Stephen Rimmer

Development of biodegradable electrospun scaffolds for dermal replacement

Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA+10% oligolactide; (3) PLLA+rhodamine and (4-6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.

Production and performance of biomaterials containing RGD peptides

The arginine-glycine-aspartic acid (RGD) sequence binds to key cell membrane structures within the integrin family. These sequences are found on several extracellular proteins that have roles in cell adhesion. Therefore, it is not surprising that with the advent of the field of tissue engineering a large amount of work has been carried out on materials functionalised with a wide variety of RGD-containing peptides. In this review we provide an overview of the field to 2007 and we include both synthesis and design strategies required to produce useful RGD functional materials.

Hyper/highly-branched polymers by radical polymerisations

Hyper branched and highly branched polymers (HB polymers) can be prepared by a variety of radical polymerisations. Here we describe the various techniques that can be used and include hybrid polymerisations that involve both radical and ring opening polymerisations. Adaptations of established techniques that use transfer to monomer are described. Then controlled radical polymerisations are outlined. Finally, some of emergent applications are reviewed with a special emphasis on applications in the life sciences.

Highly branched poly-(N-isopropylacrylamide)s with arginine–glycine–aspartic acid (RGD)- or COOH-chain ends that form sub-micron stimulus-responsive particles above the critical solution temperature

Highly branched poly(-isopropyl acrylamide)s with peptide-end groups form colloidally stable dispersions of sub-micron particles above the lower critical solution temperature.

Synthesis and properties of amphiphilic networks 2: a differential scanning calorimetric study of poly(dodecyl methacrylate-stat-2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate) networks and adhesion and spreading of dermal fibroblasts on these materials

A series of amphiphilic networks was prepared by radical copolymerisation of dodecyl methacrylate, 2,3-propandiol-1-methacrylate and ethandiol dimethacrylate. DSC studies on these materials, swollen in water. revealed that only materials containing more than 27 wt% of water displayed melting endotherms due to the melting of ice-like structures of water (freezing water). In materials that did produce a melting endotherm the peak was generally bimodal. Changing thermal history and heating rate did not effect the shape of the two peaks, nor the relative contribution of each peak to the total endothermic response. These observations and the narrow peak width of the low temperature endotherm suggested that the bimodality was an artefact of the DSC experiment and may be due to the promotion of the glass transition once a fraction of the water has frozen. The morphology of transformed human dermal fibroblasts grown on these materials was then examined by scanning electron microscopy. Compositions that contained only non-freezing water were found to allow cell adhesion and spreading. Cells with well-spread morphologies were obtained on materials containing small fractions of freezing water and dodecyl methacrylate. These fibroblasts displayed surface features such as microvilli and filapodia. However, all compositions of poly(2,3-propandiol-1-methacrylate-co-ethandiol dimethacrylate) (i.e. hydrogels that do not contain dodecyl methacrylate repeat units) were poor substrates for cell growth and examination of these materials showed that very few cells had adhered and those that did were highly rounded.

Binding bacteria to highly branched poly(N-isopropyl acrylamide) modified with vancomycin induces the coil-to-globule transition.

Binding of highly branched poly(N-isopropylacrylamide) with vancomycin end groups to Staphylococcus aureus induced a coil-to-globule phase transition. The polymers aggregated this gram-positive bacteria (but not gram-negative bacteria) over a wide range of temperatures, but cooling to 24-26 degrees C progressed the polymer-bound bacteria through a globule-to-coil phase transition, after which the bacteria were released.

Thermally responsive polymeric hydrogel brushes: synthesis, physical properties and use for the culture of chondrocytes

Hydrogel brushes are materials composed of a water-swollen network, which contains polymer chains that are grafted with another polymer. Using a thermally responsive polymer, poly(N-isopropyl acrylamide) (polyNIPAM), as the graft component we are able to maintain the critical solution temperature (Tcrit), independent of the overall composition of the material, at approximately 32°C. The change in swelling at Tcrit is a function of the amount of polyNIPAM in the system. However, there is a much smaller change in the surface contact angles at Tcrit. PolyNIPAM-based materials have generated considerable interest, as ‘smart’ substrates for the culture of cells and here, we show the utility of hydrogel brushes in cell culture. Chondrocytes attached to the hydrogel brushes and yielded viable cell cultures. Moreover, the chondrocytes could be released from the hydrogel brushes without the use of proteases by reducing the temperature of the cultures to below Tcrit to induce a change in the conformation of the polyNIPAM chain at Tcrit. The importance of the crosslink hydrogel component is illustrated by significant changes in cell attachment/cell viability as the crosslink density is changed.

Time-resolved fluorescence anisotropy studies of the temperature-induced intramolecular conformational transition of poly(N-isopropylacrylamide) in dilute aqueous solution

Abstract Time-resolved fluorescence anisotropy measurements have been performed upon an acenaphthylene-labelled (0.5 mol%) sample of poly( N -isopropylacrylamide), PNIPAM, in dilute solutions in both methanol and water as solvents. In methanol, segmental relaxation of PNIPAM follows an Arrhenius dependence upon temperature over the range investigated (279–323K) characterized by an ‘activation energy’ of 13.4(±0.5) kJ mol −1 . This is only slightly greater (by ca 2.4 kJ mol −1 ) than that of solvent flow and it is likely that specific dipolar interactions between the PNIPAM and the methanol determine the macromolecular dynamics in this solvent. In aqueous solution, the segmental mobility of PNIPAM exhibits a dramatic thermoreversible discontinuity at ca 32°C. This change in conformational behaviour occurs at the polymers lower critical solution temperature in aqueous media. This observation, supports the proposition (Winnik, F. M. Polymer 1990, 31, 2125) that the thermally-induced separation in this system occurs by a ‘dual mode’ mechanism wherein intermolecular aggregation is preceded by intramolecular conformational shrinkage of the polymer coils.

Hyperbranched poly(NIPAM) polymers modified with antibiotics for the reduction of bacterial burden in infected human tissue engineered skin

The escalating global incidence of bacterial infection, particularly in chronic wounds, is a problem that requires significant improvements to existing therapies. We have developed hyperbranched poly(NIPAM) polymers functionalized with the antibiotics Vancomycin and Polymyxin-B that are sensitive to the presence of bacteria in solution. Binding of bacteria to the polymers causes a conformational change, resulting in collapse of the polymers and the formation of insoluble polymer/bacteria complexes. We have applied these novel polymers to our tissue engineered human skin model of a burn wound infected with Pseudomonas aeruginosa and Staphylococcus aureus. When the polymers were removed from the infected skin, either in a polymer gel solution or in the form of hydrogel membranes, they removed bound bacteria, thus reducing the bacterial load in the infected skin model. These bacteria-binding polymers have many potential uses, including coatings for wound dressings.

Emulsion polymerizations in the presence of β-cyclodextrin

Poly(butyl methacrylate) particles were prepared using an emulsion polymerization procedure with β-cyclodextrin (β-CD) in place of the surfactant. The rate of polymerization was lower than in the equivalent sulphate surfactant stabilized emulsion polymerizations. Particle size and molecular weight distributions were unimodal and did not show any dependance on β-CD concentration. However, the average molecular weight was, in all β-CD containing polymerizations, significantly higher than in polymerizations in the absence of both surfactant and β-CD. β-CD had little effect on the behaviour of surfactant-containing systems. A tentative explanation, involving the enhancement of colloidal stability by poly(butyl methacrylate) included by β-CD amphiphilic polymers, is proposed.