Ranibizumab Modifies the Expression of Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood Mononuclear Cells in Patients with Exudative Age-Related Macular Degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in people over 60 years of age. Despite research, the causes of AMD remain unclear. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in AMD development, and anti-vascular endothelial growth factor therapy has revolutionized its treatment. This study aims to analyze the changes in gene expression in MMPs and TIMPS in patients with neovascular AMD before and after three doses of ranibizumab. METHODS: The study involved 29 patients with neovascular AMD treated with ranibizumab. Peripheral blood mononuclear cells were collected before treatment and 24 h after the third dose of ranibizumab. We assessed MMP and TIMP gene expression profiles through oligonucleotide microarrays and validated selected differential genes using RT-qPCR. RESULTS: A statistically significant change in the expression of six MMP- and TIMP-related genes was observed using oligonucleotide microarray. The mRNA levels of the two genes with the most significant fold changes, MMP15 and TIMP2, were then quantified using RT-qPCR. The results confirmed a statistically significant increase in MMP15 expression and a decrease in TIMP2 levels, although this change was not statistically significant in the group before and after the third dose of ranibizumab. CONCLUSION: Ranibizumab affects the systemic expression of MMP and TIMP-related genes in patients with neovascular AMD. Results from our exploratory study suggest that MMP15, in particular, may play a role in the treatment response, but further research is necessary.

Effects of 17-ß-estradiol released from shape-memory terpolymer rods on sciatic nerve regeneration after injury and repair with chitosan nerve conduit in female rats.

The aim of this study was to assess 17-ß-estradiol (E2) influence on sciatic nerve regeneration after injury followed by a repair with chitosan conduit in ovariectomized female rats. The study was performed in 2 groups (n = 16) of rats: OVChit - after excision of a fragment of the sciatic nerve, a chitosan conduit was implanted; OVChitE10 group - additionally to chitosan conduit, shape-memory terpolymer rods based on poly(L-lactide-co-glycolide- co-trimethylene carbonate) releasing 17-ß-estradiol for 20 weeks were implanted. The mean number of regenerating axons and mean fiber area were significantly greater in 17-ß-estradiol-treated animals. In this group, the infiltrate of leukocytes was diminished. The presence of 17-ß-estradiol receptors alpha and beta in motoneurons in the spinal cord were discovered. This may indicate the location where 17-ß-estradiol affects the regeneration of the injured nerve. Estradiol released from the terpolymer rods for 20 weeks could enhance, to some extent, sciatic nerve regeneration after injury, and diminish the inflammatory reaction. In the future, 17-ß-estradiol entrapped in terpolymer rods could be used in the repair of injured peripheral nerves, but there is a need for further studies.

Mechanical properties of the porcine pericardium extracellular matrix cross-linked with glutaraldehyde and tannic acid.

PURPOSE: The aim of this study was to determine the influence of factors such as temperature and glutaraldehyde (GA) concentration on the mechanical properties of porcine pericardia, in order to propose the recommended optimal conditions of a cross-linking process. It was also to verify whether tannic acid (TA), a natural cross-linking agent that stabilizes collagenous tissues by a different mechanism than GA, may positively influence the strength of pericardium. METHODS: The samples were incubated at various temperatures (4, 22, and 37°C) and GA concentration solutions (0.6, 1.5 and 3%) for 7 days. Three series were selected and additionally cross-linked with 0.3% TA for another 7 days. Mechanical properties of cross-linked pericardium samples, i.e., ultimate tensile strength (UTS) and elastic modulus (E) were measured in uniaxial tensile testing. The hyperelastic model for incompressible materials - isotropic by Ogden [24] and anisotropic by Fung [7] were utilized to describe the mechanical behaviour of treated pericardium. RESULTS: The temperature has an influence on cross-linking effects; the lowest values of UTS were reported for specimens cross-linked at 22 °C, while the mechanical properties of series treated at 4°C or 37°C were comparable. At a particular temperature of incubation, the GA concentrations have not affected the mechanical properties of tissues. The dependence between mechanical parameters and agent concentration was only observed for specimens treated with GA at 37 °C. CONCLUSIONS: The conditions of the cross-linking process affect the mechanical properties of the porcine pericardium. Room temperature (22 °C) and the concentration of 1.5% GA occurred to be ineffective. The mechanical properties of GA-treated pericardium were improved by an additional TA cross-linking.

Effects of Using Corn Dried Distillers' Grains with Solubles (cDDGS) as a Partial Replacement for Soybean Meal on the Outcomes of Pig Fattening, Pork Slaughter Value and Quality.

The present study set out to determine the effects of incorporating cDDGS into starter, grower, and finisher diets (containing 5%, 10%, and 15% of cDDGS, respectively) on growth performance, carcass and meat quality, and cost effectiveness of pig fattening. Sixty-four pigs (mean body weight of 15.0 ± 2.1 kg) were divided into two groups (n = 32) and fed a control diet (cereal-soybean meal-based) or cDGGS-containing diets (with soybean meal partially replaced with cDDGS). Live weights of pigs as well as weight gains/daily weight gains across all fattening phases did not differ between the two groups of fattener pigs studied (p > 0.05). Addition of cDDGS decreased feed intake per pig during the grower (p < 0.05) and finisher (p < 0.01) phases, and, as a result, throughout the entire fattening period (254 vs. 245 kg for control and cDDGS groups, respectively; p < 0.01). The feed conversion ratio (FCR) for the entire fattening period was significantly less for cDDGS-fed fatteners (2.77) than for controls (2.91; p < 0.05). Carcass weights, fat thickness, and meatiness did not vary between the two groups of animals (p > 0.05). Loin depth was greater in the cDDGS group by ~5 mm (p < 0.05). Slaughter value was higher for the cDDGS group (76.1% vs. 77.0%, p < 0.05). The total cost of fattening and total cost of 1 kg of body weight decreased in cDDGS compared with the control subset of fatteners by ~7% and 8% during the grower and finisher phases, respectively (p < 0.01). The simplified direct surplus per pig was approximately 63% higher for the cDDGS group. Our results indicate that even moderate inclusion of cDDGS to concentrate mixtures (or a partial replacement of soybean meal with cDDGS) may improve FCR without any substantial changes in meat and back fat characteristics as well as significantly decrease the cost of feeding and increase the profitability of pig production.

The Role of the Mechanical, Structural, and Thermal Properties of Poly(l-lactide-co-glycolide-co-trimethylene carbonate) in the Development of Rods with Aripiprazole.

In this work, we aimed to determine the role of the mechanical, structural, and thermal properties of poly(l-lactide-co-glycolide-co-trimethylene carbonate) (P(l-LA:GA:TMC)) with shape memory in the formulation of implantable and biodegradable rods with aripiprazole (ARP). Hot melt extrusion (HME) and electron beam (EB) irradiation were applied in the formulation process of blank rods and rods with ARP. Rod degradation was carried out in a PBS solution. HPLC; NMR; DSC; compression and tensile tests; molecular weight (Mn); water uptake (WU); and weight loss (WL) analyses; and SEM were used in this study. HME and EB irradiation did not influence the structure of ARP. The mechanical tests indicated that the rods may be safely implanted using a pre-filled syringe. During degradation, no unfavorable changes in terpolymer content were observed. A decrease in the glass transition temperature and the Mn, and an increase in the WU and the WL were revealed. The loading of ARP and EB irradiation induced earlier pore formation and more intense WU and WL changes. ARP was released in a tri-phasic model with the lag phase; therefore, the proposed formulation may be administered as a delayed-release system. EB irradiation was found to accelerate ARP release.

Designing Biodegradable Wafers Based on Poly(L-lactide-co-glycolide) and Poly(glycolide-co-ε-caprolactone) for the Prolonged and Local Release of Idarubicin for the Therapy of Glioblastoma Multiforme.

PURPOSE: The blood-brain barrier limits the application of idarubicin in the therapy of glioblastoma multiforme. Biodegradable, intracranial wafers with prolonged release may increase therapy efficiency. METHODS: Blank wafers, wafers containing 5% w/w and 10% w/w of idarubicin were formulated by solution casting from poly(L-lactide-co-glycolide) and poly(glycolide-co-ε-caprolactone). The following methods were used: NMR, GPC, DSC, FTIR, AFM, UV-VIS, and a viability and proliferation assay for idarubicin action (U87MG cell line). RESULTS: Wafers showed a surface with numerous immersions and hills. A lack of interactions between idarubicin and the copolymers was observed. The substance was entrapped in the matrix and released in two phases for all wafers with the appropriate bolus and maintenance dose. The burst effect was observed for all wafers, however, the biggest bolus for poly(L-lactide-co-glycolide) wafers containing 5% w/w of idarubicin was noted. The stable and steady degradation of poly(glycolide-co-ε-caprolactone) wafers containing 5% w/w of idarubicin ensures the most optimal release profile and high inhibition of proliferation. CONCLUSIONS: Copolymer wafers with idarubicin are an interesting proposition with great potential for the local treatment of glioblastoma multiforme. The release rate and dose may be regulated by the amount and kind of wafers for various effects.

Electron beam sterilization of implantable rods with risperidone and with 17-ß-estradiol: a structural, thermal and morphology study.

PURPOSE: Poly(L-lactide-co-glycolide-co-trimethylene carbonate) rods with risperidone and 17-ß-estradiol were sterilized by electron beam irradiation. The aim of the study was to assess electron beam irradiation impact on terpolymer composition, chain microstructure, glass transition temperature, molecular weight and the morphological features of rods. METHODS: Hot melt extrusion in the formulation of rods was applied. Sterilization of the rods was performed by electron beam in an electron beam accelerator (10 MeV, 360 mA, 25 kGy). The following methods in the development of rods were applied: nuclear magnetic resonance, differential scanning calorimetry, gel permeation chromatography and scanning electron microscopy. RESULTS: Sterilization influenced only glass transition temperature in blind rods and rods with risperidone. As for the other parameters, no significant changes were observed as far as a sterilization effect is concerned. However, some changes were noted after introducing drug substances and after extrusion. CONCLUSIONS: Electron beam irradiation of rods with risperidone and rods with 17-ß-estradiol is an adequate method for sterilizing implantable drug delivery systems.

Formulation of delivery systems with risperidone based on biodegradable terpolymers.

Risperidone is applied in oral dosage formulations in the treatment of mental diseases. Current trends point toward parenteral delivery systems based on poly(lactide-co-glycolide), with wafers or rods being the more attractive option than the routinely used intramuscular suspension with microparticles. The aim of our work was to study the utility of solution casting and hot melt extrusion in the formulation of wafers and rods with risperidone based on terpolymers, namely poly(lactide-co-glycolide-co-trimethylene carbonate) and poly(lactide-co-glycolide-co-ε-caprolactone). Synthesis of the terpolymers was carried out by using a non-toxic zirconium initiator and a racemic (LL/DD) or optically active form of the lactide monomer. The delivery systems were analyzed by NMR, DSC, GPC, and SEM. The release profile was monitored by HPLC. Terpolymer chain microstructure, glass transition temperature, and morphology revealed unchanged values after formulation. Solution casting resulted in a drop in molecular weight to a smaller degree than hot melt extrusion. The presence of risperidone influenced another decrease in molecular weight. Both methods are adequate for the formulation of delivery systems based on terpolymers for prolonged release of risperidone. An adequate selection of monomer composition in terpolymers allows to control the release period. Risperidone was released in three phases, however, the burst effect was observed for poly(L-lactide-co-glycolide-co-ε-caprolactone).

Shape-Memory Terpolymer Rods with 17-ß-estradiol for the Treatment of Neurodegenerative Diseases: an In Vitro and In Vivo Study.

PURPOSE: Estradiol (E2)-loaded poly(L-lactide-co-glycolide-trimethylenecarbonate) (P(L-LA:GA:TMC)) rods with shape-memory were developed for the treatment of neurodegenerative diseases. Usefulness of the extrusion method in the obtaining process was also considered. The influence of structural and surface properties during hydrolytic degradation was developed. The possible therapeutic aspect of rods with E2 was determined. METHODS: The extruded rods were incubated in a PBS solution (pH 7.4, 37°C, 240 rpm). The amount of released E2 in vitro conditions was estimated by UV-VIS method. The following methods in the degradation of rods were applied: NMR, DSC, FTIR, GPC, SEM, and optical microscopy. Changes in water uptake and weight loss were also determined. In vivo study was performed on rats. Measurements of E2 level were performed before and after ovariectomy of rats using ELISA method. A sample of tissue adjacent to the site of the rod implantation was analysed under an optical microscope. RESULTS: A stable and steady degradation process ensured zero-order release of E2. The in vivo study indicated a significant increase in the E2 level in serum after ovariectomy. Moreover, structural and surface features indicated that the extrusion method was appropriate for obtaining E2-loaded rods. CONCLUSIONS: Shape-memory P(L-LA:GA:TMC) rods with E2 are an adequate proposal for further research in the field of neurological disorders.

EFFECTS OF 300 mT STATIC MAGNETIC FIELD ON IL-8 SECRETION IN NORMAL HUMAN COLON MYOFIBROBLASTS.

Intestinal subepithelial myofibroblasts play a crucial role in the growth and development of the intestine. Colitis, small bowel injury, gastric ulcer disease and inflammatory bowel disease (IBD) accompany the increase in the count of activated myofibroblasts. In the last few years, the increasing production of electromagnetic (EMF) and static magnetic (SMF) fields due to the expanding use of electronic devices in everyday life, has led to a number of studies on the effects of these fields on living organisms. Because of its anti-inflammatory properties, EMF therapy may be of medical use as an IBD treatment. This mechanism has not been elucidated yet. In the present work normal human colon myofibroblasts were exposed to SMF with a flux density of 300 mT for 96 h and then the cells were cultured for 24 and 48 h with 25 mM sodium butyrate (NaB) and 10 mM 5-aminosalicylic acid (5-ASA) in either the presence or absence of SMF. Tumor necrosis factor α (TNF-α)--dependent IL-8 secretion was determined with ELISA kit. Cell viability was determined with XTT assay. It was shown that SMF has no effect on TNF-α--dependent IL-8 secretion in control cells and in cells cultured in the presence of 5-ASA and NaB.

Influence of selective digestion of elastin and collagen on mechanical properties of human aortas.

PURPOSE: There are two families of fibres taking part in the process of mechanical loads transfer, i.e. elastin and collagen fibres. Their number, spatial arrangement and specific properties determine the capacity of a blood vessels to resist mechanical loads resulting from the impact of blood on vessel walls. The purpose of the present paper is to define the load-bearing capacities of elastin and collagen scaffolds equivalent to natural fibre arrangements of human aorta and produced by selective digestion. METHODS: Samples of thoracic human aortas were digested by using phosphate buffer of trypsin at pH 8.0 for 22 hours in order to degrade elastin and by autoclaving followed by incubation in 90% formic acid for 22 hours. The efficacy of digestion was assessed immunohistochemically. Mechanical properties of pre-stretched native and digested samples were determined by uniaxial tensile test. RESULTS: Samples subjected to autoclaving have been successfully deprived of both types of collagen and elastin has been intact. Treatment with trypsin caused a removal of elastin and the presence of type I and IV collagen was demonstrated. Digestion of aortic samples either by formic acid or trypsin has resulted significantly decreasing mechanical properties in comparison with native samples. CONCLUSIONS: Collagen and elastin scaffold-like stuctures have been effectively produced by selective digestion of thoracic human aorta and their contribution to the load-bearing process was evaluated. Isolated collagen network are more durable and stiffer and less deformable than elastin network, hence are responsible for load-bearing process at higher strain since the range of working of elastin is at lower strain values.

Thermal properties and morphology changes in degradation process of poly(L-lactide-co-glycolide) matrices with risperidone.

Determining thermal properties and morphology seems to be useful in the analysis of release and degradation processes form polymeric materials. Risperidone is available in the formulation of a long-acting injection based on poly(D,L-lactide-co-glycolide). Currently, alternative solutions are also offered, i.e., nano- and microparticles or implants, including copolymers of lactide and glycolide. The effect of risperidone content on the properties of poly(L-lactide-co-glycolide) matrices was determined. The study also involved an assessment of the changes during degradation. Risperidone free matrices and the matrices with risperidone were obtained by solvent casting. Thermal characteristics were tested by means of differential scanning calorimetry, and the morphology was evaluated using a scanning electron microscope. Risperidone did not change significantly semi-crystalline structure of poly(L-lactide-co-glycolide) matrices. The decrease in crystallization temperature and glass transition temperature during degradation was observed. Many pores and their deformation, the widening of pore area, cracks and slits because of degradation were observed. The analysis of thermal properties and morphology allowed us to explain degradation process. Matrices exhibited stable process of degradation, which may be advantageous for development of prolonged risperidone release systems.

Supramolecular structure of human aortic valve and pericardial xenograft material: atomic force microscopy study.

Pericardial tissue (bovine or porcine), chemically stabilized with glutaraldehyde (GA), is widely used in cardiovascular surgery in the form of bioprosthetic valves. GA reacts with tissue proteins and creates inter- and intra-molecular cross-links, resulting in improved durability. However, tissue calcification and mechanical damage are still unresolved problems. The purpose of this study was to examine the surface topography of normal human aortic valve and GA-stabilized porcine pericardium tissue in order to gain comparative insight into supramolecular structure of both tissues. The analysis was focused on morphologic evaluation of collagen constituents of the tissues. Atomic force microscopy working in the contact mode in air was employed in the study. Considerable diversity in the spatial orientation of collagen fibrils for the human aortic valve and pericardial tissue were observed. It was found that different forms of collagen fibril packing, i.e. dense and "in phase" or loose, could have an impact on the collagen D-banding pattern. Stabilization with GA introduced significant changes in the surface topography of collagen fibrils and in their spatial organization on the tissue surface. Strong disturbance in the fibril's D-spacing was observed. It was also suggested, that the observed structural changes at the supramolecular level might make an important contribution to the progressive damage and calcification of the tissue. The presented results demonstrate that the AFM method can be useful for non-destructive structural characterization of heart valves and bioprosthetic heart valve material.