Yes / On 14/15th March 2016 we held a MRC/DBT funded workshop on the theme of Materials to Combat Antibiotic Resistance. The workshop was part of a continuing series of events that are part of the work of UK-India Centre for Advanced Technologies-Minimising the indiscriminate use of Antibiotics (UKICAT–MA). The following is the collection of presentations and the results of discussions highlighting key themes for future work by this group. Combating antibiotic resistance is perhaps the biggest issue facing the global community in the 21st century and no other area, with the exception perhaps of nuclear conflict, has the capacity to significantly reduce living standards and mortality rates. Key objectives identified by WHO1 in this area among five key aspects, include: Objective 4-to optimize the use of antimicrobial agents Objective 5-new medicines, diagnostic tools, vaccines and other interventions Our aims in this series of workshops are to provide an Indo-UK forum for: discussions of our advances in providing technologies to address these objectives; facilitate the interface between UK and Indian clinicians, materials and biological scientists and to identify key areas for new projects. An important aspect of the work in a global context is that by combining the UK and Indian community and clinical experiences we cover most of the scenarios that the global population might expect to encounter

Significant advancements have been made within the field of regenerative medicine over the last few decades with the goal of creating biological substitutes to mimic tissue for research and wound healing purposes. Simply put, regenerative medicine works by understanding and then manipulating the processes by which cells communicate and proliferate for healing purposes. Before valuable progress can be made in regenerative medicine, smaller steps need to be taken first, like understanding the biomaterials that are used within regenerative medicine research. Biomaterials, which are materials that interact with cells and perform a function, are used to mimic the native extracellular matrix of cell scaffolding in regenerative medicine research. Numerous types of biomaterials exist, and it is important to choose the most appropriate material for the goal at hand. Therefore, biomaterials need to be characterized before useful research with the materials can be done. An important aspect of biomaterials that can be characterized is fluid flow through the biomaterials. This is important because adequate transport of oxygen, nutrients, waste, and soluble factors are required for cell proliferation and survival.[1] Biomaterials can be characterized based on their chemical, physical, and mechanical characteristics via many different characterization methods that are discussed in this paper. The overall goal of this research is to characterize the fluid flow metrics through Micro-porous Annealed Particle (MAP) hydrogels and others using Dynamic Contrast Enhanced – Magnetic Resonance Imaging (DCE-MRI) and computational analysis of the images via MATLAB. The analysis was utilized to analyze the fluid flow through several different biomaterial types, allowing for observational comparison between biomaterial groups. Overall, this method for characterizing fluid flow through biomaterials shows promise for future use and further understanding of biomaterials' roles in regenerative medicine. / Master of Science / Regenerative medicine encompasses the use of scientific knowledge and tools to determine novel methods for generating functioning tissues and organs. Commonly, biomaterials are used to assist in this process. Biomaterials frequently function as a solid structure that houses cells and encourages cell growth, eventually leading to tissue formation. Many different types of biomaterials exist, so it is important to determine the most suitable biomaterial for each project to improve efficiency and experiment outcomes. Biomaterial properties, like stiffness or flexibility, can be determined through various scientific testing methods. An important property of biomaterials is the fluid flow through the biomaterials. Cells housed inside biomaterials require oxygen and nutrients to grow, so it is important that fluids carrying these molecules can flow through biomaterials to provide support for the cells. This paper utilizes a computational analysis method to analyze Magnetic Resonance Imaging (MRI) images of fluid flow through biomaterials. The analysis provides information on fluid flow metrics through the biomaterials, like fluid flow velocity and direction. This analysis provides a new method for understanding biomaterial properties and provides the analysis for several different biomaterials.

Selective Serotonin Reuptake Inhibitors (SSRIs) are the most frequently prescribed class of drugs worldwide and are implemented in the treatment of depression and other psychiatric disorders. SSRIs relieve depressive symptoms by modulating levels of the neurotransmitter serotonin in the brain. SSRIs block the function of the serotonin transporter, thereby increasing concentrations of extracellular serotonin. However, serotonin levels in the neurons of the brain only account for 5% while the remaining 95% is present outside the brain. Serotonin receptors and transporter are located on bone resident cells (mesenchymal stem cells (MSCs)), osteoblasts and osteoclasts, and serotonergic activity is believed to affect bone homeostasis. Consequently, alterations in serotonin levels by SSRI treatment have the potential to alter bone formation and remodeling. Clinical reports correlate increase risk of bone fractures and delayed bone healing with SSRI use. Metallic implants are commonly used as orthopedic and dental implants to fix bony defects. Surface modifications have been used to increase the level of bone to implant contact by controlling the differentiation of MSCs into an osteoblastic linage and facilitate bone production. However, it is not known if SSRIs can affect MSCs osteoblastic differentiation and bone remodeling signaling in response to microstructured biomaterials. The aims of this study were: 1) Investigate the effects of SSRIs on MSCs differentiation on microstructured titanium (Ti), 2) Determine the effects of SSRIs on bone remodeling signaling and osteoclast activation, and 3) Elucidate the effects of SSRIs on serotonin receptors and their effect on bone remodeling. To investigate this, human MSCs were grown on tissue culture polystyrene (TCPS), smooth Ti (PT) or microstructured Ti (SLA) surfaces under exposure to therapeutic concentrations of commonly prescribed antidepressants (SSRIs (fluoxetine, sertraline, paroxetine), Selective Norepinephrine Reuptake Inhibitor (SNRI) (duloxetine) and other regularly prescribed antidepressants (bupropion)) during differentiation toward osteoblasts. Osteoblastic differentiation was assessed in MSCs after treatment with the drugs (0.1μM, 1μM, 10μM) by alkaline phosphatase activity and osteocalcin levels. Antidepressant treatment decreased levels of MSC differentiation markers on microstructured Ti surfaces. Furthermore, treatment dose-dependently decreased protein levels secreted by MSCs which are important for bone formation (BMP2, VEGF, Osteoprotegerin), and increased those involved in bone resorption (RANKL). To determine the effect of SSRIs on bone remodeling signaling and osteoclast activation, human osteoclasts were either directly exposed to antidepressants or conditioned media obtained from MSCs treated with antidepressants on Ti surfaces, after which, enzymatic tartrate-resistant acid phosphatase (TRAP) activity was assessed. Antidepressants increased TRAP activity both directly and through treated MSCs, with the highest levels evident after treatment with conditioned media from MSCs on microstructured Ti surfaces. To elucidate the effects of serotonin receptors and their effect on bone remodeling, receptors were pharmacologically inhibited. Surface roughness decreased gene expression of HTR2A, HTR1B, and HTR2B, and antidepressant treatment increased their expression. Inhibition of HTR2A decreased RANKL protein levels, while inhibition of other serotonin receptors had no effect on RANKL or OPG levels. These studies suggest that antidepressants inhibit MSCs differentiation on microstructured Ti surfaces and increase levels of proteins associated with bone resorption. Additionally, our results showed that RANKL is regulated by serotonin receptor HTR2A. Taken together, our results suggest that antidepressants have a negative effect on osteoblastic differentiation, compromising bone formation and enhancing bone resorption, which can be detrimental to patients under orthopedic and dental treatment.

<p>Curiosity is a powerful tool, and combined with the ability to observe the natural world, grants humankind an unique opportunity, the opportunity to wonder why. Why do things exist?, why do they do the things they do?, why is this even possible?</p> <p>Research in our lab is focused on the basic understanding and potential application of biological materials, in particular, biological adhesives produced by marine organisms such as oysters. Oysters produce a cement-like material that is able to withstand the dynamic conditions found in coastal environments. The focus of this dissertation is to lay the basis of the characterization of new biological materials by observing and analyzing its physical properties, to measure the performance of the material in natural conditions and finally to identify the basic components that give the material the properties that we observe. The end goal of this project is to understand the properties of this material so we are able to develop a synthetic system that is able to imitate, as close as possible, what we find in nature. These results, and more importantly, the new questions that emerge from this research, provide a first look at the adhesive system of oysters leading the way to new discoveries in the future.</p>

The recreation of the complicated tissue microenvironment is essential to reduce the gap between in vitro and in vivo research. Polysaccharide-based hydrogels form excellent scaffolds to allow for three-dimensional cell culture owing to the favorable properties such as capability to absorb large amount of water when immersed in biological fluids, ability to form “smart hydrogels” by being shear-thinning and thixotropic, and eliciting minimum immunological response from the host. In this study, the biodegradable shear-thinning polysaccharide, gellan-gum based hydrogel was investigated for the conditions and concentrations in which it can be applied for the adhesion, propagation and assembly of different mammalian cell types in an unmodified state, at physiological conditions of temperature. Cell studies, to show successful propagation and assembly into three-dimensional structures, were performed in the range of hydrogels which were deemed to be optimum for cell culture and the cell types were chosen to represent each embryonic germ layer, i.e., human neural stem cells for ectoderm, human brain microvasculature cells for mesoderm, and murine β-cells for endoderm, along with a pluripotent cell line of human induced pluripotent stem cells, derived from human foreskin fibroblasts. Three-dimensional cell organoid models, to allow for gellan gum based bioprinting, were also developed using human induced pluripotent stem cells and human neural stem cells.

The purpose of this study was to evaluate the incidence of corrosion and fretting in 48 retrieved titanium-6aluminum-4vanadium and/or cobalt-chromium-molybdenum modular total hip prosthesis with respect to alloy material microstructure and design parameters. The results revealed vastly different performance results for the wide array of microstructures examined. Severe corrosion/fretting was seen in 100% of as-cast, 24% of low carbon wrought, 9% of high carbon wrought and 5% of solution heat treated cobalt-chrome. Severe corrosion/fretting was observed in 60% of Ti-6Al-4V components. Design features which allow for fluid entry and stagnation, amplification of contact pressure and/or increased micromotion were also shown to play a role. 75% of prosthesis with high femoral head-trunnion offset exhibited poor performance compared to 15% with a low offset. Large femoral heads (>32mm) did not exhibit poor corrosion or fretting. Implantation time was not sufficient to cause poor performance; 54% of prosthesis with greater than 10 years in-vivo demonstrated none or mild corrosion/fretting.

The design and selection of a biomaterial will depend on its specific application and the required properties for that application, both mechanical physicochemical properties. Biomaterials can be extremely helpful in order to treat and help the human body to heal and repair faster any kind of fracture produced in bones. Calcium phosphate scaffolds produced by sol-gel procedures have been used for this purpose with a great success regarding mechanical properties and biocompatibility. This is the reason why new techniques needs to be developed to be able to produce scaffolds in a faster way and to reach a personalized treatment to each patient. By using 3D printing techniques, a new and promising scope is open for bone tissue engineering due to the possibility of printing scaffolds with any shape and complexity through CAD design and modelling. In this project 3D printed scaffolds with a matrix combination of polymers and calcium phosphate will be produced and studied for bone tissue regeneration. Self-setting alpha tricalcium phosphate (α-TCP) based cement inks combined with polycaprolactone (PCL) were optimized, and 3D printed structure scaffolds were successfully generated by direct ink writing. Afterwards, the scaffolds were subjected to different hardening processes in order to obtain different hydroxyapatite microstructure morphologies and were characterised by different methodologies. It was demonstrated the important effect of obtaining a complete transformation from the α-TCP into hydroxyapatite in the mechanical properties. An improvement in the mechanical properties at compression was achieved with the addition of PCL within the scaffold ́s structure and a different fracture mode of the scaffolds was observed.

<p>Injectable, in-situ gelling magnetic plum pudding hydrogel composites were fabricated by entrapping superparamagnetic iron oxide nanoparticles (SPIONs) and thermosensitive N-isopropylacrylamide (NIPAM)-co–N-isopropylmethacrylamide (NIPMAM) microgels in a pNIPAM-hydrazide/carbohydrate-aldehyde hydrogel matrix. The resulting composites exhibited significant, repeatable pulsatile release of 4 kDa FITC-dextran upon exposure to an alternating magnetic field. The pulsatile release from the composites could be controlled by altering the volume phase transition temperatures of the microgel particles (with VPTTs over 37°C corresponding to improved pulsatile release) and changing the microgel content of the composite (with higher microgel content corresponding to higher pulsatile release). By changing the ratio of dextran-aldehyde (which deswells at physiological temperature) to CMC-aldehyde (which swells at physiological temperature) in the composites, bulk hydrogel swelling and thus pulsatile release could be controlled; specifically, lower CMC-aldehyde contents resulted in little to no composite swelling, improving pulsatile release. <em>In vitro</em> cytotoxicity testing demonstrated that the composite precursors exhibit little to no cytotoxicity up to a concentration of 2000 µg/mL. Together, these results suggest that this injectable hydrogel-microgel composite hydrogel may be a viable vehicle for <em>in vivo</em>, pulsatile drug delivery.<strong></strong></p> / Master of Applied Science (MASc)

Oxidative stress is a pathophysiological condition defined by an increased production of reactive oxygen species (ROS), which can result in the growth arrest of cells followed by cell disintegration or necrosis. A number of small molecule antioxidants (e.g. curcumin, quercetin and resveratrol) are capable of directly scavenging ROS, thereby short-circuiting the self-propagating oxidative stress state. However, poor solubility and rapid 1st pass metabolism results in overall low bioavailability and acts as a barrier for its use as a drug to suppress oxidative stress efficiently. To overcome this limitation, these small molecule antioxidants were covalently conjugated into poly(β-amino ester) (PβAE) cross-linked networks to formulate prodrug gel microparticles and nanoparticles (nanogels). Being hydrolytically degradable in nature, these PβAE crosslinked systems released antioxidants in their original structural form in a sustained controlled fashion. Both quercetin and curcumin-PβAE nanogels showed prolonged suppression of cellular oxidative stress induced by H2O2. Curcumin PβAE nanogels also demonstrated protection against mitochondrial oxidative stress induced by H2O2 and polychlorinated biphenyls. Curcumin-PβAE gel microparticles were also developed as a platform to treat oral mucositis through a local antioxidant delivery route. The same synthesis chemistry was transferred to formulate resveratrol PβAE gel microparticles for topical applications, to treat UV radiation induced oxidative stress. Both formulations showed suppression of induced oxidative stress. An in vivo trial with curcumin-PβAE microparticles further showed relatively reduced the severity of induced oral mucositis (OM) in hamster check pouch as compared to placebo.

Cancer metastasis directly accounts for an estimated 90% of all cancer related deaths and is correlated with the presence of malignant cells in systemic circulation. This observed relationship has prompted efforts to develop a fluid biopsy, with the goal of detecting these rare cells in patient peripheral blood as surrogate markers for metastatic disease as a partial replacement or supplement to tissue biopsies. Numerous platforms have been designed, yet these have generally failed to support a reliable fluid biopsy due to poor performance parameters such as low throughput, low purity of enriched antigen positive cells, and insufficiently low detection thresholds to detect poor expressed surface markers of target cell populations. This work describes the development of a rapid cell sorting technology called Antigen Specific Lysis (ASL) based on photo-crosslinked polymer encapsulation to isolate tumor cells in suspension. In the first study, we characterize the chemical and structural properties of the surface-initiated polymer films formed directly on mammalian cell surfaces. Coated populations are shown to remain highly viable after coating formation. Biomolecular transport is examined though film coatings on cellular substrates using fluorescent, time-resolved confocal microscopy and diffusivity estimates are generated for these materials. In the next study, a lysis-based cell isolation platform is described in which marker positive cells can be specifically coated in a heterogeneous cell suspension. Anionic surfactants lyse virtually 100% of uncoated cells while fully encapsulated cells remain protected, and are then easily collected by centrifugation. We report that purified cells are released from polymeric coatings to yield viable and functional populations. We monitor cell response throughout the isolation process by multiple techniques, and report viability >80% after the sorting process. Lastly, we examine the response of process yield on the level of photoinitiator loading on target populations. Streptavidin-fluorochrome loading was quantitatively assessed on a panel of markers, both epithelial and mesenchymal, on representative model breast and lung cancer cells. We report that ASL is fundamentally capable of achieving 50-60% yield which is promising for fluid biopsy applications. Finally, both EpCAM and metastatic targeting strategies are then compared to covalently biotinylated samples to inform future robust targeting strategies.