance, remodeling, and disease onset in connective tissues. Harnessing the relationship between mechanical signals and how cells remodel their surrounding extracellular matrix would provide new insights into the fundamental processes of wound healing and fibrosis and also assist in the creation of custom-tailored tissue equivalents for use in regenerative medicine. In 3D tissue models, uniaxial cyclic stretch has been shown to stimulate the synthesis and crosslinking of collagen while increasing the matrix density, fiber alignment, stiffness, and tensile strength in the direction of principal stretch. Unfortunately, the profound fiber realignment in these systems render it difficult to differentiate between passive effects and cell-mediated remodeling. Further, these previous studies generally focus on a single level of stretch magnitude and duration, and they also investigate matrix remodeling under a homogeneous strain conditions. Therefore, these studies are not sufficient to establish key information regarding stretch-dependent remodeling for use in tissue engineering and also do not simulate the complex mechanical environment of connective tissue. We first developed a novel in vitro model system using equibiaxial stretch on fibrin gels (early models of wound healing) that enabled the isolation of mechanical effects on cell-mediated matrix remodeling. Using this system we demonstrated that in the absence of in-plane alignment, stretch stimulates fibroblasts to produce a stronger tissue by synthesizing collagen and condensing their surrounding matrix. We then developed dose-response curves for multiple aspects of tissue remodeling as a function of stretch magnitude and duration (intermittent versus continuous stretch). Our results indicate that both the magnitude and the duration per day of stretch are important factors in mechanically induced cell activity, as evidenced by dose-dependent responses of several remodeling metrics in response to these two parameters (UTS, matrix stiffness, collagen content, cell number). In addition, we found that cellularity, collagen content, and resistance to tension increased when the tissues were mechanically loaded intermittently as opposed to continuously. Finally, we developed a novel model system that produces non-homogeneous strain distribution, allowing for the simultaneous study of strain gradients, strain anisotropy, and strain magnitude in 2D and 3D. Establishing a system that produces complex strain distributions provides a more accurate model of the mechanical conditions found in connective tissue, and also allows for the investigation of cellular adaptations to a changing mechanical environment. "

"In this thesis, a cell traction force microscopy method is developed for measuring traction forces of connective tissue cells. This method includes an improved methodology in traction force microscopy of live cells cultured on an elastic substrate. Tissue cells, such as skin and muscle cells respond to the mechanical stimuli of their microenvironment by adhering to their substrate and exerting forces on the proteins of the extracellular matrix (ECM). These forces are called cell traction forces. Fibroblasts are grown on polyacrylamide (PA) gels embedded with fluorescent beads and coated with different types of ECM ligands. Traction forces of NIH 3T3 fibroblasts are calculated from the measured deformations of PA gels by using a 3-D finite element method. The advantages of this method compared to the traditional methods of cell traction force microscopy (CTFM) are that this method takes into account the finite thickness of the substrate by applying a 3-D FEM analysis to reduce the errors of using an infinite half space approximation for a substrate with a finite thickness and that it uses a novel method for embedding the substrate with fluorescent markers that decreases the measurement uncertainties. In our approach fluorescent beads were embedded on the top of substrate instead of getting mixed with the gel. This decreases the effect of out-of-focus fluorescent beads on the measured deformation fields which enhances the accuracy of cell traction force measurements."

Kok Dick Shun , Louis. / Thesis (Ph.D.)--Chinese University of Honbg Kong, 1997. / Includes bibliographical references (p. i-xiii). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

In der vorliegenden Arbeit konnte die essentielle Beteiligung von Caspasen im Zelltodmodell der AKR 2B-Mausfibroblasten nachgewiesen und ihre Aktivitäten charakterisiert werden. AKR 2B-Mausfibroblasten stellen eine subklonierte und gut charakterisierte Zellinie dar, in der durch Entzug des Serums der Zelltod induziert wird. Während des Zelltods sterben innerhalb von 6h etwa 50 Prozent einer dichtearretierten Kultur. Die überlebenden Zellen bleiben von diesem Mangelzustand für mindestens weitere 48h unbeeinflußt, benötigen aber zum Überleben eine Proteinneusynthese. Der Zelltod zeigt für eine Apoptose typische morphologische Veränderungen der Zelle, obwohl apoptotische Charakteristika, wie die oligonukleosomale Fragmentierung der DNA oder die Aufnahme der zerfallenen Zelle durch benachbarte Zellen, ausbleiben. Mittels unterschiedlicher Methoden konnte die Expression von mRNA aller für den apoptotischen Prozeß bekannten relevanten Caspasen in den AKR 2B-Mausfibroblasten nachgewiesen werden. Die Caspasen-1, -2, -3, -6 und -9 liegen in ihrer zymogenen Form konstitutiv in den Zellen vor. Mit Ausnahme der Caspase-9 konnte die durch Serumentzug induzierte Spaltung dieser Caspasen in Untereinheiten und somit ihre Aktivierung nicht detektiert werden. Die wesentliche Beteiligung dieser Cystein-Proteasen wurde jedoch durch den protektiven Effekt spezifischer Inhibitoren und den Nachweis ihrer spezifischen Aktivität bestimmt. Die Charakterisierung dieser enzymatischen Aktivitäten lieferte Hinweise zur Identität der aktivierten Caspasen. Neben einer konstitutiven VEIDase- und IETDase-Aktivität wird 3h nach Entzug des Serums eine DEVDase maximal aktiviert. Das Gemisch an Caspase-Aktivitäten wird durch eine DEVDase dominiert. Diese Aktivität wird zum größten Teil durch nur ein Enzym gestellt, wie durch eine Affinitätsmarkierung und 2D-Gelelektrophorese gezeigt wurde. KM- und Ki-Wert-Bestimmungen der DEVDase deuten darauf hin, daß dieses Enzym typische Effektoreigenschaften, wie die der Caspase-3, besitzt. Daneben werden Lamine während des Zelltods in AKR 2B-Mausfibroblasten abgebaut, was auf eine aktivierte Caspase-6 hinweist. Die enzymatischen Charakteristika dieser Protease weichen aber von den in AKR 2B-Mausfibroblasten festgestellten Werten deutlich ab, so daß man ihr nur eine untergeordnete Rolle im Caspasen-Gemisch zuordnen kann. Eine mehrfach chromatographische Reinigung der Aktivität bietet die beste Grundlage für eine anschließende Sequenzierung der Caspase mit dem Ziel ihrer Identifizierung. Durch die Expression des viralen Caspase-Inhibitors CrmA konnte eine tragende Rolle der Caspase-8 und damit des Rezeptor-vermittelten Weges in der Initiierung des apoptotischen Programms in AKR 2B-Mausfibroblasten ausgeschlossen werden. Gleiches gilt für den mitochondrial-vermittelten Weg, für dessen Beteiligung, bis auf die Spaltung der Caspase-9, keine Hinweise vorliegen. Der Weg, der zur Aktivierung der DEVDase führt, ist Ziel gegenwärtiger Untersuchungen. Substanzen, die Signalwege aktivieren PDGF-BB, TPA, Forskolin und 8Br-cAMP) oder auch Substanzen, deren Verbindung zu Signalwegen noch weitgehend offen ist, schützen die Zellen vor dem Zelltod. Der protektive Effekt dieser Signalwege konzentriert sich in einem Konvergenzpunkt, der auf noch unbekannte Weise die Aktivierung der Effektor-Caspasen blockiert. Die Identität dieses Konvergenzpunktes und von ihm ausgehenden protektiven Weges ist Ziel weiterer Untersuchungen. So ist es möglicherweise dieser Weg, der zum Überleben von 50 Prozent der AKR 2B-Mausfibroblasten während des Serumentzugs wesentlich beiträgt. / In the work presented here, the essential involvement of caspases in the cell death of AKR 2B-fibroblasts could be proved as well as their activities could be characterized. Confluent AKR 2B-fibroblasts, a good characterized and subcloned cell line, rapidly disintegrate after serum deprivation. Dying of the cells ceases after 6 hours with a survival of 50 per cent. These surviving cells remain uneffected for additional 48 hours which is dependent on neo-protein biosynthesis. During cell death AKR 2B-fibroblasts show morphological changes characteristically for apoptosis, even though typical features like oligonucleosomal DNA fragmentation is absent. Using different approaches the expression of mRNA of all known caspases, which are believed to be involved in apoptosis essentially, was successfully detected in AKR 2B-fibroblasts. Caspases-1, -2, -3, -6 and -9 were constitutively expressed as zymogens. With the exception of Caspase-9, the processing into their active subunits induced by serum removal could not be detected. At least their considerable importance during cell death of AKR cells could be proved by using specific caspase inhibitors and by determination of their specific activity. The characterization of that activity gave some hints for the identity of activated caspases. Beside constutive VEIDase and IETDase activities, a DEVDase reaches its maximum 3 hours after the onset of apoptosis. The present mixture of caspase activity is dominated by this DEVDase, which seems to be represented by just one enzyme, as shown by affinity labeling and 2D-SDS-PAGE. Determinations of KM- and Ki-values lead to the conclusion, that this enzyms has typical effector caspase characteristics, like caspase-3. Cleavage of lamins during cell death of the fibroblasts indicate that a caspase-6 became active. However, the known characteristics of caspase-6 are different of that found in AKR 2B cells, so that it may play just a minor role in the caspase mixture. Established repeated purification steps by chromatography, offers best conditions for protein sequencing and identification of the active caspase. The involvement of the receptor mediated pathway could be excluded by an overexpression of CrmA , a cowpox virus derived Caspase inhibitor; also there are no hints for an involvement of the mitochondria mediated pathway, except of caspase-9 cleavage. Pathways which lead to DEVDase activation are of major interests in present and future. Stimulation of signal pathways by PDGF-BB, TPA, Forskolin and 8Br-cAMP and others agents protect the fibroblasts from death. The stimulated pathways converge in one point up stream of effector-caspase activation. The identification of this point and its regulatory properties is a future goal, which will maybe lead to an understanding of processes responsible for surviving of 50 per cent of AKR 2B-Mausfibroblasten during serum removal.

Ovaries maintain and produce functional female gametes, oocytes, for fertilisation. Oocytes develop inside cellular assemblies, the ovarian follicles, before birth and can reside there for up to 50 years in the human. Despite recent inroads, the precise mechanisms of initial follicle recruitment and growth remain unclear. Although the pituitary gonadotrophins play a role in this developmental process, locally produced factors have been implicated strongly in initiation of follicle growth. It is known that fibroblast growth factor 2 (FGF2) is a powerful mitogen for follicular granulosa cells in culture and initial studies undertaken in this project were successful in detecting FGF2 gene expression in ovarian biopsies from fertile healthy women. To further elucidate which cells were expressing FGF2, laser microdissection was employed to isolate differentially staged follicle populations. Real-time RT-PCR was used to quantify mRNA in relation to follicle development. Decreasing levels of FGF2 expression were detected as follicles developed. Non-radioactive in situ hybridisation confirmed FGF2 mRNA localisation in granulosa cells of preantral follicles. FGF2 protein localisation was assessed with immunohistochemistry; two primary antibodies raised against different fragments of human FGF2 were used. Both antibodies detected FGF2 in the oocyte cytoplasm of putative non-growing follicles, whereas only one of the antibodies showed additional reactivity to the basement membrane region of these same follicles. These results suggest different isoforms of FGF2 may localise specifically to different cellular sites. Follicle stimulating hormone receptor (FSHR) gene expression was also investigated in follicles using laser microdissection, real-time RT-PCR and in situ hybridisation. FSHR mRNA was detected in all follicle populations, including the smallest putative non-growing follicles. Disparity to other published works was attributed to the position of primer annealing, and thus the ability to detect alternatively spliced transcripts. In conclusion, the work presented here provides evidence that FGF2 and FSHR are present in small follicles and that their actions may be stimulatory or inhibitory to initial follicle recruitment.