Gail A. McFarland

A highly elastic tissue sealant based on photopolymerised gelatin

Gelatin is widely used as a medical biomaterial because it is readily available, cheap, biodegradable and demonstrates favourable biocompatibility. Many applications require stabilisation of the biomaterial by chemical crosslinking, and this often involves derivatisation of the protein or treatment with cytotoxic crosslinking agents. We have previously shown that a facile photochemical method, using blue light, a ruthenium catalyst and a persulphate oxidant, produces covalent di-tyrosine crosslinks in resilin and fibrinogen to form stable hydrogel biomaterials. Here we show that various gelatins can also be rapidly crosslinked to form highly elastic (extension to break >650%) and adhesive (stress at break >100 kPa) biomaterials. Although the method does not require derivatisation of the protein, we show that when the phenolic (tyrosine-like) content of gelatin is increased, the crosslinked material becomes resistant to swelling, yet retains considerable elasticity and high adhesive strength. The reagents are not cytotoxic at the concentration used in the photopolymerisation reaction. When tested in vivo in sheep lung, the photopolymerised gelatin effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. The elastic properties, thermal stability, speed of curing and high tissue adhesive strength of this photopolymerised gelatin, offer considerable improvement over current surgical tissue sealants.

Multifunctional Polymer Coatings for Cell Microarray Applications

Biocompatible coatings with suitable chemistries for the immobilization of biomolecules are increasingly in demand, as they can be applied in a wide range of biomedical applications. In particular, multifunctional polymer coatings displaying reactive functional groups for the immobilization of specific biological factors that can influence the cellular response while at the same time exhibiting low nonspecific protein adsorption and cell attachment properties have the potential to significantly advance the fields of biomaterials and regenerative medicine. In this study, multifunctional polymer surface chemistries were developed for a cell microarray application with the aim of screening cellular interactions with surface immobilized factors. Coatings were prepared by the deposition of an allylamine plasma polymer pinning layer followed by the deposition of random copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). Coatings were characterized by X-ray photoelectron spectroscopy (XPS), infrared spectroscopy, ellipsometry, and contact angle measurements. A variety of proteins as well as synthetic polymers were printed onto copolymer-coated slides using a high-precision contact microarrayer. Printing conditions were optimized for a fluorescently labeled model protein in regard to the temperature, humidity, pin geometry, concentration, and pH of the printing solution. Finally, the suitability of the surface chemistry for the evaluation of cellular responses to surface immobilized factors in a microarray format was demonstrated using HeLa cells.

High Refractive Index Polysiloxane as Injectable, In Situ Curable Accommodating Intraocular Lens

Functionalised siloxane macromonomers, with properties designed for application as an injectable, in situ curable accommodating intraocular lens (A-IOL), were prepared via re-equilibration of a phenyl group-containing polysiloxane of very high molecular weight with octamethylcyclotetrasiloxane (D₄) and 2,4,6,8-tetra(n-propyl-3-methacrylate)-2,4,6,8-tetramethyl-cyclotetrasiloxane (D₄(AM)) in toluene using trifluoromethanesulfonic acid as a catalyst. Hexaethyldisiloxane was used as an end group to control the molecular weight of the polymer. The generated polymers had a consistency suitable for injection into the empty lens capsule. The polymers contained a low ratio of polymerisable groups so that, in the presence of a photo-initiator, they could be cured on demand in situ within 5 min under irradiation of blue light to form an intraocular lens within the lens capsule. All resulting polysiloxane soft gels had a low elastic modulus and thus should be able to restore accommodation. The pre-cure viscosity and post-cure modulus of the generated polysiloxanes were controlled by the end group and D₄(AM) concentrations respectively in the re-equilibration reactions. The refractive index could be precisely controlled by adjusting the aromatic ratio in the polymer to suit such application as an artificial lens. Lens stretching experiments with both human and non-human primate cadaver lenses of different ages refilled with polysiloxane polymers provided a significant increase in amplitude of accommodation (up to 4 D more than that of the respective natural lens). Both in vitro cytotoxicity study using L929 cell lines and in vivo biocompatibility study in rabbit models demonstrated the non-cytotoxicity and ocular biocompatibility of the polymer.

The use of corneal organ culture in biocompatibility studies

This study investigated the potential of a corneal organ culture system in the evaluation of polymers for ophthalmic devices that require epithelialisation. Two different polymers were tested in lenticule form to explore the sensitivity of this in vitro assay. Polycarbonate and perfluoropolyether-based lenticules were surgically implanted into bovine corneas and compared with a parallel series of sham-wounded corneas. Following surgery, all corneas were maintained in an air/liquid organ culture system for up to 8 days during which time they were evaluated clinically to monitor the rate of epithelial growth across the lenticule surface (implanted) or wound bed (sham). Data showed differences in the kinetics of epithelial migration according to the underlying surface with full epithelialisation of the sham series occurring on day 5+/-0.5, the perfluoropolyether lenticules on day 6+0.5 and polycarbonate lenticules on day 8+/-0.5. Histology revealed differences in the structure and morphology of the migrating and stable epithelium in each series of corneas. The differential response of the corneal epithelium was related to the physiochemical characteristics of the natural (sham) or synthetic (perfluoropolyether or polycarbonate) substrata which the epithelium could detect when maintained in organ culture. This assay system has utility for screening candidate polymers for certain ophthalmic applications.

One step multifunctional micropatterning of surfaces using asymmetric glow discharge plasma polymerization

Micropatterning of surfaces with varying chemical, physical and topographical properties usually requires a number of fabrication steps. Herein, we describe a micropatterning technique based on plasma enhanced chemical vapour deposition (PECVD) that deposits both protein resistant and protein repellent surface chemistries in a single step. The resulting multifunctional, selective surface chemistries are capable of spatially controlled protein adhesion, geometric confinement of cells and the site specific confinement of enzyme mediated peptide self-assembly.

Biocompatibility and modification of the protein‐based adhesive secreted by the Australian frog Notaden bennetti

When provoked, Notaden bennetti frogs secrete a proteinaceous exudate, which rapidly forms a tacky and elastic glue. This material has potential in biomedical applications. Cultured cells attached and proliferated well on glue-coated tissue culture polystyrene, but migrated somewhat slower than on uncoated surfaces. In organ culture, dissolved glue successfully adhered collagen-coated perfluoropolyether lenses to debrided bovine corneas and supported epithelial regrowth. Small pellets of glue implanted subcutaneously into mice were resorbed by surrounding tissues, and all of the animals made a full recovery. An initial but transient skin necrosis at the implant site was probably caused by some of the potentially toxic metabolites present in the frog secretion; these include sterols and carotenoids, as well as fatty alcohols, aldehydes, ketones, acids, and aromatic compounds. Removal of the carotenoid pigments did not significantly alter the glues material properties. In contrast, peroxidase treatment of dissolved glue introduced unnatural crosslinks between molecules of the major protein (Nb-1R) and resulted in the formation of a soft hydrogel, which was very different to the original material.

The architecture of a collagen coating on a synthetic polymer influences epithelial adhesion

The current study sought to identify a collagen coating methodology for application to polymer surfaces that would provide for the development of adhesive structures responsible for the sustained adhesion of corneal epithelial tissue. We compared an uncoated microporous polycarbonate surface and equivalent surfaces coated with either covalently immobilized collagen I or chemically crosslinked collagen I gel in a corneal explant outgrowth assay over 21 days. Electron microscopy was used to examine the formation of hemidesmosomes, basal lamina, and anchoring fibrils at the tissue-polymer interface. The crosslinked collagen gel preparation supported the overlying epithelial tissue across the pore openings and allowed for the formation of identifiable basal lamina, hemidesmosomes, and anchoring fibrils between the epithelial tissue and the polymer surface. Hemidesmosomal plaque, but no basal lamina or anchoring fibril formation, occurred on the uncoated surface or on that coated with covalently immobilized collagen I. We propose that the collagen matrix provided by the crosslinked collagen gel was reorganized by the epithelial tissue and that this, combined with the secretion of ECM molecules, served to limit the diffusion of basement membrane components, which permitted an increase in the local concentration of these molecules, which favored the assembly of epithelial adhesive structures.

The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion

Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.

Microarrays for the evaluation of cell-biomaterial surface interactions

The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.