Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Observation of solution-induced corneal staining with fluorescein, rose bengal and lissamine green

Ever since sodium fluorescein (‘fluorescein’ [FL]) was first used to investigate the ocular surface over a century ago, the term ‘staining’ has been taken to mean the presence of ocular surface fluorescence [1]. This term has not been necessarily taken to infer any particular mechanism of causation, and indeed, can be attributed to a variety of possible aetiologies [2]. In recent times, there has been considerable interest in a form of ocular surface fluorescence seen in association with the use of certain combinations of soft contact lenses and multipurpose solutions. The first clinical account of this phenomenon was reported by Jones et al. [3], which was followed by a more formal investigation by the same author in 2002 [4]. Jones et al described this appearance as a ‘classic solution-based toxicity reaction’. Subsequently, this appearance has come to be known as ‘solution-induced corneal staining’ or more recently by the acronym ‘SICS’ [5]. The term SICS is potentially problematic in that from a cell biology point of view, there is an inference that ‘staining’ means the entry of a dye into corneal epithelial cells. Morgan and Maldonado-Codina [2] noted there was no foundation of solid scientific literature underpinning our understanding of the true basic causative mechanisms of this phenomenon; since that time, further work has been published in this field [6] and [7] but questions still remain about the precise aetiology of this phenomenon...

Tissue engineered prefabricated vascularized flaps

BACKGROUND.: Microvascular free tissue transfer has become increasingly popular in the reconstruction of head and neck defects, but it also has its disadvantages. Tissue engineering allows the generation of neo-tissue for implantation, but these tissues are often avascular. We propose to combine tissue-engineering techniques together with flap prefabrication techniques to generate a prefabricated vascularized soft tissue flap. METHODS: Human dermal fibroblasts (HDFs) labeled with fluorescein diacetate were static seeded onto polylactic-co-glycolic acid-collagen (PLGA-c) mesh. Controls were plain PLGA-c mesh. The femoral artery and vein of the nude rat was ligated and used as a vascular carrier for the constructs. After 4 weeks of implantation, the constructs were assessed by gross morphology, routine histology, Masson trichrome, and cell viability determined by green fluorescence. RESULTS: All the constructs maintained their initial shape and dimensions. Angiogenesis was evident in all the constructs with neo-capillary formation within the PLGA-c mesh seen. HDFs proliferated and filled the interyarn spaces of the PLGA-c mesh, while unseeded PLGA-c mesh remained relatively acellular. Cell tracer study indicated that the seeded HDFs remained viable and closely associated to remaining PLGA-c fibers. Collagen formation was more abundant in the constructs seeded with HDFs. CONCLUSIONS: PLGA-c, enveloped by a cell sheet composed of fibroblasts, can serve as a suitable scaffold for generation of a soft tissue flap. A ligated arteriovenous pedicle can serve as a vascular carrier for the generation of a tissue engineered vascularized flap.

The synthesis of novel nitroxides and their application as small-molecule antioxidants and profluorescent probes for oxidative stress

The detection and potential treatment of oxidative stress in biological systems has been explored using isoindoline-based nitroxide radicals. A novel tetraethyl-fluorescein nitroxide was synthesised for its use as a profluorescent probe for redox processes in biological systems. This tetraethyl system, as well as a tetramethyl-fluorescein nitroxide, were shown to be sensitive and selective probes for superoxide in vitro. The redox environment of cellular systems was also explored using the tetramethylfluorescein species based on its reduction to the hydroxylamine. Flow cytometry was employed to assess the extent of nitroxide reduction, reflecting the overall cellular redox environment. Treatment of normal fibroblasts with rotenone and 2-deoxyglucose resulted in an oxidising cellular environment as shown by the lack of reduction of the fluorescein-nitroxide system. Assessment of the tetraethyl-fluorescein nitroxide system in the same way demonstrated its enhanced resistance to reduction and offers the potential to detect and image biologically relevant reactive oxygen species directly. Importantly, these profluorescent nitroxide compounds were shown to be more effective than the more widely used and commercially available probes for reactive oxygen species such as 2’,7’-dichlorodihydrofluorescein diacetate. Fluorescence imaging of the tetramethyl-fluorescein nitroxide and a number of other rhodamine-nitroxide derivatives was undertaken, revealing the differential cellular localisation of these systems and thus their potential for the detection of redox changes in specific cellular compartments. As well as developing novel methods for the detection of oxidative stress, a number of novel isoindoline nitroxides were synthesised for their potential application as small-molecule antioxidants. These compounds incorporated known pharmacophores into the isoindoline-nitroxide structure in an attempt to increase their efficacy in biological systems. A primary and a secondary amine nitroxide were synthesised which incorporated the phenethylamine backbone of the sympathomimetic amine class of drugs. Initial assessment of the novel primary amine derivative indicated a protective effect comparable to that of 5-carboxy-1,1,3,3- tetramethylisoindolin-2-yloxyl. Methoxy-substituted nitroxides were also synthesised as potential antioxidants for their structural similarity to some amphetamine type stimulants. A copper-catalysed methodology provided access to both the mono- and di-substituted methoxy-nitroxides. Deprotection of the ethers in these compounds using boron tribromide successfully produced a phenolnitroxide, however the catechol moiety in the disubstituted derivative appeared to undergo reaction with the nitroxide to produce quinone-like degradation products. A novel fluoran-nitroxide was also synthesised from the methoxy-substituted nitroxide, providing a pH-sensitive spin probe. An amino-acid precursor containing a nitroxide moiety was also synthesised for its application as a dual-action antioxidant. N-Acetyl protection of the nitroxide radical was necessary prior to the Erlenmeyer reaction with N-acetyl glycine. Hydrolysis and reduction of the azlactone intermediate produced a novel amino acid precursor with significant potential as an effective antioxidant.

How optometrists record corneal staining

Purpose: The aim of this study was to determine current approaches adopted by optometrists to the recording of corneal staining following fluorescein instillation. Methods: An anonymous ‘record-keeping task’ was sent to all 756 practitioners who are members of the Queensland Division of Optometrists Association Australia. This task comprised a form on which appeared a colour photograph depicting contact lens solution-induced corneal staining. Next to the photograph was an empty box, in which practitioners were asked to record their observations. Practitioners were also asked to indicate the level of severity of the condition at which treatment would be instigated. Results: Completed task forms were returned by 228 optometrists, representing a 30 per cent response rate. Ninety-two per cent of respondents offered a diagnosis. The most commonly used descriptive terms were ‘superficial punctate keratitis’ (36 per cent of respondents) and ‘punctate staining’ (29 per cent). The level of severity and location of corneal staining were noted by 69 and 68 per cent of respondents, respectively. A numerical grade was assigned by 44 per cent of respondents. Only three per cent nominated the grading scale used. The standard deviation of assigned grades was � 0.6. The condition was sketched by 35 per cent of respondents and two per cent stated that they would take a photograph of the eye. Ten per cent noted the eye in which the condition was being observed. Opinions of the level of severity at which treatment for corneal staining should be instigated varied considerably between practitioners, ranging from ‘any sign of corneal staining’ to ‘grade 4 staining’. Conclusion: Although most practitioners made a sensible note of the condition and properly recorded the location of corneal staining, serious deficiencies were evident regarding other aspects of record-keeping. Ongoing programs of professional optometric education should reinforce good practice in relation to clinical record-keeping.

Multiple analysis of three common genetic alterations associated with thrombophilia

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia. (C) 2000 Lippincott Williams and Wilkins.

Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore’s usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially “light up” in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.

Ocular surface changes with short-term contact lens wear

Contact lenses are a common method for the correction of refractive errors of the eye. While there have been significant advancements in contact lens designs and materials over the past few decades, the lenses still represent a foreign object in the ocular environment and may lead to physiological as well as mechanical effects on the eye. When contact lenses are placed in the eye, the ocular anatomical structures behind and in front of the lenses are directly affected. This thesis presents a series of experiments that investigate the mechanical and physiological effects of the short-term use of contact lenses on anterior and posterior corneal topography, corneal thickness, the eyelids, tarsal conjunctiva and tear film surface quality. The experimental paradigm used in these studies was a repeated measures, cross-over study design where subjects wore various types of contact lenses on different days and the lenses were varied in one or more key parameters (e.g. material or design). Both, old and newer lens materials were investigated, soft and rigid lenses were used, high and low oxygen permeability materials were tested, toric and spherical lens designs were examined, high and low powers and small and large diameter lenses were used in the studies. To establish the natural variability in the ocular measurements used in the studies, each experiment also contained at least one “baseline” day where an identical measurement protocol was followed, with no contact lenses worn. In this way, changes associated with contact lens wear were considered in relation to those changes that occurred naturally during the 8 hour period of the experiment. In the first study, the regional distribution and magnitude of change in corneal thickness and topography was investigated in the anterior and posterior cornea after short-term use of soft contact lenses in 12 young adults using the Pentacam. Four different types of contact lenses (Silicone hydrogel/ Spherical/–3D, Silicone Hydrogel/Spherical/–7D, Silicone Hydrogel/Toric/–3D and HEMA/Toric/–3D) of different materials, designs and powers were worn for 8 hours each, on 4 different days. The natural diurnal changes in corneal thickness and curvature were measured on two separate days before any contact lens wear. Significant diurnal changes in corneal thickness and curvature within the duration of the study were observed and these were taken into consideration for calculating the contact lens induced corneal changes. Corneal thickness changed significantly with lens wear and the greatest corneal swelling was seen with the hydrogel (HEMA) toric lens with a noticeable regional swelling of the cornea beneath the stabilization zones, the thickest regions of the lenses. The anterior corneal surface generally showed a slight flattening with lens wear. All contact lenses resulted in central posterior corneal steepening, which correlated with the relative degree of corneal swelling. The corneal swelling induced by the silicone hydrogel contact lenses was typically less than the natural diurnal thinning of the cornea over this same period (i.e. net thinning). This highlights why it is important to consider the natural diurnal variations in corneal thickness observed from morning to afternoon to accurately interpret contact lens induced corneal swelling. In the second experiment, the relative influence of lenses of different rigidity (polymethyl methacrylate – PMMA, rigid gas permeable – RGP and silicone hydrogel – SiHy) and diameters (9.5, 10.5 and 14.0) on corneal thickness, topography, refractive power and wavefront error were investigated. Four different types of contact lenses (PMMA/9.5, RGP/9.5, RGP/10.5, SiHy/14.0), were worn by 14 young healthy adults for a period of 8 hours on 4 different days. There was a clear association between fluorescein fitting pattern characteristics (i.e. regions of minimum clearance in the fluorescein pattern) and the resulting corneal shape changes. PMMA lenses resulted in significant corneal swelling (more in the centre than periphery) along with anterior corneal steepening and posterior flattening. RGP lenses, on the other hand, caused less corneal swelling (more in the periphery than centre) along with opposite effects on corneal curvature, anterior corneal flattening and posterior steepening. RGP lenses also resulted in a clinically and statistically significant decrease in corneal refractive power (ranging from 0.99 to 0.01 D), large enough to affect vision and require adjustment in the lens power. Wavefront analysis also showed a significant increase in higher order aberrations after PMMA lens wear, which may partly explain previous reports of "spectacle blur" following PMMA lens wear. We further explored corneal curvature, thickness and refractive changes with back surface toric and spherical RGP lenses in a group of 6 subjects with toric corneas. The lenses were worn for 8 hours and measurements were taken before and after lens wear, as in previous experiments. Both lens types caused anterior corneal flattening and a decrease in corneal refractive power but the changes were greater with the spherical lens. The spherical lens also caused a significant decrease in WTR astigmatism (WRT astigmatism defined as major axis within 30 degrees of horizontal). Both the lenses caused slight posterior corneal steepening and corneal swelling, with a greater effect in the periphery compared to the central cornea. Eyelid position, lid-wiper and tarsal conjunctival staining were also measured in Experiment 2 after short-term use of the rigid and SiHy contact lenses. Digital photos of the external eyes were captured for lid position analysis. The lid-wiper region of the marginal conjunctiva was stained using fluorescein and lissamine green dyes and digital photos were graded by an independent masked observer. A grading scale was developed in order to describe the tarsal conjunctival staining. A significant decrease in the palpebral aperture height (blepharoptosis) was found after wearing of PMMA/9.5 and RGP/10.5 lenses. All three rigid contact lenses caused a significant increase in lid-wiper and tarsal staining after 8 hours of lens wear. There was also a significant diurnal increase in tarsal staining, even without contact lens wear. These findings highlight the need for better contact lens edge design to minimise the interactions between the lid and contact lens edge during blinking and more lubricious contact lens surfaces to reduce ocular surface micro-trauma due to friction and for. Tear film surface quality (TFSQ) was measured using a high-speed videokeratoscopy technique in Experiment 2. TFSQ was worse with all the lenses compared to baseline (PMMA/9.5, RGP/9.5, RGP/10.5, and SiHy/14) in the afternoon (after 8 hours) during normal and suppressed blinking conditions. The reduction in TFSQ was similar with all the contact lenses used, irrespective of their material and diameter. An unusual pattern of change in TFSQ in suppressed blinking conditions was also found. The TFSQ with contact lens was found to decrease until a certain time after which it improved to a value even better than the bare eye. This is likely to be due to the tear film drying completely over the surface of the contact lenses. The findings of this study also show that there is still a scope for improvement in contact lens materials in terms of better wettability and hydrophilicity in order to improve TFSQ and patient comfort. These experiments showed that a variety of changes can occur in the anterior eye as a result of the short-term use of a range of commonly used contact lens types. The greatest corneal changes occurred with lenses manufactured from older HEMA and PMMA lens materials, whereas modern SiHy and rigid gas permeable materials caused more subtle changes in corneal shape and thickness. All lenses caused signs of micro-trauma to the eyelid wiper and palpebral conjunctiva, although rigid lenses appeared to cause more significant changes. Tear film surface quality was also significantly reduced with all types of contact lenses. These short-term changes in the anterior eye are potential markers for further long term changes and the relative differences between lens types that we have identified provide an indication of areas of contact lens design and manufacture that warrant further development.

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21waf1/cip1

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

Prevalence of papillary changes and folliculosis of the palpebral conjunctiva in asymptomatic Chinese children

Purpose To determine the prevalence of papillary changes of the upper palpebral conjunctiva and folliculosis of the lower palpebral conjunctiva in Chinese children with no history of contact lens wear. Method Ninety-nine subjects (aged 6–15 years old) who were interested in a myopia control study were screened for papillary changes and folliculosis of the palpebral conjunctiva. Photodocumentation was performed under white and blue light (after the application of fluorescein) with a yellow filter and the photographs were graded by a group of practitioners according to a pre-set grading scale. Analysis was performed with the subjects divided into groups according to gender and age. Results More than 48% of the subjects had clinically significant (≥Grade 3) papillary changes in the upper palpebral conjunctiva. The prevalence of significant folliculosis in the lower lid was about 33%. The prevalence of significant papillary changes and folliculosis were similar between genders. No differences were observed between younger (age ≤ 10 years old) and older (age > 10 years old) in papillary changes but younger subjects showed a higher prevalence of folliculosis. Conclusions The prevalences of clinically significant papillary changes and folliculosis of unknown aetiology are high in Chinese children.

The Ghrelin axis : does it have an appetite for cancer progression?

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHSR), is a peptide hormone with diverse physiological roles. Ghrelin regulates GH release, appetite and feeding, gut motility, and energy balance and also has roles in the cardiovascular, immune, and reproductive systems. Ghrelin and the GHSR are expressed in a wide range of normal and tumor tissues, and a fluorescein-labeled, truncated form of ghrelin is showing promise as a biomarker for prostate cancer. Plasma ghrelin levels are generally inversely related to body mass index and are unlikely to be useful as a biomarker for cancer, but may be useful as a marker for cancer cachexia. Some single nucleotide polymorphisms in the ghrelin and GHSR genes have shown associations with cancer risk; however, larger studies are required. Ghrelin regulates processes associated with cancer, including cell proliferation, apoptosis, cell migration, cell invasion, inflammation, and angiogenesis; however, the role of ghrelin in cancer is currently unclear. Ghrelin has predominantly antiinflammatory effects and may play a role in protecting against cancer-related inflammation. Ghrelin and its analogs show promise as treatments for cancer-related cachexia. Further studies using in vivo models are required to determine whether ghrelin has a role in cancer progression.

Putting vital stains in context

While vital staining remains a cornerstone in the diagnosis of ocular disease and contact lens complications, there are many misconceptions regarding the properties of commonly used dyes by eye-care practitioners and what is and what is not corneal staining after instillation of sodium fluorescein. Similarly, the proper use and diagnostic utility of rose Bengal and lissamine green B, the other two ophthalmic dyes commonly used for assessing ocular complications, have similarly remained unclear. Due to the limitations of vital stains for definitive diagnosis, concomitant signs and symptoms in addition to a complete patient history are required. Over the past decade, there have been many reports of a type of corneal staining—often referred to as solution-induced corneal staining (SICS)—that is observed with the use of multipurpose solutions in combination with soft lenses, more specifically silicone hydrogel lenses. Some authors believe that SICS is a sign of lens/solution incompatibility; however, new research shows that SICS may be neither a measure of lens/solution biocompatibility nor ‘true’ corneal staining, as that observed in pathological situations. A large component of SICS may be a benign phenomenon, known as preservative-associated transient hyperfluorescence (PATH). There is a lack of correlated signs and/or symptoms with SICS/PATH. Several properties of SICS/PATH, such as appearance and duration, differentiate it from pathological corneal staining. This paper reviews the properties of vital stains, their use and limitations in assessment of the ocular surface, the aetiology of corneal staining, characteristics of SICS/PATH that differentiate it from pathological corneal staining and what the SICS/PATH phenomenon means for contact lens-wearing patients.

Synthesis and toxicological evaluation of a chitosan‑L‑leucine conjugate for pulmonary drug delivery applications

Herein are reported the synthesis of a conjugate of chitosan with L-leucine, the preparation of nanoparticles from both chitosan and the conjugate for use in pulmonary drug delivery, and the in vitro evaluation of toxicity and inflammatory effects of both the polymers and their nanoparticles on the bronchial epithelial cell line, BEAS-2B. The nanoparticles, successfully prepared both from chitosan and the conjugate, had a diameter in the range of 10−30 nm. The polymers and their nanoparticles were tested for their effects on cell viability by MTT assay, on trans-epithelial permeability by using sodium fluorescein as a fluid phase marker, and on IL-8 secretion by ELISA. The conjugate nanoparticles had a low overall toxicity (IC50 = 2 mg/mL following 48 h exposure; no induction of IL-8 release at 0.5 mg/mL concentration), suggesting that they may be safe for pulmonary drug delivery applications.

Scheimpflug imaging of the post lens tear film during contact lens wear

A new imaging methodology is described to visualise the post lens tear film (PLTF) during contact lens wear. A rotating-Scheimpflug camera in combination with sodium fluorescein allows evaluation of the PLTF for different contact lens modalities, including mini-scleral, rigid gas permeable (RGP) and soft contact lenses. This imaging technique provides an extension of the instrument’s current functionality. The potential advantages and limitations of the technique are discussed.