Dissertations / Theses on the topic 'Wound healing assay'

Tato diplomová práce se zabývá popisem principů kultivace embryonálních fibroblastových buněk myší (3T3), lidských endoteliálních buněk odebraných z pupečníkové žily (HUVEC) a epiteliálních buněk vaječníku čínského křečka (CHO) v mikrofluidních systémech simulujících kapiláry. Byly provedeny literární rešerše v oblasti realizací experimentu “Scratch Wound Healing Assay” v mikrofluidních systémech s použitím fibroblastů a endotheliálních buněk. V práci jsou dále popsány principy konfokální a fluorescenční mikroskopie a metody zpracování obrazů pro sledování buněčné migrace. Experimentální nastavení pro mikrofluidní realizaci “Scratch Wound Healing Assay” s použitím trypsinu – EDTA pro vytvoření rýhy, a konfokálního mikroskopu Leica TCS SP8 X pro následující snímání pořízených dat bylo navrženo a otestováno s dostatečným počtem opakování. Vhodný algoritmus pro analýzu buněčné migrace byl napsán v programovacím prostředí Matlab. Závěrem této práce je diskuze získaných výsledků.

Collective cell migration is critical for various physiological and pathological processes. In vitro wound healing assay has been widely used to study collective cell migration due to its technical simplicity and ability of revealing the complexity of collective cell migration. This project studies the function and importance of leader cells, the cells pulling cell monolayer migrating into free space, in endothelium and skin epithelial regeneration via plasma lithography enhanced in vitro wound healing assay. Despite leader cells have been identified in in vitro wound healing assays, little is known about their regulation and function on collective cell migration. First, I investigated the role of leader cells in endothelial cell collective migration. I found that the leader cell density is positively related with the cell monolayer migration rates. Second, we used this knowledge to study the effects of arsenic treatment on skin regeneration via in vitro wound healing assay. We found that low concentration of arsenic treatment can accelerate the keratinocyte monolayer migration. We further found that arsenic affected cell migration by modulating leader cell density through Nrf2 signaling pathway. As a conclusion of these studies, we evaluated the function of leader cells in collective cell migration, and elucidated the mechanism of arsenic treatment on skin regeneration.

Collective behaviour is critical to a variety of biological and ecological processes, including tumour invasion, wound healing and spreading of invasive species. This thesis investigated mathematical models of collective cell behaviour, with an aim to develop techniques for applying these models to experimental data to obtain quantitative insight from experiments, and to develop novel models that accurately incorporate cellular mechanisms. We determined various appropriate techniques to extract quantitative information about cell movement and cell proliferation, given particular experimental data. We also developed novel mathematical models that accurately describe the average behaviour of cells undergoing birth, death and movement.

Collective cell spreading is frequently observed in development, tissue repair and disease progression. Mathematical modelling used in conjunction with experimental investigation can provide key insights into the mechanisms driving the spread of cell populations. In this study, we investigated how experimental and modelling frameworks can be used to identify several key features underlying collective cell spreading. In particular, we were able to independently quantify the roles of cell motility and cell proliferation in a spreading cell population, and investigate how these roles are influenced by factors such as the initial cell density, type of cell population and the assay geometry.

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

The investigation of complex biological processes, such as wound healing, cell migration, cancer cell invasion, and gene regulatory networks can be benefited tremendously by novel biosensing techniques with high stability and spatiotemporal resolution. In particular, molecular probes with qualities including high stability, sensitivity, and specificity are highly sought-after for long-term monitoring of gene expression in individual cells. Among different single-cell analysis techniques oligonucleotide optical probes is a promising detection method to monitor the dynamics of cellular responses. Herein, the design and optimization of double-stranded LNA probes are first investigated. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is highly stable in living cells and is capable of detecting changes in gene expression induced by external stimuli. Using dsLNA probes we then demonstrate the novel approaches to monitor the spatiotemporal gene expression response during cell injury. Our results also suggest a potential autoregulatory role of Nrf2 in injury induced EMT. We also show that the signaling level of dsLNA probe can serve as a molecular signature for the leader cells near the wound which allows us to track the behaviors of leader cells during collective cell migration. Finally multimodal GNR-LNA approach is proposed to map spatiotemporal gene expression profile and reveal dynamic characteristics of heat shock response in photothermal operations.

碩士
國立陽明大學
生醫光電研究所
100
There are several wound healing assays based on scratching, solid barrier and liquid barrier. However, none of them can represent the actual micro-environment which represents the direction of flow and EF toward the center of the wound. It has been suggested that wound healing is related to electric fields. Recently, Min Zhao et al. found the electrical signal also regulates the wound re-epithelialization. The disruption of epithelial barrier short-circuits the trans-epithelial potential and then creates a lateral endogenous electric field. The field has already been proofed as an important cue for guiding the migration direction of the fibroblasts, macrophages and keratinocytes in response to wounding site of a monolayer in vitro. This induced directional movement of cells toward the cathode or the anode under direct current electric field is so called electrotaxis. In this abstract, we propose a modified wound-healing-assay chip for studying electric field-assisted wound healing process. In preliminary test, we adopt NIH/3T3 fibroblast cell line to demonstrate the feasibility of our chip.

碩士
國立成功大學
分子醫學研究所
91
Wound healing is one of the most frequently used methods to study cell motility. A monolayer of cells is scratch-wounded and cells alongside the wound would proliferate and migrate to fill up the denuded area. The area change or the wound closure distance is considered to be a measurement of cell motility. However, the rate of wound closure may not be a true measurement of cell motility. By measuring the distance of wound closure, we found that high-density monolayers of T24 cells (a bladder cancer cell line) showed faster wound-closure rates than low-density ones, which, by conventional interpretation, implied that T24 cells at higher cell densities would have greater cell motilities. To clarify such an observation, we investigated the details of wound healing with our long-term, time-lapse recording system, which was able to record and depict the migration path of a single cell through the entire healing process. Only the first few rows of cells behind the wounded edge contributed to wound closure. These cells showed better moving directionality (toward the direction of wound closure) at higher cell-densities, explaining the greater wound-closure rate, whereas the average lengths of the migration paths are the same in high- and low-density experiments. The lengths of migration paths over a period of time are the better measurement of cell motility, whereas the wound-closure rate represents the combinational effect of cell motility and directionality. The effects of mitomycin C and β-Glycyrrhetinic acid on wound-closure rate and cell motility in wound healing were further investigated. It had been suggested that in order to minimize the effect of cell proliferation on wound healing, the proliferation activity should be inhibited or the assay time should be kept as short as possible. However, we found that inhibition of cell proliferation by mitomycin C treatment may affect cell motility in a short period of time. Gap junctional communication was thought to play a role in wound healing. By inhibition of gap junction with GCA, we found that the percentage of forward moving cells as well as the migration rate significantly decreased. In the second part of the study, we applied the long-term time-lapse recording system to the functional analyses of genes through transient transfections. A preliminary procedure was established. Using a construct to co-express green fluorescent protein and EMP2 in NIH3T3 cells, we established a procedure to evaluate potential effects of EMP2 on cell morphology, viability, apoptosis, membrane ruffling and cell motility. The procedure could be utilized as a rapid screening test for gene functions.

Yes
Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re‐epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re‐epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome.
University of Manchester Strategic Fund; Wellcome Trust; BBSRC; Ministry of Higher Education, Malaysia Universiti; Sains Malaysia

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctorate of Philosophy.
Senecio serratuloides is widely used for wound healing in South Africa but minimal information regarding its efficacy is available. Furthermore toxic pyrrolizidine alkaloids may be present. The following investigation sought firstly to evaluate the efficacy and safety of Senecio serratuloides in a porcine wound model; secondly to assess for a potential mechanism and finally isolate and identify fractions in in-vitro assays. Assessment of Efficacy and Safety Materials and Methods: Deep partial thickness and full thickness wounds were created on 9 pigs. Treatment included an occlusive dressing (negative control), activated carbon, or the Senecio preparation. Wounds were monitored using photographic documentation, pH measurement and histological analysis (skin thickness and collagen content). Toxicity was monitored on blood and liver samples. Results and Discussion: Efficacy of Senecio serratuloides was established with a significantly thicker epidermis, maximal at day 7 post-operative, 2 days before the controls. Effects on collagen content was negligible with no toxicity detected. Mechanistic investigation Materials and Methods: Wound fluid was analysed for IL-10, IL-12, IL-1β, IL-6, IL-8, TNF-α using flow cytometry based assays. Tyrosine phosphorylation and cellular proliferation was assessed using dual immunofluorescence staining. Results and Discussion: IL-1β levels were significantly greater in the Senecio treatment. Tyrosine phosphorylation increased to day 9 post-operative where it stabilised in all groups. In the same period, cellular proliferation was sustained in the Senecio treated wounds but not in the controls. Keratinocyte proliferation was identified as the target for in-vitro assays. Extraction, Isolation and Partial Identification using In-vitro Proliferation Assays. Materials and Methods: The plant was fractionated using solid phase extraction cartridges. Keratinocytes were grown under standard conditions in 96-well plates. Cellular proliferation was assessed spectrophotometrically using a resazurin dye technique. Active fractions were analysed using gas chromatography and mass spectrometry. Results and Discussion: Identified fractions increased the rate of proliferation by 300- 400%. Potential lead compounds were identified. Importantly, pyrrolizidine alkaloids could not be detected. Conclusion Senecio serratuloides is efficacious in treating deep partial thickness wounds without inducing liver toxicity. Sustained keratinocyte proliferation linked to tyrosine phosphorylation may be an underlying mechanism. Although successful, in-vitro detection of active fractions requires further characterisation.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
The skin is the first line of defense of the human body. When a wound is formed there is a disruption of the cellular and anatomic continuity of a tissue and therefore greater susceptibility to infections and other types of agressions. Wound healing is a complex and dynamic process, which can be divided into four phases: Homeostasis; Inflammatory;Proliferative; Remodeling. When wound healing don’t occur in a regularly way due to a proliferative disorder of the dermis, hypertrophic scars or keloids may be formed. Wound healing depends of a correct blood perfusion and oxygenation of the wound, a nutrition without nutritional deficiencies and the absence of infection. Diabetes is a pathology associated with diminished healing capacity and defects in the microcirculation, so wounds have high risk of becoming chronic. The age is another important factor, it is estimated that the total amount of collagen in the dermis decreases by 1% per year.Natural products have shown good potential in wound treatment. For example, the honey has several antibacterial properties due to the high sugar content, low pH and the ability of generation of hydrogen peroxide. Leptospermum scoparium honey has excellentantibacterial properties due to the high level of the reactive dicarbonyl methylglyoxal, a potent inhibitor of bacterial growth. Also, various types of plant extracts and/or plant isolates have demonstrated good efficacy. Rosa rubiginosa oil demonstrated a good regeneration of the tissues in post-surgical wounds. The aerial parts of Centella asiatica are rich in pentacyclic triterpenoids which are responsible for the cicatrizing properties. Centella asiatica and Aloe vera demonstrated efficay in the treatment of chronic wounds. Curcuma longa has marked anti-inflammatory and antioxidant properties what leads to a decrease in oxidative stress around the wound. Several studies have shown the ability of curcumin to increase wound contraction and accelerate the healing process.In conclusion, the phytotherapy has demonstrated enormous potencial in treating wounds either as na adjuvante or as an alternative to conventional medicine with demonstrated efficacy in vitro and in vivo assays and in clinical trials.
A pele é a primeira linha de defesa do corpo humano. Quando surge uma ferida há uma descontinuidade celular e anatómica de um tecido e por isso maior suscetibilidade a infeções e outros tipos de agressões. A cicatrização da ferida é um processo fisiológico complexo e dinâmico, que se pode dividir em quatro fases: Homeostase; Inflamatória;Proliferativa;Remodelação. Quando a cicatrização não ocorre de forma regular devido a uma desordem proliferativa da derme podem-se formar cicatrizes hipertróficas ou queloides. A cicatrização da ferida depende de uma correta perfusão sanguínea e oxigenação da ferida, de uma alimentação sem carências nutricionais e da ausência de infeção. A diabetes é uma patologia associada à diminuta capacidade de cicatrização e a defeitos na microcirculação,pelo que as feridas têm um risco elevado de se tornar crónicas. A idade é outro fator importante, estimando-se que a quantidade de colagénio da derme diminui aproximadamente1% por ano.Os produtos de origem natural têm demonstrado um bom potencial no tratamento deferidas. Por exemplo, o mel apresenta várias propriedades antibacterianas que se devem aoelevado teor em açúcar, baixo pH, e a capacidade de gerar peróxido de hidrogénio. Destaca-seo mel de Leptospermum scoparium que apresenta excelentes propriedades antibacterianasdevido aos elevados níveis de metilglioxal dicarbonil reativo, um potente inibidor bacteriano.Também vários tipos de extratos vegetais e/ou compostos isolados de plantas têmdemonstrado uma boa eficácia. O óleo de Rosa rubiginosa aplicado continuadamente em feridas pós-cirúrgicas demonstrou uma boa regeneração dos tecidos. As partes aéreas de Centella asiatica são ricas em triterpenoides pentacíclicos, responsáveis pela sua ação cicatrizante. Tanto a Centella asiatica como o Aloe vera demonstraram ser eficazes no tratamento de feridas crónicas. A Curcuma longa apresenta propriedades anti-inflamatórias e antioxidantes marcantes, diminuindo o stress oxidativo em redor da ferida. Vários estudos demonstraram a capacidade da curcumina em aumentar a contração de feridas e acelerar o processo de cicatrização de feridas.Assim, a fitoterapia tem demonstrado um enorme potencial no tratamento de feridas quer como adjuvante, quer como alternativa à medicina convencional, com eficácia demonstrada em ensaios in vitro e in vivo e em ensaios clínicos.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.
May 2006