Light Confocal Scanning Microscopy Research Articles

Observation of white spot lesions using swept source optical coherence tomography (SS-OCT): in vitro and in vivo study.

This study aimed to assess swept source optical coherence tomography (SS-OCT) for in vitro and in vivo detection of enamel white spot lesion (WSL). WSLs without surface breakdown on 33 extracted human posterior teeth were non-invasively scanned using SSOCT. The teeth were then cross-sectioned and imaged under confocal laser scanning microscope (CLSM) and light microscopy (LM). SS-OCT cross-sectional images were compared with CLSM and LM. WSL shapes in SS-OCT images closely corresponded to those of LM. There were significant correlations (p<0.001) in WSLs depth between SS-OCT and LM (r=0.92), SS-OCT and CLSM (r=0.80) and CLSM and LM (r=0.85). Six WSLs were also evaluated clinically using SS-OCT; clear in-depth images of these natural WSLs were obtained in vivo. SS-OCT appears to be an effective tool for observation of the internal structure of WSLs, enabling quantitative assessment of WSL depth. Such data can be considered in the clinical management of WSLs.

Clinical, Functional and Structural Studies of Humans and Merino Sheep Corneas from Two Different Regions of Argentina

Objective: The purpose of our work was to study and compare the cornea and tear film of people and sheep inhabiting in a region of Argentinean Patagonia (PATG), and in a geographically and climatically different region in the Argentinean Pampa (CAPT). Methods: Using cross sectional study, corneal examinations were performed in people and Merino sheep inhabiting PATG and CAPT regions, respectively. All people completed a questionnaire related to work activity, diet, and the use of hats or sunglasses during their life. Eyes from all participants were examined with a portable handheld slitlamp biomicroscope (BM) to evaluate corneal appearance, epithelium integrity, and transparency. Later on, in a subgroup of participants we studied eyeblinking frequency (EBF), ocular surface staining (FS), breakup time (BUT), Schirmer Tear Test (STT), corneal structure using confocal laser scanning microscopy (CLSM) and light microscopy (LM), and concentrations of serum ascorbate (sAA). Results: BM studies revealed numerous cases of Climatic Droplets Keratopathy (CDK) only in people inhabiting the PATG region. CLSM studies confirmed typical punctiform deposits at the Bowman´s layer in CDK patients living in that region. CLSM images from sheep did not show any abnormalities at the Bowman´s layer but exhibited small hyper reflective dots at the epithelium only in animals pasturing in the PATG region. FS and EBF mean values were significant higher in sheep grazing in the PATG region (p <0.05). No differences were found in surface eye tests when people from both regions were analyzed. Low sAA levels were found only in CDK people living in PATG region. Conclusions: CDK is a multi-factorial disease not only associated to harsh climate. We provide data that low sAA levels can play a role in its genesis in Patagonia patients’... Sheep pasturing in this region with a high prevalence of CDK have high concentration of sAA and did not present any sub epithelial corneal abnormalities.

Osteochondral lesions in distal tarsal joints of Icelandic horses reveal strong associations between hyaline and calcified cartilage abnormalities

Osteochondral lesions in the joints of the distal tarsal region of young Icelandic horses provide a natural model for the early stages of osteoarthritis (OA) in low-motion joints. We describe and characterise mineralised and non-mineralised osteochondral lesions in left distal tarsal region joint specimens from twenty-two 30±1 month-old Icelandic horses. Combinations of confocal scanning light microscopy, backscattered electron scanning electron microscopy (including, importantly, iodine staining) and three-dimensional microcomputed tomography were used on specimens obtained with guidance from clinical imaging. Lesion-types were described and classified into groups according to morphological features. Their locations in the hyaline articular cartilage (HAC), articular calcified cartilage (ACC), subchondral bone (SCB) and the joint margin tissues were identified and their frequency in the joints recorded. Associations and correlations between lesion-types were investigated for centrodistal joints only. In centrodistal joints the lesion-types HAC chondrocyte loss, HAC fibrillation, HAC central chondrocyte clusters, ACC arrest and ACC advance had significant associations and strong correlations. These lesion-types had moderate to high frequency in centrodistal joints but low frequencies in tarsometatarsal and talocalcaneal-centroquartal joints. Joint margin lesion-types had no significant associations with other lesion-types in the centrodistal joints but high frequency in both the centrodistal and tarsometatarsal joints. The frequency of SCB lesion-types in all joints was low. Hypermineralised infill phase lesion-types were detected. Our results emphasise close associations between HAC and ACC lesions in equine centrodistal joints and the importance of ACC lesions in the development of OA in low-motion compression-loaded equine joints.

Studying bacterial hydrophobicity and biofilm formation at liquid–liquid interfaces through interfacial rheology and pendant drop tensiometry

Bacterial adsorption to interfaces is a key factor in biofilm formation. One major limitation to understanding biofilm formation and development is the accurate measurement of bacterial cell adhesion to hydrophobic interfaces. With this study, bacterial attachment and biofilm growth over time at water–oil interface was monitored through interfacial rheology and tensiometry. Five model bacteria (Pseudomonas putida KT2442, Pseudomonas putida W2, Salmonella typhimurium, Escherichia coli, and Bacillus subtilis) were allowed to adsorb at the water–oil interface either in their non-growing or growing state. We found that we were able to observe the initial kinetics of bacterial attachment and the transient biofilm formation at the water–oil interface through interfacial rheology and tensiometry. Electrophoretic mobility measurements and bacterial adhesion to hydrocarbons (BATH) tests were performed to characterize the selected bacteria. To validate interfacial rheology and tensiometry measurements, we monitored biofilm formation utilizing both confocal laser scanning microscopy and light microscopy. Using this combination of techniques, we were able to observe the elasticity and tension development over time, from the first bacterial attachment up to biofilm formation.

Cartilage damage involving extrusion of mineralisable matrix from the articular calcified cartilage and subchondral bone

Arthropathy of the distal articular surfaces of the third metacarpal (Mc3) and metatarsal (Mt3) bones in the Thoroughbred racehorse (Tb) is a natural model of repetitive overload arthrosis. We describe a novel pathology that affects the articular calcified cartilage (ACC) and subchondral bone (SCB) and which is associated with hyaline articular cartilage degeneration. Parasagittal slices cut from the palmar quadrant of the distal condyles of the left Mc3/Mt3 of 39 trained Tbs euthanized for welfare reasons were imaged by point projection microradiography, and backscattered electron (BSE) scanning electron microscopy (SEM), light microscopy, and confocal scanning light microscopy. Mechanical properties were studied by nanoindentation. Data on the horses' training and racing career were also collected. Highly mineralised projections were observed extending from cracks in the ACC mineralising front into the hyaline articular cartilage (HAC) up to two-thirds the thickness of the HAC, and were associated with focal HAC surface fibrillation directly overlying their site. Nanoindentation identified this extruded matrix to be stiffer than any other mineralised phase in the specimen by a factor of two. The presence of projections was associated with a higher cartilage Mankin histology score (P<0.02) and increased amounts of gross cartilage loss pathologically on the condyle (P<0.02). Presence of projections was not significantly associated with: total number of racing seasons, age of horse, amount of earnings, number of days in training, total distance galloped in career, or presence of wear lines.

Design and evaluation of baicalin-containing in situ pH-triggered gelling system for sustained ophthalmic drug delivery

Baicalin has been reported to have anti-inflammatory and anti-cataract effects on eye tissues, but it has a low bioavailability partly due to its poor stability of baicalin, the special anatomic structure and efficient protective mechanism of eyes. The aim of this study was to investigate the correlation between the stability of baicalin and in situ pH-triggered gelling system. Carbopol ® 974P (0.3%, w/v) was used as the gelling agent combined with hydroxypropylmethylcellulose E4M (0.6%, w/v) which acted as a viscosity enhancing agent. In vitro and in vivo evaluations were performed using several techniques, namely confocal scanning light microscopy analysis, rheometry, Gamma scintigraphic technique and microdialysis method. The rheological behavior showed a significant enhancement in gel strength under physiological conditions, and the formulation provided sustained release of the drug over an 8-h period. In elimination studies, the radioactivity of formulation was always higher than that of the control solution. Additionally, the AUC and C max values were 6.1-fold and 3.6-fold higher than those of the control solution, respectively. The results demonstrated that an in situ pH-triggered gelling system have better ability to keep baicalin stable and retain drug release than marketed baicalin eye drops to enhance the ocular bioavailability.

Effects of filler-matrix morphology on mechanical properties of corn starch–zein thermo-moulded films

The effects of filler-matrix morphology on mechanical behaviour of thermo-moulded films based on native or amorphous corn starch as matrix, and zein (sizes of <100, <250, and >500μm) as filler were investigated. The morphology and mechanical behaviour were characterized by Confocal Light Scanning Microscopy and three point bending test, respectively. For native starch blends, the zein network was relatively dense when initial zein size increased from 100 to 500μm. However, small initial zein size favored continuous morphology for amorphous starch blends. It is noted that the stress at rupture values of native starch blends decreased when zein size increased, indicating relatively compact zein network damaged the stress of starch matrix. On the contrary, the variations of the stress at rupture values of amorphous starch blends were not significantly different, attributing to the heterogeneous morphology of incomplete zein network dispersed in the amorphous starch matrix.

Preparation of epidermal growth factor (EGF) conjugated iron oxide nanoparticles and their internalization into colon cancer cells

Epidermal growth factor (EGF) was conjugated with carboxymethyldextran (CMDx) coated iron oxide magnetic nanoparticles using carbodiimide chemistry to obtain magnetic nanoparticles that target the epidermal growth factor receptor (EGFR). Epidermal growth factor modified magnetic nanoparticles were colloidally stable when suspended in biological buffers such as PBS and cell culture media. Both targeted and non-targeted nanoparticles were incubated with CaCo-2 cancer cells, known to overexpress EGFR. Nanoparticle localization within the cell was visualized by confocal laser scanning microscopy and light microscopy using Prussian blue stain. Results showed that targeted magnetic nanoparticles were rapidly accumulated in both flask-shaped small vesicles and large circular endocytic structures. Internalization patterns suggest that both clathrin-dependent and clathrin-independent receptors mediated endocytosis mechanisms are responsible for nanoparticle internalization.

Selection and application of M cell targeting peptide ligand to induce immune responses after mucosal immunization of the ligand-fused antigen (39.31)

Abstract Mucosa is the most successful application area of vaccination through the induction of the systemic and mucosal immune responses. In this study, we have searched for reliable peptides which could be used as mucosal adjuvant to potentiate the orally introduced antigen to induce the immune response. Using the phage display library, we have selected peptides which can bind onto cultured M cell model using Caco-2 and Raji co-culture system which had been confirmed by UEA-1, WGA, and M cell specific antibody by confocal scanning light microscopy. Interestingly, one of the selected peptides showed a high homology with outer membrane protein of Yersinia enterocolitica-like strains which are well-known human pathogen causing various gastrointestinal systemic syndromes and capable of invading and destroying M cells. In order to confirm the role of selected peptide to enhance the immune response induction after oral administration, fusion proteins between a peptide ligand and EGFP or VP1 antigen of foot-and-mouth disease were produced and tested in ex vivo intestinal loop and in vivo mouse systems. (This work was supported by the grant from ARPC.)

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization.

BackgroundGemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.ResultsUsing phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as well as DNA confirmed the behaviour of the nucleoid and nucleoid envelope during cell division. Electron microscopy of cryosubstituted cells confirmed deductions from light microscopy concerning nucleoid presence in relation to the stage of budding, and showed that the nucleoid was observed to occur in both mother and bud cells only at later budding stages. It further suggested that nucleoid envelope formed only after the nucleoid was translocated into the bud, since envelopes only appeared in more mature buds, while naked nucleoids occurred in smaller buds. Nucleoid envelope appeared to originate from the intracytoplasmic membranes (ICM) of both mother cell and bud. There was always a connecting passage between mother cell and bud during the budding process until separation of the two cells. The division cycle of the nucleated planctomycete G. obscuriglobus appears to be a complex process in which chromosomal DNA is transported to the daughter cell bud after initial formation of the bud, and this can be performed repeatedly by a single mother cell.ConclusionThe division cycle of the nucleated planctomycete G. obscuriglobus is a complex process in which chromosomal nucleoid DNA is transported to the daughter cell bud after initial formation of a bud without nucleoid. The new bud nucleoid is initially naked and not surrounded by membrane, but eventually acquires a complete nucleoid envelope consisting of two closely apposed membranes as occurs in the mother cell. The membranes of the new nucleoid envelope surrounding the bud nucleoid are derived from intracytoplasmic membranes of both the mother cell and the bud. The cell division of G. obscuriglobus displays some unique features not known in cells of either prokaryotes or eukaryotes.

A confocal laser scanning and conventional wide field light microscopy study of Classopollis from the Toarcian‐Aalenian of the Fuentelsaz section (Spain)

A morphological study of three different types of dispersed Circumpolles pollen grains from the Toarcian‐Aalenian of the Fuentelsaz section (Spain) was performed using conventional wide field and confocal laser scanning microscopy (CSLM). The CSLM data allowed scanning electron microscope‐like three‐dimensional reconstructions to be made and showed that the most part of the studied pollen have taxonomic features characteristic of the Mesozoic genus Classopollis. At the species level, Classopollis torosus and Classopollis classoides were identified. The third form studied is characterized by the presence of Ubisch bodies adhering to the pollen wall.

Variations in articular calcified cartilage by site and exercise in the 18-month-old equine distal metacarpal condyle

To interrelate articular calcified cartilage thickness, mineralisation density, tidemark count and tidemark linear accretion rate by site in the equine third metacarpal distal condyle. To determine the effects of exercise during early life on articular calcified cartilage. Six of 12 pasture-raised Thoroughbred horses were exercised from 10 days old. Calcein labels were given 19 and 8 days prior to euthanasia at 18 months old. Osteochondral specimens were cut from the distal third metacarpal condyle and imaged using confocal scanning light microscopy (CSLM) and quantitative backscattered electron scanning electron microscopy (qBSE). Articular calcified cartilage thickness and total thickness mineralisation density were measured on montaged qBSE image sets, and inter-label mineralisation density, tidemark count and linear accretion rate measured on registered CSLM-qBSE image pairs. Calcified cartilage thickness, mineralisation density, tidemark count and linear accretion rate varied significantly between sites. Regions with thinner calcified cartilage had greater linear accretion rates, hence rapid chondroclastic resorption. Mineralisation density was positively correlated with linear accretion rate. Fewer multiple tidemarks were counted in regions with greater linear accretion rates. Lag time between the tidemark and cement line was estimated (180 days; in the range of 0-648 days). Exercise had little effect on measured parameters. The major determinant of articular calcified cartilage thickness is the rate of chondroclastic resorption, not tidemark linear accretion rate. Our evidence supports coupled, mechanosensitive regulation of chondroclastic resorption and linear accretion rate in articular calcified cartilage. Exercising pasture-reared foals causes little additional adaptation in distal third metacarpal articular calcified cartilage.

Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.

Effects of variation in the palm stearin: Palm olein ratio on the crystallisation of a low- trans shortening

Changes in the rheological properties during crystallisation and in the crystal size and morphology of blends containing rapeseed oil with varying percentages of palm stearin (POs) and palm olein (POf) have been studied. The crystals formed from all three blends were studied by confocal laser scanning microscopy, light microscopy and environmental scanning electron microscopy, which revealed the development of clusters of 3–5 individual elementary “spherulites” in the early stages of crystallisation. The saturated triacylglycerol content of the solid crystals separated at the onset of crystallisation was much greater than that in the total fat. Fat blends with a higher content of palm stearin had a more rapid nucleation rate when observed by light microscopy, and this caused an earlier change in the rheological properties of the fat during crystallisation. Using a low torque amplitude (0.005 Pa, which was within the linear viscoelastic region of all samples studied) and a frequency of 1 Hz, the viscoelastic properties of melted fat during cooling were studied. All samples, prior to crystallisation, showed weak viscoelastic liquid behaviour ( G″, loss modulus > G′, storage modulus). After crystallisation, a more “solid like” behaviour was observed ( G′ similar to or greater than G″). The blend having the highest concentration of POs was found to have the earliest onset of crystallisation (27% w/w POs; 12 mins, 22% w/w POs; 13.5 mins, 17% w/w POs, 15 mins, respectively). However, there were no significant differences in the time to the point when G′ became greater than G′ among the three blends.

Relationships between texture, mechanical properties and structure of cornflakes

Abstract Hominies and flours derived from four corn varieties were prepared according to industrial recipes and processed as petals by extrusion cooking (SME = 150 J g−1, Tdie = 130 °C) and a batch thermal process (100 °C, 50 min). Flake texture was evaluated by crushing bulk petals in a Kramer cell and comparing measurements to those obtained for commercial cornflakes, previously graded by a trained sensory analysis panel. Depending on the corn variety, various results were found in regard to crispness. Bending moduli of dense extruded materials (Es from 0.5 to 2.1 GPa) were found to be correlated to the texture of petals derived from the same blends. Results obtained at different scales showed that the expanded microstructure of flakes, determined by X-ray tomography, and the morphology of their constitutive materials, deduced from confocal scanning light microscopy and RVA, primarily depended on the process used rather than on the corn variety. Adhesion between protein aggregates and amorphous starch matrix was inferred from the mechanical properties involved in texture.

The expression of prostate stem cell antigen in human clear cell renal cell carcinoma: a quantitative reverse transcriptase‐polymerase chain reaction analysis

To analyse the gene expression level of prostate stem cell antigen (PSCA) in human clear cell renal cell carcinoma (CC-RCC) and its relationship with conventional clinicopathological manifestations, to evaluate its prognostic value for patient outcome, and to determine the effect of PSCA on the progression of CC-RCC. We quantified PSCA mRNA level in human RCC cell lines (ACHN, A704, KPK-1, Caki-1, and Caki-2) and in 154 surgical tissue samples (81 from CC-RCC, 73 from normal kidney) using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The findings were analysed in relation to clinicopathological factors. Immunohistochemical expression was examined using confocal laser scanning light-microscopy. PSCA was overexpressed in all RCC cell lines. PSCA mRNA levels were significantly higher in CC-RCC than in normal kidney tissue samples (P < 0.001), in G2-G3 than in G1 tumours (P = 0.028), and in advanced disease (T3-T4) than in organ-confined (T1-T2) tumours (P = 0.016). There was significantly higher PSCA mRNA expression in patients with M1 than in those with M0 disease (P = 0.029). Patients in whom the lesions had high PSCA expression levels had a significantly worse prognosis than those with low PSCA expression levels (P = 0.044). Using immunohistochemical analysis there was markedly greater PSCA expression in CC-RCC than in normal kidney, and in advanced-disease high-grade tumours than in organ-confined low-grade tumours. A significant correlation was detected in the gene expression level of PSCA with histological grade, clinicopathological stage and prognosis in CC-RCC. Our data indicate that PSCA is associated with carcinogenesis and progression of CC-RCC.

Rheology and microstructure of gluten and soya-based o/w emulsions

Highly concentrated oil-in-water (o/w) emulsion stabilised by means of gluten and soya protein isolate (SPI) at low pH have been characterized by means of linear dynamic viscoelasticity and droplet size distribution analysis (DSD). The microstructure of these emulsions has been characterized at a colloidal level by using confocal laser scanning microscopy (CLSM) and light microscopy (LM). These emulsions always exhibited a behaviour characteristic of highly flocculated emulsions with a mechanical spectrum showing a well-developed plateau region. DSD results generally showed log normal bimodal profiles. Microstructure images revealed occurrence of a close packing of droplets with a broad distribution of sizes participating in the formation of a three dimensional flocculated network. The Mason model of elasticity of compressed emulsions has been used to correlate viscoelastic and microstructural parameters giving adequate fitting but underestimating the elastic properties obtained for the highest concentration of gluten. These deviations may be explained in terms of an enhancement of the elastic network formed in the aqueous phase in which the glutenin fraction must play an important role.

Approaches to quantification of microstructure for model lipid systems

AbstractSemisolid fat samples with different solid fat contents and microstructures were prepared by crystallization of mixtures of model lipid systems containing high‐melting and lowmelting lipids and analyzed for microstructural properties. Microstructure images were acquired by confocal scanning light microscopy and showed fat crystals or fat crystal flocs combined with their surrounding continuous phase to constitute microstructural units and a microstructural network that was formed through their interaction. Fat crystal flocs and their centroids, microstructure units, and their interface boundaries were identified by image analysis. Several methods to quantify microstructure were compared. A new concept was introduced: the microstructure density, defined as the number of microstructural units per unit volume of the system. Also, the Richardson plot and particle counting methods (PCM) were used to find the fractal dimension of the crystal network. The Euler characteristic and nearest neighbor features of the microstructure were obtained as well by use of custom‐developed programs. The different metrics of semisolid lipid microstructure were compared in terms of their physical meaning, means of acquisition, and consistency. The results showed that the quantitative microstructure parameters obtained from different approaches, except the fractal dimesion determined by the PCM, can identify both differences and similarities of microstructural characteristics in the model lipid systems studied in this work.

Combining confocal and BSE SEM imaging for bone block surfaces

The present report presents a method for the correlation of qualitative and quantitative BSE SEM imaging with confocal scanning light microscopy (CSLM) imaging modes applied to bone samples embedded in PMMA. The SEM has a proper digital scan generator: we leave the BSE image unchanged, and match the CSLM image to it, because the CSLM scan mechanism is not digital, though the signal is digitised. Our overlapping program uses a linear transformation matrix which projects one system to the other, calculated by finding three corresponding points in BSE and CSLM pictures. BSE images are empty where cells and osteoid are present. Fluorescence mode CSLM fills in these gaps. The combination images enhance our understanding of what is going on - and re-establish the need for good cellular preservation.

In Vitro and ex Vivo Intestinal Tissue Models to Measure Mucoadhesion of Poly (Methacrylate) and N-Trimethylated Chitosan Polymers

The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC). The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers. Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29. Human mucus-producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. pDMAEMA displayed similar levels of mucoadhesion and lower levels of bioadhesion than a chitosan derivative and it was not cytotoxic. pDMAEMA decreased FD-4 flux in the presence of mucus, whereas TMC increased it. The combination of mucus and methacrylate polymers appears to increase barrier function of the apical membrane.