Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment.

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Phytochemical Screening and Bioactivity Studies of Endophytes Cladosporium sp. Isolated from the Endangered Plant Vateria Indica Using In Silico and In Vitro Analysis.

Vateria indica is persistent tree used in Unani sources for the medication and classified as critically endangered. Thus, endophytes for alternative methods to explore these endangered Plants having rich source pharmaceuticals' active molecules for drug development and production. Endophytes comprises unexplored microbes as a potential source of rich pharmaceutically bioactive compounds attributable to their relationship with the host. In the current study, we have isolated endophyte fungi Cladosporium from the plant Vateria indica and performed phytochemical screening of its ethanolic extract to detect the phytochemicals using thin layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), UV-visible spectrophotometry (UV-VIS), and Fourier transform infrared spectroscopy (FTIR). GC-MS analysis revealed the presence of an anticancer compound hydroxymethyl colchicine, antioxidant compound benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro in endophyte fungal extract of plant Vateria indica. Moreover, in silico analysis of bioactive compounds identified by GC-MS analysis using the Autodock Vina and SwissADME confirmed excellent anticancer activity methanone, [4-amino-2-[(phenylmethyl) amino]-5-thiazolyl] (4-fluorophenyl)- and hydroxymethyl colchicine against 6VO4 (Bfl-1 protein) as per Lipinski rule. Furthermore, we also demonstrated the excellent antioxidant of endophytic extract compared to plant extract by DPPH and ABTS assay, as well as antimicrobial activity against both Gram (+ ve) and Gram (- ve) bacteria. Moreover, the endophytic extract also showed its antimitotic activity with a mitotic index of 65.32, greater than the plant extract of 32.56 at 10 mg/ml. Thus endophytic fungi Cladosporium species isolated from plant Vateria indica might be used as a potential source for phytochemical anticancer hydroxymethyl colchicine, an antioxidant benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro.

In situ microemulsion-gel obtained from bioadhesive hydroxypropyl methylcellulose films for transdermal administration of zidovudine.

This study aims to develop in situ microemulsion-gel (ME-Gel) obtained from hydroxypropyl methylcellulose (HPMC) films for transdermal administration of Zidovudine (AZT). Firstly, HPMC films containing propylene glycol (PG) and eucalyptus oil (EO) were obtained and characterized. Later, a pseudo-ternary phase diagram composed of water, EO, tween 80 and PG was obtained and one microemulsion (ME) with a similar proportion of the film components was obtained. ME was transformed in ME-Gel by the incorporation of HPMC. Finally, HPMC films were hydrated with Tween 80 solution to yield in situ ME-Gel and its effect on AZT skin permeation was compared with HPMC film hydrated with water (F5hyd). The results showed that the ME and ME-Gel presented a droplet size of 16.79 and 122.13 µm, respectively, polydispersity index (PDI) < 0.39 and pH between 5.10 and 5.40. The incorporation of HPMC resulted in viscosity about 2 times higher than the use of ME. The presence of AZT did not alter the formulation properties. The in situ ME-Gel promoted a two-fold increase in the permeated amount of AZT compared to F5hyd. The results suggest that it was possible to obtain an ME-Gel in situ from HPMC films and that its effect on transdermal permeation of AZT was significant.

In vitro digestibility of highly concentrated methylcellulose O/W emulsions: rheological and structural changes.

The changes in structure during the digestion of highly concentrated methyl cellulose (MC) O/W emulsions and of hydrated MC were investigated. The effect of human saliva and in vitro stomach digestion was attributed to a dilution effect, rather than to pH or pepsin activity. After in vitro intestine incubation, a decrease in viscoelasticity and an increase in fat globule size were observed. The fat released after the digestion of the MC emulsion was 49.8% of the initial fat, indicating the existence of a big physical impediment. In comparison with an O/W whey protein emulsion with fat content equal to the fat released during the MC emulsion digestion, a 12% reduction in free fatty acid formation was found, which indicates that the decrease in fat bioaccessibility in the MC emulsion should be attributed not only to a physical effect against fat release but also to a further impediment related to the fat digestion process. Fat released quantification informs about the physical retention of fat in the emulsion matrix structure. Enzymes may not act if fat is not released and solubilized. Free fatty acid quantification is the real indicator of fat digestion, but contrary to the total fat released, it is affected by a wide variety of enzymatic factors, which should be considered for the correct comparison of systems of different properties, for example systems where the amount of fat release during the digestion may be different or initially unknown.

Impact of dietary fibers [methyl cellulose, chitosan, and pectin] on digestion of lipids under simulated gastrointestinal conditions.

A simulated in vitro digestion model was used to elucidate the impact of dietary fibers on the digestion rate of emulsified lipids. The influence of polysaccharide type (chitosan (cationic), methyl cellulose (non-ionic), and pectin (anionic)) and initial concentration (0.4 to 3.6% (w/w)) was examined. 2% (w/w) corn oil-in-water emulsions stabilized by 0.2% (w/w) Tween-80 were prepared, mixed with polysaccharide, and then subjected to an in vitro digestion model (37 °C): initial (pH 7.0); oral (pH 6.8; 10 min); gastric (pH 2.5; 120 min); and, intestinal (pH 7.0; 120 min) phases. The impact of polysaccharides on lipid digestion, ζ-potential, particle size, viscosity, and stability was determined. The rate and extent of lipid digestion decreased with increasing pectin, methyl cellulose, and chitosan concentrations. The free fatty acids released after 120 min of lipase digestion were 46, 63, and 81% (w/w) for methyl cellulose, pectin, and chitosan, respectively (3.6% (w/w) initial polysaccharide), indicating that methyl cellulose had the highest capacity to inhibit lipid digestion, followed by pectin, and then chitosan. The impact of the polysaccharides on lipid digestion was attributed to their ability to induce droplet flocculation, and/or due to their interactions with molecular species involved in lipid hydrolysis, such as bile salts, fatty acids, and calcium. These results have important implications for understanding the influence of dietary fibers on lipid digestion. The control of lipid digestibility within the gastrointestinal tract might be important for the development of reduced-calorie emulsion-based functional food products.

Arginine, soy isoflavone and hydroxypropylmethylcellulose have protective effects against obesity in broiler breeder hens fed on high-energy diets.

1. The present study was undertaken to determine the effects of arginine, soy isoflavone (ISF) and hydroxypropylmethylcellulose (HPMC) on obesity in broiler breeder hens. 2. A total of 320 Cobb 500 hens, 45 weeks of age, were assigned to 64 floor pens. The experiment was conducted as a completely randomised design in a factorial arrangement (2 × 2 × 2 × 2) with 4 replicates of 5 hens in each pen. Factors included two concentrations of HPMC (0 and 1%), two concentrations of arginine (8.4 and 12 g/kg), two concentrations of ISF (zero and three times more than that present in basal diets) and two contents of energy (11.7 and 14.6 MJ/kg). Performance criteria and blood characteristics of hens were measured during the experimental period. Expression of genes involved in lipid metabolism was determined in the liver at 55 weeks of age. 3. Hens given high-energy diets showed increased BW (body weight), ovary weight and abdominal fat pad and enhanced plasma glucose, triglyceride (TG), cholesterol, haemoglobin, haematocrit and low lymphocyte percentages. The expression of malic enzyme, peroxisome proliferator-activated receptor-α (PPARα), peroxisome proliferator-activated receptor-γ (PPARγ) and inducible nitric oxide (iNOS) increased and sterol regulatory element binding protein-1c (SREBP1c) decreased with increasing energy content of diets. Arginine addition decreased TG, cholesterol and A1-c haemoglobin concentration and increased PPARα, PPARγ and iNOS expression. Inclusion of ISF and HPMC decreased BW, egg weight, plasma TG, cholesterol and increased egg production and also enhanced PPARγ and iNOS expression. Significant interactions were observed between energy concentration and ISF and HPMC on BW. 4. The results of the current study revealed that ISF, HPMC and arginine have beneficial effects on controlling the metabolism of obese broiler breeder hens and using a mix of these products minimises the harmful effects of obesity.

Preparation and characterization of shellac-coated anthocyanin pectin beads as dietary colonic delivery system.

SCOPE: Anthocyanins are connected with various biological activities. A promising way to enhance the availability of anthocyanins for in situ effects in the lower intestine is colon-specific delivery. METHODS AND RESULTS: Shellac and shellac/hydroxypropyl methylcellulose (HPMC) coated anthocyanin amidated pectin beads as dietary colonic delivery systems were successfully prepared by ionotropic gelation and fluid bed Wurster coating with aqueous shellac solution. Release characteristics, studied in vitro and ex vivo using simulated gastric fluid (SGF), ileostomy fluid and colostomy fluid (CF) revealed a retardation of anthocyanins during simulated passage of stomach and ileum as well as the desired release of pigments in the colon. Coating level was identified as an important parameter. By addition of 5 or 15% of the water-soluble polysaccharide HPMC to the shellac film, resistance in SGF was increased due to the plasticizer properties of the polymer. Incorporation of 15% HPMC (w/w based on shellac) into the shellac film additionally led to increased anthocyanin diffusivity and complete release as well as degradation of the formulation in CF. CONCLUSION: In the used in vitro and ex vivo model system mimicking the human intestinal transit, the potential of shellac and shellac/HPMC coated anthocyanin amidated pectin beads as dietary colon targeting systems was demonstrated.

Aceclofenac topical dosage forms: in vitro and in vivo characterization.

Aceclofenac is a new generation non-steroidal anti-inflammatory drug showing effective anti-inflammatory and analgesic properties. It is available in the form of tablets of 100 mg. Importance of aceclofenac as a NSAID has inspired development of topical dosage forms. This mode of administration may help avoid typical side effects associated with oral administration of NSAIDs, which have led to its withdrawal. Furthermore, aceclofenac topical dosage forms can be used as a supplement to oral therapy for better treatment of conditions such as arthritis. Ointments, creams, and gels containing 1% (m/m) aceclofenac have been prepared. They were tested for physical appearance, pH, spreadability, extrudability, drug content uniformity, in vitro diffusion and in vitro permeation. Gels prepared using Carbopol 940 (AF2, AF3) and macrogol bases (AF7) were selected after the analysis of the results. They were evaluated for acute skin irritancy, anti-inflammatory and analgesic effects using the carrageenan-induced thermal hyperalgesia and paw edema method. AF2 was shown to be significantly (p < 0.05) more effective in inhibiting hyperalgesia associated with inflammation, compared to AF3 and AF7. Hence, AF2 may be suggested as an alternative to oral preparations.

Design and evaluation of transdermal drug delivery system for curcumin as an anti-inflammatory drug.

The purpose of this research was to develop a matrix-type transdermal therapeutic system containing herbal drug, curcumin (CUR), with different ratios of hydrophilic (hydroxyl propyl methyl cellulose K4M [HPMC K4M]) and hydrophobic (ethyl cellulose [EC]) polymeric systems by the solvent evaporation technique. Different concentrations of oleic acid (OA) were used to enhance the transdermal permeation of CUR. The physicochemical compatibility of the drug and the polymers was also studied by differential scanning calorimetry (DSC) and infrared (IR) spectroscopy. The results suggested no physicochemical incompatibility between the drug and the polymers. Formulated transdermal films were physically evaluated with regard to drug content, tensile strength, folding endurance, thickness, and weight variation. All prepared formulations indicated good physical stability. In vitro permeation studies of formulations were performed by using Franz diffusion cells. The results followed Higuchi kinetics, and the mechanism of release was diffusion-mediated. Formulation prepared with hydrophilic polymer containing permeation enhancer showed best in vitro skin permeation through rat skin as compared with all other formulations. This formulation demonstrated good anti-inflammatory activity against carrageenan-induced oedema in Wistar albino rats similar to standard formulation.

Esterification reactions catalyzed by lipases immobilized in organogels: effect of temperature and substrate diffusion.

Rhizomucor miehei lipase was immobilized in hydroxy(propylmethyl) cellulose or agar gels containing lecithin or AOT microemulsions. The effect of the diffusion of substrates and products to this catalyst was studied, as well as the effect of temperature on the initial rate of ester synthesis. The composition of the gel affects the reaction rate due to mass transport phenomena. The apparent activation energies were higher for the systems based on agar, independently of the microemulsion used, and lower for the systems based on AOT microemulsions, independently of the polymer used.

Fish oil supplementation in pregnancy modifies neonatal progenitors at birth in infants at risk of atopy.

Dietary n-3 polyunsaturated fatty acids (PUFA) may represent a mode of allergy prevention. Cord blood (CB) CD34+ hemopoietic progenitors are altered in infants at risk of atopy. We therefore studied the effects of dietary n-3 PUFA supplementation during pregnancy on numbers and function of progenitors in neonates at high risk of atopy. In a double-blind study, atopic, pregnant women were randomized to receive fish oil capsules or placebo from 20 wk gestation until delivery. At birth, CB CD34+ cells were isolated and analyzed by flow cytometry for expression of cytokine (IL-5Ralpha, IL-3Ralpha, granulocyte/macrophage colony-stimulating factor Ralpha) or chemokine (CXCR4 and CCR3) receptors. CB cells were also cultured in methylcellulose assays for eosinophil/basophil colony-forming cells. At age 1 y, infants were clinically assessed for atopic symptoms and skin tests. Percentages of CB CD34+ cell numbers were higher after n-3 PUFA than placebo. Co-expression of cytokine or chemokine receptors on CD34 cells was not altered by n-3 PUFA supplementation. However, there were significantly more IL-5-responsive CB eosinophil/basophil colony forming units (Eo/B-CFU) in the fish oil, compared with the control, group. Overall, there was a positive association between CD34+ cells and IL-5-responsive Eo/B-CFU in CB and 1 y clinical outcomes, including atopic dermatitis and wheeze. Dietary n-3 PUFA supplementation during pregnancy in atopic mothers alters infant cord blood hemopoietic progenitor phenotype. This may have an impact on development of atopic disease.

A novel pH-dependent gradient-release delivery system for nitrendipine II. Investigations of the factors affecting the release behaviors of the system.

Nitrendipine, a dihydropyridine calcium antagonist, was used as a poorly water-soluble model drug. To improve its dissolution rate and extend the therapeutic period in vivo as well, a novel pH-dependent gradient-release drug delivery system for nitrendipine having a solid dispersed matrix structure was developed. Four factors, i.e. the amount of excipients, the pH of the dissolution medium, the rotating speed of the paddle of the dissolution apparatus and the particle size of the microspheres, all of which affect the drug-release behavior of the pH-dependent microspheres of the system were investigated in detail. The release profiles of the pH-dependent drug delivery system under simulated gastrointestinal tract pH conditions were also investigated. The results showed that the release rate of drug from the microspheres increased on increasing the amount of respective pH-dependent polymers formulated. Due to the fact that the active drug was incorporated in pH-dependent polymers and was present in a solid dispersion state in the microspheres, the release rate of the drug from the microspheres depended on the dissolution rate of the polymers, which was mainly influenced by the pH of dissolution medium, whereas the rotating speed of the paddle and the particle size of the microspheres had only a relatively minor effect. The release behavior of the system under simulated gastrointestinal tract conditions exhibited obvious gradient-release characteristics, showing that the release rate of the active drug could be controlled efficiently before the microspheres reached the appropriate region of the gut for absorption. These findings suggest that the pH-dependent drug delivery system could be fabricated by using present microspheres.

Double-layered mucoadhesive tablets containing nystatin.

The objective of this work was to design a mucoadhesive tablet with a potential use in the treatment of oral candidosis. A 2-layered tablet containing nystatin was formulated. Lactose CD (direct compression), carbomer (CB), and hydroxypropylmethylcellulose (HPMC) were used as excipients. Tablets were obtained through direct compression. Properties such as in vitro mucoadhesion, water uptake, front movements, and drug release were evaluated. The immediate release layer was made of lactose CD (100 mg) and nystatin (30 mg). The CB:HPMC 9:1 mixture showed the best mucoadhesion properties and was selected as excipient for the mucoadhesive polymeric layer (200 mg). The incorporation of nystatin (33.3 mg) in this layer affected the water uptake, which, in turn, modified the erosion front behavior. Nystatin showed a first-order release. The polymeric layer presented an anomalous kinetic (n = 0.82) when this layer was individually evaluated. The mucoadhesive tablet formulated in this work releases nystatin quickly from the lactose layer and then in a sustained way, during approximately 6 hours, from the polymeric layer. The mixture CB:HPMC 9:1 showed good in vitro mucoadhesion. A swelling-diffusion process modulates the release of nystatin from this layer. A non-Fickian (anomalous) kinetic was observed.

Retrograde spread of therapeutic enemas in patients with inflammatory bowel disease.

Little is known about the factors that determine the extent of dispersion of enema solutions in the colon. To unravel some of the determinants we evaluated a consecutive series of patients with left-sided colitis. 40 ml enema solutions, viscosity 0.062 Pa.s (62 cP) at 37 degrees C were labelled with 10 MBq 99m-technetium human serum albumin microcolloid. Scintigraphic imaging was performed in 35 patients until 2 hours after administration of the enema. In 8 of the 16 patients with limited retrograde spread the study was repeated after doubling the volume (80 ml). We conclude that the extent of dispersion of an enema 0.062 Pa.s solution is highly variable. The basic fluid component for therapeutic 40 ml enemas (viscosity 0.062 Pa.s) reaches the affected area in patients with left sided colitis only in 40% of the cases. Increasing the volume of the enema can be an effective way to increase the retrograde spread up to the affected area in patients with limited retrograde spread. Scintigraphic imaging is a simple and reliable method of checking whether an enema conforms to the requirements of medical treatment. Scintigraphic imaging lasting for 1 hour after administration of the enema appears to suffice.

The nutrient requirements of MRC-5 human diploid cells.

Nutrients were assayed by their effect on maximum cell yield (maximum cell population less inoculum) of MRC-5 cells previously maintained in Eagle's basal medium with 10% (v/v) serum, trypsinised, centrifuged and washed. When nonlimiting amounts of iron, methylcellulose and a 68-component supplement were included in our defined medium, cell yields obtained were equivalent to those obtained with 2 to 3% whole serum. Growth then became limited by serum growth factors which, when serum was fractionated by the low temperature ethanol procedure, appeared to precipitate with the alpha-globulins, although the distribution of activity varied from batch to batch of serum. Column chromatography (DEAE and concanavalin A-Sepharose) of serum resulted in much (20 to 45%) loss of growth factor activity during exhaustive dialysis or Sephadex G-25 desalting suggesting that the growth factor may be a diffusible molecule bound in serum to a large protein carrier.

Trace-metal requirements of NCTC clone 929 strain L cells.

The effects of iron, zinc, copper, manganese, and Methocel on the growth of L cells in a synthetic medium were studied. The medium was iron-deficient unless a supplement of 1.0 g/liter Methocel HG (4,000 centipoises), Methocel ash equivalent to 1.0 g/liter Methocel, or 1.0 microM FeSO4 was added. Cells grew faster in the presence of FeSO4 or Methocel ash (generation time = 40 hours) than of Methocel (generation time = 110 hours), but Methocel was needed to protect cells from mechanical destruction. Treating the growth medium with Dowex A-1 chelating resin produced a medium deficient in both iron and zinc. The iron requirement in this medium varied between 0.6 and 1.4 microM, depending on the concentrations of other cations added to the medium. A copper concentration of at least 0.4 microM was beneficial as it reduced the amount of iron necessary for cell growth. No manganese was required in this medium; any added manganese produced an inhibition of cell growth. Additional iron could reverse this inhibition. The minimum iron requirement for the production of L cells was 3 nmoles/mg cell nitrogen produced. Approximately 0.6 microM zinc was required for maximum cell growth in resin-treated medium.