Induction of morphological and physiological reactions of primary skin cells by bio-inspired nano- and microstructured substrates

Im Forschungsgebiet Biomaterialien ist die Entwicklung und Optimierung von Materialien für biologische Anwendungen wie beispielsweise Implantate und Prothesen von zentraler Bedeutung. Gegenwärtig liegt der Fokus auf bioinspirierten und biomimetischen Strategien, wobei die Kenntnis spezifischer Zell-Material-Wechselwirkungen im Mittelpunkt steht. Dieses Wissen ist auch für das Tissue Engineering essentiell, um Zellen funktionell zu kultivieren oder Stammzellnischen nachzuahmen. In dieser Arbeit wurden bioinspirierte Oberflächen hergestellt, die angelehnt an Proteine der extrazellulären Matrix globulär oder fibrillär strukturiert waren. Die Funktionalisierung der Substrate erfolgte mit Amino- oder Carboxygruppen. Mit Hilfe von REM, AFM, ESCA, Ellipsometrie und Kontaktwinkelmessungen ließen sich die erzeugten, modifizierten Substrate charakterisieren. MALDI-TOF MS diente zur Evaluierung der Proteinadsorption, die einen entscheidenden Einfluss auf zelluläre Reaktionen haben kann. Nach erfolgreicher Herstellung und Charakterisierung der Substrate wurden primäre humane Keratinozyten und Fibroblasten auf den Oberflächen kultiviert. Es zeigten sich deutliche und unterschiedliche Reaktionen der Zel¬len in Bezug auf ihre Morphologie und Physiologie. Keratinozyten verfügten auf rauen globulären und fibrillären Substraten über langgestreckte Zellkörper, die cha¬rakteristisch für migrierende Zellen sind. Fibroblasten richteten sich bei den rauen fibrillären Substraten parallel zu den Rillen aus. Diese Resultate verdeutlichen den Einfluss von bioinspirierten Geometrien, da mit der erzeugten fibrillären Strukturierung große Kollagen-Fibrillen nachgeahmt wurden, an die sich Fibroblasten in vivo anordnen. Fokalkontakte, die für die feste Verankerung adhärenter Zellen wichtig sind, waren auf allen Oberflächen nachzuweisen. Eine deutlich höhere Viabilität und Proliferation von Keratinozyten auf amino-modifizierten Substraten ergaben die WST-Messungen. Die Ausbildung von Zell-Zell Kontakten wurde bei keinem Zelltyp durch die Strukturierung und Funktionalisierung beeinträchtigt. Bei der Evaluierung des Differenzierungszustandes primärer Keratinozyten kamen auf allen Oberflächen über 95% früh differenzierte, Cytokeratin 14 exprimierende Zellen vor. Der als epidermaler Stammzellmarker diskutierte Transkriptionsfaktor p63 war in einzelnen Zellen auf allen Substraten nachzuweisen. Tendenziell schienen besonders Keratinozyten auf carboxy-modifizierten Oberflächen und am Rand von Kolonien diesen Marker zu besitzen, wohingegen hoch proliferative Zellen im Zentrum der Kolonien vorlagen. Eine Bestimmung der Funktio¬nalität von Fibroblasten erfolgte über den Nachweis der Kollagen Typ I Produktion. Die Syn¬these dieses Proteins war auf strukturierten und funktionalisierten Substraten nicht eingeschränkt. Bei REM-Aufnahmen zeigten sich Unterschiede in der Ausbildung von sensorischen Aktin-Ausstülpungen, den Filopodien. Kera¬tinozyten auf rauen Oberflächen bildeten deutlich weniger dieser Strukturen aus. Diese Arbeit liefert neue Erkenntnisse bezüglich zelltypspezifischer Material-Wechselwirkungen. Neben der Herstellung, Funktionalisierung und Charakterisierung von bioinspirierten Substraten konnten morphologische und physiologische Reaktionen primärer humaner Keratinozyten und Fibroblasten induziert werden.Research in biomaterial sciences focus on the development and optimization of materials for biological applications, e.g. implants and prostheses. Main topics are bioinspired or biomimetic strategies. Knowledge of specific cell-material interaction is crucially important, especially for the cultivation of functional cells and the in vitro imitation of stem cell niches in tissue engineering. Within the scope of this dissertation bio-inspired globular or fibrillar structured substrates were generated, modelled after extracellular matrix proteins. Surfaces were functionalized with amino- or carboxy groups. The produced modified substrates were evaluated by SEM, AFM, XPS, ellipsometry and contact angle measurements. For characterizing the protein adsorption, which is essential for cell adhesion, MALDI-TOF MS was performed. After their successful generation and characterization, pri¬mary human keratinocytes and fibroblasts were cultivated on the modified substrates. Different morphological and physiological reactions of keratinocytes and fibroblasts could be determined. On rough globular or fibrillar substrates the cell shape of keratinocytes was elongated, which is characteristic for migrating cells. Fibroblasts strongly aligned to rough fibrillar structures, which had similar dimensions to collagen fibrils in vivo. This result underlines the impact of bio-inspired geometries and dimensions in biomaterial sciences. The formation of focal adhesions, which is necessary for strong attachment to the surface, could be observed for adherent cells on all substrates. Based on WST-measurements a higher viability and proliferation were determined for keratinocytes cultured on amino-modified surfaces. Functionalization and structuring of the material interfaces did not have any negative effects on the formation of cell-cell contacts. Regarding the differentiation of keratino-cytes, more than 95% of the adherent cells on all substrates were very early differentiated keratinocytes, which express cytokeratin 14. P63 is discussed as an epidermal stem cell marker and could be detected in several keratinocytes on all sub-strates. While more p63 positive cells seemed to be at carboxy-modified substrates and at the edge of the colonies, highly proliferative keratinocytes were located in the centre of the colonies. Functionality of fibroblasts was verified by staining of collagen type I production. Fibroblasts showed no change in the protein syntheses on structured and functionalized surfaces. With SEM differences in the development of sensory actin-structures, so called filopodia were found. Keratinocytes on rough sub-strates had notably less of these structures than adherent cells on planar surfaces. The results give new insights into cell-material interactions of selected primary human skin cells. Bioinspired substrates were generated, functionalized and characterized. With these modified surfaces morphological and physiological reactions of keratinocytes and fibroblasts could be induce

How Mechanical and Physicochemical Material Characteristics Influence Adipose-Derived Stem Cell Fate

Adipose-derived stem cells (ASCs) are a subpopulation of mesenchymal stem cells. Compared to bone marrow-derived stem cells, they can be harvested with minimal invasiveness. ASCs can be easily expanded and were shown to be able to differentiate into several clinically relevant cell types. Therefore, this cell type represents a promising component in various tissue engineering and medical approaches (e.g., cell therapy). In vivo cells are surrounded by the extracellular matrix (ECM) that provides a wide range of tissue-specific physical and chemical cues, such as stiffness, topography, and chemical composition. Cells can sense the characteristics of their ECM and respond to them in a specific cellular behavior (e.g., proliferation or differentiation). Thus, in vitro biomaterial properties represent an important tool to control ASCs behavior. In this review, we give an overview of the current research in the mechanosensing of ASCs and current studies investigating the impact of material stiffens, topography, and chemical modification on ASC behavior. Additionally, we outline the use of natural ECM as a biomaterial and its interaction with ASCs regarding cellular behavior

Osteoclast formation within a human co-culture system on bone material as an in vitro model for bone remodeling processes

Bone remodeling can be mimicked in vitro by co-culture models. Based on bone cells, such co-cultures help to study synergistic morphological changes and the impact of materials and applied substances. Hence, we examined the formation of osteoclasts on bovine bone materials to prove the bone resorption functionality of the osteoclasts in three different co-culture set-ups using human monocytes (hMCs) and (I) human mesenchymal stem cells (hMSCs), (II) osteogenic differentiated hMSCs (hOBs), and (III) hOBs in addition of soluble monocyte-colony stimulating factor (M-CSF) and cytokine receptor activator of NFκB ligand (RANKL). We detected osteoclast-specific actin morphology, as well as the expression of cathepsin K and CD51/61 in single cells in set-up II and in numerous cells in set-up III. Resorption pits on bone material as characteristic proof of functional osteoclasts were not found in set-up I and II, but we detected such resorption pits in set–up III. We conclude in co-culture models without M-CSF and RANKL that monocytes can differentiate into osteoclasts that show the characteristic actin structures and protein expression. However, to receive functional bone resorbing osteoclasts in vitro, the addition of M-CSF and RANKL is needed. Moreover, we suggest the use of bone or bone-like materials for future studies evaluating osteoclastogenesis

EGF and hydrocortisone as critical factors for the co-culture of adipogenic differentiated ASCs and endothelial cells

In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose-derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available