Most cell types will only survive to form viable cultures in vitro if they are in contact with a surface. Commonly ‘tissue culture polystyrene’ – a special grade of polystyrene – is used for this purpose. Techniques for preparing a wide range of types of surface chemistry have been developed at Sheffield to improve (or prevent) cells from growing on them. Additionally, bioactive surface to prevent inflammation have been created.
The most clinically advanced of these new materials –‘Myskin™’ – is now used to deliver human keratinocyte cultures to non-healing wounds and severe burns patients in the clinic.
Above right: A batch of Myskin™ plastic patches ready to receive keratinocytes for culture, after which they will be used in the clinic.
Plasma polymerised surface chemistries
Plasma polymerisation is a technique in which a surface is exposed to a low-pressure plasma containing monomer radicals which condense on the surface to form a pinhole-free polymeric film. The advantages of this technique are:
- That the film can be deposited on a very wide range of substrates
- It requires minimal surface pre-preparation
- It leaves no holes, so the patient’s body never comes into contact with the underlying material
- While the polymerisation process is random, some control over the final surface chemistry is possible through careful selection of monomer
Above right: Photograph of a plasma polymerisation. The glowing plasma completely envelopes the samples being treated, depositing a pinhole-free film.
This technique allows materials with good engineering or mechanical properties to have their surface chemistry changed to tune their biocompatibility. For example – medical grade PVC modified to encourage colonisation by keratinocytes is now marketed as Myskin™.
In tissue engineering we usually want cells to colonise a surface, but for many applications, such as contact lenses or medical sensors, we want to dissuade cells from settling, and prevent a build-up of proteinaceous plaque that would cause irritation or result in device failure.
Above left: Nerve cells do not adhere to allyll amine surfaces, but have high affinity for acrylic acid (middle).
Relatively simple modifications to the plasma polymerisation technique allow us to deposit chemical gradients or patterns. These can vary between extreme surface chemistries. In the ‘TONY’ image, a hydrophillic (cell adherent) surface has been laid down over a hydrophobic (non-adherent) surface through a mask. This left a pattern which has been preferentially colonised by cells in culture.
Above right: Cells colonising a surface, part of which has been coated with a hydrophilic / hydrophobic pattern leaving a preferential surface for cells to adhere. Royal Institution Christmas Lectures 2002, ‘Smart Stuff’, Prof. Tony Ryan OBE, Keratinocytes on an acrylic acid / octadiene pattern – John Haycock / BD Biosciences.
Frequently there is a requirement for amplifying cells in vitro, prior to use in a clinical procedure. This necessitates removing cells from their substrate without damaging them, or growing the cells in suspension – a technique that has not met with a great deal of success. Both techniques offer the opportunity for preparing tissues without any contaminating foreign materials, so are of considerable interest.
Recently at Sheffield, ‘smart’ hydrogels have been prepared that can change their cell binding characteristics in response to changes in environmental conditions. Of particular interest are polymer gels that exhibit a lower critical solution temperature (LCST) close to the human physiological temperature. These materials exhibit a stepwise change from an open hydrophilic structure to a tightly coiled hydrophobic structure over a very short temperature range.
Above right: Electron micrograph of cells growing on different block-copolymer gel surfaces. The polymers differ only in the proportions of the two component blocks used: (a) has a high density of cells, while (b) has no adherent cells. Scale bar 5µm.
Careful design of the polymers has resulted in the preparation of a polymer support that acts as a high-density surface when pelleted. On cooling, an initially hydrophilic gel that has good affinity for cells suddenly changes to a hydrophobic form that ejects the cells from its surface. The cells are then readily collected from suspension in the growth medium. Such gels are also being developed as scaffolds for tissue culture and for protein purification.
Above: The chemical structure of a calixarene, in this instance composed of four phenyl rings (nomenclature calixarene) linked to form a basket shape. The lower rim has hydroxyl groups pointing downwards, these form a strong bond to many surfaces. The upper ring can be modified (replacing R) to give a range of chemical functionalities – in this instance an anti-inflammatory peptide, which when adhered to a surface inhibits inflammatory signaling in vitro.
Basket shaped calixarenes have a rim of hydroxyl groups which bind strongly to many surfaces. The R groups on the opposite side to the rim can be replaced with a wide range of chemical groups. A solution of the synthetic calixarene, bearing the chemical group(s), can be applied directly to the surface to be treated, which adsorbs a layer of these molecules.
We are developing calixarenes with short anti-inflammatory peptide sequences. One of the sequences of interest has been shown in isolation to suppress the inflammatory response of cells in vitro. With these calixarenes coating the surface of biomaterials, it is intended that the immune response to implantable devices can be controlled, and the incidence of acute or chronic inflammation reduced.
Above: A self-assembled monolayer shown in diagrammatic form. The sulphide head group (blue) binds strongly to gold and silver substrates. The length of the alkyl chain (grey) can be varied. A second group (R, shown in red) is then presented on the surface. UV light can then be used to pattern discreet regions by photoxidation, removal and the introduction of different chemical groups. The sequence of images to the right shows the assembly of a photopatterned SAM.
The coating of a surface with a self-assembled monolayer provides a precisely controlled surface chemistry. Techniques developed by scientists at Sheffield use long chain alkyl thiols. The terminal thiol group binds tightly to metals such as silver and gold, giving rise to a strongly adherent surface film. Great chemical versatility can be introduced by including a second functional group, such as hydroxyl or carboxylic acid, at the other end of the alkyl chain.
These surfaces are laid down as a single molecule coverage, but patterns can be introduced either by the dip pen technique, or by photolithographic techniques. These rely on the reaction of the thiol group with ozone when exposed to UV light. The product can then be washed off the metal substrate, leaving bare metal. If the substrate is subsequently exposed to a second alkyl thiol, this will stick to exposed metal. The second alkyl thiol can have a different functional group to the first, giving rise to chemical patterns in the surface.