Stuart L. James

A review of tissue-engineered skin bioconstructs available for skin reconstruction

Situations where normal autografts cannot be used to replace damaged skin often lead to a greater risk of mortality, prolonged hospital stay and increased expenditure for the National Health Service. There is a substantial need for tissue-engineered skin bioconstructs and research is active in this field. Significant progress has been made over the years in the development and clinical use of bioengineered components of the various skin layers. Off-the-shelf availability of such constructs, or production of sufficient quantities of biological materials to aid rapid wound closure, are often the only means to help patients with major skin loss. The aim of this review is to describe those materials already commercially available for clinical use as well as to give a short insight to those under development. It seeks to provide skin scientists/tissue engineers with the information required to not only develop in vitro models of skin, but to move closer to achieving the ultimate goal of an off-the-shelf, complete full-thickness skin replacement.

Gelatin-fibrinogen cryogel dermal matrices for wound repair: Preparation, optimisation and in vitro study.

Macroporous sponge-like gelatin-fibrinogen (Gl-Fg) scaffolds cross-linked with different concentrations (0.05-0.5%) of glutaraldehyde (GA) were produced using cryogelation technology, which allows for the preparation of highly porous scaffolds without compromising their mechanical properties, and is a more cost-efficient process than freeze-drying. The produced Gl-Fg-GA(X) scaffolds had a uniform interconnected open porous structure with a porosity of up to 90-92% and a pore size distribution of 10-120 microm. All of the obtained cryogels were elastic and mechanically stable, except for the Gl-Fg-GA(0.05) scaffolds. Swelling kinetics and degradation rate, but not the porous structure of the cryogels, were strongly dependent on the degree of cross-linking. A ten-fold increase in the degree of cross-linking resulted in an almost 80-fold decrease in the rate of degradation in a solution of protease. Cryogels were seeded with primary dermal fibroblasts and the densities observed on the surface, plus the expression levels of collagen types I and III observed 5 days post-seeding, were similar to those observed on a control dermal substitute material, Integra. Fibroblast proliferation and migration within the scaffolds were relative to the GA content. Glucose consumption rate was 3-fold higher on Gl-Fg-GA(0.1) than on Gl-Fg-GA(0.5) cryogels 10 days post-seeding. An enhanced cell motility on cryogels with reducing GA crosslinking was obtained after long time culture. Particularly marked cell infiltration was seen in gels using 0.1% GA as a crosslinker. The scaffold started to disintegrate after 42 days of in vitro culturing. The described in vitro studies demonstrated good potential of Gl-Fg-GA(0.1) scaffolds as matrices for wound healing.

Characterisation and performance of hydrogel tissue scaffolds

Porosity over a broad range (typically 0.001–300 μm in diameter) of tissue scaffolds provides appropriate conditions for diffusion and adsorption of small molecules and macromolecules, migration of cells through the scaffold, and adequate cell proliferative capacity. Characterisation of pores over this large range poses a problem especially when analysing soft polymer hydrogels, as no one methodology can adequately cover the entire range. This work describes a combined technique used for evaluation of the porous structure of a collagen hydrogel (dermal substitute Integra®) on the basis of NMR-cryoporometry (sensitive to nanopores) and confocal laser scanning microscopy (CLSM) imaging (sensitive to macropores). Thermodesorption of water, diffusion of proteins through a collagen membrane, migration and growth of normal primary human skin fibroblasts, and the interaction kinetics of 3T3 mouse fibroblast cells (using a quartz crystal microbalance) with collagen were analysed with respect to the porous structure of the material. The contribution to the total porosity of pores with a diameter of less than 100 nm is low, at approximately 3–5%, a figure estimated using the methods described above. However, these pores are the main contributor to the specific surface area (S ≈ 120 m2 g−1) as larger diameter macropores, with diameters of 20–200 μm, have a much lower surface area at S ≈ 0.4 m2 g−1 relative to their large pore volume V = 14.4 cm3 g−1.

Measurement of Pore Size and Porosity of Tissue Scaffolds

Tissue engineering involves seeding the patient’s cells on to a three-dimensional temporary scaffold. It is becoming increasingly obvious that cells used to seed the scaffold have very specific requirements concerning the morphology and chemistry of the surface of the scaffold and its interconnectivity. A range of techniques has been examined in relation to key measurements such as pore size and porosity. Since capillary flow porometry measures a pore solely at its most constricted point, the method is unable to provide characterisation of other aspects of the pore. Scanning Electron Microscopy is limited to examining surface pores in ‘stiff’ scaffolds. Although cryo-SEM reduces the amount of ice-induced damage generated in ‘soft’ scaffolds upon freezing, the technique is limited to the same constraints. Images produced via scanning confocal microscopy are probably more representative of the true structure of the scaffold than that implied by cryo-SEM, although due to the diffuse nature of the image it is difficult to measure pore sizes.

Methodologies for assessing local surface texture features that are relevant to cell attachment

In this paper, we describe techniques for extracting features from surface topography data, gathered by a 3D-microscopy system, on a length scale that is relevant for cell attachment. The feature parameters considered include standard surface roughness parameters applied to the complete surface as well as new feature parameters designed to quantify local variations in surface topography potentially influencing cell behaviour. Methodologies have been developed both to determine the degree of homogeneity or isotropy of a surface and to compare the topographies of different samples. The approaches followed include wavelet decomposition and linear and nonlinear filtering techniques. The analysis has been used to investigate the correlation between osteoblast cell attachment and structural features of titanium-coated surfaces representative of orthopaedic implants. The results confirm that there is a discernible correlation between cell orientation and the underlying surface lay.

Rheological Properties and Hydration of Airway Mucus

The protective functions of mucus may chiefly be defined by its barrier and transport properties. These properties are largely, but not exclusively, determined by the physical characteristics of the mucus which, in turn, may be most usefully considered as its characteristic behaviour under physical stress, that is, its rheological properties [1]. Abnormalities in the rheological properties of respiratory mucus are reliable indicators of suboptimal transport properties. Increases in viscoelastic parameters, common in cystic fibrosis, for example, lead to poor mucociliary clearance, whilst it has been suggested that a reduction in viscoelastic parameters, a situation possibly found in fucosidosis, will also result in impaired clearance [2].

An in vitro evaluation of fibrinogen and gelatin containing cryogels as dermal regeneration scaffolds

Macroporous cryogels containing mixtures of two key components of the dermal extracellular matrix, fibrinogen and collagen-derived gelatin, were evaluated for use as dermal tissue regeneration scaffolds. The infiltration of human dermal fibroblasts into these matrices was quantitatively assessed in vitro using a combination of cell culture and confocal laser scanning microscopy. The extent of cellular infiltration, as measured by the number of cells per distance travelled versus time, was found to be positively correlated with the fibrinogen concentration of the cryogel scaffolds; a known potentiator of cell migration and angiogenesis within regenerating tissue. An analysis of the proteins expressed by infiltrating fibroblasts revealed that the cells that had migrated into the interior portion of the scaffolds expressed predominantly F-actin along their cytoplasmic stress fibres, whereas those present on the periphery of the scaffolds expressed predominantly α-smooth muscle actin, indicative of a nonmotile, myofibroblast phenotype associated with wound contraction. In conclusion, the cryogels produced in this study were found to be biocompatible and, by alteration of the fibrinogen content, could be rendered more amenable to cellular infiltration.

Real-time monitoring of cellular integration within bulk soft tissue scaffold materials

A non-invasive, real-time acoustic method for the monitoring of cellular integration within commercial collagen-based dermal replacement scaffolds is reported for the first time. An unexpectedly high degree of acoustic energy transfer through heavily hydrated thick film (up to 0.5 mm) sections of collagen/glycosaminoglycan scaffold material intimately associated with a quartz crystal sensor allowed quantitative resonant frequency measurements upon application of fibroblast cell suspensions to the material. Changes in resonant frequency and energy dissipation were commensurate with cellular interaction with the gel.

Use of Poly (ε-Lysine) Dendrons: A Strategy Targeting Bacterial Quorum Sensing and Biofilm Formation

Pseudomonas aeruginosa is recognised as a major aetiological agent of nosocomial infections, which are associated with multiple-antibiotic resistance. Among many of its important virulence factors is its ability to form biofilms on the surfaces of implantable medical devices and to produce toxic metabolites, pyocyanin, via an intercellular cell density-dependent signalling system of communication. In this study, poly (-lysine) dendrons composed of increasingly branching generations were synthesised, characterised, and examined for their effects on virulence factor production in P. aeruginosa. The results show that these hyperbranched poly (-lysine) dendrons, in particular the 3rd generation, can increase the efficacy of a conventional antibiotic, ciprofloxacin, and reduce pyocyanin production, with marginal effects on the rate of bacterial replication, suggesting that the observed effects are not due to dendron toxicity. Furthermore, dendron and ciprofloxacin coadministration was identified as the most effective strategy which highlights the potential of peptide-based dendrons as quorum sensing inhibitors.

Affinity Binding Macroporous Monolithic Cryogel as a Matrix for Extracorporeal Apheresis Medical Devices

Cytapheresis is an extracorporeal separation technique widely used in medicine for elimination of specific classes of blood cells from circulating blood. It has been shown recently to have clinical efficacy in various disease states, such as leukaemia, autoimmune disorders, rheumatoid arthritis, renal allograft rejection and sickle–cell anaemia. The current study was undertaken to produce an affinity-binding column, based upon a macroporous monolithic cryogel with a structure of interconnected pores, with pore size and low flow resistance potentially suitable for use in cytapheresis. The affinity column was produced from poly (2-hydroxyethyl methacrylate) PHEMA cryogels synthesized by free radical polymerization at -12°C. This study involved assessing haemolytic potential, and functionalisation of polymer matrix with biological ligands. Haemolytic potential of poly (2-hydroxyethyl methacrylate) cryogel was established by measuring free haemoglobin after blood filtration through the column. The anti-human albumin (antibody) was chemically coupled to the epoxy derivatised monolithic cryogels and the binding efficiency of anti-human albumin (antibody) to the cryogel was determined. Our results show that approximately 100% of Red blood cells passed through the column with no evidence of haemolysis found in blood eluted. It was found that ~82% of human serum albumin was retained on the monolithic IgG anti-human albumin cryogel matrix. The obtained results suggest that poly (2-hydroxyethyl methacrylate) monolithic cryogel is a non-haemolytic material (haemocompatible matrix) capable of functionalisation with antibody and thus can be an appropriate matrix for use in extracorporeal apheresis system.