Staphylococcus aureus binding proteins for prevention of orthopaedic implant-related infections

Orthopaedic implant infections are an increasing problem and management commonly involves implant removal with serious consequences. Biofilm-forming Staphylococci are the most common causative organisms, with Staphylococcus aureus being the most virulent and MRSA increasingly involved. Initial bacterial adhesion is a crucial event in biofilm formation and infection establishment. Directing host antibody against bacterial factors involved in adhesion and biofilm formation may significantly inhibit infection establishment on biomaterials. Two recombinant S. aureus-derived binding proteins (FnBP, IsdA) were investigated as potential vaccine antigens and resultant antibody was assessed to determine whether immune inhibition of bacteria-ligand binding can significantly impact on attachment to plasma-conditioned biomaterial surfaces, in the presence of other bacterial ligands. Adhesion of homologous and heterologous (clinical MRSA) S. aureus to plasma-conditioned steel was significantly reduced (~50% average reduction, p & #60;0.0001) when pre-exposed to anti-rFnBP-A antiserum that was 50-fold more dilute than the actual titre from immunisation. Inhibition was related to ligand presence and not staphylococcal Protein A. Reduced adhesion was not observed with an FnBP-mutant strain, indicating specific inhibitory antibody involvement, and demonstrating the potential of rFnBP-A for prevention of S. aureus implantrelated infection. Adhesion-inhibitory activity was also observed with a purified IgG-fraction of rIsdA antiserum but this activity appeared to be masked by non-IsdA-related interactions when non-IgG-purified antiserum was assessed

Rising influence of synthetic biology in regenerative medicine

Synthetic biology is an emerging area of research that combines the investigative nature of biology with the constructive nature of engineering. Despite the field being in its infancy, it has already aided the development of a myriad of industrially and pharmaceutically useful compounds, devices and therapies and is now being applied within the field of regenerative medicine. By combining synthetic biology with regenerative medicine, the engineering of cells and organisms offers potential avenues for applications in tissue engineering, bioprocessing, biomaterial and scaffold development, stem cell therapies and even gene therapies. This review aims to discuss how synthetic biology has been applied within these distinct areas of regenerative medicine, the challenges it faces and any future possibilities this exciting new field may hold

Visualizing medium and biodistribution in complex cell culture bioreactors using in vivo imaging

There is a dearth of technology and methods to aid process characterization, control and scale-up of complex culture platforms that provide niche micro-environments for some stem cell-based products. We have demonstrated a novel use of 3d in vivo imaging systems to visualize medium flow and cell distribution within a complex culture platform (hollow fiber bioreactor) to aid characterization of potential spatial heterogeneity and identify potential routes of bioreactor failure or sources of variability. This can then aid process characterization and control of such systems with a view to scale-up. Two potential sources of variation were observed with multiple bioreactors repeatedly imaged using two different imaging systems: shortcutting of medium between adjacent inlet and outlet ports with the potential to create medium gradients within the bioreactor, and localization of bioluminescent murine 4T1-luc2 cells upon inoculation with the potential to create variable seeding densities at different points within the cell growth chamber. The ability of the imaging technique to identify these key operational bioreactor characteristics demonstrates an emerging technique in troubleshooting and engineering optimization of bioreactor performance

A scaled-down model for the translation of bacteriophage culture to manufacturing scale

Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial-resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1-20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large-scale bioreactors (up to 50 L) containing different bacteriophage-bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi-liter large-scale bioreactors is challenging in itself, but performing this for individual phage-host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage-bacterial host bioprocesses (T4 - Escherichia coli; Phage K - Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake-flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale-up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550-fold increase in bacteriophage yield and these improvements successfully translated to the large-scale cultures

Antimicrobial resistance mechanisms and potential synthetic treatments

In 1928, penicillin was discovered, changing the field of modern medicine as it provided an opportunity to treat microbial infections. Since then, microorganisms such as bacteria have evolved and now have the ability to resist a wide variety of agents that might otherwise prevent their growth. By 2050, it is estimated that around 10 million lives each year will be lost due to these bacteria. This article provides an insight into how bacteria resist antibiotics and potential new methods of treating these organisms

Bioinspired poly(vinylidene fluoride) membranes with directional release of therapeutic essential oils

Here, the morphology of polypore fungi has inspired the fabrication of poly(vinylidene fluoride) (PVDF) membranes with dual porosity by nonsolvent-induced phase separation (NIPS). The fruiting body of such microorganisms is constituted of two distinct regions, finger- and sponge-like structures, which have been successfully mimicked by controlling the coagulation bath temperature during the NIPS process. The use of water at 10 °C as coagulant resulted in membranes with the highest finger-like/sponge-like ratio (53% of the total membrane thickness), while water at 90 °C allowed the formation of macrovoid-free membranes. The microchannels and the asymmetric porosity were used to enhance the oil sorption capacity of the PVDF membranes and to achieve directional release of therapeutic essential oils. These PVDF membranes with easily tuned asymmetric channel-like porosity and controlled pore size are ideal candidates for drug delivery applications

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture

Aim: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of ‘quality-by-design’ tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Materials & methods: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Results & conclusion: Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such ‘quality-by-design’ type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes

Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34 hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture

Background aims: Economic ex vivo manufacture of erythrocytes at 10 cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. Methods: CD34 cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. Results: The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. Conclusions: Erythroid differentiation chronology is partly determined by the heterogeneous CD34 progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps. © 2013 International Society for Cellular Therapy

Precision manufacturing for clinical-quality regenerative medicines

Innovations in engineering applied to healthcare make a significant difference to people's lives. Market growth is guaranteed by demographics. Regulation and requirements for good manufacturing practice—extreme levels of repeatability and reliability—demand high-precision process and measurement solutions. Emerging technologies using living biological materials add complexity. This paper presents some results of work demonstrating the precision automated manufacture of living materials, particularly the expansion of populations of human stem cells for therapeutic use as regenerative medicines. The paper also describes quality engineering techniques for precision process design and improvement, and identifies the requirements for manufacturing technology and measurement systems evolution for such therapies

Qualification of academic facilities for small-scale automated manufacture of autologous cell-based products

Academic centres, hospitals and small companies, as typical development settings for UK regenerative medicine assets, are significant contributors to the development of autologous cell-based therapies. Often lacking the appropriate funding, quality assurance heritage or specialist regulatory expertise, qualifying aseptic cell processing facilities for Good Manufacturing Practice (GMP) compliance is a significant challenge. The qualification of a new Cell Therapy Manufacturing Facility (CTMF) with automated processing capability, the first of its kind in a UK academic setting, provides a unique demonstrator for the qualification of small-scale, automated facilities for GMP compliant manufacture of autologous cell-based products in these settings. This paper shares our experiences in qualifying the CTMF, focussing on our approach to streamlining the qualification effort, the challenges, project delays and inefficiencies we encountered and the subsequent lessons learned