Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation

David C. Bassett *, Armend G. Håti , Thor B. Melø , Bjørn T. Stokke and Pawel Sikorski
Biophysics and Medical Technology, Department of Physics, Norwegian University of Science and Technology, Trondheim, NO-7491, Norway. E-mail: david.bassett@ntnu.no

Abstract

Currently there are limitations to gelation strategies to form ionically crosslinked hydrogels, derived in particular from a lack of control over the release kinetics of crosslinking ions, which severely restrict applications. To address this challenge, we describe a new approach to form hydrogels of ionotropic polymers using competitive displacement of chelated ions, thus making specific ions available to induce interactions between polymer chains and form a hydrogel. This strategy enables control of ion release kinetics within an aqueous polymer solution and thus control over gelation kinetics across a wide range of pH. The described technique simplifies or facilitates the use of ionotropic hydrogels in a range of applications, such as 3D printing, microfluidic-based cell encapsulation, injectable preparations and large scale bubble and solid free mouldable gels. We investigate a range of chelator-ion combinations and demonstrate this powerful method to form hydrogels across a wide range of pH and µm–cm length scales. We highlight our findings by applying this gelation strategy to some of the more challenging hydrogel application areas using alginate and polygalacturonate as model polymer systems.

Reference: David Bassett, Armend Gazmeno Håti, Thor Melø, Bjørn Torger Stokke, Pawel Sikorski. Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation. Journal of Materials Chemistry B 2016.

Tunable high aspect ratio polymer nanostructures for cell interfaces

Author affiliations

Abstract

Nanoscale topographies and chemical patterns can be used as synthetic cell interfaces with a range of applications including the study and control of cellular processes. Herein, we describe the fabrication of high aspect ratio nanostructures using electron beam lithography in the epoxy-based polymer SU-8. We show how nanostructure geometry, position and fluorescence properties can be tuned, allowing flexible device design. Further, thiol–epoxide reactions were developed to give effective and specific modification of SU-8 surface chemistry. SU-8 nanostructures were made directly on glass cover slips, enabling the use of high resolution optical techniques such as live-cell confocal, total internal reflection and 3D structured illumination microscopy to investigate cell interactions with the nanostructures. Details of cell adherence and spreading, plasma membrane conformation and actin organization in response to high aspect ratio nanopillars and nanolines were investigated. The versatile structural and chemical properties combined with the high resolution cell imaging capabilities of this system are an important step towards the better understanding and control of cell interactions with nanomaterials.

Reference: Kai Sandvold Beckwith, Simon Phillip Cooil, Justin W Wells and Pawel  Sikorski. Tunable High Aspect Ratio Polymer Nanostructures for Cell Interfaces.Nanoscale 2015.

Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox

Sindre H. Bjørnøy, Stefan Mandaric, David C. Bassett, Andreas K.O. Åslund, Seniz Ucar, Jens-Petter Andreassen, Berit L. Strand, Pawel Sikorski.

Department of Physics, NTNU, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Department of Chemical Engineering, NTNU, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Department of Biotechnology, NTNU, Norwegian University of Science and Technology, 7491 Trondheim, Norway

 

Abstract

Due to their large water content and structural similarities to the extracellular matrix, hydrogels are an attractive class of material in the tissue engineering field. Polymers capable of ionotropic gelation are of special interest due to their ability to form gels at mild conditions. In this study we have developed an experimental toolbox to measure the gelling kinetics of alginate upon crosslinking with calcium ions. A reaction–diffusion model for gelation has been used to describe the diffusion of calcium within the hydrogel and was shown to match experimental observations well. In particular, a single set of parameters was able to predict gelation kinetics over a wide range of gelling ion concentrations. The developed model was used to predict the gelling time for a number of geometries, including microspheres typically used for cell encapsulation. We also demonstrate that this toolbox can be used to spatiotemporally investigate the formation and evolution of mineral within the hydrogel network via correlative Raman microspectroscopy, confocal laser scanning microscopy and electron microscopy.

 

Reference: Sindre H Bjornoy, Stefan Mandaric, David C Bassett, Andreas KO Åslund, Seniz Ucar, Jens-Petter Andreassen, Berit L Strand, Pawel Sikorski. Gelling kinetics and in situ mineralization of alginate hydrogels: a correlative spatiotemporal characterization toolbox. Acta Biomaterialia 2016.

 

Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

Marianne Sandvold Beckwith, Kai Sandvold Beckwith, Pawel Sikorski, Nan Tostrup Skogaker, Trude Helen Flo , Øyvind Halaas

Department of Physics, NTNU, Trondheim, Norway; Department of Laboratory Medicine, Children’s and Women’s Health, NTNU, Trondheim, Norway; Centre of Molecular Inflammation Research, Norwegian University of Science and Technology (NTNU), Trondheim, Norway, Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway

 

 

Abstract

Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.

 

Reference: Beckwith, MS; Beckwith, KS; Sikorski, P;  Skogaker, NT; Flo, TH; Halaas, Ø. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomograph. PLOS ONE. 2015.


Patterned cell arrays and patterned co-cultures on polydopamine-modified poly(vinyl alcohol) hydrogels

Kai M Beckwith and Pawel Sikorski, Department of Physics, Norwegian University of Science and Technology

Abstract

Live cell arrays are an emerging tool that expand traditional 2D in vitro cell culture, increasing experimental precision and throughput. A patterned cell system was developed by combining the cell-repellent properties of polyvinyl alcohol hydrogels with the cell adhesive properties of self-assembled films of dopamine (polydopamine).

It was shown that polydopamine could be patterned onto spin-cast polyvinyl alcohol hydrogels by microcontact printing, which in turn effectively patterned the growth of several cell types (HeLa, human embryonic kidney, human umbilical vein endothelial cells (HUVEC) and prostate cancer). The cells could be patterned in geometries down to single-cell confinement, and it was demonstrated that cell patterns could be maintained for at least 3 weeks. Furthermore, polydopamine could be used to modify poly(vinyl alcohol) in situ using a cell-compatible deposition buffer (1 mg mL−1 dopamine in 25 mM tris with a physiological salt balance). The treatment switched the PVA hydrogel from cell repellent to cell adhesive. Finally, by combining microcontact printing and in situ deposition of polydopamine, patterned co-cultures of the same cell type (HeLa/HeLa) and dissimilar cell types (HeLa/HUVEC) were realized through simple chemistry and could be studied over time. The combination of polyvinyl alcohol and polydopamine was shown to be an attractive route to versatile, patterned cell culture experiments with minimal infrastructure requirements and low complexity.

Reference: Kai M Beckwith and Pawel Sikorski. Patterned cell arrays and patterned co-cultures on polydopamine-modified poly(vinyl alcohol) hydrogels. Biofabrication 2013, 5 045009 doi:10.1088/1758-5082/5/4/045009.


A transparent nanowire-based cell impalement device suitable for detailed cell–nanowire interaction studies

Florian Mumm, Kai M. Beckwith, Sara Bonde, Karen L. Martinez, and Pawel Sikorski. Department of Physics, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
Bio-Nanotechnology Laboratory, Department of Chemistry, University of Copenhagen, Universitetsparken 5, Copenhagen, DK-2100, Denmark.

Abstract

A method to fabricate inexpensive and transparent nanowire impalement devices is invented based on CuO nanowire arrays grown by thermal oxidation. By employing a novel process the nanowires are transferred to a transparent, cell-compatible epoxy membrane. Cargo delivery and detailed cell-nanowire interaction studies are performed, revealing that the cell plasma membrane tightly wraps the nanowires, while cell membrane penetration is not observed. The presented device offers an efficient investigation platform for further optimization, leading towards a simple and versatile impalement delivery system.

This research includes a combination of nanofabrication, device fabrication and cell experiments. NTNU Nanolab played a critical role characterization of nanowires (STEM), integration of nanowires in polymer based impalement devices (lithography, thin film deposition and sputtering), device characterization (SEM and STEM). Cell experiments and confocal microscopy was performed at the Department of Physics.

Reference: A Transparent Nanowire-Based Cell Impalement Device Suitable for Detailed Cell-Nanowire Interaction Studies. Small 2013. 9, 2, 263–272. DOI: 10.1002/smll.201201314.


The Structure of Cross-β Tapes and Tubes Formed by an Octapeptide, αSβ1

Kyle L. Morris, Shahin Zibaee, Lin Chen, Michel Goedert, Pawel Sikorski,  Louise C. Serpell.

School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, BN1 9QG (UK). MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH (UK). Department of Physics, Norwegian University of Science and Technology, Trondheim (Norway). Centre for Materials Discovery, University of Liverpool, Crown Street, Liverpool, L69 7ZD (UK).

Abstract

The αSβ1 peptide, a fragment of α-synuclein, assembles into flat tapes consisting of a peptide bilayer, which can be modelled based on the cross-β structure found in amyloid proteins. The tapes are stabilized by hydrogen bonding, whilst the amphiphilic nature of the peptide results in the thin bilayer structure. To further stabilize the structure, these tapes may twist to form helical tapes, which subsequently close into nanotubes.

Reference: The Structure of Cross-β Tapes and Tubes Formed by an Octapeptide, αSβ1. Angewandte Chemie International Edition 2013. 52, 8, 2279–2283. DOI: 10.1002/anie.201207699.

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