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DDC: 570 × Subject: heparin ×

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    Geometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization
    (San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.
    To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.
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    Techniques for RNA extraction from cells cultured in starPEG-heparin hydrogels
    (London : Royal Society Publishing, 2021) Jaeschke, Anna; Harvey, Nicholas R.; Tsurkan, Mikhail; Werner, Carsten; Griffiths, Lyn R.; Haupt, Larisa M.; Bray, Laura J.
    Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.