Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 mu M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1 alpha) at a concentration of 10 mu M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury

Modulation of Functional Characteristics of Mesenchymal Stromal Cells by Acellular Preparation of Porcine Hemoglobin

Exploring the potential usage of the acellular preparation of porcine hemoglobin (PHb) isolated from slaughterhouse blood as a cell culture media component, we have tested its effects on the functional characteristics of stromal cells of mesodermal origin. Human peripheral blood mesenchymal stromal cells (PB-MSCs) were used in this study as a primary cell model system, along with three mouse cell lines (ATDC5, MC3T3-E1, and 3T3-L1), which represent more uniform model systems. We investigated the effect of PHb at concentrations of 0.1, 1, and 10 μM on these cells’ proliferation, cycle, and clonogenic and migratory potential, and found that PHb’s effect depended on both the cell type and its concentration. At the lowest concentration used (0.1 μM), PHb showed the least evident impact on the cell growth and migration; hence, we analyzed its effect on mesenchymal cell multilineage differentiation capacity at this concentration. Even under conditions that induce a specific type of MSC differentiation (cultivation in particular differentiation media), PHb modulated chondrogenic, osteogenic, and adipogenic differentiation, making it a potential candidate for a supplement of MSC culture. Through a model of porcine hemoglobin, these findings also contribute to improving the knowledge of extracellular hemoglobin’s influence on MSCs in vivo

An overview of encapsulation technologies for food applications

Encapsulation is a process to entrap active agents within a carrier material and it is a useful tool to improve delivery of bioactive molecules and living cells into foods. Materials used for design of protective shell of encapsulates must be food-grade, biodegradable and able to form a barrier between the internal phase and its surroundings. Among all materials, the most widely used for encapsulation in food applications are polysaccharides. Proteins and lipids are also appropriate for encapsulation. Spray drying is the most extensively applied encapsulation technique in the food industry because it is flexible, continuous, but more important an economical operation. Most of encapsulates are spray-dried ones, rest of them are prepared by spray-chilling, freeze-drying, melt extrusion and melt injection. Molecular inclusion in cyclodextrins and liposomal vesicles are more expensive technologies, and therefore, less exploited. There are number of reasons why to employ an encapsulation technology and this paper reviews some of them. For example, this technology may provide barriers between sensitive bioactive materials and the environment, and thus, to allow taste and aroma differentiation, mask bad tasting or smelling, stabilize food ingredients or increase their bioavailability. One of the most important reasons for encapsulation of active ingredients is to provide improved stability in final products and during processing. Another benefit of encapsulation is less evaporation and degradation of volatile actives, such as aroma. Furthermore, encapsulation is used to mask unpleasant feelings during eating, such as bitter taste and astringency of polyphenols. Also, another goal of employing encapsulation is to prevent reaction with other components in food products such as oxygen or water. In addition to the above, encapsulation may be used to immobilize cells or enzymes in food processing applications, such as fermentation process and metabolite production processes. There is an increasing demand to find suitable solutions that provide high productivity and, at the same time, satisfy an adequate quality of the final food products. This paper aims to provide a short overview of commonly used processes to encapsulate food actives

Diesel production by fast pyrolysis of miscanthus giganteus, well-to-pump analysis using the greet model

In this paper "well-to-pump" environmental analysis of pyrolytic diesel from Miscanthus gigantheus is performed. The average annual yield of Miscanthus from III-V year of cultivation on 1 ha of chernozem soil in Serbia (23.5 t) is considered as an input for the process. Two pyrolytic diesel pathways are considered: distributed pyrolytic pathway with external hydrogen production (from natural gas) and integrated pyrolytic pathway with internal hydrogen production (from pyrolytic oil). and are compared to a conventionally produced diesel pathway. The results of the analysis reveal that integrated-internal pyrolytic diesel pathway has lowest resources consumption and lowest pollutant emissions. Compared to conventionally produced diesel, integrated-internal pyrolysis pathway consumes 80% less of fossil fuels, and 92% more of renewables, has 90% lower global warming potential, 30% lower terrestrial acidification potential but 38% higher particulate matter formation potential. Compared to the distributed-external pathway, 88% less fossil fuels, and 36% less renewables are consumed in the integrated-internal pathway, global warming potential is 97% lower, terrestrial acidification is 20% lower, and particulate matter formation is 49% lower. Nevertheless, this pathway has high coal and hydroelectrical power consumption due to electricity production and high emissions of particulate matter, CO2, SOx, and N2O. Another drawback of this production pathway is the low yield of diesel obtained (38% lower than in distributed-external pathway). With this regard, it is still hard to designate production of diesel from fast pyrolysis of Miscanthus as a more environmentally friendly replacement of the conventional production diesel pathway

Poboljšanje metaboličke aktivnosti bakterije Lactobacillus plantarum 564 pomoću elektroporacije

The exposure of bacterial cells to pulsed electric fields (PEF) leads to the reversible formation of pores in the cell membrane if an applied energy is below the critical level. Therefore, the effect of electric field pulses with amplitudes below 14 kV/cm and the applied energy up to 12.2 J/cm3 on the growth of Lactobacillus plantarum 564 cells was investigated. After PEF treatments, the growth of lactobacilli in De Man-Rogosa-Sharpe broth at 37 °C was monitored by isothermal calorimetry, absorbance and plate counts. All the applied treatments resulted in a higher growth rate of PEF-treated cells during early and mid-log phase, especially bacterial samples treated with lower field intensities (1.3–5.5 J/cm3). The transport of ions and molecules through the cell membrane (which facilitates the growth of electroporated lactobacilli) was particularly evident in the mid-exponential growth phase, where the doubling time was reduced more than 3 times after the exposure to electric pulses of 5.5 J/cm3. The heat production rate during the growth of electroporated cells was also higher, indicating the enhanced metabolic activity of PEF-treated cells. Moreover, the electroporated cells had a better acidification ability than the untreated ones. It can be summarized that the applied PEF treatments with an energy input of below 12 J/cm3 potentially induce reversible electroporation of the cell membrane, which has a positive impact on the growth and metabolic activity of the cells of lactobacilli.Izlaganje bakterija pulsirajućim električnim poljima uzrokuje reverzibilno stvaranje pora na staničnoj membrani, ako je energija električnog polja ispod kritične razine. Stoga je istražen utjecaj pulsirajućih električnih polja na rast stanica bakterije Lactobacillus plantarum 564 primjenom polja jačine do 12.2 J/cm3, tj. amplituda manjih od 14 kV/cm. Rast laktobacila u De Man-Rogosa-Sharpe bujonu na 37 °C nakon tretmana praćen je pomoću izotermalne kalorimetrije, te mjerenjem apsorbancije i ukupnog broja stanica. Utvrđeno je da su stanice izložene pulsirajućem električnom polju brže rasle tijekom rane i srednje logaritamske faze, što je naročito bilo izraženo kod stanica tretiranih poljima niskog intenziteta (1.3-5.5 J/cm3). Transport iona i molekula kroz staničnu membranu (što olakšava rast elektroporiranih laktobacila) bio je posebno izražen tijekom eksponencijalne faze rasta, kad je generacijsko vrijeme stanica tretiranih pulsevima energije od 5.5 J/cm3 bilo trostruko kraće. Tijekom rasta tretiranih stanica oslobođena je veća količina topline, što znači da se metabolička aktivnost stanica povećala nakon tretmana. Osim toga, elektroporirane su stanice jače zakiseljavale sredinu. Iz dobivenih se rezultata može zaključiti da pulsirajuća električna polja jačine manje od 12.2 J/cm3 uzrokuju reverzibilnu elektroporaciju stanične membrane, što ima pozitivan učinak na rast i metaboličku aktivnost stanica laktobacila

Analysis of metal ion release from biomedical implants

Metallic biomaterials are commonly used for fixation or replacement of damaged bones in the human body due to their good combination of mechanical properties. The disadvantage of metals as implant materials is their susceptibility to corrosion and metal ion release, which can cause serious health problems. In certain concentrations metals and metal ions are toxic and their presence can cause diverse inflammatory reactions, genetic mutations or even cancer. In this paper, different approaches to metal ion release examination, from biometallic materials sample preparation to research results interpretation, will be presented. An overview of the analytical techniques, used for determination of the type and concentration of released ions from implants in simulated biofluids, is also given in the paper

Optimizacija procesa izolovanja hemoglobina iz goveđih eritrocita kontrolisanom hemolizom

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts). Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 83% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The results show that isolation process yielded predominantly to oxyhemoglobin. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min.Dugi niz godina, i kod nas i u svetu, razvijaju se tehnološki postupci za izolovanje strukturno očuvanog i biološki aktivnog hemoglobina koji bi mogao da se koristi kao zamenik za krv, kao izvor biološki visokoaktivnog hemskog gvožđa u prevenciji anemije kod ljudi i životinja, ili kao reagens u dijagnostici. U ovom radu optimizovan je postupak za izolovanje hemoglobina iz eritrocita poreklom iz otpadne klanične goveđe krvi sa ciljem dobijanja preparata hemskog gvožđa za prevenciju anemije kod životinja. Testirana je osetljivost goveđih eritrocita na osmotsku lizu sa ciljem definisanja optimalnog puferskog sistema za efikasno izvođenje kontrolisane hemolize. Dobijeni rezultati su pokazali da goveđi eritrociti imaju povećanu osmotsku osetljivost u odnosu na humane eritrocite, a da je optimalan puferski sistem za izvođenje njihove kontrolisane hemolize 35 mM natrijum-fosfatni/NaCl pufer pH 7,2-7,4. Kontrolisana hemoliza sa optimizovanim puferskim sistemom je izvedena u membranskom reaktorskom sistemu i ostvaren je prinos hemoglobina od 83±12%. Tokom ovog procesa nije došlo do oštećenja membrane eritrocita, a intaktne membrane praznih eritrocita bi se mogle koristiti za inkapsulaciju biološki aktivnih supstanci

Immobilization of Horseradish Peroxidase on Magnetite-Alginate Beads to Enable Effective Strong Binding and Enzyme Recycling during Anthraquinone Dyes’ Degradation

The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/gcarrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/gcarrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H2O2, 200 mg of HRP-MABs (8.9 mg/gcarrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment

Inkapsulacija resveratrola u sferične čestice na bazi hidrogelova dozvoljenih za upotrebu u hrani

The paper reports about the preparation and characterization of hydrogel particles containing liposomes loaded with resveratrol as an active compound. The materials used for preparation of the particles were chosen to be suitable for food industry. Different polymer concentrations affect particles shape, size, size distribution, as well as the release kinetics of resveratrol. The diameter of particles varied from 360 to 754 μm, while the narrow size distribution was observed for all types of particles. Release studies were performed in Franz diffusion cell and the results showed the prolonged release of resveratrol from all samples, but the sample with the highest content of polymer (2.5% w/w) in particular stood out. The research provides useful information about liposomes containing active compound encapsulated in hydrogel matrices and offers the basis for its application in the food industry.Ovaj rad daje podatke o pripremi i karakterizaciji čestica koje sadrže lipozome sa inkapsuliranom aktivnom komponentom resveratrolom. Komponente koje ulaze u sastav ovih čestica odabrane su tako da mogu jednostavno da se primene u prehrambenoj industriji. Prikazan je uticaj različitih koncentracija početnih rastvora polimera čija upotreba je dozvoljena u hrani, a samim tim i njihove viskoznosti na veličinu formiranih čestica, njihov oblik i raspodelu veličina, ali i na otpuštanje resveratrola iz ovih složenih sistema. Prečnik čestica bio je između 360 i 754 μm, dok je uska raspodela veličina detektovana u svim uzorcima. Otpuštanje resveratrola praćeno je u Francovoj difuzionoj ćeliji gde su rezultati ukazali na produženo oslobađanje resveratrola u svim uzorcima. Ipak, uzorak koji je imao najveći udeo polimera u početnom rastvoru (2,5% w/w) najsporije je otpuštao aktivnu komponentu. Ovi rezultati daju korisne podatke o kompleksnim sistemima gde je aktivna komponenta inkapsulirana u lipozome dalje obložena polimerom čime doprinose potencijalnoj aplikaciji ovih i sličnih sistema u prehrambene proizvode