106 research outputs found
Nuclear staining and relative distance for quantifying epidermal differentiation in biomarker expression profiling
No full textAbstract Background The epidermal physiology results from a complex regulated homeostasis of keratinocyte proliferation, differentiation and death and is tightly regulated by a specific protein expression during cellular maturation. Cellular in silico models are considered a promising and inevitable tool for the understanding of this complex system. Hence, we need to incorporate the information of the differentiation dependent protein expression in cell based systems biological models of tissue homeostasis. Such methods require measuring tissue differentiation quantitatively while correlating it with biomarker expression intensities. Results Differentiation of a keratinocyte is characterized by its continuously changing morphology concomitant with its movement from the basal layer to the surface, leading to a decreased average nuclei density throughout the tissue. Based thereon, we designed and evaluated three different mathematical measures (nuclei based, distance based, and joint approach) for quantifying differentiation in epidermal keratinocytes. We integrated them with an immunofluorescent staining and image analysis method for tissue sections, automatically quantifying epidermal differentiation and measuring the corresponding expression of biomarkers. When studying five well-known differentiation related biomarkers in an epidermal neck sample only the resulting biomarker profiles incorporating the relative distance information of cells to the tissue borders (distance based and joint approach) provided a high-resolution view on the whole process of keratinocyte differentiation. By contrast, the inverse nuclei density approach led to an increased resolution at early but heavily decreased resolution at late differentiation. This effect results from the heavy non-linear decay of DAPI intensity per area, probably caused by cytoplasmic growth and chromatin decondensation. In the joint approach this effect could be compensated again by incorporating distance information. Conclusion We suppose that key mechanisms regulating tissue homeostasis probably depend more on distance information rather than on nuclei reorganization. Concluding, the distance approach appears well suited for comprehensively observing keratinocyte differentiation.</p
Reconstructing protein networks of epithelial differentiation from histological sections
No full textFor systems biology of complex stratified epithelia like human epidermis, it will be of particular importance to reconstruct the spatiotemporal gene and protein networks regulating keratinocyte differentiation and homeostasis
Spatial quantification and classification of skin response following perturbation using organotypic skin cultures
No full textAbstract
Motivation: For a mechanistic understanding of skin and its response to an induced perturbation, systems biology is gaining increasing attention. Unfortunately, quantitative and spatial expression data for skin, like for most other tissues, are almost not available.
Results: Integrating organotypic skin cultures, whole-slide scanning and subsequent image processing provides bioinformatics with a novel source of spatial expression data. We here used this approach to quantitatively describe the effect of treating organotypic skin cultures with sodium dodecyl sulphate in a non-corrosive concentration. We first measured the differentiation-related spatial expression gradient of Heat-Shock-Protein 27 in a time series of up to 24 h. Secondly, a multi-dimensional tissue classifier for predicting skin irritation was developed based on abstract features of these profiles. We obtained a high specificity of 0.94 and a sensitivity of 0.92 compared with manual classification. Our results demonstrate that the integration of tissue cultures, whole-slide scanning and image processing is well suited for both the standardized data acquisition for systems biological tissue models and a highly robust classification of tissue responses.
Contact: [email protected]
Supplementary information: Supplementary data are available at Bioinformatics online.</jats:p
Controlling osteoblastmorphology and proliferation via surface micro-topographies of implant biomaterials
No full textCurrent research on surface modifications has yielded advanced implant biomaterials. Various implant surface modifications have been shown to be promising in improving bone target cell response, but more comprehensive studies whether certain implant surface modifications can directly target cell behavioural features such as morphogenesis and proliferation are needed. Here, we studied the response of primary alveolar bone cells on various implant surface modifications in terms of osteoblast morphology and proliferation in vitro. Analyses of surface modifications led to surface-related test parameters including the topographical parameters micro-roughness, texture aspect and surface enlargement as well as the physicochemical parameter surface wettability. We compared osteoblast morphology and proliferation towards the above-mentioned parameters and found that texture aspect and surface enlargement but not surface roughness or wettability exhibited significant impact on osteoblast morphology and proliferation. Detailed analysis revealed osteoblast proliferation as a function of cell morphology, substantiated by an osteoblast size- and morphology-dependent increase in mitotic activity. These findings show that implant surface topography controls cell behavioural morphology and subsequently cell proliferation, thereby opening the road for cell instructive biomaterials
Dynamic release kinetics and biological impact of leachables from 3d-printed oral devices: an integrated in vitro and computational exposure model
No full textResearch has highlighted the release of monomers and leachables from additively manufactured (AM) oral devices, raising concerns about their potential biological impact. The oral cavity's dynamic epithelial system necessitates exposure models that accurately reflect real-world conditions. Traditional static models often overestimate or underestimate patient exposure, failing to predict in vivo risks effectively. To address this gap, we developed an advanced dynamic oral tissue exposure model that simulates the release kinetics of leachables, saliva flow, and gingival tissue perfusion. This dynamic approach, integrated with an in vitro human gingival keratinocyte (HGK) model, was applied for the first time in this study. We quantified urethane dimethacrylate (UDMA) release from AM biomaterials through extraction experiments, generating data for computational modeling. The model revealed that dynamic in vivo monomer exposure peaks at specific time points before declining, a pattern not captured by static calculations. In vitro analysis showed that UDMA exposure inhibited metabolic activity and reduced Ki-67 expression in HGK cultures at micromolar concentrations. While inhibitory in vitro concentrations exceeded predicted in vivo estimates, low-dose effects on Ki-67 expression were still observed. These findings suggest that although calculated UDMA exposure remains sub-cytotoxic, it may still induce sensitizing effects. Overall, the dynamic exposure model introduced in this study represents a significant advancement in risk assessment, offering more accurate predictions of the biological effects of leachables and contributing to the safety evaluation of AM biomaterials.Statement of significanceAdditively manufactured (AM) oral devices are a significant source of monomer release into the oral cavity, raising concerns about tissue exposure. Traditional static models provide limited or inaccurate risk estimates due to the cavity’s dynamic nature. In this study, we developed a dynamic oral tissue exposure model that estimates in vivo-relevant monomer and leachable concentrations in saliva and oral mucosa while integrating an in vitro gingival keratinocyte model to assess biological effects. The model provides key insights into predicted in vivo exposure to monomers and leachables, improving in vitro evaluations of biological effects. Overall, it serves as a valuable risk assessment tool for the research community by enhancing predictions of patient exposure to potential monomers and leachables, thereby supporting AM biomaterial safety
Connective-tissue fibroblasts established on micropillar interfaces are pivotal for epithelial tissue morphogenesis
No full textPolydimethylsiloxane (PDMS) pillar arrays are applied as a biomechanical microenvironment to establish gingival connective-tissue fibroblasts (GCTFs) and to further analyze the pivotal role of GCTFs in epithelial-tissue morphogenesis. GCTFs are known to exert successful adhesion and growth on fibronectin immobilized on pillar heads, over time, concomitant with the increased gene expression of vimentin and collagen type-I. GCTF-populated pillar arrays clearly reveal that epithelial-tissue morphogenesis of immortalized human gingival keratinocytes (IHGKs), co-cultured for 7 and 14 days, parallels the in vivo phenotype more closely, when compared with GCTF-free control arrays. This in vivo-like phenotype is substantiated by higher mRNA levels for keratin 1, involucrin and filaggrin differentiation markers. Furthermore, it is reflected by a tissue-specific protein orientation of the aforementioned molecules, and also of the cell-to-cell contact forming desmoplakin and the basement membrane constituents, laminin-5, laminin-1/10, and collagen type-IV. These experiments suggest that the in vivo-like phenotype of the IHGK is governed by the GCTFs growing on the micropillar interfaces. Moreover, they form the basis for the optimization or neogeneration of biomaterials by varying predefined microenvironmetal parameters to achieve an in vivo-like cell growth and differentiation, indispensable for tissue morphogenesis during regeneration
Early keratinocyte differentiation on micropillar interfaces
No full textWe employed topographical patterning to analyze early keratinocyte differentiation on top of microfabricated pillar arrays. Fibronectin immobilized on pillar "heads" yielded a nucleus-associated granular keratin 1 (K1) pattern in immortalized human gingival keratinocytes (IHGK) at pillar interspaces of 14 mum. Decreasing distances of 11and 8 mum revealed cytoplasmic extension of the early differentiation marker K1 on poly(dimethylsiloxane) (PDMS) pillars. The most extensive cytoplasmic K1 protein distribution noted at the smallest pillar scale coincided with higher ratios of K1 mRNA gene transcription. These experiments suggest that early keratinocyte differentiation was governed by the topographical characteristics of the pillar pattern. Moreover, they form the basis to study cell functions such as differentiation in a defined topologically structured environment
Charakterisierung der Hydrogele GelNB/S und GelNB/DTT für das 3D Bioprinting von Knochen-, Knorpel- und Fettersatzgeweben
No full textIn dieser Arbeit wurden zwei semi-synthetische Hydrogele für das Tissue Engineering charakterisiert: GelNB/S (mit drei Vernetzungsgraden Low, Medium und High) und GelNB/DTT. Während das letzte eine unzureichende ASCs-Biokompatibilität aufwies, zeigten alle anderen Vernetzungsgrade des Hydrogels GelNB/S eine Eignung als Trägermatrix für die ASCs im Bereich des Tissue Engineering. Je nach Vernetzungsgrad, ließen sich signifikante Unterschiede in physikalisch-chemischen Eigenschaften der Hydrogele beobachten. Das GelNB/S Low zeigte die geringste, Medium – eine mittlere und High – die höchste Hydrogelsteifigkeit. Eine Reihe von Differenzierungsexperimenten wurde vorgenommen und der Einfluss der Matrixsteifigkeit auf die ASCs untersucht. Die verwendeten Stammzellen wurden osteogen, chondrogen und adipogen in GelNB/S Low, Medium und High differenziert. Gemäß den histologischen Ergebnissen wies das Hydrogel GelNB/S High die stärkste Fähigkeit auf, die Osteogenese der ASCs zu unterstützen. Die Mineralisierung der ECM nahm proportional zur Steifigkeitsstufe der Gele ab. Die chondrogene Differenzierung wurde mittels RT-PCR dargestellt. Im GelNB/S Medium fand die stärkste Genexpression der Knorpelmarker Aggrecan und COMP statt. Gemäß den lichtmikroskopischen und DIA-Untersuchungen wurde die adipogene Differenzierung der ASCs im Hydrogel GelNB/S Low am besten unterstützt. Darüber hinaus lässt sich die Ausprägung der Differenzierung bzw. die ECM-Produktion der ASCs mittels Steifigkeitsänderung des Hydrogels GelNB/S regulieren. Des Weiteren wurde in der vorliegenden Arbeit das Hydrogel mit dem höchsten Speichermodul GelNB/S High mittels eines 3D-Biodruckers gedruckt. Die Zellüberlebensrate in den gedruckten Konstrukten zeigte keinen Unterschied zu den in manuell hergestellten Hydrogelen. Zusammenfassend lässt sich sagen, dass das neuartige Hydrogel GelNB/S, welches sich mit drei unterschiedlichen Vernetzungsgraden herstellen lässt, sehr gute in vitro Biokompatibilität gegenüber humanen ASCs aufweist. Darüber hinaus ist das Hydrogel hervorragend geeignet für die Kultivierung der Zellen sowie für die Unterstützung der osteogenen, adipogenen und chondrogenen Differenzierungsfähigkeit der ASCs. Diese Eigenschaften zusammen mit einer ausgezeichneten Druckbarkeit ermöglichen den Einsatz des Hydrogels GelNB/S im Tissue Engineering bzw. 3D-Bioprinting für die Herstellung von artifiziellen humanen Geweben
Untersuchung des E-Moduls der humanen Cornea
No full textIn dieser Arbeit wurden mittels Nanoidentation die biomechanischen Eigenschaften der menschlichen Hornhaut analysiert. Bei knapp 60 menschlichen Hornhäuten erfolgten insgesamt fast 10.000 Messungen, womit eine Karte des oberflächennahen Elastizitätsmoduls erstellt wurde. Der Nanoindenter zeichnete bei Aufbau einer kontrollierten Belastung den Vorschub der Messspitze im Gewebe auf, womit der E-Modul entsprechend der Hertz-Formel berechnet werden konnte.Hierbei zeigte sich ein vom Zentrum in Richtung Peripherie, im Wesentlichen in alle Richtungen gleich abfallender, zentripetaler Gradient. Passend hierzu konnten während der Indentation, vor allem in der Limbusregion und der Sklera, mehr Flüssigkeitsverschiebungen (engl. Creep) im Gewebe als zentral beobachtet werden. Die Ergebnisse werden in Zusammenhang mit der anatomischen Kollagenstruktur der Hornhaut diskutiert und tragen so zur weiteren Gewebecharakterisierung und dem Verständnis der komplexen biomechanischen Eigenschaften der Hornhaut bei
Fabrication of magnetically actuated micropillars combining micromolding and C,H insertion crosslinking
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