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'fences' a Cell Migration Assay System for 24-Well Tissue Culture Plates

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  • Migration assay for endothelial cells in multiwells. Application to studies on the effect of opioids.
    Fischer EG, Stingl A, Kirkpatrick CJ
    J.Immunol.Meth. (1990) 128: 235-239
    An assay system is described which permits rapid and effective evaluation of endothelial cell repair, using cells growing in a monolayer. With this method it was possible to obtain highly significant results. For example, endothelial growth factor and heparin, significantly enhanced cell migration and/or proliferation, whereas beta-endorphin, an endogenous opioid, had no effect on the migration and/or proliferation of human umbilical vein endothelial cells. This model may be used to study the cell migration of a variety of cell types which under certain experimental conditions (e.g., irradiation) do not proliferate.

  • Implication de l'endosome de recyclage dans la migration cellulaire in vivo.
    Assaker G
    M.Sc. thesis, Université de Montréal (2009)   [Gloria Assaker] .
        [Résumé / Abstract]
    Au cours de l’ovogenèse chez la mouche du vinaigre: Drosophila melanogaster, un groupe de cellules folliculaires appelées cellules de bord, migrent à travers les cellules nourricières pour atteindre l’ovocyte. Cet événement, nécessitant la transition épithéliomésenchymateuse (TEM), la réorientation, puis l’arrêt, ressemble à la formation de métastases.
    L’endocytose est un régulateur clé de plusieurs événements polarisés, y compris la migration cellulaire. En effet, différentes protéines impliquées dans la migration, comme les intégrines et les E-cadhérines (cadhérines épithéliales), sont régulées par transport à travers les endosomes. De même, l’endocytose restreint au front de migration l’activité des récepteurs tyrosine kinases (RTKs) qui guident les cellules de bord dans leur mouvement. Cependant les mécanismes moléculaires de cette restriction spatiale de l’activité des RTKs demeurent largement inconnus. Nous avons testé l’implication du trafic vésiculaire à travers la machinerie d’endocytose, dans la migration dirigée des cellules de bord, car ce système est facilement accessible pour l’expression de protéines et l’analyse de mutants. Nous avons commencé par confirmer une observation précédente du rôle de l’endosome précoce dans la migration des cellules de bord. Ensuite, nous avons identifié l’endosome de recyclage (ER) comme un régulateur clé de cette migration. En effet, nous avons démontré que l’expression dans les cellules de bord d’une forme dominante négative de Rab11, la petite GTPase régulant le transport vésiculaire à travers l’ER, bloque la migration ou entraîne de sévères défauts de migration dans environ 80% des chambres d’oeufs examinées. De plus, nous observons par immunofluorescence une relocalisation de l’activité des RTKs alors que d’autres protéines de migration ne sont pas affectées par Rab11 dominant négatif. Ce résultat a été par la suite confirmé par une interaction génétique entre Rab11 et les RTKs. D’autre part, nous avons montré que le complexe exocyste, un effecteur de Rab11, est impliqué dans la migration des cellules de bord. Nous avons trouvé par microscopie confocale en tissu fixé et par microscopie en temps réel que Sec15, un composant de ce complexe, est polarisé, de façon Rab11- dépendante, dans des vésicules qui s’accumulent au front de migration tout au long du mouvement des cellules de bord. De plus, la perte de l’activité de Sec15 perturbe à son tour la migration. Ainsi, toutes ces données démontrent le rôle fondamental d’un cycle d’endo- exocytose dans le maintien des RTKs actifs au niveau du front de migration des cellules de bord le long de leur mouvement.

    During Drosophila melanogaster's oogenesis, a cluster of folllicle cells, called border cells, perform an invasive migration through the surrounding nurse cells to reach the oocyte. This event resembles metastasis formation since it requires epithelial- mesenchymal transition, reorientation and arrest.
    Endocytosis plays a fundamental role in many polarized processes, including cell migration, since different migration proteins, like integrins and E-cadherins traffic through the endocytic pathway. Furthermore, receptor tyrosine kinases (RTKs) that guide border cells during their migration are regulated by endocytosis, although the mechanisms involved are largely unknown. We tested the implication of vesicular trafficking through the endocytic machinery, in border cells' directed migration, because this system is easily accessible for protein expression and mutant analysis.
    We first confirmed previous observation that trafficking through the early endosome is necessary for border cells migration, and then we identified the recycling endosome as a key compartment for this migration. Indeed, we showed that overexpression in border cells of a dominant negative form of Rab11, the small GTPase regulating vesicular trafficking through the recycling endosome, blocks migration or leads to severe migration defects in about 80% of examined egg chambers. Furthermore, using immunofluorescence, we observed a relocalization of RTKs activity, whereas other migration proteins were not redistributed upon dominant negative Rab11 expression. This result was further confirmed by a genetic interaction between Rab11 and RTKs. Moreover, we showed that the exocyst complex, an effector of Rab11, is also involved in border cells migration. We found by using confocal microscopy of fixed tissues and time-lapse microscopy of living egg chambers, that Sec15, a member of this complex, is distributed in vesicles which are polarized, in a Rab11- dependent manner, throughout border cells migration. In addition, loss of Sec15 also impairs migration. Together these data demonstrate a fundamental role for an endo- exocytic cycle in the maintenance of active RTKs at the leading edge of border cells during their migration.
  • Quantitative analysis of autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration using a silicon template compartmentalization technique.
    Augustin-Voss HG, Pauli BU
    Exp.Cell Res. (1992) 198(2):221-227  
    Autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration was quantitatively analyzed using a two-dimensional, two-compartment coculture system. Silicon templates were used to subdivide 35-mm tissue culture dishes into two separate compartments. Endothelial cells were grown to confluence in the inner compartment and released from growth arrest by removal of the silicon template. The distance of endothelial cell outgrowth from the monolayer was measured in 24-h intervals. Endothelial cells from different vascular beds migrated with different migration rates (large vessel endothelial cells > hemangioendothelioma cells > microvessel endothelial cells). Prior coating of tissue culture wells with fibronectin, type I collagen, or type IV collagen and increasing serum concentrations strongly enhanced endothelial cell migration. Seeding tumor cells into the outer compartment prior to removal of the silicon template permitted the direct coculture analysis of tumor cell-induced endothelial cell migration. Microvascular endothelial cell migration was stimulated in a tumor cell number-dependent fashion, whereas large vessel endothelial cells could not consistently be stimulated by coculture with tumor cells. It is concluded that silicon templates offer a useful approach for the quantitative study of migration of anchorage-dependent cells, permitting follow-up measurements over several days, the study of matrix effects, and the direct coculture analysis of cell migration.
  • Improving skin integration around long-term percutaneous devices using fibrous scaffolds in a reconstructed human skin equivalent model.
    Bolle ECL, Bartnikowski N, Haridas P, Parker TJ, Fraser JF, Gregory SD, Dargaville TR
    J.Biomed.Mater.Res. (2019) B: 1-12   | DOI: 10.1002/jbm.b.34428
    The interface between synthetic percutaneous devices and skin is a common area for bacterial infection, which may ultimately result in failure of the device. Better integration of percutaneous devices with skin may help reduce infection rates due to the creation of a dermal seal. However, the mismatch in material and chemical properties of devices and skin presents a challenge for closing the dermal gap at the skindevice interface. Here, we have used a tissue engineering approach to tissue integration by creating a highly fibrous poly(e-caprolactone) scaffold using melt electrowriting and seeding this with dermal fibroblasts, followed by maturation and insertion into a full-thickness defect made in an ex vivo skin model. The integration of seeded scaffolds was compared with controls including a non-seeded scaffold and a polymer tube with a smooth surface. Dermal fibroblast inclusion in the scaffold and epidermal upgrowth versus downgrowth/marsupialization around the device were used as measures of integration. Based on these measures, almost all pre-seeded scaffolds performed better than both the non-seeded scaffolds and smooth tubes. The hypothesis is that the fibroblasts act as a barrier to epithelial downward migration, and provide healthy tissue for nascent epidermal development.
  • Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture
    Borg DJ, Dawson RA, Leavesley DI, Hutmacher DW, Upton Z, Malda J
    J.Biomed.Mater.Res. (2009) 88A: 184-194   [Danielle J. Borg et al.] .
    Skin cells for transplantation are routinely prepared by growing patient keratinocytes in a semi-defined cocktail of growth factors, including serum and feeder cells. However, these reagents require substantial risk remediation and can contribute to transplant rejection. Microcarrier culture is an emerging technology that may allow the elimination of feeder cells whilst facilitating expansion of cultured keratinocytes. However, the behavior of keratinocytes in microcarrier culture and the potential of these cells to form an epidermis have been poorly defined. We characterized freshly isolated human keratinocytes cultured on CultiSpher®-G1 microcarriers in the absence of murine feeder cells and assessed the potential of the keratinocytes to form an epidermis in an in vitro model. In a single passage, keratinocytes multiplied 44.9-fold in microcarrier-bioreactor culture in 17 days, whereas two-dimensional cultures reached confluence in 9 days and only expanded 7.4-fold. Histological characterization of keratinocytes on the microcarriers revealed that the cells were randomly distributed within these porous structures, however, not all pores contained cells. High-resolution microcomputed tomography imaging of the microcarriers confirmed limited interconnectivity of the pores. Immunoreactivity of specific epidermal markers was confirmed during cell expansion via immunohistochemistry. Despite the expression of differentiation markers, microcarrierexpanded keratinocytes retained the capacity to form an epidermis, as was evaluated using an in vitro human skin equivalent model. The epidermis formed by microcarrierexpanded keratinocytes in this model exhibited morphology similar to native skin. Significantly, the microcarrier technique successfully eliminates the need for a feeder cell layer and hence facilitates development of an improved culture system.
  • Investigating the potential of Oxymatrine as a psoriasis therapy.
    Chen Q, Zhou H, Yang Y, Chi M, Xie N, Zhang H, Deng X, Leavesley D, Shib H, Xie Y.
    Chemico-Biological Interactions 2017, 271:59-66  
    Psoriasis vulgaris is a chronic inflammatory skin disease, stubbornly intractable, with substantial consequences for patient physical and mental welfare. Approaches currently available to treat psoriasis are not satisfactory due to undesirable side-effects or expense. Psoriasis is characterized by hyperproliferation and inflammation. Oxymatrine, an active component extracted from Sophora flavescens, has been demonstrated to possess anti-proliferation, anti-inflammatory, anti-tumorigenic, immune regulation and pro-apoptotic properties. This investigation presents a detailed retrospective review examining the effect of Oxymatrine on psoriasis and investigates the mechanisms underlying patient responses to Oxymatrine. We confirm that Oxymatrine administration significantly reduced the Psoriasis Area Severity Index score, with high efficacy compared to the control group. In addition, we have found that Oxymatrine significantly inhibits the viability, proliferation and differentiation of human keratinocyte in vitro. Immunohistochemical analysis indicates Oxymatrine significantly suppresses the expression of Pan-Cytokeratin, p63 and keratin 10. The results indicate that the suppression of p63 expression may lead to the anti-proliferation effect of Oxymatrine on human skin keratinocytes. Oxymatrine does not affect the formation of basement membrane, which is very important to maintain the normal function of human skin keratinocytes. In summary, Oxymatrine offers an effective, economical, and safe treatment for patients presenting with intractable psoriasis vulgaris.
  • Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death.
    Cheng G, Zielonka J, McAllister DM, Mackinnon AC, Joseph1 J, Dwinell MB, Kalyanaraman B
    BMC Cancer 2013, 13:285-298   [Gang Cheng et al.] .
    Background: Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells.
    Methods: In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed.
    Results: Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer.
    Conclusions: We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.
  • Morphine reduces local cytokine expression and neutrophil infiltration after incision.
    Clark JD, Shi X, Li X, Qiao Y, Liang D, Angst MS, Yeomans DC
    Molecular Pain (2007) 3:28-39   [J David Clark et al.] .
    Inflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines.
  • Wild type mesenchymal cells contribute to the lung pathology of lymphangioleiomyomatosis.
    Clements D, Dongre A, Krymskaya VP, Johnson SR
    PLoS ONE (2015) 10;5:e0126025.   [Debbie Clements et al.] .
    Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.
  • Measurement of cell adhesion and migration using phosphor-screen autoradiography.
    Dalton BA, Dziegielewski M, Johnson G, Underwood PA, Steele JG
    BioTechniques (1996) 21,2:298-303   [B. Ann Dalton et al.] .
    This study demonstrates the rise of phosphor-screen autoradiography as a means of measuring cell adhesion and cell expansion on polymer surfaces. The method has particular merit in cases where a specific substrate may be opaque or biochemically incompatible with colorimetric assay methodologies. With the phosphor-screen autoradiography method. there was a linear relationship between cell number and quantitated radioactivity The technique has also been validated by comparison with a colorimetric assay of adhesion conducted for attachment to conventional culture substrata. The data supported the view that the use of phosphor-screen autoradiography was a valid method for detecting cell attachment, and it gave equivalent results to the colorimetric assay. Furthermore, a comparison between phosphor-screen autoradiography and a dye-staining method showed that this technique can be used as a means of quantifying cellular expansion over surfaces.
  • Polymer surface chemistry and bone cell migration.
    Dalton BA, McFarland CD, Gengenbach TR, Griesser HJ, Steele JG
    J.Biomater.Sci.Polym.Ed. (1998) 9,8:781-799
    Implant devices for orthopaedic applications may be improved if the surface of the biomaterial provides for osteointegration. To understand the effect of hydrophilicity on colonisation by human bone derived (HBD) cells, we compared untreated polystyrene (PS) and a sulfuric acid-treated PS surface for mechanisms of cell migration. The chemical composition of the acid-treated PS surface was analysed by monochromatic X-ray photoelectron spectroscopy and found to contain various oxidatively produced groups and a minor amount of sulfonate groups. It was found that migration of HBD cells on both PS and acid-treated PS surface was dependent on the presence of vitronectin (Vn) and was higher on the hydrophilic acid-treated surface. Minimal migration of HBD cells occurred on either surface in the absence of Vn, even when fibronectin was present in the culture medium. Using radiolabelled protein, it was shown that Vn adsorption onto the acid-treated surface was two to three fold greater than that on the hydrophobic PS. When HBD cells were seeded onto a patterned surface in a medium containing Vn, the cells preferentially colonised the hydrophilic region and few, if any, cells traversed the haptotactic boundary from the hydrophilic to the hydrophobic side. Thus the enhanced HBD cell migration seen on the acid-treated PS compared with the untreated PS surface and the haptotactic boundary phenomenon, relate to Vn adsorption.
  • Modulation of epithelial tissue and cell migration by microgrooves.
    Dalton BA, Walboomers XF, Dziegielewski M, Evans MDM, Taylor S, Jansen JA, Steele JG
    J.Biomed.Mater.Res. (2001) 56,2:195-207
    We used a polystyrene substratum to study the response of migrating epithelium to 1- or 5-m depth microgrooves with groove/ridge widths of 1, 2, 5, or 10 m. The migration of a tissue sheet was enhanced along the microgrooves, while migration across the microgrooves was inhibited. Changing the depth of the microgrooves had a greater effect on migration than alteration of the groove/ridge width. The migration of epithelial cells from a confluent monolayer culture followed a similar pattern to that of intact epithelial tissue. Cellular extensions generally followed the microgroove direction by tracking along the top of the ridges or following the ridge walls, as revealed by scanning electron microscopy. Actin filaments within the basal cell layer of the tissue were aligned with the microgrooves, unlike filaments in the superficial layers that did not appear to be affected by the presence of underlying microgrooves. The basal cell layer of the tissue conformed to the contours of the microgroove following migration. However, the ultrastructure of the tissue above the ridges resembled that of tissue on a flat surface. We concluded that surface microgrooves have the potential to direct the migration of immediately adjacent epithelial tissue, the effect of which is to guide epithelial tissue on the surface of implanted biomaterials.
  • Migration mechanisms: corneal epithelial tissue and dissociated cells.
    Dalton BA, Steele JG
    Exp.Eye Res. (2001) 73,6:797-814
    The migratory mechanism of intact bovine corneal epithelial tissue and individual corneal epithelial cells over synthetic surfaces in vitro were compared. In migrating tissue, adhesion between component cells was demonstrated by immunostaining for desmoplakin and identification of desmosomes by electron microscopy. The apparent intermeshing of microtubules within the tissue and interdigitation of cytoplasmic membranes showed the close association of cells. Portions of the advancing edge of the tissue contained actin filaments that were orientated parallel to the leading tissue front. These filaments appeared to span adjacent cells suggesting that migration partially involved the contraction of the actin cable, similar to the 'purse-string' mechanism originally identified in the closure of fetal skin wounds. Intact actin filaments and microtubules were necessary to maintain optimum migration rates for tissue and cells. However, tissue morphology was not dependent on microtubule integrity. During the migration of individual epithelial cells, no staining for desmoplakin was observed and there were clear divisions between the microtubules of adjacent cells. Actin filaments tended to be arranged parallel to the direction of cell movement.Therefore, migration of epithelial tissue sheets over synthetic surfaces occurs by mechanisms that differ from the migration of individual epithelial cells. Model systems based on the migration of intact tissue would give a more realistic assessment of the suitability of a material for biomaterial applications than the use of separate epithelial cells.
  • Stimulation of epithelial tissue migration by certain porous topographies is independent of fluid flux.
    Dalton BA, McFarland GA, Steele JG
    J.Biomed.Mater.Res. (2001) 56,1:83-92
    A surface with columnar pores 0.1 or 0.4 m in diameter is shown to have a novel effect on the migration of corneal epithelial tissue sheets; migration is stimulated in a nondirectional manner with respect to migration over a planar, nonporous surface (Dalton, Evans, McFarland, and Steele, J Biomed Mater Res 1999;45:384-394; Steele, Johnson, McLean, Beumer, and Griesser, J Biomed Mater Res 2000;50:475-482). By blind-ending the pores, we show that this increase in tissue migration is not dependent on fluid flux through the pores and so appears to occur as a result of surface topography. From transmission electron micrographs, the migrating tissue appears to form either close contacts or focal adhesions at the edge of some pore channels; we speculate that this may provide a fulcrum for the enhanced migration. Scanning electron micrographs suggest that within tissue that migrates over the surfaces that contain blind-ended pores, the cells are more extensively spread than those in tissue migrating on a planar surface. The migration of disaggregated epithelial cells is enhanced on surfaces that contain 0.1- or 0.4-m-diameter pores (compared with a planar surface), and this is similarly independent of fluid flux.
  • Bedeutung der therapeutischen Zielstruktur EpCAM (CD326) für die Progression von Ösophaguskarzinomen. [The relevance of the therapeutic target structure EpCAM (CD326) for the progression of esophageal carcinoma.]
    Driemel C, Panagiotidou P, Hoffmann I, Schumacher S, Luca AC, Pietsch J, Knoefel WT, Stoecklein NH
    in: Chirurgisches Forum und DGAV Forum 2010, Deutsche Gesellschaft für Chirurgie (2010) 39:35-36
    Introduction. EpCAM is an epithelial homophilic cell adhesion molecule that is de novo expressed in esophageal cancer. It is considered as one of the best-characterised tumour-related antigens and is an important target for molecular cancer therapies. However, the function of EpCAM in tumour progression is still poorly understood. Because esophageal cancer (ECA) is highly metastatic, we have tested in cell-based experiments whether EpCAM contributes to this aggressive phenotype.
    Material and Methods. We used a cohort of 7 human ECA cell lines (4 squamous cell carcinoma and 3 adenocarcinoma cell lines) that express EpCAM at different levels and which served us as a model system. To measure the potential effects of the loss of EpCAM expression, we used a lentiviral pGIPZ shRNAmir system with two different sh-RNAs directed against EpCAM and one control shRNA vector. The EpCAM-suppression of the transduced cells was tested by quantitative RT-PCR and Western blot. Cell lines with an 80 % reduction of EpCAM expression were further analysed. We used the »Fence-assay« to investigate the migration. The tumour cell invasion was assessed with a commercially available Matrigel-coated Transwell system.
    Results. The migration of EpCAMshRNA- transduced squamous cell carcinoma cells was reduced by 30-50 % compared to tumour cells transduced with the control vector. A 3-4 % reduction of the invasion was observed. Both, the reduction in migration and invasion was statistically significant. Conclusion. Our data indicate a relevance of EpCAM in the progression of ECA. In view of the absence of effective systemic therapies for ECA, EpCAM appears to be an extremely attractive therapeutic target for adjuvant therapies. Therefore, our cell-based findings of EpCAM function are also of clinical relevance.
  • Analysis methods of human cell migration. (Review)
    Entschladen F, Drell TL, Lang K, Masur K, Palm D, Bastian P, Niggemann B, Zaenker KS
    Exper.Cell Res. (2005) 307:418-426   [Frank Entschladen et al.] .
    The autonomous migration of specialized cells is an essential characteristic in both physiological and pathological functions in the adult human organism. Leukocytes, fibroblasts, and stem cells, but also tumor cells, are thus the subject of intense investigation in a broad range of research fields. Awide spectrum of methods have therefore been established to analyze chemokinetic and chemotactic cell migration, ranging from easy-to-handle two-dimensional surface migration assays to highly specialized three-dimensional and intravital analysis methods. It is now manifest that the results obtained with these various migration assays substantially differ. This review therefore gives an overview of the migration assays which are currently in use, describes the methods, and critically enlightens the particular advantages and disadvantages of each method.
  • Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.
    Evans J, Infusini G, McGovern J, Cuttle L, Webb A, Nebl T, Milla L, Kimble R, Kempf M, Andrews CJ, Leavesley D, Salamonsen LA
    FASEB J (2019) in press   [Jemma Evans et al.] .
    Repair after damage is essential for tissue homeostasis. Postmenstrual endometrial repair is a cyclical manifestation of rapid, scar-free, tissue repair taking ~3-5 d. Skin repair after wounding is slower (~2 wk). In the case of chronic wounds, it takes months to years to restore integrity. Herein, the unique 'rapid-repair' endometrial environment is translated to the 'slower repair' skin environment. Menstrual fluid (MF), the milieu of postmenstrual endometrial repair, facilitates healing of endometrial and keratinocyte 'wounds' in vitro, promoting cellular adhesion and migration, stimulates keratinocyte migration in an ex vivo human skin reconstruct model, and promotes re-epithelialization in an in vivo porcine wound model. Proteomic analysis of MF identified a large number of proteins: migration inhibitory factor, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were selected for further investigation. Functionally, they promote repair of endometrial and keratinocyte wounds by promoting migration. Translation of these and other MF factors into a migration-inducing treatment paradigm could provide novel treatments for tissue repair.
  • Focal adhesion dynamics are altered in schizophrenia.
    Fan Y, Abrahamsen G, Mills R, Calderón CC, Tee JY, Leyton L, Murrell W, Cooper-White J, McGrath JJ, Mackay-Sim A
    Biological Psychiatry (2013) 74(6):418-426
    -Background- Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem/progenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics.
    -Methods- Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics.
    -Results- Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells.
    -Conclusions- The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.
  • In vitro models for investigating keratinocyte responses to ultraviolet B radiation.
    Fernandez TL
    PhD thesis, Queensland University of Technology (2013)   [Tara L. Fernandez] .
    Australia experiences extreme levels of solar ultraviolet radiation (UVR), as well as increasing rates of skin cancer incidence among the Australian population. Strong associations between chronic unprotected sun exposure (in particular the UVB radiation wavelength) and photocarcinogenesis have been established. However, major knowledge gaps still exist in our understanding of the biology of the cutaneous photoresponse, in particular the complex cellular and molecular mediators involved in resisting and repairing UVB-induced damage. Due to the intricate nature of human skin, the lack of complete, accessible and biologically-relevant experimental models to investigate the human photoresponse has limited in vitro investigations into these processes.
    Keratinocytes are the primary cell type of the epidermis, which functions as a barrier against environmental hazards. Due to their frequent exposure to genotoxic insults like UVB radiation, keratinocytes are equipped with complex protective and repair strategies against photodamage, including the formation of DNA photolesions. Intercellular interactions between keratinocytes and dermal fibroblasts and the role of this dermal-epidermal crosstalk in modulating keratinocyte responses to UVB radiation have been relatively uncharacterised. These fibroblasts are known to synthesize various factors with potent effects on basal keratinocytes, such as stimulating proliferation, regulating epidermal regeneration and homeostasis. Importantly, the association of these fibroblast-secreted factors with the acute keratinocyte UVB radiation response and potentially their role in epidermal photocarcinogenesis need to be further investigated.
    The overriding aim of this thesis was therefore to characterise and develop 2-dimensional (2D) and 3D in vitro skin models as platforms for investigating the responses of human keratinocytes to UVB radiation. A tissue-engineered human skin equivalent (HSE) model consisting of primary human keratinocytes cultured on a de-epidermised dermal (DED) scaffold has been established in our laboratory as an organotypic model to study aspects of skin biology. Importantly, this HSE-KC skin model (comprised of a stratified epidermal layer with primary keratinocytes on a DED scaffold) has been previously demonstrated to retain several structural and functional properties of the native epidermis. Hence, in this thesis, the characterisation and validation of the HSE-KC skin model as a tool to study keratinocyte responses to UVB radiation experimentally are reported. Significantly, the HSE-KC was shown to possess several biological parallels to ex vivo skin, such as the expression of several epidermal protein markers. Furthermore, histological and immunohistochemical analysis of irradiated HSE-KC constructs showed the generation of key markers of the epidermal photoresponse, including the formation of DNA photolesions, apoptotic sunburn cells, increased synthesis of inflammatory cytokines and shifts in epidermal proliferation and differentiation patterns.
    To study the paracrine interactions between dermal fibroblasts and keratinocytes, the novel application of a cell co-culture system to study the influence of fibroblasts on modulating keratinocyte responses to UVB radiation is described herein. Using this previously undescribed technique, I report the enhancement of keratinocyte viability, an inhibition of UVB radiation-induced apoptosis and the enhancement of DNA repair in cells co-cultured with fibroblasts prior to irradiation. Next, this HSE-KC model was further developed and characterised as a model of epidermal-dermal interactions during the acute UVB irradiation response via the incorporation of human dermal fibroblasts into the DED component, termed the HSE-KCF. This construct was utilised as a novel in vitro 3D model for studying the influence of epidermal-dermal crosstalk on photodamage and repair in keratinocytes post-irradiation. The protective effect of fibroblasts previously described in the abovementioned 2D co-culture model was also observed in the epidermis of these HSE-KCF models. Hence, the molecular mechanisms surrounding this fibroblast-induced keratinocyte protection during the UVB irradiation response were further analysed. Importantly, the presence of fibroblasts in the HSE-KCF skin constructs modulated the expression and activation of elements of the UVB radiation-induced apoptotic cascade including Bcl-2-associated death promoter (Bad) and caspase-3, but not of cleaved poly (ADP-ribose) polymerase (PARP).
    To further investigate this fibroblast-mediated protection of UVB-irradiated keratinocytes, the effect of insulin-like growth factor-I (IGF-I) on cellular photoresponses was also analysed. The insulin-like growth factor (IGF) system plays a central role in dermal-epidermal interaction during the homeostatic maintenance of the epidermis and during wound healing. In the studies presented in this thesis, the modulation of keratinocyte photoresponses to UVB radiation by IGF-I-mediated activation of the insulin-like growth factor receptor type 1 (IGF-IR) and the resulting downstream pathways were reported. Significantly, IGF-IR activation: 1) enhanced keratinocyte viability; 2) suppressed UVB radiation-induced apoptosis; 3) accelerated the rate of UVR-induced photolesion removal; 4) increased the activation of p53; 4) modulated cell cycle progression; and 5) enhanced the activation of DNA damage response pathways. Notably, these results elucidate several mechanisms through which the IGF-I system may regulate appropriate epidermal responses to UVB radiation exposure, through the use of a novel in vitro photobiology culture system. These findings have implications not only for broadening our understanding of the involvement of fibroblast-keratinocyte interactions in the initiation of NMSCs, but also as a potential therapeutic or protective strategy against cutaneous photodamage and photocarcinogenesis.
    In summary, this project has characterised and validated previously undescribed primary cell-based in vitro platforms for photobiology research. The 2D Transwell® and 3D organotypic HSE-KC and HSE-KCF described herein have numerous possible applications in studying the biology of the cutaneous photoresponse. Significantly, the impact of fibroblast-keratinocyte interactions, in particular via the IGF-I system as reported in this thesis, has highlighted potential targets for the possible development of novel therapeutic interventions against the harmful effects of UVB radiation exposure.
  • Characterization of a human skin equivalent model to study the effects of ultraviolet B radiation on keratinocytes.
    Fernandez TL, van Lonkhuyzen DR, Dawson RA, KimlinMG, Upton Z.
    Tissue Engineering Part C: Methods (2014) 20(7):588-598
    The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.
  • Opioid influence on the adherence of granulocytes to human umbilical vein endothelial cells in vitro.
    Fischer EG, Stingl A, Kirkpatrick CJ.
    Cell Biol.Int.Rep. (1990) 14,9:797-804
    Recent studies revealed the existence of opioid receptors on human polymorphonuclear leukocytes (hPMN) and reported the effects of endogenous opioids on hPMN migration and adherence on glass or serum coated glass. Extending these studies, two different assay systems served to quantify the two basic events of adherence: attachment and spreading. hPMN in suspension were allowed to settle under the influence of beta-endorphin on human umbilical vein endothelial cells. After 30 and 240 sec the number of attached cells was enhanced 2.5-fold. Studying the spreading of cells, beta-endorphin increased the area 1.5-fold. Since adherence precedes the migration of hPMN through the endothelial layer towards foci of inflammation, the results suggest a modulatory role of endogenous opioids in defence mechanisms.
  • Surface topography can interfere with epithelial tissue migration.
    Fitton JH, Dalton BA, Beumer G, Johnson G, Griesser HJ, Steele JG
    J.Biomed.Mater.Res. (1998) 42,2:245-257
    Corneal epithelial tissue migration over the surface of a synthetic polymer can be inhibited by pores in the substrate. The effects of this substrate topography upon epithelial tissue migration were studied in vitro. Membranes of different porosities and structures were used to provide two series of surfaces having a graded increase in discontinuities: cellulose nitrate/acetate membranes with a tortuous network of pores, and track-etched polycarbonate membranes with columnar pores. Corneal epithelial tissue outgrowth was inhibited by increased pore size, and for both series of membranes, outgrowth was completely halted on membranes with mean diameter of the pores 0.9 µm at the pore densities measured. On the track-etched membranes with pores of <0.9 µm diameter, tissue outgrowth could be partially 'rescued' by coating with fibronectin or collagen, but above this size, the inhibition predominated. The effect of porosity of the track-etched membranes upon the migration of dissociated epithelial cells was also examined. Although migration of these cells was reduced on membranes having pore sizes larger than 0.9 µm, it was not completely inhibited even on membranes of 2.3-µm pore diameter. Therefore, tissue movement of adult stratified epithelium may be inhibited by specific surface topographies, and in this assay system, epithelial tissue outgrowth was more affected than was the migration of dissociated epithelial cells.
  • Evaluation of polyvinyl alcohol composite membranes containing collagen and bone particles.
    Hameed N, Glattauer V, Ramshaw JAM
    J Mech Behav Biomed Mater (2015) 48:38-45
    Composite biomaterials provide alternative materials that improve on the properties of the individual components and can be used to replace or restore damaged or diseased tissues. Typically, a composite biomaterial consists of a matrix, often a polymer, with one or more fillers that can be made up of particles, sheets or fibres. The polymer matrix can be chosen from a wide range of compositions and can be fabricated easily and rapidly into complex shapes and structures. In the present study we have examined three size fractions of collagen-containing particles embedded at up to 60% w/w in a poly(vinyl alcohol) (PVA) matrix. The particles used were bone particles, which are a mineral-collagen composite and demineralised bone, which gives naturally cross-linked collagen particles. SEM showed well dispersed particles in the PVA matrix for all concentrations and sizes of particles, with FTIR suggesting collagen to PVA hydrogen bonding. The Tg of membranes shifted to a slightly lower temperature with increasing collagen content, along with a minor amount of melting point depression. The modulus and tensile strength of membranes were improved with the addition of both particles up to 10 wt%, and were clearly strengthened by the addition, although this effect decreased with higher collagen loadings. Elongation at break decreased with collagen content. Cell adhesion to the membranes was observed associated with the collagen particles, indicating a lack of cytotoxicity.
  • Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model.
    Haridas P, McGovern JA, McElwain DLS, Simpson MJ. (2017)
    PeerJ 5:e3754 https://doi.org/10.7717/peerj.3754 [Parvathi Haridas et al.]
    Background Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.Methods 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.Results Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at.
  • Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue.
    Haridas P, Browning AP, McGovern JA, McElwain DLS, Simpson MJ.
    BMC Systems Biology (2018) 12:34-48 [Parvathi Haridas et al.]
    Melanoma can be diagnosed by identifying nests of cells on the skin surface. Understanding the processes that drive nest formation is important as these processes could be potential targets for new cancer drugs. Cell proliferation and cell migration are two potential mechanisms that could conceivably drive melanoma nest formation. However, it is unclear which one of these two putative mechanisms plays a dominant role in driving nest formation. Results We use a suite of three-dimensional (3D) experiments in human skin tissue and a parallel series of 3D individual-based simulations to explore whether cell migration or cell proliferation plays a dominant role in nest formation. In the experiments we measure nest formation in populations of irradiated (non-proliferative) and non-irradiated (proliferative) melanom a cells, cultured together with primary keratinocyte and fibroblast cells on a 3D experimental human skin model. Results show that nest size depends on initial cell number and is driven primarily by cell proliferation rather than cell migration. Conclusions Nest size depends on cell number, and is driven primarily by cell proliferation rather than cell migration. All experimental results are consistent with simulation data from a 3D individual based model (IBM) of cell migration and cell proliferation.
  • Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.
    Haridas P, Penington CJ, McGovern JA, McElwain DLS, Simpson MJ.
    J Theor. Biol. (2017) 423:13-25 [Parvathi Haridas et al.]
    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects.
  • In vitro characterisation of melanoma progression in a melanoma skin equivalent model.
    Haridas P.
    Thesis Queensland University of Technology (2018) [Parvathi Haridas]
    Melanoma is a fatal form of skin cancer which progresses in an orchestrated pattern in human skin. In this thesis, we focus on three phases of melanoma progression: radial growth phase (RGP) where melanoma cells are generally confined to the epidermis of the skin; vertical growth phase (VGP) where melanoma cells invade into the dermis of the skin; and the metastatic phase where melanoma cells enter the blood stream and spread to other parts of the body. Characterising these phases of melanoma in vitro is important to investigate disease progression. The principal aim of this thesis is to quantify key features of melanoma progression, these include: melanoma cell migration; melanoma cell proliferation; melanoma cell invasion; and melanoma nest formation using two-dimensional (2D) and three-dimensional (3D) assays. We first investigate a reliable melanoma-specific marker and provide evidence that S100 is a sensitive marker that identifies melanoma cell lines: WM35 (RGP); WM793 (VGP); and SK-MEL-28 (metastatic) used in this project. Also included in this thesis is a demonstration of the difficulty of identifying a certain melanoma cell line, MM127. The most commonly used melanoma-associated markers failed to identify this cell line. We further investigate the rates of melanoma cell migration and cell proliferation using 2D co-culture assays. Since fibroblasts are thought to play an important role in cancer progression, the result from the cross-talk between melanoma cells and primary fibroblast cells could provide insightful information about their influence on the rates of spatial expansion. However, results from this study provide evidence that a more multicellular, heterogeneous, 3D environment might be necessary to examine the cross-talk between skin cells and melanoma cells. Since, cell-cell interactions are known to differ in an environment with more than two cell types, we characterise melanoma progression by constructing a 3D melanoma skin equivalent (MSE) model which resembles human skin in vivo and present our quantitative results of melanoma invasion in a time course pattern. Lastly, we use the 3D MSE model to identify and report our findings that proliferation and increased initial cell number drives melanoma nest formation. The outcomes of this project provide a foundation for 3D melanoma research and future in vitro assays are essential to fully understand the potential of this 3D model for clinical purposes.
  • Effect of extracellular-matrix proteins on vascular smooth-muscle cell phenotype.
    Hayward IP, Bridle KR, Campbell GR, Underwood PA, Campbell JH
    Cell Biol.Internatl. (1995) ( 19,9:727-734 ) 19,10:839-846
    The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (V(v)myo) in their cytoplasm. Laminin was best at maintaining SMC with a high V(v)myo (V(v)myo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest V(v)myo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high V(v)myo phenotype.
  • A novel pulsatile bioreactor for mechanical stimulation of tissue engineered cardiac constructs.
    Hollweck T, Akra B, Häussler S, äberfuhr, Schmitz C, Pfeifer S, Eblenkamp M, Wintermantel E, Eissner G
    J.Funct.Biomater. (2011) 2:107-118   [Trixi Hollweck et al.] .
    After myocardial infarction, the implantation of stem cell seeded scaffolds on the ischemic zone represents a promising strategy for restoration of heart function. However, mechanical integrity and functionality of tissue engineered constructs need to be determined prior to implantation. Therefore, in this study a novel pulsatile bioreactor mimicking the myocardial contraction was developed to analyze the behavior of mesenchymal stem cells derived from umbilical cord tissue (UCMSC) colonized on titanium-coated polytetrafluorethylene scaffolds to friction stress. The design of the bioreactor enables a simple handling and defined mechanical forces on three seeded scaffolds at physiological conditions. The compact system made of acrylic glass, Teflon®, silicone, and stainless steel allows the comparison of different media, cells and scaffolds. The bioreactor can be gas sterilized and actuated in a standard incubator. Macroscopic observations and pressure-measurements showed a uniformly sinusoidal pulsation, indicating that the bioreactor performed well. Preliminary experiments to determine the adherence rate and morphology of UCMSC after mechanical loadings showed an almost confluent cellular coating without damage on the cell surface. In summary, the bioreactor is an adequate tool for the mechanical stress of seeded scaffolds and offers dynamic stimuli for pre-conditioning of cardiac tissue engineered constructs in vitro.
  • Ein neues kardiales Stammzellimplantat zur Therapie kardiomyodegenerativer Erkrankungen.
    Hollweck T
    Dr.-Ing. thesis, Universität München (2011)   [Trixi Hollweck] .
    Das kardiale Tissue Engineering ist ein vielversprechender Ansatz zur Wiederherstellung von geschädigtem Herzmuskelgewebe nach einem Myokardinfarkt. Neben anderen Stammzellquellen hat der Transfer von mesenchymalen Stammzellen aus dem Nabelschnurgewebe (UCMSC) zur ischämischen Region des Herzens möglicherweise einen positiven Einfluss auf die Geweberegeneration. UCMSC stehen in großen Mengen ohne medizinische Intervention zur Verfügung, zeigen Selbsterneuerungspotential, immunologische Naivität und Multipotenz. In der vorliegenden Arbeit wurde daher die Differenzierbarkeit von UCMSC zu Kardiomyozyten durch den Vergleich verschiedener publizierter Protokolle untersucht. Die kardiomyogene Differenzierung von UCMSC (cUCMSC) wurde dabei durch die Behandlung mit 5-Azazytidin und Oxytocin sowie durch die Formierung von <embryoid bodies> induziert. Umfangreiche morphologische und immunzytochemische Analysen von cUCMSC mit einem Panel an kardialen Markern zeigte eine effektivere kardiomyogene Differenzierung durch Oxytocin als durch 5-Azazytidin oder durch die Formierung von <embryoid bodies>. cUCMSC wiesen einen Kardiomyozyten-ähnlichen Phänotyp auf und exprimierten kardiale Proteine. Nach Verifizierung der UCMSC als geeignete Zellquelle zur Therapie kardiomyodegenerativer Erkrankungen, wurden im weiteren Verlauf der vorliegenden Arbeit Untersuchungen zur geeigneten Verabreichungsform der Zellen angestellt. Eine Alternative zur Zellinjektion, die wenig Retention am Zielort gewährleistet, ist die Implantation von besiedelten Zellträgern auf die ischämische Region des Herzens. Es wurde die Besiedelungseffizienz von unbeschichteten und titanisierten Zellträgern aus expandierten Polytetrafluorethylen (ePTFE) unterschiedlicher Struktur mit UCMSC untersucht. Das GORE-TEX® Dualmesh® (DM) weist im Vergleich zu der planaren Oberfläche des GORE-TEX® kardiovaskulären Patches (CVP) eine gerippte Oberfläche auf. Die Untersuchung der Anwachsrate, Vitalität und Proliferation von UCMSC zeigte, daß titanisierte Zellträger, unabhängig von der Oberflächenstruktur, unbeschichteten Zellträgern überlegen sind. Mikroskopische Aufnahmen zeigten eine untypische, kugelförmige Gestalt von UCMSC auf unbeschichteten Zellträgern. UCMSC auf titanisierten Zellträgern zeigten dagegen ihre charakteristische spindelförmige Morphologie und eine homogene Besiedelung des titanisierten CVP. Zur Untersuchung der mechanischen Belastbarkeit von UCMSC auf titanisierten CVP, wie sie der Myokardkontraktion entspricht, wurde ein pulsatiler Bioreaktor entwickelt. Das Design des Bioreaktors ermöglicht eine einfache Handhabung und eine definierte mechanische Beanspruchung dreier besiedelter Zellträger im Parallelbetrieb unter physiologischen Bedingungen. Orientierende Versuche zur Ermittlung der Anwachsrate und Morphologie von UCMSC nach mechanischer Belastung zeigten eine nahezu konfluente zelluläre Beschichtung auf titanisierten CVP. UCMSC nach mechanischer Belastung wiesen dabei im Vergleich zu UCMSC ohne mechanische Beanspruchung keine Schädigungen der Zelloberfläche auf. Die Titanisierung von klinisch zugelassenem CVP ermöglicht somit die Retention von UCMSC unter statischen und dynamischen Bedingungen und stellt daher eine potentielle Zellverabreichungsform zur Wiederherstellung von geschädigtem Herzmuskelgewebe dar.
  • Cell migration rate on poly(ε‐caprolacton)/poly(ethylene glycol) diblock copolymers and correlation with the material sliding angle.
    Hsu S-h, Tang C-M, Chiu J-J, Liao T-C, Lin C-C, Iwata H
    Macromol. Biosci. (2007) 7,4:482-490  
    The nanostructure of a biomaterial surface has strong influence on cell behavior. The migration of cells on nanostructured surfaces, however, has not been investigated so far. In this study, we used PCL/PEG diblock copolymers as model surfaces to examine the effect of nanoislands on migration of different cells, including fibroblasts and endothelial cells. The water sliding angle of the substrates was measured. The cell migration rate was examined under a real-time optical microscope. It was found that a greater cell migration rate correlated with the smaller sliding angle of the substrate.
  • Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents.
    Kairuz E, Upton Z, Dawson RA, Malda J .
    Wound Repair Regeneration (2007) 15,2:266-274   [Evette Kairuz et al.] .
    The crucial role of oxygen during the complex process of wound healing has been extensively described. In chronic or nonhealing wounds, much evidence has been reported indicating that a lack of oxygen is a major contributing factor. Although still controversial, the therapeutic application of hyperbaric oxygen (HBO) therapy can aid the healing of chronic wounds. However, how HBO affects reepithelization, involving processes such as keratinocyte proliferation and differentiation, remains unclear. We therefore used a three-dimensional human skin-equivalent (HSE) model to investigate the effects of daily 90-minute HBO treatments on the reconstruction of an epidermis. Epidermal markers of proliferation, differentiation, and basement membrane components associated with a developing epidermis, including p63, collagen type IV, and cytokeratins 6, 10, and 14, were evaluated. Morphometric analysis of hematoxylin and eosin-stained cross sections revealed that HBO treatments significantly accelerated cornification of the stratum corneum compared with controls. Protein expression as determined by immunohistochemical analysis confirmed the accelerated epidermal maturation. In addition, early keratinocyte migration was enhanced by HBO. Thus, HBO treatments stimulate epidermal reconstruction in an HSE. These results further support the importance of oxygen during the process of wound healing and the potential role of HBO therapy in cutaneous wound healing.
  • Differential expansion of human endothelial monolayers on basement membrane and interstitial collagens, laminin and fibrinectin in vitro.
    Kirkpatrick CJ, Kampe M, Fischer EG, Rixen H, Richter H, Mittermayer C
    Pathobiology (1990) 58,4:221-225
    In this study the ability of a human endothelial cell monolayer to expand over specific components of the basement membrane and extracellular matrix was investigated over a 5-day period. The method was intended as a model to study the mechanisms of endothelial regeneration. All components were coated onto sterile coverslips at a concentration of 10 µg/ml. The highest expansion was obtained on fibronectin, laminin and collagen type III, all three being statistically significantly greater than on the uncoated control surface (0.002 > p > 0.0001). Collagens types I and IV and a high molecular weight fragment mixture of type IV (IV-F, consisting of 75, 120 and 140 kD fragments) elicited approximately similar expansion rates, significantly higher than the control (0.02 > p > 0.003), although significantly lower (approximately 15%) than collagen type III, fibronectin and laminin (p < 0.001). The high monolayer expansion on collagen type III is surprising, as it is a relatively minor biosynthetic product of the endothelial cell. It could, however, be of significance in wound healing, in which endothelial cells come into contact with this interstitial collagen. In addition, the similar results obtained with collagens IV and IV-F indicate that expansion of the endothelial monolayer is not dependent on the integrity of the tetrameric structure of type-IV collagen.
  • In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane.
    Kirkpatrick CJ, Kampe M, Rixen H, Fischer EG, Ruchartz D, Mittermayer C
    Virchows Arch.Abt.B. Cell Pathol. (1990) 58:207-213
    The purpose of the present study was to observe the expansion of a monolayer of endothelial cells over specific components of the basement membrane. This was performed in vitro in a monolayer expansion assay over 5 days. The control surface was uncoated glass in the form of coverslips. Test substances were coated at a concentration of 10 micrograms/ml. The highest expansion was obtained with a high molecular weight fragment mixture of collagen type IV (IV-F, consisting of 75, 120 and 140 KD fragments), followed by fibronectin. Collagens type I, III and IV tetramer gave similar results, less than fibronectin or collagen type IV-F, although all of the above basement membrane coatings promoted expansion significantly above that of the control (P less than 0.01). The poorest expansion was obtained with laminin, which was significantly less than the control. The pentapeptide GRGDS, related to the fibronectin cell binding region, gave expansion significantly below that of the intact fibronectin molecule, as did the intact collagen type IV molecule compared with type IV-F (P less than 0.025). This indicates that sequences of the fibronectin molecule other than the cell binding sequence may be involved in promoting endothelial cell expansion. In addition, the integrity of the collagen type IV molecule does not appear necessary for this effect. On the contrary, the higher movement on IV-F may represent an inherent repair mechanism in damaged endothelium. Autoradiographic studies show that endothelial cell proliferation at the expanding front is involved in the migration assay.
  • Endothelial cells under mechanical and humoral stress.
    Klosterhalfen B, Rixen H, Mittermayer Ch, Kirkpatrick CJ, Richter H
    Biomechanical Transport Processes - NATO ASI Series Volume (1990) 193:313-322
    Endothelial cells are the interface between blood components and the vessel wall. Alterations of these vascular cells provide changing conditions of the vessel wall surface. Considerations about blood-vessel wall interactions have to include the pathology of the endothelium. Complex in vivo and simplified in vitro models are necessary to advance in our knowledge about the pathophysiology of these particular cells. Two studies in our institute, an in vivo as well as an in vitro model, are described and their results discussed.
  • Suppression of corneal neovascularization by culture supernatant of human amniotic cells.
    Kobayashi N, Kabuyama Y, Sasaki S, Kato K-i, Homma Y
    Cornea (2002) 21,1:62-67   [Namie Kobayashi et al.] .
    -Purpose- To examine the applicability of culture supernatant of human amniotic cells on basic fibroblast growth factor (bFGF)-induced corneal neovascularization. -Methods- Human amniotic epithelial and mesenchymal cells (AC) were obtained from human amniotic membranes by digesting with collagenase and maintained in serum-containing medium. The AC preparations predominantly contained cytokeratin-positive cells (91.2 ± 3.1%, n = 4). The culture supernatant was prepared by cultivating AC in serum-free medium for 24 hours. Neovascularization was obtained by a micropocket assay with Hydron pellets containing bFGF. Migration assay was carried out by a double-chamber method using human umbilical vein endothelial cells (HUVEC). Cell growth assay was done by MTT assay using HUVEC. -Results- Basic fibroblast growth factor-induced corneal neovascularization was significantly reduced by administration of AC culture supernatant. When either control or AC culture supernatant was administered three times per day for 10 days, the area with neovascularization was 13.2 ± 3.2 mm 2 and 4.0 ± 1.4 mm 2 for the control and AC culture supernatant-treated eyes, respectively (n = 7, p = 0.021). AC culture supernatant strongly inhibited bFGF-induced migration and cell growth of HUVEC. -Conclusions- Amniotic cell culture supernatant contains potent inhibitors of neovascularization. This effect is explained in part by inhibition of migration and cell growth of vascular endothelial cells. AC culture supernatant may be applicable for treatment of corneal diseases with neovascularization.
  • Effect of thiolated glycosaminoglycans on the behavior of breast cancer cells: toward the development of in vitro models of cancer.
    Köwitsch A, Chhalotre A, Groth T
    Internatl J Artificial Organs (2018) 40(1):31-39  
    The influence of extracellular matrix components like glycosaminoglycans (GAG) or adhesive proteins on the migration of cancer cells and the progression of tumorigenesis remains a challenging task. Therefore, this study aims to give insight into the interaction of cancer cells exhibiting different metastatic potential (MDA-MB-231, MDA-MB-468) with surface immobilized GAG interacting with serum proteins like fibronectin. Methods Model substrata were covalently coated with different thiolated GAG (hyaluronan (HA), chondroitin sulfate (CS), heparin (Hep)) and investigated for the adsorption of fibronectin (FN) with surface plasmon resonance. Then, adhesion of breast cancer cells in the presence of and without serum proteins was studied. Further, the outgrow behavior of confluent cancer cells was examined with the help of cell migration chambers and single-cell migration with time-lapse microscopy. Results FN adsorption revealed that the Hep-coated surfaces were able to adsorb significantly more protein than CS and HA. Generally, initial adhesion of breast cancer cells on GAG-coated substrata was inhibited for HA- and CS-coated substrata in the presence of serum proteins for both cell lines in comparison to serum-free conditions. The cell size was also significantly decreased by the influence of serum proteins. The outgrow studies clearly confirmed the different migration speed of both cancer cells while single-cell migration was particularly enhanced on HA-coated surfaces. Conclusions The results reveal that adsorption of serum proteins (e.g. albumin) possess an inhibiting effect on the adhesion of breast cancer cells and that single-cell migration is enhanced for both breast cancer cell lines on HA.
  • In vitro cell migration and invasion assays. (Review)
    Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschläger M, Dolznig H
    Mutation Research/Reviews in Mutation Research (2013) 752,1:10-24   [N. Kramer et al.] .
    Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.
  • Signalling mechanisms of SDF-induced endothelial cell proliferation and migration.
    Kuhlmann CRW, Schaefer CA, Reinhold L, Tillmanns H, Erdogan A
    Biochemical Biophysical Res.Communications (2005) 335:1107-1114   [Christoph R.W. Kuhlmann et al.] .
    The aim of our study was to investigate the effect of stromal-derived factor-1-alpha (SDF-1-alpha) on endothelial angiogenic effects. SDF-1-alpha (50 ng/ml) increased the number of cultured endothelial cells from 33,653 ± 1183 to 55,398 ± 2741, which significantly reduced by adding the BK(Ca)-inhibitor iberiotoxin, or the endothelial nitric oxide synthase-blocker, L-NMMA (n = 24, p < 0.05). Using the 'Fences'-assay a significant increase of HUVEC migration induced by SDF-1-alpha was reported, which was blocked by the addition of iberiotoxin or L-NMMA (n = 12, p < 0.05). BK(Ca) open-state probability (NPo) was analysed using the patch-clamp technique and NPo was increased from 0.003 (control) to 0.052 (SDF-1-alpha; n = 10, p < 0.05). NO synthesis was measured using a cGMP-radioimmunoassay. A significant increase of cGMP levels from 0.952 pmol/mg protein to 2.179 pmol/mg protein was observed, that was abolished by L-NMMA and significantly reduced by iberiotoxin (n=15, p<0.05). SDF-1-alpha increases endothelial proliferation and migration involving the activation of BK(Ca) and an increased production of NO.
  • Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current.
    Kuhlmann CRW, Scharbrodt W, Schaefer CA, Most AK, Backenköhler U, Neumann T, Tillmanns H, Waldecker B, Erdogan A, Wiecha J
    J.Molecular Cellular Cardiol. (2005) 38,2:315-322  
    The inward rectifier K+ current (K(ir)) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate K(ir) and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endothelium-dependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates K(ir) and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic K(ir), which were blocked by extracellular barium (100 µmol/l). Perfusion with nicotine (1 nmol/l-10 µmol/l) revealed a dose-dependent reduction of K(ir). The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 µmol/l) still significantly reduced K(ir) (n = 8; P < 0.01). Cell counts revealed that bFGF-mediated proliferation of HUVEC was significantly inhibited when using 1-10 µmol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration--examined using the 'Fences-Migration-Assay'--was significantly reduced by 10 mumol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of K(ir) seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation.
  • Osteoblast alignment, elongation and migration on grooved polystyrene surfaces patterned by Langmuir-Blodgett lithography.
    Lenhert S, Meier M-B, Meyer U, Chi L, Wiesmann HP
    Biomaterials (2005) 26,5: 563-570   [Steven Lenhert et al.] .
    Topographically patterned surfaces are known to influence cellular behavior in a controllable manner. However, the relatively large surface areas (several cm2) required for many biomaterial applications are beyond the practical limits of traditional lithography. Langmuir-Blodgett lithography, a recently developed method, was used to fabricate regularly spaced grooves of different depths (50 and 150 nm) with a periodicity of 500 nm over several square centimeter on silicon surfaces. These topographies were transferred into polystyrene surfaces by means of nanoimprinting. Primary osteoblasts were cultured on the patterned polymer surfaces. They were observed to align, elongate and migrate parallel to the grooves. The combination of Langmuir-Blodgett lithography with nanoimprinting enables the fabrication of large, nanostructured surface areas on a wide spectrum of different biomaterials. Osteoblasts show a significant anisotropic behavior to these surfaces, which can enhance cell settlement on the surface or be used to direct tissue generation on the biomaterial interface.
  • Fabrication and characterisation of degradable biosynthetic hydrogels for cell encapsulation: development of a new method for protein incorporation.
    Lim KS.
    PhD thesis, University of New South Wales (2014)   [Khoon S. Lim] .
    Biosynthetic hydrogels which have tailorable physical properties as well as the desired biological attributes to support cellular interaction have emerged as potential biomaterials for cell encapsulation. These hydrogels are normaUy fabricated by incorporating biological polymers into a synthetic hydrogel network. However, long term use of these matrices requires the biological molecules to be covalently bound into the network. This stable integration can be achieved by chemically modifying the biological polymer with short linear polymer chains, such as poly(ethylene glycol) (PEG), or functional moieties like acrylate and methacrylates. However, these chemical functionalisation processes may impose degradation and denaturation of the biological molecules, as well as disrupting the bioactive side groups that are required for cellular interactions.
    Therefore, the overall aim of this research is to covalently incorporate biological molecules into synthetic hydrogels without the need of prior chemical modification. A visible light polymerisation system consisting of ruthenium and persulphate that was previously shown to crosslink proteins through their phenolic residues was employed. It was shown that by grafting phenolic containing moieties, such as tyramine onto poly(vinyl alcohol) (PVA-Tyr), the resultant PVA-Tyr was able to be crosslinked in a similar manner to proteins using the ruthenium/persulphate system. The physical properties of the hydrogels were tailorable through varying the nominal macromer concentration. Non-chemically modified gelatin was successfully covalently integrated into the PVA-Tyr hydrogels, without affecting the base characteristics (mass loss, swelling and degradation profile) of PVA-Tyr, but also retained the bioactivity to support cells in 2D culture (fibroblasts, endothelial, Scbwann cells).
    Fibroblasts were also encapsulated inside the PVA-Tyr gels, where it was showed that the presence of the antioxidative protein, sericin was needed to ensure survival of the cells during the photoencapsulation process. However, both sericin and gelatin were required synergistically to facilitate long-term 3D cell growth, proliferation and function. The encapsulated cells were able to form clusters and interconnected networks, as well as remained metabolically active after 21 days in culture. This work has demonstrated a novel method to covalently incorporate proteins in their native state into synthetic hydrogels.
  • Optimization of crosslinking parameters for biosynthetic poly(vinyl-alcohol)-tyramine hydrogels.
    Lim KS, Ramaswamy Y, Alves MH, Green RA, Poole-Warren LA, Martens PJ.
    IFMBE Proceedings 51, Springer International Publishing Switzerland (2015)   [Khoon S Lim et al.] .
    Photo-polymerizable hydrogels have been widely researched as tissue engineering matrices. When designing a new photo-crosslinkable, biosynthetic hydrogel system, a number of parameters need to be optimized, such as the polymerization conditions and amount of biological polymer included. This study aimed to investigate the crosslinking parameters (i.e., choice of initiator, light intensity and irradiation time), as well as the biological polymer (i.e., gelatin) content, for a degradable tyramine functionalized poly(vinyl alcohol) (PVA-Tyr) system. This PVA-Tyr can be photocrosslinked using a visible light initiated process composed of ruthenium (Ru) and persulfate compounds. Comparison of ammonium persulfate (APS) and sodium persulfate (SPS) showed that SPS supported fabrication of higher quality gels at lower concentrations than APS. The initiator concentration and irradiation conditions that were found to produce the best quality PVATyr gels were 2 mM Ru/20 mM SPS and 3 minutes of 15 mW/cm2 of visible light. Moreover, incorporation of gelatin into the PVA-Tyr gels successfully facilitated attachment of Schwann cells on the gels. The Schwann cells were able to survive and proliferate over 3 days on the PVA-Tyr/gelatin gels. Overall, this study showed that PVA-Tyr gels have high potential as biomaterials for tissue engineering applications.
  • Der Einfluss der extrazellulären Matrix auf den Phänotyp, die Gen- und Proteinexpression und die EGFR-Inhibition bei kolorektalen Tumorzelllinien.
    Luca AC
    PhD thesis, Heinrich-Heine-Universität Düsseldorf (2011)   [Anna Clarissa Luca] .
    The cultivation of permanent tumour cell lines represents the most important approach to conduct mechanistic studies on human tumour cells in vitro under controlled conditions. The cultivation of tumour cells as a two-dimensional (2D) monolayer on plastic substrates is still the most commonly used cultivation method although the cells are completely separated from their natural environment. Therefore, culture systems have been developed that allow a more realistic tumour modelling in vitro. In this context, the extracellular matrix (ECM) model appears to be particularly interesting because the ECM is an integral component of the natural environment of tumour cells in vivo interacting with the cells and providing them with essential signals for cell polarity, growth and cell survival. The aim of this study was to establish an ECM culture model for representative, permanent colorectal cancer (CRC) cell lines and to further systematically investigate the impact of the two different cell culture systems in cell biological and molecular terms. For the ECM culture (<on-top> assay), cells were embedded in laminin-rich extracellular matrix (lrECM). All cell lines formed tumour-like structures with a typical as well as reproducible morphology in the lrECM <on-top> model. The 3D morphologies were not correlated to the 2D phenotype of the CRC cells lines. Two different spheroid types were identified: a grape-like morphology with loose cell-cell interactions and a compact growing, mass-like spheroid. Interestingly, all grape-like spheroids were generated from cell lines derived from metastases, while all cell lines derived from primary tumours formed compact growing spheroids. A correlation between the migratory or the invasive capacity and the two different morphology types was not observed. Grape-like spheroids showed a significantly reduced overall cell proliferation compared to the compact growing spheroid type in the lrECM <on-top> assay. The observed, extensive morphological alterations in the lrECM <on-top> assay compared to 2D culture on plastic substrata were associated with measurable expression changes on the transcription- and protein-level. This was shown by the analysis of selected genes that play a crucial role in ECM-cell-interaction, cell-cell-adhesion und cell proliferation. In order to investigate the observed changes at the transcriptional level more globally, a transcriptome analysis of all seven investigated CRC cell lines was performed using Agilent Arrays. Hierarchal cluster analysis separated clearly the two culture conditions for every cell line according to their expression profiles demonstrating the considerable impact of the lrECM <on-top> culture on the cell lines. A set of 225 significantly differentially regulated genes was identified. Fourteen of these genes are known interaction partners and most of them are involved in cell proliferation and apoptosis. This observation might explain the reduction of cell proliferation by 10-30% that was noted under lrECM <on-top> conditions. Furthermore, upon lrECM culture condition a reduced EGFR expression as well as a differential activation within the EGFR-pathway was observed. This resulted in a statistically significant decreased response to anti-EGFR therapy, which is from a clinical point of view a relevant finding considering the importance of EGFR for CRC therapy. Taken together, the lrECM <on-top> cell culture model seems to represent a more physiological in vitro CRC tumour model. Of course, this model cannot represent all CRC progression steps from tumour initiation to metastasis. However, it should be a suitable model for the development of micrometastases, a most critical step for metastasis formation. In addition, the present work provides a first insight into the molecular changes associated with the interaction of CRC tumour cell lines with the lrECM. Since this lrECM model could be easily extended to a high-throughput scale, it could represent an effective in vitro strategy for preclinical therapeutic studies to test new adjuvant therapeutic substances under more realistic conditions.
  • Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.
    Luca AC, Mersch S, Deenen R, Schmidt S, Messner I, Schäfer K-L, Baldus SE, Huckenbeck W, Piekorz RP, Knoefel WT, Krieg A, Stoecklein NH
    PLoS ONE (2013) 8,3: e59689.   [Anna Clarissa Luca] doi:10.1371/journal.pone.0059689 .
    Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.
  • The effect of amphiphilic siloxane oligomers on fibroblast and keratinocyte proliferation and apoptosis.
    Lynam E, Xie Y, Loli B, Dargaville TR, Leavesley DI, George G, Upton Z
    J.Biomed.Mater.Res.Part A (2010) 95A(2):620-631   [Emily Lynam] .
    The formation of hypertrophic scars (HSF) is a frequent medical outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS) or apoptosis-inducing agents, including bleomycin. Although widely used, knowledge regarding SGS and their mode of action is limited. Preliminary research has shown that small amounts of amphiphilic silicone present in SGS have the ability to move into skin during treatment. We demonstrate herein that a commercially available analogue of these amphiphilic siloxane species, the rake copolymer GP226, decreases collagen synthesis on exposure to cultures of fibroblasts derived from HSF. By size exclusion chromatography, GP226 was found to be a mixture of siloxane species, containing five fractions of different molecular weight. By studies of collagen production, cell viability and proliferation, it was revealed that a low molecular weight fraction (fraction IV) was the most active, reducing the number of viable cells present after treatment and thereby reducing collagen production as a result. On exposure of fraction IV to human keratinocytes, viability and proliferation were also significantly affected. HSF undergoing apoptosis after application of fraction IV were also detected via real-time microscopy and by using the TUNEL assay. Taken together, these data suggests that these amphiphilic siloxanes could be potential non-invasive substitutes to apoptotic-inducing chemical agents that are currently used as scar treatments.
  • Growth factor-induced cell migration: biology and methods of analysis.
    Manske M, Bade EG
    Int.Rev.Cytol. (1994) 155:49-96
    Cell migrations are at the basis of metazoan life. Starting with the migration of the sperm and ending with the invasive behavior of malignant tumor cells that finally kill their host, cell translocations over considerable dis-tances are an essential part of the dynamics of higher eukaryotes. The implantation and development of the embryo, the development of the nervous system and the gonads as well as metamorphosis and, in the adult organism, the renewal of surface epithelia, all require a precise control of cell migration at multiple stages. These precise and cell-specific controls are altered or lost when tumor cells become invasive and metastatic.
    Much of our current information on cell migration has been obtained from in vitro studies with embryonic and tumor cells. Malignant tumors have provided 'motile' and 'immortalized' cells, and have also been the source of proteins that were initially isolated as 'growth' (i.e., proliferation-stimulating) factors. Surprisingly, however, their role as migration modulators was recognized only very gradually. The first growth factor known, nerve growth factor (NGF), discovered as a secreted product of a malignant tumor, and the transforming growth factors (TGFs), first detected in the conditioned media of cultured malignant tumor cells are now known to have multiple functions as modulators of cell proliferation and migration. The concept of angiogenesis as a crucial step in tumor development stimulated the search for and analysis of factors that modulate the proliferation and migration of endothelial cells.
    Our current knowledge of cell migration and its regulation has been obtained mainly through the analysis of cell culture systems. Accordingly, a brief review of a number of commonly used systems is included (Section Since some systems might have been involuntarily overlooked, the authors would appreciate comments on these omissions. ...
  • Salt-leached porous scaffolds functionalized with VEGF for Islets of Langerhans transplantation.
    Marchioli G, Hertsig D, Engelse M, de Koning E, Karperien M, Moroni L, van Apeldoorn A
    in: A journey towards an extrahepatic islet delivery device with a tissue engineering toolbox in hand (2016) Thesis: University of Twente, Enschede, Chap.4   [Giulia Marchioli]
    Cell migrations are at the basis of metazoan life. Starting with the migration of the sperm and ending with the invasive behavior of malignant tumor cells that finally kill their host, cell translocations over considerable dis-tances are an essential part of the dynamics of higher eukaryotes. The implantation and development of the embryo, the development of the nervous system and the gonads as well as metamorphosis and, in the adult organism, the renewal of surface epithelia, all require a precise control of cell migration at multiple stages. These precise and cell-specific controls are altered or lost when tumor cells become invasive and metastatic.
    Much of our current information on cell migration has been obtained from in vitro studies with embryonic and tumor cells. Malignant tumors have provided 'motile' and 'immortalized' cells, and have also been the source of proteins that were initially isolated as 'growth' (i.e., proliferation-stimulating) factors. Surprisingly, however, their role as migration modulators was recognized only very gradually. The first growth factor known, nerve growth factor (NGF), discovered as a secreted product of a malignant tumor, and the transforming growth factors (TGFs), first detected in the conditioned media of cultured malignant tumor cells are now known to have multiple functions as modulators of cell proliferation and migration. The concept of angiogenesis as a crucial step in tumor development stimulated the search for and analysis of factors that modulate the proliferation and migration of endothelial cells.
    Our current knowledge of cell migration and its regulation has been obtained mainly through the analysis of cell culture systems. Accordingly, a brief review of a number of commonly used systems is included (Section Since some systems might have been involuntarily overlooked, the authors would appreciate comments on these omissions. ...
  • Albumin-binding surfaces: synthesis and characterization.
    McFarland CD, Jenkins M, Griesser HJ, Chatelier RC, Steele JG, Underwood PA
    J.Biomater.Sci.Polym.-Ed. (1998) 9,11:1207-1225
    The nature of the proteinaceous film deposited on a biomaterial surface following implantation is a key determinant of the subsequent biological response. To achieve selectivity in the formation of this film, monoclonal antibodies have been coupled to a range of solid substrates using avidin-biotin technology. Antibody clones varied in their antigen-binding activity following insertion of biotin groups into lysine residues. Biotinylated antibodies coupled to solid substrates via an immobilized avidin bridge retained their biological activity. During immobilization of avidin a significant proportion of the protein molecules were passively adsorbed rather than covalently attached to the surface. This loosely bound material could be removed by stringent elution procedures which resulted in a surface density of 5.4 pmol avidin cm(-2). Although these conditions would be harsh enough to denature monoclonal antibodies, they did not destroy the biotin-binding activity of the residual surface-coupled avidin, enabling the subsequent immobilization of biotinylated antibodies. The two-step immobilization technique allowed the use of gentle protein modification procedures, reduced the risk of surface-induced denaturation and removed loosely bound material from the surface. The versatility of the technique encourages its application to a wide range of immobilization systems where retention of biological activity is a key requirement
  • Albumin-binding surfaces: in vitro activity.
    McFarland CD, De Filippis C, Jenkins M, Tunstell A, Rhodes NP, Williams DF, Steele JG
    J.Biomater.Sci.Polym.Ed. (1998) 9,11:1227-1239
    Immobilized monoclonal antibodies (Mabs) have been used to attract specific molecules to a solid surface from complex mixtures such as blood, plasma or serum, thereby directing the response to the modified substrate, a key goal in rational biomaterial design. The nature of the Mab dictated the nature of the response: anti-albumin antibodies were used to prevent cell and platelet adhesion in vitro, whilst anti-fibronectin Mabs promoted attachment. Patterned surfaces could be formed, bearing Mabs that generated adhesive and non-adhesive regions. Fibrinogen adsorption from plasma showed a Vroman peak on unmodified control polymer, which was reduced by 64% in the presence of surface-bound anti-albumin Mab. Immobilization of a control Mab reduced fibrinogen adsorption only slightly, implying an albumin-mediated effect. In static tests, platelet adhesion from human platelet rich plasma was significantly reduced by the immobilization of anti-HSA Mab when compared to the untreated FEP surface (p < 0.0001). This effect was also seen with citrated blood flowing through Mab-treated polyurethane tubing at a shear rate of 132 s(-1) (p=0.034). Since platelets and proteins (as blood, plasma or serum) were introduced to the surface simultaneously, the generation of a defined protein film must have been sufficiently rapid as to shape the platelet or cell response.
  • Protein adsorption and cell attachment to patterned surfaces.
    McFarland CD, Thomas CH, DeFilippis C, Steele JG, Healy KE
    J.Biomed.Mater.Res. (1999) 49,2:200-210
    To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bone-derived cells (HBDC) to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS). At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin (Vn) from serum onto the EDS domains. In contrast, fibronectin (Fn) was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. in this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function.
  • Attenuated kallikrein-related peptidase activity disrupts desquamation and leads to stratum corneum thickening in human skin equivalent models.
    McGovern JA, Meinert C, de Veer SJ, Hollier BG, Parker TJ, Upton Z .
    Br.J.Dermatol. (2016) 176:145-158   [Jacqui Anne McGovern et al.] .
    -Background- Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation, however human skin equivalent (HSE) models are known to excessively differentiate. In native tissue, proteases such as kallikrein-related peptidase (KLK) 5, and KLK7 cleave the extracellular components of corneodesmomes; proteins corneodesmosin (CDSN), desmocollin 1 (DSC1) and desmoglein 1 (DSG1), loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors; skin-derived antileukoproteinase (SKALP), and lympho-epithelial Kazal-type-related inhibitor (LEKTI) which also inhibits KLK5, localising protease activity to the stratum corneum.
    -Objectives- To investigate the mechanisms which inhibit the desquamation cascade in HSE models.
    -Methods- Human skin tissue and HSE models were investigated using gene microarray, real-time PCR, immunohistochemistry, and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in both HSEs and native skin, in situ and gel zymography was performed.
    -Results- Histological analysis indicated that HSE models form a well-organised epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin.
    -Conclusions- These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosomes degradation in the HSE model epidermis.
  • Investigating epidermogenesis in a human skin equivalent model.
    McGovern JA
    PhD thesis, Queensland University of Technology (2012)   [Jacqui Anne McGovern] .
    Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy.
    There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment.
    The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment an a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.
  • Optimization of (phenylmethylidene)-hydantoins as prostate cancer migration inhibitors: SAR-directed design, synthesis, and pharmacophore modeling.
    Mudit M, El Sayed KA
    Chemistry & Biodiversity (2011) 8,8:1470-1485  
    Prostate cancer is one of the most common cancer forms among males of Western countries. Natural products proved to be an unparalleled source of molecular diversity. The 4-(hydroxyphenylmethylidene)hydantoin (PMH; 1), (5Z)-5-(4-hydroxybenzylidene)imidazolidine-2,4-dione, was isolated from the Red Sea sponge Hemimycale arabica, and recently showed junctional complexes stabilization, anti-invasive, and antimetastatic activities in vitro and in vivo. The related synthetic analogue, (5Z)-5-[4-(ethylsulfanyl)benzylidene]imidazolidine-2,4-dione (2), showed several-fold-improved in vivo antimetastatic properties against the highly invasive prostate cancer. To further optimize the activity of PMHs, various ligand-based strategies were used including the extension of the structure, structural simplification, linker extension, and computer-assisted CoMFA (Comparative Molecular Field Analysis) results. These strategies yielded thirty 2nd-generation PMHs, designed based on the 1st-generation PMHs, such as 1 and 2. Wound-healing assay was selected to evaluate the in vitro anti-migratory potential of these new PMHs against the PC-3 cell line. Several active PMHs, including 10, 13, 24, 29, with nearly twelvefold enhancement of activity vs.2, were identified. Active compounds were then used to build a pharmacophore model using the SYBYL's DIStance COmparison technique (DISCOtech). Active PMHs were also screened for fragment-based drug likeness using the OSIRIS program, and an overall drug score was also calculated. Interestingly, the overall drug scores of 24 and 29 along with their anti-migratory activity were significantly greater than those of 1 and 2. In conclusion, PMHs can be the appropriate scaffolds for the urgently needed drug candidates for the control of androgen independent prostate cancer.
  • Serum-free primary human fibroblast and keratinocyte coculture.
    Mujaj S, Manton K, Upton Z, Richards S
    Tissue Eng.Part A (2010) 16,4:1407-1420  
    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins.
  • Endothelial cell migration assays
    Polytarchou C, Hatziapostolou M, Papadimitriou E .
    - in the book ;   Angiogenesis assays: A critical appraisal of current techniques
    Eds.: Staton CA, Lewis C, Bicknell R ;   John Wiley & Sons (2007)
    Migration of endothelial cells plays a central role in many multi-step and complex physiological and pathological events, such as the repair of the blood vessel wall after tissue injury, in vasculogenesis during embryonic development and in angiogenesis during tumour growth. A number of in vitro endothelial cell migration assays have been developed, in an effort to isolate endothelial cell migration from this complexity. Among these are the transfilter assay, the wound healing assay, the Teflon fence assay and the phagokinetic track assay. There is also increasing interest and effort in developing assay(s) for assessing endothelial cell migration in vivo. Such approaches inciude the aortic ring assay, the Matrigel plug assay and, most recently, the zebrafish assay. A critical appraisal of the strengths and weaknesses, as well as recent applications and developments of these assays are discussed.
  • A method to assess multiple aspects of the motile behaviour of adherent PC12 cells on applied biological substrates
    Rankin SL, Rahimtula M, Mearow KM
    J. Neurosci. Meth. (2006) 156,1-2:55-63   [Reza Riahi et al.] .
    Cellular migration is central to a wide range of biological and pathological processes in vivo. In vitro cell migration assays can be used to obtain invaluable information relating to the mechanism of cell movement, but current available methods can be limiting. Here we describe a novel motility assay that allows the simultaneous investigation of both quantitative and qualitative aspects of a population of motile cells as they move across a variety of substrates. By plating cells in a confluent monolayer on a coverslip, the monolayer can then be inverted to migrate over a larger substrate-coated coverslip, which can subsequently be reliably quantified, and subjected to immunocytochemistry and confocal imaging. This assay can be used to assess multiple aspects of motility, including distance, quantity, morphology, polarization and component colocalization. To demonstrate the utility of this assay, it was applied to the study of a stimulator of PC12 cell migration, nerve growth factor (NGF), and how this migration is influenced by the extracellular substrate, laminin. Furthermore, since mutations to the NGF receptor, TrkA, have been noted to alter the behaviour of PC12 cells in response to NGF, a PC12 subline that expresses a mutated TrkA receptor was utilized to illustrate that a Y785F mutation in the cytoplasmic tail of TrkA results in increased migration in response to the stimulus compared to the control PC12s.
  • Signalling pathways involved in the inhibition and promotion of axonal regeneratiom.
    Rankin SL
    PhD thesis, Memorial University of Newfoundland (2008) -   [Sherri L. Rankin] .
    Axonal regeneration following nerve injury requires the complex orchestration of various molecular events. Neurite outgrowth can be initiated by a variety of cues from the extracellular environment, including neurotrophins (e.g. nerve growth factor; NGF) and the extracellular matrix (ECM). Biological responses to neurotrophins are mediated by two distinct receptors: Trks, which initiate distinct signalling for the promotion of growth and survival, and p75NTR. Signalling pathways initiated by p75NTR exhibit considerable complexity and can elicit a variety of paradoxical physiological responses depending upon cellular context. Biological responses to ECM components, including laminin (LN), are mediated by a group of receptors known as integrins, which facilitate signal transmission to regulate cellular behaviour. Signalling pathways initiated by NGF and LN, have been previously reported to synergize resulting in optimized axonal regeneration of sensory neurons. The present series of studies sought to explore the molecular mechanisms underlying the enhanced growth initiated by stimulation with neurotrophies and LN, with a particular focus on the roles of the NGF receptors; specifically, TrkA phosphorylation-induced signalling cascades, and events associated with p75NTR ligand-dependent and independent signalling.
    My initial studies utilized a series of PCl2 cell derivatives expressing TrkA phosphorylation mutants, to investigate the potential role of TrkA in the regulation of p75NTR expression. I determined that TrkA played a role in the regulation of constitutive p75NTR expression, and further, controlled the up-regulation of p75NTR in response to neurotrophin stimulation. In a subsequent study, l demonstrated that this occurred via a phospholipase C y-protein kinase C 8-dependent mechanism, and confirmed the existence of this regulatory pathway in cerebellar granule neurons (CGN). I further investigated the contribution of the ECM to regenerative growth, in both its capacity to signal synergistically with the TrkA receptor for the enhancement of early signalling intermediates, and its ability to elicit growth in a neurotrophin-independent scenario. Strikingly, integrin activation in the absence of neurotrophins was responsible for the promotion of neurite outgrowth via a rapid and potent Egr-I-dependent increase in the expression of the phosphatase PTEN, which relocalized to the nucleus where it dephosphorylated transcription factor SpI, thereby decreasing its ability to bind to the p75NTR promoter, resulting in the subsequent down-regulation of p75NTR and depression of Rho activity. This novel ECM-induced signalling paradigm was also determined to occur in CGNs, and following the development of a unique motility assay. I demonstrated that interference with this cascade impaired motility, suggesting that this signalling cascade may contribute to the developmental migration of CGNs.
    p75NTR is a unique and flexible pleiotropic receptor which may promote or inhibit cell growth depending upon the presence or absence of neurotrophins. Taken together, the results of these studies detail the mechanisms involved in the up-regulation of p75NTR expression in the presence of neurotrophins, but additionally present a novel signalling paradigm initiated by the ECM for the down-regulation of the p75NTR in the absence of neurotrophin stimulation. Interestingly, both scenarios result in the promotion of neurite outgrowth and cellular motility as a result of the flexible signalling interactions of p75NTR, panicularly those involving Rho, which can directly influence cytoskeletal dynamics.
  • Advances in wound-healing assays for probing collective cell migration. (Review)
    Riahi R, Yang YL, Zhang DD, Wong PK
    J.Lab.Autom. (2012) 17,1:59-65   [Reza Riahi et al.] .   or [R R etc.] .
    Collective cell migration plays essential roles in a wide spectrum of biological processes, such as embryogenesis, tissue regeneration, and cancer metastasis. Numerous wound-healing assays based on mechanical, chemical, optical, and electrical approaches have been developed to create model <wounds> in cell monolayers to study the collective cell migration processes. These approaches can result in different microenvironments for cells to migrate and possess diverse assay characteristics in terms of simplicity, throughput, reproducibility, and multiplexability. In this review, we provide an overview of advances in wound-healing assays and discuss their advantages and limitations in studying collective cell migration.
  • Polissacarídeos sulfatados de algas marinhas: formulação de géis e análise dos seus efeitos na cicatrização em experimentos in vivo e in vitro. (engl.: Sulphate polystacarides of seaweeds: formulation of gels and analysis of their effects on wound healing in in vivo and in vitro experiments.)
    Ribeiro NA.
    Thesis: (2016) Universidade Federal Ceará, Fortaleza   [NatásSsia Albuquerque Ribeiro] .
    A utilização de algas marinhas como ferramentas potenciais para a obtenção de produtos farmacêuticos tem se mostrado uma tendência mundial e cresceu substancialmente nas duas últimas décadas. Moléculas bioativas obtidas a partir de algas marinhas tais como proteínas, carboidratos, compostos fenólicos, ácidos graxos poliinsaturados, terpenóides etc. possuem inúmeras atividades biológicas. Dentre essas moléculas, destacam-se os polissacarídeos sulfatados de algas marinhas. Este trabalho teve como objetivo avaliar a eficácia do potencial cicatrizante de géis desenvolvidos à base de polissacarídeos sulfatados das algas marinhas: Caulerpa racemosa (CrII), Cryptonemia crenulata (CcII) e Gracilaria birdiae (GbI), nativas do litoral cearense em modelos de cicatrização in vivo e in vitro. Os polissacarídeos sulfatados foram obtidos por procedimento cromatográfico de troca iônica. As macromoléculas obtidas foram avaliadas em ensaio toxicológico e analisadas quanto ao seu potencial de interferir no sistema nervoso central (SNC). Posteriormente foram utilizadas em experimentos de atividades pró-coagulante in vitro/ex vivo e antibacteriana. Em seguida os polissacarídeos sulfatados foram utilizados na formulação de géis que foram avaliados quanto à sua irritabilidade dermal durante 7 dias, onde foi analisado as reações dermatológicas macroscópicas, espessura da pele e o valor de exsudato presente no tecido. Os géis foram aplicados em feridas induzidas em camundongos sadios por trauma mecânico e avaliados quanto ao seu potencial cicatrizante durante 15 dias. Uma avaliação histopatológica foi realizada para comprovação da eficiência dos géis no tratamento das lesões. CcII foi utilizado em experimentos in vitro onde foi observado o seu potencial de aderência, proliferação e citotoxicidade utilizando células HaCaT. E por fim, CcII foi avaliado em um modelo 3D de equivalentes de pele humana (3D EPHs) durante 7 dias, onde foi analisado parâmetros como área de re-epitelização e migração lateral dos queratinócitos. Os resultados observados demonstraram que CrII, CcII e GbI apresentaram rendimentos de 20,5%; 21,3% e 23,6%, respectivamente, e mostraram-se atóxicos e sem efeitos sobre o SNC. No ensaio de atividade pró-coagulante CrII demonstrou ser pró-coagulante e CcII apresentou atividade anticoagulante no ensaio do APTT in vitro. Entretanto, estas atividades não foram confirmadas no ensaio ex vivo. O polissacarídeo de GbI não apresentou atividades anticoagulante e pró-coagulante em nenhum dos ensaios testados. CrII, CcII e GbI não apresentaram atividade antibacteriana frente às bactérias gram-negativa e gram-positiva. Os géis formulados com CrII, CcII e GbI não se mostraram irritantes em um modelo de irritação cutânea em camundongos. E quando testados em feridas, apenas o gel formulado com CcII apresentou efeito cicatrizante. Diante deste fato, CcII foi avaliada em experimentos in vitro, em que, não foi capaz de provocar a aderência, proliferação e citotoxidade em células HaCaT. CcII quando analisada no modelo 3D EPHs apresentou um efeito cicatrizante, acelerando o processo de re-epitelização e migração dos queratinócitos. Tendo em vista os resultados obtidos, CcII apresenta um potencial para formulação de um bioproduto promissor com uma função de cicatrização.
    The use of marine algae as potential tools for obtaining pharmaceuticals has shown a worldwide trend and has grown substantially over the last two decades. Bioactive Molecules obtained from seaweeds such as proteins, carbohydrates, phenolics, polyunsaturated fatty acids, terpenoids, etc. they showed numerous biological activities. Among these molecules, it highlights the sulfated polysaccharides from seaweed. This work aimed to evaluate the effect of the healing potential gels developed from sulfated polysaccharide of seaweed: Caulerpa racemosa (CrII), Cryptonemia crenulata (CcII) e Gracilaria birdiae (GbI), native of Cear in healing in vivo and in vitro models. The sulfated polysaccharides were obtained by chromatographic procedure of ion exchange. The macromolecules obtained were evaluated in toxicological testing and analyzed for their potential to interfere in the central nervous system (CNS). Later, it was used in procoagulant in vitro/ex vivo and antibacterial activity experiments. Then the sulfated polysaccharides were used in the formulation of gels that were evaluated for their dermal irritant for 7 days, which was analyzed macroscopic skin reactions, skin thickness and the amount of exudate present in the tissue. The gels were applied to wounds in healthy mice induced by mechanical trauma and evaluated for their potential healing for 15 days. A histopathological evaluation was performed to prove the efficiency of the gels in the treatment of injuries. CcII was used in in vitro experiments where it was observed its potential adhesion, proliferation and cytotoxicity using HaCaT cells. Finally, CcII was evaluated in a 3D model of human skin equivalent (3D EPHS) for 7 days, which was analyzed parameters such as re-epithelialization area and lateral migration of keratinocytes. The results obtained showed that CrII, CcII and GbI present income 20.5%; 21.3% and 23.6%, respectively, and shown to be non-toxic and without CNS effects. In the coagulant test, CrII shown to be pro-coagulant and CcII showed anticoagulant activity in APTT assay in vitro. However, these activities have not been confirmed in the ex vivo assay. The GbI polysaccharide showed no anticoagulant and procoagulant activities in any of the assays tested. CrII, CcII and GbI showed no antibacterial activity against gram-negative and gram-positive bacteria. The gels formulated with CrII, CcII and GbI were not irritating in a skin irritation model in mice. And, when tested in wounds, only the gel formulated with CcII presented healing effect. Considering this fact, CcII was evaluated in experiments in vitro where it was not able to cause adherence, proliferation and cytotoxicity in HaCaT cells. CcII when analysed in the 3D EPHs model has a healing effect, accelerating the process of re-epithelization, and keratinocytes migrating. In view of the results obtained, CcII has a promising potential for formulating bioproduct with a healing activity.
  • Development of defined media for the serum-free expansion of primary keratinocytes and human embryonic stem cells.
    Richards S, Vesley DL, Topping G, Upton Z .
    Tissue Engineering C (2008) 14,3: 221-232  
    Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulinlike growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.
  • Towards feeder-free and serum-free growth of cells.
    Richards SD .
    PhD thesis, Queensland University of Technology (2007)   [Sean D. Richards] .
    The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient.
    The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjuction with growth factors and growth factor-bindinq proteins (VN:GF combination), to promote enhanced cell migration and growth through the coactivation of inteqrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types.
    Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation.
    Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.
  • In situ formation of poly(vinyl alcohol)-heparin hydrogels for mild encapsulation and prolonged release of basic fibroblast growth factor and vascular endothelial growth factor.
    Roberts JJ, Farrugia BL, Green RA, Rnjak-Kovacina J, Martens PJ .
    J. Tissue Eng. (2016) 7(1):1-10 -   [Justine J Roberts et al.] .
    Heparin-based hydrogels are attractive for controlled growth factor delivery, due to the native ability of heparin to bind and stabilize growth factors. Basic fibroblast growth factor and vascular endothelial growth factor are heparin-binding growth factors that synergistically enhance angiogenesis. Mild, in situ encapsulation of both basic fibroblast growth factor and vascular endothelial growth factor and subsequent bioactive dual release has not been demonstrated from heparin-crosslinked hydrogels, and the combined long-term delivery of both growth factors from biomaterials is still a major challenge. Both basic fibroblast growth factor and vascular endothelial growth factor were encapsulated in poly(vinyl alcohol)-heparin hydrogels and demonstrated controlled release. A model cell line, BaF32, was used to show bioactivity of heparin and basic fibroblast growth factor released from the gels over multiple days. Released basic fibroblast growth factor promoted higher human umbilical vein endothelial cell outgrowth over 24 h and proliferation for 3 days than the poly(vinyl alcohol)-heparin hydrogels alone. The release of vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels promoted human umbilical vein endothelial cell outgrowth but not significant proliferation. Dual-growth factor release of basic fibroblast growth factor and vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels resulted in a synergistic effect with significantly higher human umbilical vein endothelial cell outgrowth compared to basic fibroblast growth factor or vascular endothelial growth factor alone. Poly(vinyl alcohol)-heparin hydrogels allowed bioactive growth factor encapsulation and provided controlled release of multiple growth factors which is beneficial toward tissue regeneration applications.
  • Development of in vitro model systems to study single and collective cell migration.
    Rolli CG .
    Dr.rer.nat. Ruperto-Carola University of Heidelberg, Germany (2011) -   [Claudio Gavino Rolli] .
    Cell migration is an essential characteristic of both physiological and pathological processes within the human body. In order to study the complex process of cell migration diferent in vitro model systems have been developed in the past. The challenge for all these assays is to provide the cells a substrate that mimics particular properties of the extracellular matrix (ECM) while a high control over experimental parameters and monitoring is desired. However, migration assays commonly used in cell biology and medical research are rather limited in the control over the architecture of the provided matrix on or through which the cells move or by the lack of adequate imaging devices to monitor cell dynamics.
    To overcome some limitations of conventional migration assays, it was the aim of this work to develop two diferent methods and employ them in order to quantify migrative behavior of cells under precisely controlled in vitro conditions.
    The first assay consists of microfabricated three dimensional (3D) scaffolds, which allow to study cell migration dynamics through confined environments via live-cell imaging. Channel structures of precisely defined dimensions were utilized to quantify the invasiveness of single cancer cells with respect to modifications of their cytoskeleton organization. In addition, dynamical migration patterns of the cells inside these confined 3D environments were analyzed and found to be significantly changed from their counterparts on at, two dimensional (2D), surfaces. Furthermore, it was shown that such microfabricated structures could be functionalized in the nanometer range with patterns of gold nanoparticles. Thus, the selective binding of ECM-derived ligand motifs, to the gold particles allows for mimicking specific features of the ECM in 3D.
  • Switchable adhesive substrates: revealing geometry dependence in collective cell behavior.
    Rolli CG, Nakayama H, Yamaguchi K, Spatz JP, Kemkemer R, Nakanishi J
    Biomaterials (2012) 33,8:2409-2418
    Collective cell migration plays a major role in cancer metastasis and wound healing, therefore, several in vitro assays for studying such behavior have been developed. Using photoswitchable surfaces, we studied collective cell expansion behavior from initially precisely controlled adhesive patterns. A non-adhesive poly(ethylene glycol) (PEG) layer is conjugated to a glass coverslip via 2-nitrobenzyl groups, which cleave upon exposure to UV light, changing the surface from non-cell-adhesive to cell-adhesive without mechanical interference. Initial cell attaching areas are generated in arbitrary shapes via projection exposure through a photomask. After a growth phase, epithelial cell sheets are released from their confinement by a second illumination allowing for collective cell expansion. Our experiments with epithelial cells show that cluster size and boundary curvature modulate the expansion of the cell sheet and the formation of leader cells. At a certain cluster size, characteristics of the expansion behavior change and cells in the core are hardly affected by the boundary release. With donut-like ring structures, we demonstrate a break in symmetry between the behavior of cells along the outer convex boundary and along the inner concave boundary. Additionally, we observe that collective migration characteristics are modulated by the initial incubation time of the cell sheet.
  • False-positive results with MTT assay.
    Rollino C, Borsa S, Bellone G, Piccoli G, Emanuelli G
    J.Immunol.Meth. (1995) 185,1:141-143
        [   -   ]
  • Der Einfluss von Antibiotika und Antiseptika auf das in vitro Migrationsverhalten von humanen Fibroblasten.
    Rossaint L
    MD thesis, Heinrich-Heine-Universität Düsseldorf (2014)   [Lena Rossaint] .
        [ Zusammenfassung ]
    Chronische Wunden sind vor allem in den westlichen Industrienationen mit großen medizinökonomischen Problemen verbunden. Einen wichtigen Beitrag zum Ablauf der Wundheilung wird von Fibroblasten geleistet. Obwohl der routinemäßige Einsatz von Antibiotika in der Behandlung der meisten chronischen Wunden ohne Vorliegen einer akuten Infektion nicht unterstützt werden kann, erhalten mehr als die Hälfte aller Patienten mit chronischen Wunden diese Therapeutika. Der Einfluss von Antibiotika auf das Migrationsverhalten von Fibroblasten wurde bisher noch nicht untersucht. Mit der vorliegen Arbeit sollte daher untersucht werden, ob und in wie weit die am häufigsten bei chronischen Wunden eingesetzten Antibiotika die Fibroblastenmigration beeinflussen. Als Migrationsmodell wurde hierfür der Fence Assay verwendet.
    Es konnte gezeigt werden, dass Antibiotika die Fibroblastenmigration und -proliferation variabel beeinflussen. So kam es unter Penicillin/Streptomycin in einer 1,5- bzw. 10-fachen in vivo Serumspitzenkonzentration, unter Vancomycin und unter Ciprofloxacin zu einer statistisch signifikanten Hemmung des Fibroblastenverhalten (p<0,05). Während es unter Ampicillin/Sulbactam, Cefuroxim, Clindamycin und Metronidazol zu keiner signifikanten Beeinflussung des Fibroblastenverhaltens kam (p>0,05), zeigten die Fibroblasten unter Moxifloxacin sogar eine gesteigerte Migration und Proliferation (p<0,05). Neben Antibiotika wurden auch Antiseptika und nutritive Faktoren bezüglich ihres Einflusses auf die Fibroblasten untersucht. Alle getesteten Antiseptika führten zu einer statistisch signifikanten Hemmung der Fibroblastenmigration und -proliferation (p<0,05). Fehlten nutritive Faktoren analog der häufig bei Patienten mit chronischen Wunden vorkommenden negativen Stickstoffbilanz und des Serumproteinmangels in Form von fötalem Kälberserum (FCS) während des Fence-Assays, kam es zu einer starken Behinderung von Fibroblastenmotilität und -wachstum (p<0,05). Auch erhöhte Glukosekonzentrationen im Medium 4,5g/l vs. 1g/l Glukose), wie sie beispielsweise bei Diabetes mellitus in vivo vorkommen können, führten zu einer herabgesetzten Migration und Proliferation (p<0,05).
    Die vorliegenden Daten weisen damit darauf hin, dass Antibiotika in der Therapie einer Wundheilungsstörung nicht unkritisch eingesetzt werden sollten, da die vorliegenden Daten nahelegen, dass einige Antibiotika einen negativen Effekt auf Motilität/Proliferation von Fibroblasten haben und damit direkt den Wundheilungsprozess stören könnten.
  • Den diabetiske makroangiopati belyst ud fra en vaskulær biologisk synsvinkel. - Hyaluronsyre og dens receptors (CD44) dannelse og betydning for karvæggen.
    Schultz K
    PhD thesis, Århus Universitet (2004) Ugeskr Læger 2004;166(10):871 .
    Diabetes mellitus er forbundet med en markant øget risiko for udvikling af kardiovaskulære sygdomme. Dette gør sig især gældende for kvinder.
    Mekanismerne bag den høje forekomst af kardiovaskulære sygdomme hos patienter med diabetes er endnu ikke klarlagt, men skyldes til dels strukturelle og funktionelle ændringer i arterievæggen og dermed også en ændring i de arterielle glatmuskelcellers biologi.
    Formålet med dette studie var at undersøge sammenhængen mellem de metabolske forandringer hos diabetespatienter og kvantiteten af DC44-receptor samt den migratoriske effekt af CD44 på humane arterielle glatmuskelceller. Det undersøges således, hvilken indflydelse insulin, glukose, IGF-1 og hGH har ved diabeteslignende betingelser på CD44-ekspression og glatmuskelcellemigration.
    In vitro-studier bekræfter, at CD44-receptor spiller en rolle for glatmuskelcellemigration ved diabeteslignende koncentrationer af glukose, insulin og IGF-1. Derfor indgår CD44-receptor sandsynligvis, når glatmuskelceller akkumuleres i intima hos patienter med diabetes. Endvidere støtter resultaterne, at insulin kan have en signifikant indflydelse på binding af HA, og derved er insulin en faktor, der modulerer vaskulære glatmuskelcellers funktion under udviklingen af makroangiopati hos personer med diabetes.
  • Quantifying the roles of cell motility and cell proliferation in a circular barrier assay.
    Simpson MJ, Treloar KK, Binder BJ, Haridas P, Manton KJ, Leavesley DI, McElwain DLS, Baker RE
    J.R.Soc. Interface (2013) 10, 20130007   [Matthew J Simpson et al.] .
    Moving fronts of cells are essential features of embryonic development, wound repair and cancer metastasis. This paper describes a set of experiments to investigate the roles of random motility and proliferation in driving the spread of an initially confined cell population. The experiments include an analysis of cell spreading when proliferation was inhibited. Our data have been analysed using two mathematical models: a lattice-based discrete model and a related continuum partial differential equation model. We obtain independent estimates of the random motility parameter, D, and the intrinsic proliferation rate, λ, and we confirm that these estimates lead to accurate modelling predictions of the position of the leading edge of the moving front as well as the evolution of the cell density profiles. Previous work suggests that systems with a high λ/D ratio will be characterized by steep fronts, whereas systems with a low λ/D ratio will lead to shallow diffuse fronts and this is confirmed in the present study. Our results provide evidence that continuum models, based on the Fisher-Kolmogorov equation, are a reliable platform upon which we can interpret and predict such experimental observations.
  • Opioid peptides and opiate alkaloids in immunoregulatory processes. (Review)
    Stefano GB, Kream RM
    Arch.Med.Sci. (2010) 6, 3: 456-460   [George B. Stefano, Richard M. Kream] .
    Among the various non-neuronal cell types known to express and utilize neuropeptides, those of the immune system have received much attention in recent years. In particular, comparative studies in vertebrates and invertebrates have shown that endogenous opioid peptides are engaged in receptor mediated autoregulatory immune and neuroendocrine processes. The majority of these immune processes are stimulatory, as determined by their effects on conformational changes indicative of immunocyte activation, cellular motility, and phagocytosis. Endogenous opioid peptides form an effective network of messenger molecules in cooperation with cytokines, opiate alkaloids, and certain regulatory enzymes (neutral endopeptidase 24.11). Peptide-mediated immunostimulatory effects observed in this system are operationally counteracted by the inhibitory effects of morphine and related opiates. Opioid/opiate signaling processes are mediated by several types of receptors with different degrees of selectivity. Among them the recently identified, opioid insensitive µ3 receptor deserves attention on account of its specificity for opiate alkaloids.
  • Targeted delivery of antirestenotic agents. (Review)
    Thomas AC 2002/2003
    PhD thesis, The University of Queensland  
    A major complication of the surgical treatment of arteries blocked by atherosclerotic plaques is restenosis - a reblockage of the artery. In an effort to prevent restenosis a number of drugs have been administered before, during, or after surgical treatment. These include anticoagulants, antithrombotics, antioxidants, antiproliferative agents, anti-inflammatory and immunosuppressive agents, as well as agents that prevent arterial vasospasm. While many of these drugs have been successful in preventing restenosis in animal models, most, if not all, have had only limited success in clinical trials. The reasons for this lack of clinical success can, in many cases, be attributed to the exact dose and administrative regime used in the animal studies not being applied in the clinical study. Complicating the clinical situation is that in order to be active, the anti-restenotic drug must be present at the site of injury both immediately following injury and for sufficient time afterwards the specific window of opportunity. If too little drug is given, or given too late, for too short at time, or in an interrupted fashion, then restenosis may still occur. A way of preventing the complications of many treatments is to deliver the anti-restenotic agents locally rather than systemically. In this manner, the anti-restenotic drug would be directed only to the site of surgery. Thus systemic effects, as well as the cost of treatment, incidence of bleeding complications and incidence of restenosis will all be minimised.
    In the studies described here, we tested the hypothesis that antibodies against cross-linked fibrin (XLF) D-dimer can be used to deliver anti-restenotic agents to areas of fibrin deposition following angioplasty injury of animal arteries. We have called this method DDART - directly delivered anti-restenosis therapy (using anti-D-dimer XLF antibodies).
    Using animal models it was found that XLF is deposited onto the artery wall within 10 minutes of injury and remains present for at least 24 weeks. The best antibody to XLF for use in the rabbit model was found to be H93.7C.1D2/48 (1D2), given at a dose of 30-40 µg/kg. 1D2 was conjugated to the anticoagulant heparin using TV-succinimidyl 3-(2-pyridyldithio)propionate. The purified lD2-heparin was administered to rabbits immediately after carotid artery injury, and changes in cross-section arterial areas measured two weeks later. The injured carotid artery of these animals had a significantly smaller percent luminal narrowing and medial area than rabbits given control agents. They also had minimal neointimal development and a smaller intima to media ratio than rabbits given saline, but not those given unconjugated 1D2. Rabbits given the conjugate had positive expansive (adaptive) remodeling of the entire artery, so that any increase in neointimal area was accommodated without loss of functional lumen.
    1D2 was also conjugated to low molecular weight heparins (LMWHs) using adipic acid dihydrazide. Two weeks after carotid artery injury, rabbits given 1D2-LMWH had a smaller percent medial area of the area inside the external elastic lamina than control rabbits, and a smaller neointima and intima to media ratio than rabbits given saline or LMWH, but not those given unconjugated 1D2. Possibly the presence of the antibody prevented further fibrin binding and thus SMC stimulation. Although a neointimal thickening was present in arteries given 1D2-LMWH conjugate, there was sufficient positive adjustment in the artery for it to be accommodated without losing luminal area. As with rabbits given lD2-heparin, the arteries of rabbits given 1D2-LMWH after injury were able to positively remodel and accommodate the (reduced) bulk of neointima.
    1D2 was then conjugated to the immunosuppressive agent rapamycin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide stabilised with A^-hydroxysuccinimide. Rapamycin has recently been shown to reduce experimental neointimal thickenings and clinical restenosis. lD2-rapamycin was given to rabbits after carotid artery injury, and the arterial areas measured 4 weeks later. The medial area in these rabbits was smaller than in control rabbits, in both injured and uninjured arteries. This resulted in an increased intima to media ratio and a decreased luminal diameter, despite the neointima developed after injury being smaller that in control arteries.
    Thus it has been shown that antibodies against XLF can site-deliver antirestenotic agents to injured areas of the artery wall. The drugs are able to influence smooth muscle cell biology in the injured region and reducing neointimal formation. It is suggested that DDART treatment may be capable of reducing the restenosis that occurs after surgical treatment of atherosclerotic vessels, without excessive systemic complications.
  • 周邊神經修復之奈米生醫材料研發 (Development of nanobiomaterials for peripheral nerve repair.)
    湯正明 (Tang Zhengming) .
    博士 - 中興大學 (Dr. thesis, Chung Hsing University) (2006) -  
    In the first part, a method to prepare hydrogel nerve conduit based on chitosan and PVA was developed. The materials was characterized and the in vitro biocompatibility was investigated. The effect on repair of peripheral nerve injury in short time was be evaluated in animal studis. The functional assessment and histological analyse demonstrated that chitosan and chitosan/PVA 70 /30 conduits were better choices for nerve regeneration.
    In the second part, PCL/ PEG diblock copolymers was used as model surfaces to examine the effect of nanoislands on attachment, proliferation and migration of fibroblasts, and endothelial cells. Our results showed that surface hydrophilicity improved with the increased PEG segments in diblock copolymers and that bacteria adhesion was inhibited by increased PEG contents. PCL-PEG 23:77 showed nanotopography on the surface. The number of adhered endothelial cells, platelets and monocytes on diblock copolymer surfaces was inhibited in PCL-PEG 77:23 and enhanced in PCL-PEG 23:77. Nevertheless, the platelet and monocyte activation on PCL-PEG 23:77 was reduced. PCL-PEG 23:77 had better cellular response as well as lower degree of platelet and monocyte activation. Moreover, the effect of nanoislands on migration of different cells including fibroblasts and endothelial cells was investigated. The water sliding angle of the substrates was measured. The cell migration rate was examined under a real-time optical microscope. It was found that a greater cell migration rate correlated with the smaller sliding angle of the substrate.
    In the third part, two types of polyurethane-gold nanocomposites were prepared. The poly(ether)urethane-gold nanocomposite at 43.5 ppm of gold showed enhanced cellular proliferation, reduced platelet and monocyte activation and much less bacterial adhesion, relative to PU alone or nanocomposites at the other Au contents, in general. This better biocompatibility was associated with the surface morphological change in the presence of Au. The oxidative degradation in nanocomposite at 43.5 ppm of gold was also inhibited. The increased oxidative stability corresponded to the greater free radical scavenging ability of the nanocomposites. Moreover, the morphology, thermal properties, biocompatibility, oxidative degradation and free radical scavenging ability of the poly(ester)urethane-gold nanocomposites were characterized in vitro. The nanocomposite containing a certain amount (43.5-65 ppm) of gold demonstrated the optimal thermal and biological properties. The nanocomposites at 43.5 ppm or 65 ppm of gold exhibited a different surface morphology confirmed by the AFM. They also showed enhanced cellular proliferation, reduced platelet and monocyte activation and much less bacterial adhesion, relative to PU alone or nanocomposites at the other Au contents, in general. This better biocompatibility was associated with the surface morphological change in the presence of Au. PU and nanocomposites were all resistant to oxidative degradation. The nanocomposites had greater free radical scavenging ability. After 19 days of rat implantation, the nanocomposites also showed lower tissue reaction indicated by the smaller fibrous capsule thickness. The significant enhancement of biocompatibility in the nanocomposites in such low Au contents (43.5-65 ppm) appeared to be a result of the extensively modified surface morphology as well as the free radical scavenging effect in the presence of Au.

    論文第一部分採用幾丁聚醣與聚乙烯醇發展為水膠型神經導管的材料,對混摻物性質及生物相容性進行研究,並評估短期對於周邊神經損傷的回復程度,由功能恢復率及組織學分析數據,證實幾丁聚醣或幾丁聚醣/聚乙烯醇導管 (70:30)是周邊神經修復較佳的選擇。考慮適當的機械強度,幾丁聚醣/聚乙烯醇導管(70:30)最適合用於周邊神經修復。
    本論文第二部分研究一奈米相分離的生物可降解高分子的細胞效應,使用聚己內酯-聚乙烯醇雙團聯式共聚合物表面,調查奈米小島對纖維母細胞及內皮細胞貼附及增生的影響。結果顯示在雙團聯式共聚合物中增加聚乙烯醇鏈段會改善表面親水性,而細菌的附著性會被抑制。PCL-PEG 23:77表面具有奈米形態,內皮細胞、血小板及單核球在PCL-PEG 77:23表面貼附的數目被抑制,而在PCL-PEG 23:77會增加。然而在PCL-PEG 23:77表面上,血小板及單核球活化的數目是減少的。如同血小板及單核球活化程度降低,PCL-PEG 23:77 對細胞具有較好的反應。另一方面,調查奈米小島對於不同種類細胞如纖維母細胞及內皮細胞移動行為之影響,其中基質的水滑動角被量測,以曠時攝影系統結合光學顯微鏡調查細胞移動速度,發現較快的細胞移動速率與基質具有較小的滑動角有關
    本論文第三部份研究奈米複合材料生物效應,製備不同形式的聚胺酯-金奈米粒子之複合材料,其中含43.5 ppm金的聚醚型聚胺酯的複合材料顯示,相對於聚胺酯或是不同含量金之聚醚型聚胺酯複合材料,具有增加細胞增生、減少血小板及單核球活化及較少的細菌附著,良好的生物相容性與複合材料表面形態變化有關。在含43.5 ppm金的聚醚型聚胺酯複合材料中發現氧化降解作用被抑制,氧化穩定性的增加與奈米複合材料較高的自由基清除能力有關。另一方面,針對聚酯型聚胺酯-金的奈米複合材料之型態、熱性質、生物相容性、氧化降解及自由基清除能力進行調查,當聚酯型聚胺酯奈米複合材料中含有定量的金奈米粒子(43.5至65 ppm)顯示理想的熱性質及生物性質。藉由原子力顯微鏡確認,在含有43.5 ppm或65 ppm金奈米粒子之聚酯型聚胺酯奈米複合材料中顯出不同的表面形態,相對於聚酯型聚胺酯或是不同濃度金的聚酯型聚胺酯複合材料,顯示出具有增加細胞增生、減少血小板及單核球活化及較少的細菌附著。聚胺酯及其奈米複合材料對氧化降解作用都具有抵抗性,但奈米複合材料有較大的自由基清除能力。植入老鼠19天後,奈米複合材料顯示有較小的纖維包膜厚度,表示具有低度的組織反應。奈米複合材料僅含有非常低量之金奈米粒子(43.5至65 ppm),但生物相容性的確顯著提高,歸因於表面形態的顯著變化及自由基清除能力的結果。
  • Nanometer thickness laser ablation for spatial control of cell attachment.
    Thissen H, Hayes JP, Kingshott P, Johnson G, Harvey EC, Griesser HJ
    Smart Mater.Struct. (2002) 11: 792-799
    We demonstrate here a new method to control the location of cells on surfaces in two dimensions, which can be applied to a number of biomedical applications including diagnostic tests and tissue engineered medical devices. Two-dimensional control over cell attachment is achieved by generation of a spatially controlled surface chemistry that allows control over protein adsorption, a process which mediates cell attachment. Here, we describe the deposition of thin allylamine plasma polymer coatings on silicon wafer and perfluorinated poly(ethylene-co-propylene) substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved in a fast, one-step procedure by nanometer thickness controlled laser ablation using a 248 nm excimer laser. X-ray photoelectron spectroscopy and atomic force microscopy were used to confirm the production of surface chemistry patterns with a resolution of approximately 1 µm, which is significantly below the dimensions of a single mammalian cell. Subsequent adsorption of the extracellular matrix proteins collagen I and fibronectin followed by cell culture experiments using bovine corneal epithelial cells confirmed that cell attachment is controlled by the surface chemistry pattern. The method is an effective tool for use in a number of in vitro and in vivo applications.
  • Development and characterisation of human skin equivalents and their potential application as a burn wound model.
    Topping G, Malda J, Dawson R, Upton Z .
    Primary Intention (2006) 14,1:14-21   [Gemma Topping et al.] .
    Wound healing is a complex physiological process; hence a reproducible in vitro model of skin provides a valuable tool to further understand the biology of dermal wound repair and to investigate techniques to improve wound healing. Human skin equivalent models (HSEs) have been proposed to serve as an in vitro model for these purposes; however, there is currently no readily available HSE model in Australia.
    In this study, we describe the production of a HSE obtained by seeding human keratinocytes onto a de-epidermised dermis, (DED) which was then submerged in medium for 3 days and subsequently cultured at the air-liquid interface for up to 20 days. The model was characterised morphologically and biochemically over the 20 days of culture at the air-liquid interface and showed histological features similar to those observed in an in vivo epidermis. Immunohistochemistry of the epidermal markers keratin 6, keratin 14, keratins 1/10/11 and the basement membrane marker collagen type IV, revealed typical differentiation.
    The HSE was then examined for its potential as a burn wound healing model. Burn wounds were created in the model and the re-epithelialisation of the wounds was followed for 6 days by keratinocyte morphology; metabolic activity was analysed every 2 days. Keratinocytes began to migrate into the wound bed after 2 days and continued to migrate for the next 4 days, suggesting that the HSEs generated may be of great value for studies of the wound healing process and for the evaluation of new therapies.
  • Multiple types of data are required to identify the mechanisms influencing the spatial expansion of melanoma cell colonies.
    Treloar KK, Simpson MJ, Haridas P, Manton KJ, Leavesley DI, McElwain DLS, Baker RE .
    BMC Systems Biology (2013) 7:137 (20p)   [Katrina K Treloar et al.] .
    Background: The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell-to-cell adhesion and cell proliferation. New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process. Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell-to-cell adhesion, despite the fact that cell-to-cell adhesion is thought to play an important role.
    Results: We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell-to-cell adhesion strength, q, and cell proliferation rate, λ, in an expanding colony of MM127 melanoma cells. Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and λ. In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q. We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q. We find that additional data relating to the degree of cell-to-cell clustering is required to provide independent estimates of q, and in turn D. In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate. In summary, we find that our experiments are best described using the range D = 161 - 243 μm2 hour-1, q = 0.3 - 0.5 (low to moderate strength) and λ = 0.0305 - 0.0398 hour-1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony.
    Conclusions: Our systematic approach to identify the cell diffusivity, cell-to-cell adhesion strength and cell proliferation rate highlights the importance of integrating multiple types of data to accurately quantify the factors influencing the spatial expansion of melanoma cell colonies.
  • Are in vitro estimates of cell diffusivity and cell proliferation rate sensitive to assay geometry ?
    Treloar KK, Simpson MJ, McElwain DLS, Baker RE .
    J.Theor.Biol. (2014) 356:71-84   [Katrina K Treloar et al.] .
    Cells respond to various biochemical and physical cues during wound-healing and tumour progression. In vitro assays used to study these processes are typically conducted in one particular geometry and it is unclear how the assay geometry affects the capacity of cell populations to spread, or whether the relevant mechanisms, such as cell motility and cell proliferation, are somehow sensitive to the geometry of the assay. In this work we use a circular barrier assay to characterise the spreading of cell populations in two different geometries. Assay 1 describes a tumour-like geometry where a cell population spreads outwards into an open space. Assay 2 describes a wound-like geometry where a cell population spreads inwards to close a void. We use a combination of discrete and continuum mathematical models and automated image processing methods to obtain independent estimates of the effective cell diffusivity, D, and the effective cell proliferation rate, λ. Using our parameterised mathematical model we confirm that our estimates of D and λ accurately predict the time-evolution of the location of the leading edge and the cell density profiles for both assay 1 and assay 2. Our work suggests that the effective cell diffusivity is up to 50% lower for assay 2 compared to assay 1, whereas the effective cell proliferation rate is up to 30% lower for assay 2 compared to assay 1.
  • Assessing the role of spatial correlations during collective cell spreading.
    Treloar KK, Simpson MJ, Binder BJ, McElwain DLS, Baker RE .
    Scientific Reports (2014) 4, Article number: 5713   [Katrina K Treloar et al.] .
    Spreading cell fronts are essential features of development, repair and disease processes. Many mathematical models used to describe the motion of cell fronts, such as Fisher's equation, invoke a mean-field assumption which implies that there is no spatial structure, such as cell clustering, present. Here, we examine the presence of spatial structure using a combination of in vitro circular barrier assays, discrete random walk simulations and pair correlation functions. In particular, we analyse discrete simulation data using pair correlation functions to show that spatial structure can form in a spreading population of cells either through sufficiently strong cell-to-cell adhesion or sufficiently rapid cell proliferation. We analyse images from a circular barrier assay describing the spreading of a population of MM127 melanoma cells using the same pair correlation functions. Our results indicate that the spreading melanoma cell populations remain very close to spatially uniform, suggesting that the strength of cell-to-cell adhesion and the rate of cell proliferation are both sufficiently small so as not to induce any spatial patterning in the spreading populations.
  • Sensitivity of edge detection methods for quantifying cell migration assays.
    Treloar KK, Simpson MJ .
    PLoS ONE (2013) 8(6):e67389   [Katrina K Treloar et al.] .
    Quantitative imaging methods to analyze cell migration assays are not standardized. Here we present a suite of twodimensional barrier assays describing the collective spreading of an initially-confined population of 3T3 fibroblast cells. To quantify the motility rate we apply two different automatic image detection methods to locate the position of the leading edge of the spreading population after 24, 48 and 72 hours. These results are compared with a manual edge detection method where we systematically vary the detection threshold. Our results indicate that the observed spreading rates are very sensitive to the choice of image analysis tools and we show that a standard measure of cell migration can vary by as much as 25% for the same experimental images depending on the details of the image analysis tools. Our results imply that it is very difficult, if not impossible, to meaningfully compare previously published measures of cell migration since previous results have been obtained using different image analysis techniques and the details of these techniques are not always reported. Using a mathematical model, we provide a physical interpretation of our edge detection results. The physical interpretation is important since edge detection algorithms alone do not specify any physical measure, or physical definition, of the leading edge of the spreading population. Our modeling indicates that variations in the image threshold parameter correspond to a consistent variation in the local cell density. This means that varying the threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1-5% of the maximum cell density.
  • Mathematical models for collective cell spreading.
    Treloar KK.
    Thesis, Queensland University of Technology (2015)   [Katrina K Treloar ] .
    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. The principle aim of this thesis is to apply mathematical modelling frameworks to new experimental data to identify and quantify several key features underlying collective cell spreading. This work is presented as a thesis by published papers and consists of five related works which may either be read as a whole or as separate entities.
    We begin by describing a set of experiments to investigate the unique roles of cell motility and cell proliferation in driving an initially confined fibroblast cell population. To interpret our experimental observations we use a combination of lattice-based discrete simulations and a related continuum partial differential equation model. We obtain independent estimates of the cell diffusivity, D, by extracting information about the location of the leading edge from experiments where cell proliferation has been suppressed in the spreading population. Independent estimates of the cell proliferation rate, λ are obtained using cell density information from experiments where cell proliferation is not inhibited in the spreading population. Previous work suggests that cell populations with a high λ/D ratio will be characterised by steep fronts, whereas systems with a low λ/D will lead to shallow diffusive fronts and we confirm this here. Our results provide evidence that standard mathematical models, based on the Fisher-Kolmogorov equation, are appropriate to interpret and predict such experimental observations.
    We extend our investigation to quantify the mechanisms driving the collective spread of melanoma cell populations. In addition to cell motility and cell proliferation mechanisms, cell-to-cell adhesion is thought to be a crucial mechanism involved in melanoma cell population spreading. Standard mathematical models often neglect cell-to-cell adhesion and it is unclear how estimates of the strength of cell-to-cell adhesion in a cell population can be extracted from experimental data. In this work, we show that multiple types of data must be integrated to independently quantify each of the three mechanisms present in the spreading melanoma cell populations. In addition, we provide a method of quantifying the strength of cell-to-cell adhesion by extracting information about the location of isolated cells in the spreading population.
    Our experimental and mathematical modelling investigation to quantify the mechanisms driving the spread of fibroblast and melanoma cell populations raises several additional questions which we examine in the final sections of the thesis. Firstly, we examine whether the location of the leading edge, which is often used to parameterise mathematical models, is sensitive to the choice of image analysis tools. We show that a standard measure of cell migration can vary by as much as 25%. In addition, we use a mathematical model to provide a physical interpretation of the location of the leading edge and find that varying the image threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1-5% of the maximum cell density. Our results suggest that it is impossible to meaningful compare previously published measures of cell migration since previously results have been obtained using different image analysis tools and the details of these tools are often not reported.
    Secondly, we explore the role of in vitro assay geometry by performing experiments in two distinct geometries. The first geometry describes a tumour-like geometry where a cell population spreads outwards into an open space. The second geometry describes a wound-like geometry where a cell population spreads inwards to close a void. Applying the same techniques to independently quantify D and λ, we find that in vitro assay geometry does affects these estimates. Our work suggests that estimates of the cell diffusivity vary by up to 50% while estimates of the cell proliferation rate vary by up to 30%.
    Finally, we test whether standard mathematical models, which assume that there is no spatial structure such as cell clustering present in the system, are appropriate to describe the spread of the melanoma cell populations considered in this work. We analyse discrete simulation using pair correlation functions to show that spatial structure can form in a spreading population of cells either through sufficiently strong cell-to-cell adhesion or sufficiently rapid cell proliferation. We use the same pair correlation functions to analyse experimental images and find that the strength of cell-to-cell adhesion is sufficiently weak and rate of cell proliferation is sufficiently slow, so as not to induce any spatial structure in the spreading populations.
    We conclude by discussing the potential to apply the experimental and modelling approaches presented here to understand other aspects underlying collective cell spreading.
  • The effect of vitronectin and other extracellular matrix molecules on endothelial expansion and plasminogen activation.
    Underwood PA, Bean PA
    Cells Mater. (1996) 6:193-207
    Endothelial recovery following procedures used to alleviate blood vessel occlusion is modulated by the local extracellular matrix upon which it has to migrate and proliferate. This extracellular material is derived from vessel wall cells, and plasma proteins which bind to the exposed surfaces. We have demonstrated that vitronectin adsorbs efficiently to tissue culture polystyrene in competition with other plasma proteins, which suggests that it may adsorb to biomaterial surfaces in vivo. We have compared the adhesion, migration and proliferation of human umbilical artery endothelial cells on surface-coated vitronectin, with other extracellular matrix molecules encountered in this environment, namely fibronectin, laminin and collagen types I and IV. Endothelial proliferation was significantly reduced on the vitronectin surface. This was correlated with an increase in the ratio of plasminogen inhibitor-1 to urokinase in the cell/matrix layer. Laminin coated surfaces limited increases in endothelial culture area, due to poorer cell spreading on this surface. Such combination of cellular responses to vitronectin and laminin would discourage endothelial recovery, and encourage smooth muscle hyperplasia in vivo. These considerations are important in the design of biomaterial surfaces to optimise endothelial recovery.
  • Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.
    Underwood PA, Bean PA, Whitelock JM
    Atherosclerosis (1998) 141(1):141-152
    Endothelial cells recovering from damage due to disease or surgical procedures come into close contact with extracellular matrix (ECM) secreted by intimal vascular smooth muscle cells (VSMCs). We have investigated these relationships using human umbilical artery endothelial cells (HUAECs) and human mammary artery VSMC in vitro. HUAEC adhesion and proliferation were significantly lower on ECM secreted by VSMC compared with HUAEC ECM or surface-coated fibronectin. Characterisation of the ECM of both cell types with monoclonal antibodies showed that the ECM secreted by VSMC contained significantly more elastin, chondroitin sulphate and collagen types I, III and V than that from HUAECs. HUAECs adhered poorly to collagen type V coated on plastic and not at all to elastin. When these proteins were co-coated with fibronectin, elastin did not inhibit migration or proliferation compared to the response on fibronectin but collagen type V significantly inhibited both. Treatment of VSMC ECM with enzymes which selectively depleted the matrix of collagen types I, III and IV, or chondroitin sulphate, had no effect on HUAEC responses to the ECM, suggesting that these molecules did not contribute to the inhibition of HUAECs. Treatment of VSMC ECM with a mixture of collagenases, selectively depleted the matrix of collagen type V, as well as types I, III and IV. Such depleted ECMs supported increased proliferation of HUAECs compared to buffer controls. Overall these results suggest that collagen V secreted into the ECM of VSMC may inhibit the recovery of adjacent endothelium
  • Rate of endothelial expansion is controlled by cell:cell adhesion.
    Underwood PA, Bean PA, Gamble JR
    Internatl.J.Biochem. Cell Biol. (2002) 34(1):55-69   [P. Anne Underwood et al.] .
    Procedures used to alleviate blood vessel occlusion result in varying degrees of damage to the vascular wall and endothelial denudation. The presence of intact, functioning endothelium is thought to be important in controlling smooth muscle cell growth, and limiting the intimal thickening which results from damage to the vessel wall. Recovery of the endothelium is commonly slow and incomplete, due in part to endothelial lateral cell:cell adhesion, which limits cell migration and proliferation. We have investigated the effect of fibroblast growth factor 2 and vascular/endothelial growth factor on the relationship between the temporal distribution of the junctional adhesion proteins, platelet/endothelial cell adhesion molecule, vascular/endothelial cadherin and plakoglobin, and cellular migration and proliferation in an in vitro model of endothelial expansion. We found that whereas cell:cell junctions were initially disturbed to similar extents by single applications of the growth factors, outward cell migration and proliferation rates were inversely correlated with the speed at which cell:cell junctions were re-established. This occurred very rapidly with vascular/endothelial growth factor treatment and more slowly with fibroblast growth factor-2, resulting in more extensive outward migration and proliferation in response to the latter. Platelet/endothelial cell adhesion molecule and vascular/endothelial cadherin appeared to be associated with cell:cell junctional control of migration and proliferation, while plakoglobin did not contribute. It was concluded that the rate of endothelial expansion in response to growth factors, is limited by the rate of re-association of junctional complexes following initial disruption.
  • Vitronectin: Growth Factor complexes hold potential as a wound therapy approach.
    Upton Z, Cuttle L, Noble A, Kempf M, Topping G, Malda J, Xie Y, Mill J, Harkin DG, Kravchuk O, Leavesley DI, Kimble RM
    J.Invest.Dermatology (2008) 128(6):1535-1544   [Zee Upton et al.] .
    Topical administration of growth factors has displayed some potential in wound healing, but variable efficacy, high doses, and costs have hampered their implementation. Moreover, this approach ignores the fact that wound repair is driven by interactions between multiple growth factors and extracellular matrix (ECM) proteins. We report herein that complexes comprising IGF and IGF-binding proteins bound to the ECM protein vitronectin (VN) significantly enhance cellular functions relevant to wound repair in human skin keratinocytes in two- and three-dimensional in vitro cell models and are active, even in the presence of wound fluid. Moreover, these responses require activation of both the IGF receptor and the VN-binding αv integrins. Further, we assessed the complexes as a topical agent in the treatment of deep dermal partial thickness burns in a porcine model. This pilot study revealed that the complexes may hold promise as a wound healing therapy. Critically, the significant responses observed in vitro and the encouraging preliminary data in vivo were obtained with nanogram doses of growth factors. This suggests that coupling delivery of growth factors to ECM proteins such as VN may ultimately prove to be a more effective strategy for developing a wound healing therapy.
  • Crossing barriers: The new dimension of 2D cell migration assays. (review, letter)
    van Horssen R, ten Hagen TLM
    J.Cell.Physol. (2010) 226:288-290   .
    In our body cells move in three dimensions, embedded in an extracellular matrix that varies in composition, density and stiffness, and this movement is fundamental to life. Next to 3D cell migration assays, representing these physiological circumstances, still we need 2D migrations assays to perform detailed studies on the contribution of matrix-components and (extra)cellular proteins to cell movements. Next to the debate on differences between 3D and 2D migration, there also are many new perspectives on the use and development of novel or modified 2D cell migration assays. Of special significance is the introduction of so-called barrier migration assays, methods that avoid cell and matrix damage, as complementation or replacement of scratch/wound healing assays. Here, we discuss the possibilities and limitations of different 2D barrier migration assays.
  • A fence barrier method of leading edge cell capture for explorative biochemical research.
    Wager LJ, Murray RZ, Thompson EW, Leavesley DI.
    Cell Adhesion & Migration (2017) 11, NO. 5, 496-503 | DOI: 10.1080/19336918.2016.1269997   or [Lucas J. Wager et al.] .
    The scratch or wound-healing assay is used ubiquitously for investigating re-epithelialisation and has already revealed the importance of cells comprising the leading edge of healing epithelial wounds. However it is currently limited to studying the effect of known biochemical agents on the tissue of choice. Here we present an adaptation that extends the utility of this model to encompass the collection of cells from the leading edge of migrating epithelial sheets making available explorative biochemical analyses. The method is scalable and does not require expensive apparatus, making it suitable for large and small laboratories alike. We detail the application of our method and exemplify proof of principle data derived from primary human keratinocyte cultures.
  • Mechanical cell competition kills cells via induction of lethal p53 levels.
    Wagstaff L, Goschorska M, Kozyrska K, Duclos G, Kucinski I, Chessel A, Hampton-O'Neil L, Bradshaw CR, Allen GE, Rawlins EL, Silberzan P,Carazo Salas RE, Piddini E.
    Nature Communications (2016) | 7:11373 | DOI: 10.1038/ncomms11373   [Laura Wagstaff et al.] .
    Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scribKD) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scribKD cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.
  • Vasoactive Peptides in Angiogenesis.
    Walsh DA, Fan T-PD
    in: The New Angiotherapy. Eds.: Fan T-PD, Kohn EC, Springer Science+Business Media, LLC (2002) :81-104
    Angiogenesis, the formation of new from old vessels, occurs physiologically during the female reproductive cycle, and pathologically during tumor growth, diabetic retinopathy, and chronic inflammation. It may be either beneficial, for example in wound repair, or detrimental, for example in tumors. Neovascularization is a complex process rather than a single event, requiring endothelial cell migration and proliferation, capillary-tube formation, and anastomosis, recruitment of mural cells, and reorganization and maturation of the vascular bed. Each stage is regulated by a variety of factors and it is essential to complete all stages in order to establish a normal vasculature. Much research has focused on factors that are upregulated during angiogenesis, such as vascular endothelial growth factor (VEGF) and its receptors, and integrin αVβ3. Upregulation of angiogenic factors and endothelial cellsurvival factors is essential to sustain angiogenesis and prevent regression of the neovasculature. In this chapter, we discuss a group of regulatory peptides, many of which are constitutively expressed in the mature vasculature, and even may be downregulated in neovasculature (Table 1, Fig. 1). None the less, these stimulate angiogenesis both in vitro and in vivo. We suggest that regulatory peptides may play important roles in the initiation of angiogenesis, before other growth factors have been upregulated. Peptides may, in addition, facilitate vascular survival (33). Constitutively produced vasoactive peptides may particularly be important for the survival of mature, appropriate blood vessels, following the downregulation of other vascular survival factors. Bradykinin and substance P are generated and released during the earliest stages of tissue injury, and it is appropriate that peptides that mediate acute inflammatory responses may simultaneously initiate processes such as angiogenesis that are essential for tissue repair. However, inappropriate initiation of angiogenesis by regulatory peptides may be a key process in several human diseases..
  • PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.
    Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, Persohn E, Rösel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, Schirner M, Thierauch KH, Schneider MR, Drevs J, Martiny-Baron G, Totzke F, Marmé D
    Cancer Res. (2000) 60:2178-2189   .
    PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor ß tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-{alpha}. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 µM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 µM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25–100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.
  • Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid
    Wood J, Bonjean K, Ruetz S, Bellahcène A, Devy L, Foidart JM, Castronovo V, Green JR
    Pharmacol.Exp.Ther. (2002) 302,3:1055-1061   [Jeanette Wood et al.] .
    Bisphosphonate drugs inhibit osteoclastic bone resorption and are widely used to treat skeletal complications in patients with tumor-induced osteolysis. We now show that zoledronic acid, a new generation bisphosphonate with a heterocyclic imidazole substituent, is also a potent inhibitor of angiogenesis. In vitro, zoledronic acid inhibits proliferation of human endothelial cells stimulated with fetal calf serum, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (IC50 values 4.1, 4.2, and 6.9 µM, respectively), and modulates endothelial cell adhesion and migration. In cultured aortic rings and in the chicken egg chorioallantoic membrane assay, zoledronic acid reduces vessel sprouting. When administered systemically to mice, zoledronic acid potently inhibits the angiogenesis induced by subcutaneous implants impregnated with bFGF [ED50, 3 µg/kg (7.5 nmol/kg) s.c.]. These findings indicate that zoledronic acid has marked antiangiogenic properties that could augment its efficacy in the treatment of malignant bone disease and extend its potential clinical use to other diseases with an angiogenic component.
  • Ex-vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model.
    Xie Y
    PhD thesis, Queensland University of Technology (2008)   [Yan Xie] .
    It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. more specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DID and shown to migrate into the DID in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound heading. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 am diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.
  • Emergent leader cells in collective cell migration in in vitro wound healing assay.
    Yang Y
    PhD thesis - University of Arizona (2014)   [Yongliang Yang] .
    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.
  • A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.
    Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ
    BMC Biotechnology (2004) 4:21-29   [Justin C. Yarrow et al.] .
    -Background- Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. -Results- We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. -Conclusions- The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.
  • Investigation of electrophoretic loading and enhanced mechanical properties of hydrogels for delivery of therapeutic proteins.
    Yeh C-L
    PhD thesis, Queensland University of Technology (2010)   [Chun-Liang Yeh] .
    Hydrogels, which are three-dimensional crosslinked hydrophilic polymers, have been used and studied widely as vehicles for drug delivery due to their good biocompatibility. Traditional methods to load therapeutic proteins into hydrogels have some disadvantages. Biological activity of drugs or proteins can be compromised during polymerization process or the process of loading protein can be really timeconsuming. Therefore, different loading methods have been investigated. Based on the theory of electrophoresis, an electrochemical gradient can be used to transport proteins into hydrogels. Therefore, an electrophoretic method was used to load protein in this study. Chemically and radiation crosslinked polyacrylamide was used to set up the model to load protein electrophoretically into hydrogels. Different methods to prepare the polymers have been studied and have shown the effect of the crosslinker (bisacrylamide) concentration on the protein loading and release behaviour. The mechanism of protein release from the hydrogels was anomalous diffusion (i.e. the process was non-Fickian). The UV-Vis spectra of proteins before and after reduction show that the bioactivities of proteins after release from hydrogel were maintained. Due to the concern of cytotoxicity of residual monomer in polyacrylamide, poly(2-hydroxyethyl- methacrylate) (pHEMA) was used as the second tested material. In order to control the pore size, a polyethylene glycol (PEG) porogen was introduced to the pHEMA. The hydrogel disintegrated after immersion in water indicating that the swelling forces exceeded the strength of the material. In order to understand the cause of the disintegration, several different conditions of crosslinker concentration and preparation method were studied. However, the disintegration of the hydrogel still occurred after immersion in water principally due to osmotic forces. A hydrogel suitable for drug delivery needs to be biocompatible and also robust. Therefore, an approach to improving the mechanical properties of the porogen-containing pHEMA hydrogel by introduction of an inter-penetrating network (IPN) into the hydrogel system has been researched. A double network was formed by the introduction of further HEMA solution into the system by both electrophoresis and slow diffusion. Raman spectroscopy was used to observe the diffusion of HEMA into the hydrogel prior to further crosslinking by γ-irradiation. The protein loading and release behaviour from the hydrogel showing enhanced mechanical property was also studied. Biocompatibility is a very important factor for the biomedical application of hydrogels. Different hydrogels have been studied on both a three-dimensional HSE model and a HSE wound model for their biocompatibilities. They did not show any detrimental effect to the keratinocyte cells. From the results reported above, these hydrogels show good biocompatibility in both models. Due to the advantage of the hydrogels such as the ability to absorb and deliver protein or drugs, they have potential to be used as topical materials for wound healing or other biomedical applications.
  • In vitro studies on inflammatory potential of biomaterials with development of anti-inflammatory strategies.
    Zhou G
    PhD thesis, Martin-Luther-Universität Halle-Wittenberg, Germany (2017)   [Guoying Zhou] .
        [Abstract / Zusammenfassung]
    Host responses towards implanted biomaterials, in particular chronic inflammation and fibrotic encapsulation, are major hindrances to the functionality and longevity of many implanted biomedical devices. To gain more knowledge about the biomaterial-induced host responses, studies are carried out here with a newly developed in vitro macrophage/fibroblast co-culture model to investigate the pro-inflammatory and pro-fibrotic potentials of model biomaterials. The co-culture system is established using a cell migration fence chamber possessing an internal and an external compartment to generate separated and mixed co-cultures before and after removal of the chamber, respectively. The novelty of the system is that it allows not only to mimic autocrine, paracrine and juxtacrine signal exchange in one and the same system, but also to study macrophage migration and fibroblast outgrowth in the presence of the other cell type in a timely and locally controlled manner. Self-assembling monolayers (SAMs) of alkylsilanes on glass substrates with terminal methyl (CH3), amine (NH2), hydroxyl (OH), and carboxyl (COOH) groups are used here as model surfaces focusing on macrophage as well as fibroblast responses in the co-culture system. The inflammatory reactions on the different SAMs are investigated regarding macrophage adhesion and migration, foreign body giant cell (FBGC) formation, ß1 integrin expression and pro-inflammatory cytokine production in the presence of fibroblasts, while the fibrotic responses are studied in terms of fibroblast attachment, spreading, proliferation, outgrowth, fibroblast to myofibroblast differentiation as well as anti-inflammatory cytokine production within the co-culture system. The results indicate that hydrophobic CH3 surfaces possess the highest potential of inducing inflammatory responses but evoke only low levels of fibrotic reactions. In contrast, hydrophilic/anionic COOH surfaces result in low levels of both inflammatory and fibrotic responses. In addition, macrophage migration regarding motility and directional movement increase in the presence of fibroblasts, while fibroblast outgrowth on different SAMs is promoted significantly in the presence of macrophages. The obtained relations between surface functionality and host responses are further used for the development of anti-inflammatory strategies. Based on these findings, the second part of the thesis aims to develop anti-inflammatory strategies using glycosaminoglycans (GAGs), which are also hydrophilic and anionic macromolecules due to the presence of carboxyl and/or sulfate groups. In addition, GAGs bind to a wide range of chemokines, cytokines, growth factors as well as enzymes, and thus can modulate events associated with inflammation. Therefore, three kinds of GAGs, namely hyaluronic acid (HA), chondroitin sulfate (CS), and heparin (Hep) are immobilized here to model material surfaces to reduce the inflammatory responses. They are either physically adsorbed on a primary poly (ethylene imine) (PEI) layer alternately with chitosan (Chi) as multilayers by the layer-by-layer (LBL) technique or covalently immobilized to amino-functionalized substrata via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linking chemistry. The anti-inflammatory properties of the GAG-modified surfaces are studied focusing on macrophage activation. Here, macrophage adhesion, spreading, fusion, and pro-inflammatory cytokine production are all significantly decreased on each GAG-modified surface at both immobilization techniques in comparison to their respective controls. However, as demonstrated the type of GAGs plays a pivotal role in modulating the inflammation in multilayers, with the Hep-Chi system showing the highest anti-inflammatory potential. The physical adsorption of GAGs during LBL technique probably allows the uptake of Hep molecules by macrophages, which could lead to a reduction of NF-κB nuclear translocation and thus further lowers inflammatory responses. Overall, this thesis not only presents a newly developed, multifunctional co-culture system to elucidate the relation between surface functionality and host responses, but also provides useful clues for future developments of implant materials with anti-inflammatory properties.

    Gewebereaktionen gegenüber implantierten Biomaterialien, insbesondere chronische Entzündungen und fibrotischen Verkapselungen, sind wesentliche Hindernisse für die Funktionalität und Langlebigkeit vieler implantierter biomedizinische Geräte. Um mehr Wissen über Biomaterial-induzierte Wirtsreaktionen zu gewinnen, werden in dieser Arbeit Untersuchungen mit einem neu entwickelten In-vitro-Makrophagen/Fibroblasten-Co-Kultur-Modell durchgeführt, um das Entzündungs-hervorrufende und fibrotische Potenzial von Modellmaterialien zu untersuchen. Das vorgestellte Co-Kultursystem wird über eine Zellmigrationsschranke realisiert, welche die Kulturfläche in eine innere und äußere Kammer separiert und somit getrennte und gemischte Co-Kulturen vor beziehungsweise nach dem Entfernen der Schranke erzeugt. Die Neuheit des Systems besteht darin, nicht nur autokrinen, parakrinen und juxtakrinen Signalaustausch in ein und demselben System zu ermöglichen, sondern auch die Makrophagen-Migration wie auch das Herauswachsen von Fibroblasten in Anwesenheit des jeweils anderen Zelltyps zeitlich und lokal gesteuert zu studieren. Selbstorganisierende Monolagen (SAMs) von Alkylsilanen auf Glassubstraten mit endständigen Methyl-(CH3), Amino-(NH2), Hydroxyl-(OH) und Carboxyl-(COOH)-Gruppen dienen hier als Modelloberflächen, um auf die Wechselwirkung von Makrophagen und Fibroblasten im Co-Kultursystem zu fokussieren. Die Entzündungsreaktionen auf den verschiedenen SAMs werden hierbei bezüglich Makrophagenadhäsion und -migration, Fremdkörper-Riesenzell-(FBGC)-Bildung, ß1-Integrin-Expression sowie entzündliche Zytokinproduktion in Gegenwart von Fibroblasten untersucht, während die fibrotischen Reaktionen in Bezug auf Fibroblastenadhäsion, -spreitung, -wachstum, das Herauswachsen aus der Migrationsschranke, die Differenzierung zu Myofibroblasten sowie die entzündungs-hemmende Zytokinproduktion betrachtet werden. Die Ergebnisse zeigen, dass hydrophobe Methyl-Oberflächen das höchste Potential für Entzündungsreaktionen besitzen, aber nur geringe fibrotische Reaktionen hervorrufen. Im Gegensatz dazu führen hydrophile/anionische Carboxyl-Oberflächen nur zu geringen entzündlichen und fibrotischen Reaktionen. Zusätzlich ist die Makrophagenmigration bezüglich Motilität und gerichteter Bewegung in Gegenwart von Fibroblasten erhöht, während das Herauswachsen von Fibroblasten auf den verschiedenen SAMs in Gegenwart von Makrophagen signifikant gefördert wird. Die hier gefundenen Zusammenhänge zwischen Oberflächenfunktionalität und Wirtsreaktionen werden daher für die Entwicklung von weiteren entzündungshemmenden Strategien verwendet. Auf Basis jener Erkenntnisse konzentriert sich der zweite Teil der Arbeit auf die Verwendung von Glykosaminoglykanen (GAGs), ebenfalls hydrophile und anionische Makromoleküle aufgrund der Anwesenheit von Carboxyl-und/ oder Sulfatgruppen, zur Entwicklung entzündungshemmender Strategien. GAGs können zudem eine Vielzahl von Chemokinen, Zytokinen, Wachstumsfaktoren, sowie Enzyme binden, und hierdurch mit einer Entzündung assoziierte Ereignisse steuern. In dieser Arbeit werden drei Arten von GAGs, Hyaluronsäure (HA), Chondroitinsulfat (CS) und Heparin (Hep), auf Modelloberflächen immobilisiert, um entzündliche Reaktionen zu reduzieren. Sie werden entweder auf einer primären Polyethylenimin-(PEI)-Schicht abwechselnd mit Chitosan (Chi) in Mehrfachschichten durch die Layer-by-Layer-(LBL)-Technik adsorbiert oder kovalent an amino-funktionalisierte Oberflächen über 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimid (EDC)/N-Hydroxysuccinimid (NHS)-Vernetzung gebunden. Die entzündungshemmenden Eigenschaften der GAG-modifizierten Oberflächen fokussieren dabei auf die Makrophagenaktivierung. Hierbei sind Makrophagenadhäsion, -spreitung, -fusion und die entzündliche Zytokinproduktion auf jeder GAG-modifizierten Oberfläche bei beiden Immobilisierungstechniken im Vergleich zu ihren jeweiligen Kontrollen signifikant verringert. Jedoch spielt die Art der GAGs innerhalb der Mehrfachschichten eine zentrale Rolle bei der Entzündungsreaktion, wobei das Hep-Chi-System das höchste entzündungs-hemmende Potential zeigt. Die physikalische Adsorption von GAGs mit der LBL-Technik ermöglicht wahrscheinlich die Aufnahme von Hep-Moleküle durch Makrophagen, was zu einer Verringerung der NF-κB nukleären Translokation führt, was wiederum weitere Entzündungsreaktionen reduziert. Insgesamt stellt diese Arbeit nicht nur ein neu entwickeltes, multifunktionales Co-Kultursystem vor, um die Beziehung zwischen Oberflächenfunktionalität und Wirtsreaktionen aufzuklären, sondern bietet auch nützliche Hinweise für die zukünftige Entwicklung von Implantatmaterialien mit entzündungshemmenden Eigenschaften.
  • A macrophage/fibroblast co-culture system using a cell migration chamber to study inflammatory effects of biomaterials.
    Zhou G, Loppnow H, Groth T
    Acta Biomaterialia (2015) 26:54-63  
    Chronic inflammatory reactions hamper the use of biomaterials after implantation. Thus, the aim of the study was to develop a novel predictive in vitro macrophage/fibroblast co-culture model based on cell migration chambers that allows a timely and locally controlled interaction of both cell types to study the inflammatory responses of biomaterials in vitro. Here, self-assembled monolayers (SAMs) with different wettability and charge properties were used as model biomaterials on which co-cultures were established by use of fence chambers having internal and external compartments. This allowed establishing separated and mixed co-cultures of both cell types before and after removal of the chamber, respectively. The key advantages of this novel co-culture model included not only to establish a timely-resolved study of cytokine release, but also the ability to assess individual macrophage migration in both macrophage mono-cultures and co-cultures. All inflammatory reactions in terms of macrophage adhesion, macrophage migration, foreign body giant cell (FBGC) formation, β1 integrin expression and pro-inflammatory cytokine production were found strongly surface property dependent. The results show that the hydrophobic CH3 surface caused the strongest inflammatory reactions, whereas the hydrophilic/anionic COOH surface caused the least inflammatory response, indicating low and high biocompatibility of the surfaces, respectively. Most importantly, we found that both macrophage motility and directional movement were increased in the presence of fibroblasts in co-cultures compared with macrophage mono-cultures. Overall, the novel co-culture system provides access to a range of parameters for studying inflammatory reactions and reveals how material surface properties affect the inflammatory responses.
  • In vitro study of the host responses to model biomaterials via a fibroblast/macrophage co-culture system.
    Zhou G, Liedmann A, Chatterjeea C, Groth T.
    Biomaterials Sci. (2017) 5(1):141-152  
    Surface properties are believed to play important roles in initial inflammatory and subsequent wound healing/fibrotic responses after implantation of biomaterials. To investigate the surface property effect in mediating these host responses, we used an in vitro fibroblast/macrophage co-culture model established with a cell migration chamber, and a series of self-assembling monolayers (SAMs) bearing different terminal groups as model surfaces to study the effect of surface properties on macrophage fusion, fibroblast attachment, spreading morphology, proliferation, outgrowth, as well as pro-(interleukin-6) and anti-(interleukin-10) inflammatory cytokine production, expression of ED-A fibronectin (FN) and alpha-smooth muscle actin (α-SMA). The obtained results show that the hydrophobic CH3 surface caused high levels of inflammatory but low levels of wound healing/fibrotic responses, while the hydrophilic/anionic COOH surface resulted in both low levels of inflammatory and wound healing/fibrotic responses. Interestingly, the hydrophilic OH surface was found to possess a low potential of inducing inflammatory responses but high potential of inducing wound healing/fibrotic responses. These results reveal that the extent of inflammation and wound healing/fibrosis might not be always related in vitro. However, more important is the observation of the macrophage contributions in facilitating the wound healing and fibrotic responses by up-regulation of fibroblast outgrowth, cytokine production as well as ED-A FN and a-SMA expression. Overall, by linking the surface properties to cell activities with our established fibroblast/macrophage co-culture system, we could provide an useful model system for in vitro studies to design more biocompatible biomaterials for various biomedical and tissue engineering applications.
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