Developing tissue engineered models of oral mucosa and oral cancer to study novel therapeutic and diagnostic techniques

In the UK in 2008 over 1,800 people died from oral cancer. Despite advances in surgery and therapy the survival rates for those diagnosed with oral cancer have not significantly improved over the past 20 years. There is a need for both better detection and treatment. Early detection could reduce these mortality rates but unfortunately diagnosing oral cancer, which is often symptomless, in the early stages of the disease is incredibly challenging. More targeted treatments for oral cancer are also needed which can be administered to the site of disease with higher efficiency and accuracy to reduce side effects and allow higher concentrations of therapeutic agents to be delivered to tumour cells. This project used a tissue engineered oral mucosa model to develop models of oral cancer progression. These oral cancer models incorporated many pathological features of oral cancer progression including features seen in dysplastic epithelia, carcinoma in situ and early invasive squamous cell carcinomas. Multi-cellular tumour spheroids were created from an oral cancer cell line to model solid expanding tumour masses. The tissue engineered models of oral mucosa and the multi-cellular tumour spheroids were used to test the behaviour of polymersomes, a novel drug delivery system, in three dimensional tissues. Polymersome distribution and penetration in the tissue engineered models was examined over time, to investigate the potential of polymersomes to deliver therapeutic agents into and/or across the oral mucosa and into solid expanding tumours. Drug delivery agents that are able to reach the central hypoxic region of solid expanding tumours are particularly important as these cells are often resistant to both radio- and chemotherapy and correlate with poor patient prognosis. In addition, we explored the potential of polymersomes to cross the oral epithelial permeability barrier and act as delivery vehicles for topical delivery of therapies for oral mucosal diseases and as an alternative to parenteral administration for the systemic delivery of drugs. Results showed good penetration of polymersomes into the oral mucosa and the multi-cellular tumour spheroids demonstrating the potential to develop these drug delivery vehicles to deliver anti-cancer drugs and other therapeutic agents in the future. The tissue engineered models of cancer were next utilised to test four non-invasive diagnostic technologies. These included a cell metabolism marker, impedance spectroscopy, Fourier transform infra-red and optical coherence tomography. The results obtained from these different techniques showed varying degrees of promise with the images from OCT demonstrating that this technology has real diagnostic potential. The 3D models proved useful test-beds for some but not all of these diagnostic imaging techniques. They provide convenient models to tackle some of the key issues in the preclinical development of novel diagnostic technologies for oral cancer and oral dysplastic lesions.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Oral human papillomavirus infection in England and associated risk factors: a case control study.

Objectives - This study was conducted to determine the prevalence of and associated risk factors for infection with oral high-risk human papillomavirus (HR-HPV) in adult participants within England, and to explore any association with oral mucosal buccal epithelial cell and whole blood folate concentration. Design - This was an observational study to determine oral HR-HPV prevalence in the study population. A case-control study was performed to explore the association between infection and folate status. Setting - This study was conducted in Sheffield, United Kingdom between April 2013 and August 2014. Participants - Seven hundred participants, aged 18-60 yr, were recruited from university students (n=179), university and hospital staff (n=163), dental hospital patients (n=13), Sexual Health Sheffield patients (n=122) and the general public (n=223). Interventions - Participants completed a lifestyle and sexual behaviour questionnaire, provided an oral rinse and gargle sample for the detection of oral HR-HPV and an oral mucosal buccal epithelial cell sample for the measurement of oral mucosal buccal epithelial cell folate. A blood sample was collected for measurement of whole blood folate concentration. Outcome measures - The prevalence of oral HR-HPV infection in the study population was the primary outcome measure. Secondary outcome measures included associations between risk factors, folate status and infection. Results - The prevalence of oral HR-HPV infection in this cohort was 2.2% (15/680) with 0.7% (5/680) positive for HPV16 or HPV18. Twenty samples were excluded due to insufficient material for HPV detection. Participants with oral HR-HPV infection were more likely to be a former smoker, and have a greater number of sexual and oral sexual partners. Folate status was not linked to likelihood of HPV infection. Conclusions - The prevalence of oral infection with HR-HPV in adult men and women in Sheffield in the north of England was low. Smoking and sexual behaviour were associated with HR-HPV positivity

A micro-incubator for cell and tissue imaging

International audienceA low-cost micro-incubator for the imaging of dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment which can be altered to study the response of cell monolayers for several days as well as relatively thick tissue samples and tissue engineered epithelial tissues in experiments lasting several hours. Samples within the incubator are contained in a sterile cavity closed by a gas permeable membrane. The incubator can be positioned in any direction and used on an inverted as well as on an upright microscope. The temperature is regulated with a Peltier system controlled with a sensor positioned close to the sample to be able to compensate for any changes in temperature. Rapid changes in the environment can be applied to the sample because of the fast response of the Peltier system and the sample's adaptations to induced changes in the environment can be monitored. To evaluate the performance of the micro-incubator we report on studies using cultured cells in monolayers, on monolayers of cells stretched to breaking point on a distensible membrane, on cells in open 3D fibrous scaffolds and on fluorescently labelled polymersome penetration into 3D tissue engineered oral mucosa

Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation

Fucoidan Inhibition of Osteosarcoma Cells Is Species and Molecular Weight Dependent.

Fucoidan is a brown algae-derived polysaccharide having several biomedical applications. This study simultaneously compares the anti-cancer activities of crude fucoidans from Fucus vesiculosus and Sargassum filipendula, and effects of low (LMW, 10-50 kDa), medium (MMW, 50-100 kDa) and high (HMW, >100 kDa) molecular weight fractions of S. filipendula fucoidan against osteosarcoma cells. Glucose, fucose and acid levels were lower and sulphation was higher in F. vesiculosus crude fucoidan compared to S. filipendula crude fucoidan. MMW had the highest levels of sugars, acids and sulphation among molecular weight fractions. There was a dose-dependent drop in focal adhesion formation and proliferation of cells for all fucoidan-types, but F. vesiculosus fucoidan and HMW had the strongest effects. G1-phase arrest was induced by F. vesiculosus fucoidan, MMW and HMW, however F. vesiculosus fucoidan treatment also caused accumulation in the sub-G1-phase. Mitochondrial damage occurred for all fucoidan-types, however F. vesiculosus fucoidan led to mitochondrial fragmentation. Annexin V/PI, TUNEL and cytochrome c staining confirmed stress-induced apoptosis-like cell death for F. vesiculosus fucoidan and features of stress-induced necrosis-like cell death for S. filipendula fucoidans. There was also variation in penetrability of different fucoidans inside the cell. These differences in anti-cancer activity of fucoidans are applicable for osteosarcoma treatment

Surfactant-free gelatin-stabilised biodegradable polymerised high internal phase emulsions with macroporous structures

High internal phase emulsion (HIPE) templating is a well-established method for the generation of polymeric materials with high porosity (>74%) and degree of interconnectivity. The porosity and pore size can be altered by adjusting parameters during emulsification, which affects the properties of the resulting porous structure. However, there remain challenges for the fabrication of polyHIPEs, including typically small pore sizes (∼20–50 μm) and the use of surfactants, which can limit their use in biological applications. Here, we present the use of gelatin, a natural polymer, during the formation of polyHIPE structures, through the use of two biodegradable polymers, polycaprolactone-methacrylate (PCL-M) and polyglycerol sebacate-methacrylate (PGS-M). When gelatin is used as the internal phase, it is capable of stabilising emulsions without the need for an additional surfactant. Furthermore, by changing the concentration of gelatin within the internal phase, the pore size of the resulting polyHIPE can be tuned. 5% gelatin solution resulted in the largest mean pore size, increasing from 53 μm to 80 μm and 28 μm to 94 µm for PCL-M and PGS-M respectively. In addition, the inclusion of gelatin further increased the mechanical properties of the polyHIPEs and increased the period an emulsion could be stored before polymerisation. Our results demonstrate the potential to use gelatin for the fabrication of surfactant-free polyHIPEs with macroporous structures, with potential applications in tissue engineering, environmental and agricultural industries

A ruthenium(II) polypyridyl complex disrupts actin cytoskeleton assembly and blocks cytokinesis

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) “RuRuPhen” blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

BACKGROUND: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. RESULTS: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. CONCLUSION: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations

Polymersome-Mediated Delivery of Combination Anticancer Therapy to Head and Neck Cancer Cells: 2D and 3D in Vitro Evaluation

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors

Combined mathematical modelling and experimentation to predict polymersome uptake by oral cancer cells

This study is motivated by understanding and controlling the key physical properties underlying internalisation of nano drug delivery. We consider the internalisation of specific nanometre size delivery vehicles, comprised of self-assembling amphiphilic block copolymers, called polymersomes that have the potential to specifically deliver anticancer therapeutics to tumour cells. The possible benefits of targeted polymersome drug delivery include reduced off-target toxic effects in healthy tissue and increased drug uptake by diseased tissue. Through a combination of in vitro experimentation and mathematical modelling, we develop a validated model of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway, incorporating receptor binding, clustering and recycling. The model predicts how the characteristics of receptor targeting, and the size and concentration of polymersomes alter uptake by tumour cells. The number of receptors per cell was identified as being the dominant mechanism accounting for the difference between cell types in polymersome uptake rate. From the Clinical Editor - This article reports on a validated model developed through a combination of in vitro experimentation and mathematical modeling of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway. The model incorporates receptor binding, clustering, and recycling and predicts how the characteristics of receptor targeting, the size and concentration alter polymersome uptake by cancer cells