Publications by authors named "Sheila MacNeil"

221 Publications

Assessing the immunosuppressive activity of alginate-encapsulated mesenchymal stromal cells on splenocytes.

Artif Cells Nanomed Biotechnol 2022 Dec;50(1):168-176

Centre for Biological Engineering, Wolfson School of Mechanical, Electrical and Manufacturing Engineering, Loughborough, UK.

Mesenchymal stromal cells (MSCs) show immunosuppressive effects both cell-to-cell contact (direct) with immune cells and by producing paracrine factors and extracellular vesicles (indirect). A key challenge in delivering this therapeutic effect is retaining the MSCs at the site of injection. One way to address this is by encapsulating the MSCs within suitable biomaterial scaffolds. Here, we assess the immunosuppressive effect of alginate-encapsulated murine MSCs on proliferating murine splenocytes. Our results show that MSCs are able to significantly suppress splenocyte proliferation by ∼50% the indirect mechanism and almost completely (∼98%) the direct mechanism. We also show for the first time that MSCs as monolayers on tissue culture plastic or encapsulated within alginate, when physically isolated from the splenocytes transwells, are able to sustain immunosuppressive activity with repeated exposure to fresh splenocytes, for as long as 9 days. These results indicate the need to identify design strategies to simultaneously deliver both modes of MSC immunosuppression. By designing cell-biomaterial constructs with tailored degradation profiles, we can achieve a more sustained (avoiding MSCs migration and apoptosis) and controlled release of both the paracrine signals and eventually the cells themselves enabling efficient MSC-based immunosuppressive therapies for wound healing.
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http://dx.doi.org/10.1080/21691401.2022.2088547DOI Listing
December 2022

Generation and use of functionalised hydrogels that can rapidly sample infected surfaces.

MethodsX 2022 17;9:101684. Epub 2022 Apr 17.

University of Bradford, Bradford UK.

This paper outlined our method for developing polymer-linked contact lens type materials for rapid detection and differentiation of Gram-positive, Gram-negative bacteria and fungi in infected corneas. It can be applied to both model synthetic or ex-vivo corneal models and has been successfully trialed in an initial efficacy tested animal study. First a hydrogel substrate for the swab material is selected, we have demonstrated selective swabs using a glycerol monomethacrylate hydrogel. Alternatively any commercial material with carboxylic acid functional groups is suitable but risks nonspecific adhesion. This is then functionalised via use of N-hydroxysuccinimide reaction with amine groups on the specified highly branched polymer ligand (either individually gram negative, gram positive or fungal binding polymers or a combination of all three can be employed for desired sensing application). The hydrogel is then cut into swabs suitable for sampling, used, and then the presence of gram positive, game negative and fungi are disclosed by the sequential addition of dyes (fluorescent vancomycin, fluorescein isothiocyanate and calcofluor white). In summary this method presents: Method to produce glycerol monomethacrylate hydrogels to minimize nonspecific binding Methods of attaching pathogen binding highly branched polymers to produce selective hydrogel swabs Method for disclosing bound pathogens to this swab using sequential dye addition.
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http://dx.doi.org/10.1016/j.mex.2022.101684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9078998PMC
April 2022

A novel characterisation approach to reveal the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh.

RSC Adv 2021 Oct 27;11(55):34710-34723. Epub 2021 Oct 27.

Department of Materials Science and Engineering, University of Sheffield Sir Robert Hadfield Building, Mappin Street UK

Polypropylene (PP) surgical mesh, used successfully for the surgical repair of abdominal hernias, is associated with serious clinical complications when used in the pelvic floor for repair of stress urinary incontinence or support of pelvic organ prolapse. While manufacturers claim that the material is inert and non-degradable, there is a growing body of evidence that asserts PP fibres are subject to oxidative damage and indeed explanted material from patients suffering with clinical complications has shown some evidence of fibre cracking and oxidation. It has been proposed that a pathological cellular response to the surgical mesh contributes to the medical complications; however, the mechanisms that trigger the specific host response against the material are not well understood. Specifically, this study was constructed to investigate the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh. To do this we used a novel advanced spectroscopical characterisation technique, secondary electron hyperspectral imaging (SEHI), which is based on the collection of secondary electron emission spectra in a scanning electron microscope (SEM) to reveal mechanical-chemical reactions within PP meshes.
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http://dx.doi.org/10.1039/d1ra05944kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9042683PMC
October 2021

A physiologically relevant, estradiol-17β [E2]-responsive in vitro tissue-engineered model of the vaginal epithelium for vaginal tissue research.

Neurourol Urodyn 2022 04 21;41(4):905-917. Epub 2022 Mar 21.

Department of Materials Science and Engineering, Kroto Research Institute, University of Sheffield, Sheffield, UK.

Aims: There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research.

Methods: Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air-liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins.

Results: Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17β [E ] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E concentrations (100-400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression.

Conclusion: In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT.
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http://dx.doi.org/10.1002/nau.24908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9313856PMC
April 2022

Semi-interpenetrating Polyurethane Network Foams Containing Highly Branched Poly(-isopropyl acrylamide) with Vancomycin Functionality.

ACS Appl Bio Mater 2021 05 27;4(5):4319-4327. Epub 2021 Apr 27.

Polymer and Biomaterials Chemistry Laboratories, University of Bradford, Bradford, West Yorkshire BD 7 1DP, U.K.

Highly branched poly(-isopropylacrylamide) (HB-PNIPAM), functionalized with vancomycin at the chain ends, acted as a bacterial adhesive and was incorporated into polyurethane foams to form semi-interpenetrating networks. PNIPAM was labeled with a solvatochromic dye, Nile red. It was found that the thermal response of the polymer was dependent on the architecture, and temperature-dependent color changes were observed within the foam. The foams had open pore structures, and the presence of HB-PNIPAM substantially reduced the shrinkage of the foam as the temperature was increased up to 20 °C. The foams were selectively adhesive for (Gram-positive bacteria) compared to (Gram-negative bacteria), and the presence of was indicated by increased fluorescence intensity (590-800 nm).
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http://dx.doi.org/10.1021/acsabm.1c00116DOI Listing
May 2021

Tuning Electrospun Substrate Stiffness for the Fabrication of a Biomimetic Amniotic Membrane Substitute for Corneal Healing.

ACS Appl Bio Mater 2021 07 29;4(7):5638-5649. Epub 2021 Jun 29.

School of Dentistry, University of Sheffield, Sheffield S10 2TA, United Kingdom.

Corneal blindness is the fourth most common cause of vision impairment worldwide with a high incidence in global south countries. A recently developed surgical technique for treating corneal blindness is simple limbal epithelial transplantation (SLET), which uses small pieces of healthy limbal tissue (limbal explants) delivered to the damaged eye using the human amniotic membrane (AM) as a carrier. SLET relies on the use of tissue banks for the AM that reduces the availability of the technique. Replacing the AM with a synthetic membrane is key to making SLET more accessible to those who need it. Previous research has demonstrated the suitability of electrospun poly(lactide--glycolide) (PLGA) scaffolds as AM substitutes, and here, we report how these membranes can be tailored to mimic fundamental AM mechanical properties. To modify the stiffness of PLGA electrospun membranes, we explored different electrospinning solvent systems (1,1,1,3,3,3,-hexafluoroisopropanol (HFIP), dichloromethane (DCM), chloroform, and ,-dimethylformamide (DMF)) and the use of plasticizers (PEG and glycerol). PEG was found to reduce stiffness from 60 MPa to around 4 MPa, approaching the values shown by the native AM. The biocompatibility of membranes with and without PEG was found to be comparable, and cell outgrowth from rabbit/porcine explants was successfully observed on the materials after 3 weeks. This research underpins the manufacture of next-generation fibrous biomimetic membranes that will ultimately be used as amniotic membrane substitutes for biomedical applications including SLET.
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http://dx.doi.org/10.1021/acsabm.1c00436DOI Listing
July 2021

A Dinuclear Osmium(II) Complex Near-Infrared Nanoscopy Probe for Nuclear DNA.

J Am Chem Soc 2021 12 22;143(48):20442-20453. Epub 2021 Nov 22.

Institute of Physical Chemistry, Helmholtzweg 4, 07743 Jena, Germany; Institute of Photonic Technology Jena e.V., Albert-Einstein-Straße 9, 07749 Jena, Germany.

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic Os complexes containing the Os(TAP) fragment is reported. This method was used to synthesize the dinuclear Os complex, [{Os(TAP)}tpphz] (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λ = 780 nm) is due to a low-lying Os → TAP based MCLT state. Cell-based studies revealed that unlike its Ru analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.
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http://dx.doi.org/10.1021/jacs.1c10325DOI Listing
December 2021

Developing Wound Dressings Using 2-deoxy--Ribose to Induce Angiogenesis as a Backdoor Route for Stimulating the Production of Vascular Endothelial Growth Factor.

Int J Mol Sci 2021 Oct 23;22(21). Epub 2021 Oct 23.

Department of Materials Science & Engineering, Kroto Research Institute, University of Sheffield, Sheffield S3 7HQ, UK.

2-deoxy--Ribose (2dDR) was first identified in 1930 in the structure of DNA and discovered as a degradation product of it later when the enzyme thymidine phosphorylase breaks down thymidine into thymine. In 2017, our research group explored the development of wound dressings based on the delivery of this sugar to induce angiogenesis in chronic wounds. In this review, we will survey the small volume of conflicting literature on this and related sugars, some of which are reported to be anti-angiogenic. We review the evidence of 2dDR having the ability to stimulate a range of pro-angiogenic activities in vitro and in a chick pro-angiogenic bioassay and to stimulate new blood vessel formation and wound healing in normal and diabetic rat models. The biological actions of 2dDR were found to be 80 to 100% as effective as VEGF in addition to upregulating the production of VEGF. We then demonstrated the uptake and delivery of the sugar from a range of experimental and commercial dressings. In conclusion, its pro-angiogenic properties combined with its improved stability on storage compared to VEGF, its low cost, and ease of incorporation into a range of established wound dressings make 2dDR an attractive alternative to VEGF for wound dressing development.
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http://dx.doi.org/10.3390/ijms222111437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583821PMC
October 2021

Thiolene- and Polycaprolactone Methacrylate-Based Polymerized High Internal Phase Emulsion (PolyHIPE) Scaffolds for Tissue Engineering.

Biomacromolecules 2022 03 3;23(3):720-730. Epub 2021 Nov 3.

Department of Materials Science and Engineering, University of Sheffield, Kroto Research Institute, Sheffield S3 7HQ, United Kingdom.

Highly porous emulsion templated polymers (PolyHIPEs) provide a number of potential advantages in the fabrication of scaffolds for tissue engineering and regenerative medicine. Porosity enables cell ingrowth and nutrient diffusion within, as well as waste removal from, the scaffold. The properties offered by emulsion templating alone include the provision of high interconnected porosity, and, in combination with additive manufacturing, the opportunity to introduce controlled multiscale porosity to complex or custom structures. However, the majority of monomer systems reported for PolyHIPE preparation are unsuitable for clinical applications as they are nondegradable. Thiol-ene chemistry is a promising route to produce biodegradable photocurable PolyHIPEs for the fabrication of scaffolds using conventional or additive manufacturing methods; however, relatively little research has been reported on this approach. This study reports the groundwork to fabricate thiol- and polycaprolactone (PCL)-based PolyHIPE materials via a photoinitiated thiolene click reaction. Two different formulations, either three-arm PCL methacrylate (3PCLMA) or four-arm PCL methacrylate (4PCLMA) moieties, were used in the PolyHIPE formulation. Biocompatibility of the PolyHIPEs was investigated using human dermal fibroblasts (HDFs) and human osteosarcoma cell line (MG-63) by DNA quantification assay, and developed PolyHIPEs were shown to be capable of supporting cell attachment and viability.
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http://dx.doi.org/10.1021/acs.biomac.1c01129DOI Listing
March 2022

Decellularised extracellular matrix decorated PCL PolyHIPE scaffolds for enhanced cellular activity, integration and angiogenesis.

Biomater Sci 2021 Oct 26;9(21):7297-7310. Epub 2021 Oct 26.

Department of Materials Science and Engineering, University of Sheffield, Kroto Research Institute, Sheffield, S3 7HQ, UK.

Wound healing involves a complex series of events where cell-cell and cell-extracellular matrix (ECM) interactions play a key role. Wounding can be simple, such as the loss of the epithelial integrity, or deeper and more complex, reaching to subcutaneous tissues, including blood vessels, muscles and nerves. Rapid neovascularisation of the wounded area is crucial for wound healing as it has a key role in supplying oxygen and nutrients during the highly demanding proliferative phase and transmigration of inflammatory cells to the wound area. One approach to circumvent delayed neovascularisation is the exogenous use of pro-angiogenic factors, which is expensive, highly dose-dependent, and the delivery of them requires a very well-controlled system to avoid leaky, highly permeable and haemorrhagic blood vessel formation. In this study, we decorated polycaprolactone (PCL)-based polymerised high internal phase emulsion (PolyHIPE) scaffolds with fibroblast-derived ECM to assess fibroblast, endothelial cell and keratinocyte activity and angiogenesis in chick chorioallantoic membrane (CAM) assays. Our results showed that the inclusion of ECM in the scaffolds increased the metabolic activity of three types of cells that play a key role in wound healing and stimulated angiogenesis in CAM assays over 7 days. Herein, we demonstrated that fibroblast-ECM functionalised PCL PolyHIPE scaffolds appear to have great potential to be used as an active wound dressing to promote angiogenesis and wound healing.
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http://dx.doi.org/10.1039/d1bm01262bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8547328PMC
October 2021

Delivery of Bioactive Compounds to Improve Skin Cell Responses on Microfabricated Electrospun Microenvironments.

Bioengineering (Basel) 2021 Jul 27;8(8). Epub 2021 Jul 27.

Bioengineering and Health Technologies Group, The School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, UK.

The introduction of microtopographies within biomaterial devices is a promising approach that allows one to replicate to a degree the complex native environment in which human cells reside. Previously, our group showed that by combining electrospun fibers and additive manufacturing it is possible to replicate to an extent the stem cell microenvironment (rete ridges) located between the epidermal and dermal layers. Our group has also explored the use of novel proangiogenic compounds to improve the vascularization of skin constructs. Here, we combine our previous approaches to fabricate innovative polycaprolactone fibrous microtopographical scaffolds loaded with bioactive compounds (2-deoxy-D-ribose, 17β-estradiol, and aloe vera). Metabolic activity assay showed that microstructured scaffolds can be used to deliver bioactive agents and that the chemical relation between the working compound and the electrospinning solution is critical to replicate as much as possible the targeted morphologies. We also reported that human skin cell lines have a dose-dependent response to the bioactive compounds and that their inclusion has the potential to improve cell activity, induce blood vessel formation and alter the expression of relevant epithelial markers (collagen IV and integrin β1). In summary, we have developed fibrous matrixes containing synthetic rete-ridge-like structures that can deliver key bioactive compounds that can enhance skin regeneration and ultimately aid in the development of a complex wound healing device.
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http://dx.doi.org/10.3390/bioengineering8080105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8389211PMC
July 2021

Proof-of-concept study of electrospun PLGA membrane in the treatment of limbal stem cell deficiency.

BMJ Open Ophthalmol 2021 26;6(1):e000762. Epub 2021 Jul 26.

Cornea and Innovation, Dr Shroff's Charity Eye Hospital Delhi, New Delhi, India.

Objective: The aim of this study was to assess the safety of poly-lactic co-glycolic acid (PLGA) electrospun membranes as carriers for limbal tissue explants for treatment of limbal stem cell deficiency (LSCD).

Methods And Analysis: Approval was obtained for a first in-man study from the Drug Controller General of India. PLGA membranes were applied to the affected eye of five patients after removal of the vascular pannus. Simple limbal epithelial transplantation was performed and limbal explants were secured on the membrane using fibrin glue followed by a bandage contact lens. Patients were followed up for 1 year with ocular exams including slit lamp exam, corneal thickness measurements, intraocular pressure measurements and recording of corneal vascularisation and visual acuity. Systemic examinations included pain grading, clinical laboratory assessment, blood chemistry and urine analysis at baseline, 3 and 6 months after surgery.

Results: PLGA membranes completely degraded by 8 weeks post-transplantation without any infection or inflammation. In all five patients, the epithelium regenerated by 3 months. In two in five patients, there was a sustained two-line improvement in vision. In one in five patients, the vision improvement was limited due to an underlying stromal scarring. There was recurrence of pannus and LSCD in two in five patients 6 months after surgery which was not attributable to the membrane. The ocular surface remained clear with no epithelial defects in three in five subjects at 12 months.

Conclusion: PLGA electrospun membranes show promise as carrier for limbal epithelial cells in the treatment of LSCD.
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http://dx.doi.org/10.1136/bmjophth-2021-000762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8314696PMC
July 2021

Simulation of the process of angiogenesis: Quantification and assessment of vascular patterning in the chicken chorioallantoic membrane.

Comput Biol Med 2021 09 12;136:104647. Epub 2021 Jul 12.

Associated Laboratory for Energy, Transports and Aeronautics (LAETA - INEGI), Rua Dr. Roberto Frias, 400, 4200-465, Porto, Portugal; Faculty of Engineering of the University of Porto (FEUP), Mechanical Engineering Department, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal. Electronic address:

Angiogenesis, the formation of new blood vessels from pre-existing ones, begins during embryonic development and continues throughout life. Sprouting angiogenesis is a well-defined process, being mainly influenced by vascular endothelial growth factor (VEGF). In this study, we propose a meshless-based model capable of mimicking the angiogenic response to several VEGF concentrations. In this model, endothelial cells migrate according to a diffusion-reaction equation, following the VEGF gradient concentration. The chick chorioallantoic membrane (CAM) assay was used to model the branching process and to validate the obtained numerical results. To analyse the angiogenic response, the total vessel number and the total vessel length presented in the CAM images and in the simulations for all the VEGF concentrations tested were quantified. In both the CAM assay and simulation, the treatments with VEGF increased the total vessel number and the total vessel length. The obtained quantitative results were very similar between the two methodologies used. The proposed model accurately simulates the capillary network pattern concerning its structure and morphology, for the lowest VEGF concentration tested. For the highest VEGF concentration, the capillary network predicted by the model was less accurate when compared to the one presented in the CAM assay but this may be explained by changes in blood vessel width at higher VEGF concentrations. This remains to be tested.
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http://dx.doi.org/10.1016/j.compbiomed.2021.104647DOI Listing
September 2021

The Use of Microfabrication Techniques for the Design and Manufacture of Artificial Stem Cell Microenvironments for Tissue Regeneration.

Bioengineering (Basel) 2021 Apr 23;8(5). Epub 2021 Apr 23.

Bioengineering and Health Technologies Group, The School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, UK.

The recapitulation of the stem cell microenvironment is an emerging area of research that has grown significantly in the last 10 to 15 years. Being able to understand the underlying mechanisms that relate stem cell behavior to the physical environment in which stem cells reside is currently a challenge that many groups are trying to unravel. Several approaches have attempted to mimic the biological components that constitute the native stem cell niche, however, this is a very intricate environment and, although promising advances have been made recently, it becomes clear that new strategies need to be explored to ensure a better understanding of the stem cell niche behavior. The second strand in stem cell niche research focuses on the use of manufacturing techniques to build simple but functional models; these models aim to mimic the physical features of the niche environment which have also been demonstrated to play a big role in directing cell responses. This second strand has involved a more engineering approach in which a wide set of microfabrication techniques have been explored in detail. This review aims to summarize the use of these microfabrication techniques and how they have approached the challenge of mimicking the native stem cell niche.
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http://dx.doi.org/10.3390/bioengineering8050050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146165PMC
April 2021

Fabrication of Topographically Controlled Electrospun Scaffolds to Mimic the Stem Cell Microenvironment in the Dermal-Epidermal Junction.

ACS Biomater Sci Eng 2021 06 27;7(6):2803-2813. Epub 2021 Apr 27.

Bioengineering and Health Technologies Group, The School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.

The use of microfabrication techniques for the development of innovative constructs for tissue regeneration is a growing area of research. This area comprises both manufacturing and biological approaches for the development of smart materials aiming to control and direct cell behavior to enhance tissue healing. Many groups have focused their efforts on introducing complexity within these innovative constructs via the inclusion of nano- and microtopographical cues mimicking physical and biological aspects of the native stem cell niche. Specifically, in the area of skin tissue engineering, seminal work has reported replicating the microenvironments located in the dermal-epithelial junction, which are known as rete ridges. The rete ridges are key for both stem cell control and the physiological performance of the skin. In this work, we have introduced complexity within electrospun membranes to mimic the morphology of the rete ridges in the skin. We designed and tested three different patterns, characterized them, and explored their performance in vitro, using 3D skin models. One of the studied patterns (pattern B) was shown to aid in the development of an in vitro rite-ridgelike skin model that resulted in the expression of relevant epithelial markers such as collagen IV and integrin β1. In summary, we have developed a new skin model including synthetic rete-ridgelike structures that replicate both morphology and function of the native dermal-epidermal junction and that offer new insights for the development of smart skin tissue engineering constructs.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01775DOI Listing
June 2021

Economic, clinical and social impact of simple limbal epithelial transplantation for limbal stem cell deficiency.

Br J Ophthalmol 2022 07 9;106(7):923-928. Epub 2021 Mar 9.

Innovation Department, Dr Shroff's Charity Eye Hospital Delhi, New Delhi, Delhi, India

Aims: To report the global uptake of simple limbal epithelial transplantation (SLET) and compare the economic, clinical and social outcomes of SLET with those of cultured limbal epithelial transplantation (CLET).

Methods: A comprehensive literature review and an online survey of eye surgeons were conducted to understand the efficacy and current uptake of SLET surgery. A de novo economic model was developed to estimate the cost savings with SLET compared with CLET. Our economic analysis is conducted from an Indian perspective, as this is where the technique originated. A scenario analysis using the UK cost data and a user-friendly Excel model is included to allow users to input the costs from their setting to estimate the cost savings with using SLET compared with using CLET RESULTS: The anatomical success with SLET in adults (72.6% (range 62%-80%)) was the same as CLET (70.4% (range 68%-80.9%)). For children, the outcome for SLET (77.8% (range 73%-83%)) was better than with CLET (44.5% (range 43%-45%)). In response to our informal questionnaire, 99 surgeons reported to have performed SLET on 1174 patients in total. They appreciated that SLET negates the requirement for costly tissue engineering facilities. Results of economic analysis suggested that SLET provided an estimated cost-savings of US$6470.88 for adults and US$6673.10 for children. In broad terms, the cost of SLET is approximately 10% of the cost of CLET for adults and 8% for children.

Conclusion: SLET offers a more accessible and financially attractive alternative to CLET to treat limbal stem cell deficiency.
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http://dx.doi.org/10.1136/bjophthalmol-2020-318642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9234414PMC
July 2022

Branched amphotericin functional poly(-propyl acrylamide): an antifungal polymer.

R Soc Open Sci 2021 Jan 13;8(1):201655. Epub 2021 Jan 13.

Polymer and Biomaterial Chemistry Laboratories, School of Chemistry and Biosciences, University of Bradford, Bradford BD7 1DP, UK.

Branched poly(-propylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of , and (minimal inhibitory concentration ranged from 5 to 500 µg ml) but was not effective against an AmB resistant strain of nor against . The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α and interleukin-1 (at 0.5 mg ml).
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http://dx.doi.org/10.1098/rsos.201655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890487PMC
January 2021

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis.

Cells 2020 11 10;9(11). Epub 2020 Nov 10.

Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK.

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. and are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. , and corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.
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http://dx.doi.org/10.3390/cells9112450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696224PMC
November 2020

Sprouting Angiogenesis: A Numerical Approach with Experimental Validation.

Ann Biomed Eng 2021 Feb 24;49(2):871-884. Epub 2020 Sep 24.

Associated Laboratory for Energy, Transports and Aeronautics (LAETA - INEGI), Rua Dr. Roberto Frias, 400, 4200-465, Porto, Portugal.

A functional vascular network is essential to the correct wound healing. In sprouting angiogenesis, vascular endothelial growth factor (VEGF) regulates the formation of new capillaries from pre-existing vessels. This is a very complex process and mathematical formulation permits to study angiogenesis using less time-consuming, reproducible and cheaper methodologies. This study aimed to mimic the chemoattractant effect of VEGF in stimulating sprouting angiogenesis. We developed a numerical model in which endothelial cells migrate according to a diffusion-reaction equation for VEGF. A chick chorioallantoic membrane (CAM) bioassay was used to obtain some important parameters to implement in the model and also to validate the numerical results. We verified that endothelial cells migrate following the highest VEGF concentration. We compared the parameters-total branching number, total vessel length and branching angle-that were obtained in the in silico and the in vivo methodologies and similar results were achieved (p-value smaller than 0.5; n = 6). For the difference between the total capillary volume fractions assessed using both methodologies values smaller than 15% were obtained. In this study we simulated, for the first time, the capillary network obtained during the CAM assay with a realistic morphology and structure.
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http://dx.doi.org/10.1007/s10439-020-02622-wDOI Listing
February 2021

2-deoxy-d-ribose (2dDR) upregulates vascular endothelial growth factor (VEGF) and stimulates angiogenesis.

Microvasc Res 2020 09 25;131:104035. Epub 2020 Jun 25.

Department of Materials Science & Engineering, Kroto Research Institute, University of Sheffield, Sheffield, UK. Electronic address:

Background: Delayed neovascularisation of tissue-engineered (TE) complex constructs is a major challenge that causes their failure post-implantation. Although significant progress has been made in the field of angiogenesis, ensuring rapid neovascularisation still remains a challenge. The use of pro-angiogenic agents is an effective approach to promote angiogenesis, and vascular endothelial growth factor (VEGF) has been widely studied both at the biological and molecular levels and is recognised as a key stimulator of angiogenesis. However, the exogenous use of VEGF in an uncontrolled manner has been shown to result in leaky, permeable and haemorrhagic vessels. Thus, researchers have been actively seeking alternative agents to upregulate VEGF production rather than exogenous use of VEGF in TE systems. We have previously revealed the potential of 2-deoxy-d-ribose (2dDR) as an alternative pro-angiogenic agent to induce angiogenesis and accelerates wound healing. However, to date, there is not any clear evidence on whether 2dDR influences the angiogenic cascade that involves VEGF.

Methods: In this study, we explored the angiogenic properties of 2dDR either by its direct application to human aortic endothelial cells (HAECs) or when released from commercially available alginate dressings and demonstrated that when 2dDR promotes angiogenesis, it also increases the VEGF production of HAECs.

Results: The VEGF quantification results suggested that VEGF production by HAECs was increased with 2dDR treatment but not with other sugars, including 2-deoxy-l-ribose (2dLR) and d-glucose (DG). The stability studies demonstrated that approximately 40-50% of the 2dDR had disappeared in the media over 14 days, either in the presence or absence of HAECs, and the reduction was higher when cells were present. The concentration of VEGF in the media also fell after day 4 associated with the reduction in 2dDR.

Conclusion: This study suggests that 2dDR (but not other sugars tested in this study) stimulates angiogenesis by increasing the production of VEGF. We conclude 2dDR appears to be a practical and effective indirect route to upregulating VEGF for several days, leading to increased angiogenesis.
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http://dx.doi.org/10.1016/j.mvr.2020.104035DOI Listing
September 2020

Bioengineering Vascular Networks to Study Angiogenesis and Vascularization of Physiologically Relevant Tissue Models .

ACS Biomater Sci Eng 2020 06 29;6(6):3513-3528. Epub 2020 Apr 29.

Department of Materials Science and Engineering, Kroto Research Institute, University of Sheffield, Sheffield S3 7HQ, United Kingdom.

Angiogenesis assays are essential for studying aspects of neovascularization and angiogenesis and investigating drugs that stimulate or inhibit angiogenesis. To date, there are several and angiogenesis assays that are used for studying different aspects of angiogenesis. Although assays are the most representative of native angiogenesis, they raise ethical questions, require considerable technical skills, and are expensive. assays are inexpensive and easier to perform, but the majority of them are only two-dimensional cell monolayers which lack the physiological relevance of three-dimensional structures. Thus, it is important to look for alternative platforms to study angiogenesis under more physiologically relevant conditions . Accordingly, in this study, we developed polymeric vascular networks to be used to study angiogenesis and vascularization of a 3D human skin model . Our results showed that this platform allowed the study of more than one aspect of angiogenesis, endothelial migration and tube formation, when combined with Matrigel. We successfully reconstructed a human skin model, as a representative of a physiologically relevant and complex structure, and assessed the suitability of the developed platform for studying endothelialization of the tissue-engineered skin model.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304666PMC
June 2020

Identification of a fibrin concentration that promotes skin cell outgrowth from skin explants onto a synthetic dermal substitute.

JPRAS Open 2020 Sep 21;25:8-17. Epub 2020 Apr 21.

Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, North Campus, Broad Lane, Sheffield, S1 7HQ UK.

Background: Our overall objective is to develop a single-stage in-theatre skin replacement by combining small explants of skin with a synthetic biodegradable dermal scaffold. The aim of the current study is to determine the concentration of fibrin constituents and their handling properties for both adhering skin explants to the scaffold and encouraging cellular outgrowth to achieve reepithelialization.

Methods: Small skin explants were combined with several concentrations of thrombin (2.5,4.5,and 6.5 I.U) and fibrinogen (18.75,67, and 86.5 mg/ml), cultured in Green's media for 14 days and cellular outgrowth was measured using Rose Bengal staining. They were also cultured on electrospun scaffolds for 14 and 21 days. Hematoxylin and eosin (H&E) staining was undertaken to visualize the interface between skin explants and scaffolds and metabolic activity and collagen production were assessed.

Results: A thrombin/fibrinogen combination of 2.5 I. U/ml /18.75 mg/ml showed significantly greater cell viability as assessed by Rose Bengal stained areas at days 7 and 14. This was also seen in DAPI images and H&E stains skin explant/scaffold constructs. Fibrin with a concentration of thrombin 2.5 I.U./ml took 5-6 min to set, which is convenient for distributing skin explants on the scaffold.

Conclusion: The study identified concentrations of thrombin (2.5 I.U/ml) and fibrinogen (18.75 mg/ml), which were easy to handle and aided the retention of skin explants and permitted cell outgrowth from explants.
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http://dx.doi.org/10.1016/j.jpra.2019.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260611PMC
September 2020

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.

J Vis Exp 2020 05 12(159). Epub 2020 May 12.

Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield; Department of Chemical and Biological Engineering, University of Sheffield;

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.
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http://dx.doi.org/10.3791/61156DOI Listing
May 2020

Developing affordable and accessible pro-angiogenic wound dressings; incorporation of 2 deoxy D-ribose (2dDR) into cotton fibres and wax-coated cotton fibres.

J Tissue Eng Regen Med 2020 07 17;14(7):973-988. Epub 2020 Jun 17.

Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS University Islamabad Lahore Campus, Lahore, Pakistan.

The absorption capacity of cotton dressings is a critical factor in their widespread use where they help absorb wound exudate. Cotton wax dressings, in contrast, are used for wounds where care is taken to avoid adhesion of dressings to sensitive wounds such as burn injuries. Accordingly, we explored the loading of 2-deoxy-D-ribose (2dDR), a small sugar, which stimulates angiogenesis and wound healing in normal and diabetic rats, into both types of dressings and measured the release of it over several days. The results showed that approximately 90% of 2dDR was released between 3 and 5 days when loaded into cotton dressings. For wax-coated cotton dressings, several methods of loading of 2dDR were explored. A strategy similar to the commercial wax coating methodology was found the best protocol which provided a sustained release over 5 days. Cytotoxicity analysis of 2dDR loaded cotton dressing showed that the dressing stimulated metabolic activity of fibroblasts over 7 days confirming the non-toxic nature of this sugar-loaded dressings. The results of the chick chorioallantoic membrane (CAM) assay demonstrated a strong angiogenic response to both 2dDR loaded cotton dressing and to 2dDR loaded cotton wax dressings. Both dressings were found to increase the number of newly formed blood vessels significantly when observed macroscopically and histologically. We conclude this study offers a simple approach to developing affordable wound dressings as both have the potential to be evaluated as pro-active dressings to stimulate wound healing in wounds where management of exudate or prevention of adherence to the wounds are clinical requirements.
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http://dx.doi.org/10.1002/term.3072DOI Listing
July 2020

Author Correction: Regenerative medicine and injection therapies in stress urinary incontinence.

Nat Rev Urol 2020 Nov;17(11):650

Royal Hallamshire Hospital, Glossop Road, Sheffield, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41585-020-0340-xDOI Listing
November 2020

Pre-Seeding of Simple Electrospun Scaffolds with a Combination of Endothelial Cells and Fibroblasts Strongly Promotes Angiogenesis.

Tissue Eng Regen Med 2020 08 23;17(4):445-458. Epub 2020 May 23.

Tissue Engineering Group, Department of Materials Science and Engineering, Kroto Research Institute, University of Sheffield, North Campus Broad Lane, Sheffield, S3 7HQ, UK.

Background: Introduction of pro-angiogenic cells into tissue-engineered (TE) constructs (prevascularisation) is a promising approach to overcome delayed neovascularisation of such constructs post-implantation. Accordingly, in this study, we examined the contribution of human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts (HDFs) alone and in combination on the formation of new blood vessels in ex-ovo chick chorioallantoic membrane (CAM) assay.

Methods: Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and polycaprolactone (PCL) were first examined in terms of their physical, mechanical, and biological performances. The effect of gelatin coating and co-culture conditions on enhancing endothelial cell viability and growth was then investigated. Finally, the angiogenic potential of HDMECs and HDFs were assessed macroscopically and histologically after seeding on simple electrospun PHBV scaffolds either in isolation or in indirect co-culture using an ex-ovo CAM assay.

Results: The results demonstrated that PHBV was slightly more favourable than PCL for HDMECs in terms of cell metabolic activity. The gelatin coating of PHBV scaffolds and co-culture of HDMECs with HDFs both showed a positive impact on HDMECs viability and growth. Both cell types induced angiogenesis over 7 days in the CAM assay either in isolation or in co-culture. The introduction of HDMECs to the scaffolds resulted in the production of more blood vessels in the area of implantation than the introduction of HDFs, but the co-culture of HDMECs and HDFs gave the most significant angiogenic activity.

Conclusion: Our findings showed that the in vitro prevascularisation of TE constructs with HDMECs and HDFs alone or in co-culture promotes angiogenesis in implantable TE constructs.
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http://dx.doi.org/10.1007/s13770-020-00263-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392995PMC
August 2020

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Eur Respir J 2020 06 18;55(6). Epub 2020 Jun 18.

Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. , primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.
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http://dx.doi.org/10.1183/13993003.01200-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301290PMC
June 2020

Multifunctional Copper-Containing Mesoporous Glass Nanoparticles as Antibacterial and Proangiogenic Agents for Chronic Wounds.

Front Bioeng Biotechnol 2020 31;8:246. Epub 2020 Mar 31.

School of Clinical Dentistry, University of Sheffield, Sheffield, United Kingdom.

The physiological wound healing process involves a cascade of events which could be affected by several factors resulting in chronic, non-healing wounds. The latter represent a great burden especially when bacterial biofilms are formed. The rise in antibiotic resistance amongst infectious microorganisms leads to the need of novel approaches to treat this clinical issue. In this context, the use of advanced biomaterials, which can enhance the physiological expression and secretion of the growth factors involved in the wound healing process, is gaining increasing attention as a robust and appealing alternative approach. Among them, mesoporous glasses are of particular interest due to their excellent textural properties and to the possibility of incorporating and releasing specific therapeutic species, such as metallic ions. One of the most attractive therapeutic ions is copper thanks to its proangiogenic and antibacterial effects. In this contribution, copper containing mesoporous glass nanoparticles were proposed as a multifunctional device to treat chronic wounds. The developed nanoparticles evidenced a very high specific surface area (740 m/g), uniform pores of 4 nm and an almost total release of the therapeutic ion within 72 h of soaking. The produced nanoparticles were biocompatible and, when tested against Gram positive and Gram negative bacterial species, demonstrated antibacterial activity against both planktonic and biofilm bacteria in 2D cell monolayers, and in a 3D human model of infected skin. Their proangiogenic effect was tested with both the aortic ring and the chick chorioallantoic membrane assays and an increase in endothelial cell outgrowth at a concentration range between 30 and 300 ng/mL was shown. Overall, in this study biocompatible, multifunctional Cu-containing mesoporous glass nanoparticles were successfully produced and demonstrated to exert both antibacterial and proangiogenic effects.
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http://dx.doi.org/10.3389/fbioe.2020.00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136418PMC
March 2020

Characterization of Ocular Clinical Isolates of from Non-Contact Lens Related Keratitis Patients from South India.

Microorganisms 2020 Feb 15;8(2). Epub 2020 Feb 15.

Prof. Brien Holden Eye Research Centre, LV Prasad Eye Institute, Hyderabad 500034, India.

is the most common Gram-negative organism causing bacterial keratitis. utilizes various virulence mechanisms to adhere and colonize in the host tissue. In the present study, we examined virulence factors associated with thirty-four clinical isolates collected from keratitis patients seeking care at L V Prasad Eye Institute, Hyderabad. The virulence-associated genes in all the isolates were genotyped and characteristics such as antibiotic susceptibility, biofilm formation, swarming motility, pyoverdine production and cell cytotoxicity were analyzed. All the isolates showed the presence of genes related to biofilm formation, alkaline proteases and elastases; however, there was a difference in the presence of genes related to the type III secretion system (T3SS). A higher prevalence of genotype was noted in the drug-resistant isolates. All the isolates were capable of forming biofilms and more than 70% of the isolates showed good swarming motility. Pyoverdine production was not associated with the T3SS genotype. In the cytotoxicity assay, the presence of or both resulted in higher cytotoxicity compared to the absence of both the genes. Overall, our results suggest that the T3SS profile is a good indicator of virulence characteristics and the isolates lacking the effector genes may have evolved alternate mechanisms of colonization in the host.
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http://dx.doi.org/10.3390/microorganisms8020260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074794PMC
February 2020

A Dinuclear Ruthenium(II) Complex Excited by Near-Infrared Light through Two-Photon Absorption Induces Phototoxicity Deep within Hypoxic Regions of Melanoma Cancer Spheroids.

J Am Chem Soc 2020 03 25;142(10):4639-4647. Epub 2020 Feb 25.

Materials Science & Engineering, University of Sheffield, Mappin St, Sheffield S1 3JD, U.K.

The dinuclear photo-oxidizing Ru complex [{Ru(TAP)}(tpphz)] (TAP = 1,4,5,8- tetraazaphenanthrene, tpphz = tetrapyrido[3,2-:2',3'-:3″,2''-:2‴,3'''-]phenazine), , is readily taken up by live cells localizing in mitochondria and nuclei. In this study, the two-photon absorption cross section of is quantified and its use as a two-photon absorbing phototherapeutic is reported. It was confirmed that the complex is readily photoexcited using near-infrared, NIR, and light through two-photon absorption, TPA. In 2-D cell cultures, irradiation with NIR light at low power results in precisely focused phototoxicity effects in which human melanoma cells were killed after 5 min of light exposure. Similar experiments were then carried out in human cancer spheroids that provide a realistic tumor model for the development of therapeutics and phototherapeutics. Using the characteristic emission of the complex as a probe, its uptake into 280 μm spheroids was investigated and confirmed that the spheroid takes up the complex. Notably TPA excitation results in more intense luminescence being observed throughout the depth of the spheroids, although emission intensity still drops off toward the necrotic core. As can directly photo-oxidize DNA without the mediation of singlet oxygen or other reactive oxygen species, phototoxicity within the deeper, hypoxic layers of the spheroids was also investigated. To quantify the penetration of these phototoxic effects, was photoexcited through TPA at a power of 60 mW, which was progressively focused in 10 μm steps throughout the entire -axis of individual spheroids. These experiments revealed that, in irradiated spheroids treated with , acute and rapid photoinduced cell death was observed throughout their depth, including the hypoxic region.
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http://dx.doi.org/10.1021/jacs.9b11313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146853PMC
March 2020
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