Hybrid and Composite Scaffolds Based on Extracellular Matrices for Cartilage Tissue Engineering

Cartilage consists of chondrocytes and a special extracellular matrix (ECM) having unique biochemical, biophysical, and biomechanical properties that play a critical role in the proliferation and differentiation of cells inherent to cartilage functions. Cartilage tissue engineering (CTE) requires recreating these microenvironmental physicochemical conditions to lead to chondrocyte differentiation from stem cells. ECM-derived hybrid scaffolds based on chondroitin sulfate, hyaluronic acid, collagen, and cartilage ECM analogs provide environments conducive to stem cell proliferation. In this review, we describe hybrid scaffolds based on these four cartilage ECM derivatives; we also categorize these scaffolds based on the methods used for their preparation. The use of hybrid scaffolds is increasing in CTE to address the complexity of cartilage tissue. Thus, a comprehensive review on the topic should be a useful guide for future research. Scaffolds fabricated from extracellular matrix (ECM) derivatives are composed of conducive structures for cell attachment, proliferation, and differentiation, but generally do not have proper mechanical properties and load-bearing capacity. In contrast, scaffolds based on synthetic biomaterials demonstrate appropriate mechanical strength, but the absence of desirable biological properties is one of their main disadvantages. To integrate mechanical strength and biological cues, these ECM derivatives can be conjugated with synthetic biomaterials. Hence, hybrid scaffolds comprising both advantages of synthetic polymers and ECM derivatives can be considered a robust vehicle for tissue engineering applications. © Copyright 2019, Mary Ann Liebert, Inc., publishers 2019

The effect of heating, glycation and mutations on structural, interactions and allergenicity of bovine b-lactoglobulin

La b-lactoglobuline (b-Lg) est la protéine la plus abondante du lactosérum du lait de vache et des autres mammifères, elle appartient à la famille des lipocalines. Toutefois, elle est absente dans le lait de primates. C est une protéine globulaire d'environ 18 kDa formant une structure en feuillet b (appelé calice). Chaque monomère contient deux ponts disulfures et une cystéine en position 121 (C121). Ce groupement thiol libre joue un rôle important dans l'agrégation de la b-Lg induite par la chaleur et, éventuellement, dans le maintien de sa stabilité de conformation. La fonction biologique de la b-Lg n'est pas claire, mais son rôle potentiel dans le transport des acides gras dans le tube digestif a été suggéré. La b-Lg est connue comme l'un des allergènes majeurs dans le lait. Parmi les traitements technologiques le chauffage est le plus couramment appliqué au cours des procédés de transformation du lait. Malgré la fréquence d'utilisation du chauffage, les effets des traitements thermiques de la b-Lg sur sa dénaturation et sa reconnaissance par les IgE de patients ne sont pas décrits. La fixation des IgE de patients allergiques au lait de vache à la b-Lg native, et à la b-Lg chauffée dans la gamme de température de 65-95 C, a été étudiée par ELISA. La b-Lg a été soumise à des modifications au cours du chauffage en présence des sucres réducteurs par la réaction de Maillard. La fixation des IgE de patients à la b-Lg glycosylée a également été mesurée. La perte de structures tertiaires et secondaires de b-Lg par chauffage au dessus de 75 C a entraîné une diminution de sa reconnaissance par les IgE, dont les réactivités différent entre les patients. L'intensité de la baisse de la liaison IgE a également été observée dans le cas des b-Lg fortement glycosylées. L'expression au niveau la levure Pichia pastoris d'un mutant de l'espèce bovine b- Lg, dans lequel le groupement C121 a été changé en Ser (C121S) a été réalisée au cours de cette étude. La mutation C121S bloque complètement l'agrégation irréversible induite par un traitement thermique. La fixation des IgE, obtenus de patients allergiques au lait de vache, avec la b-Lg native, b-Lg type sauvage et la b-Lg mutée C121S a également été étudiée par ELISA. La b-Lg peut complexer des lipides tels que les acides butyrique et l'acide oléique ainsi que d autres variété de ligands. La sérotonine (5-hydroxytryptamine, 5-HT) est un composé important qu'on trouve chez les animaux et les plantes. Elle est connue par ses diverses fonctions, y compris la régulation de l'humeur, l'appétit, le sommeil, la contraction musculaire, et certaines fonctions cognitives comme la mémoire et l'apprentissage. Dans la dernière partie de cette étude, l'interaction de la sérotonine et la sérotonine arachidonyl (AA-5HT) avec b-Lg a été étudiée par dichroïsme circulaire (CD) et par fluorescence. Ces deux ligands interagissent avec b-Lg pour former des complexes équimolaires. La constante d affinité du complexe sérotonine/b-Lg est compris entre 105 et 106 M-1, tandis que celle du complexe AA-5HT/b-Lg est comprise entre 104 et 105 M-1. Les constantes d affinités observées sont plus élevés en milieu hydro-éthanolique (25 % EtOH). Selon les résultats des UV proche et lointain en CD, ces ligands n'ont aucune influence apparente sur la structure secondaire de la b-Lg, mais ils déstabilisent partiellement la structure tertiaire. La liaison avec la b-Lg pourrait expliquer l'un des mécanismes périphériques de la régulation de la sérotonine et de ses dérivés au niveau de l'intestin des animaux nourris au lait.b-Lactoglobulin (b-Lg) is a lipocalin, which is the major whey protein of cow milk and the milk of other mammals. However, it is absent from milk of primates. This globular protein of about 18 kDa is folded forming a -barrel (or calyx) structure. Each monomer contains two disulphide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of b-Lg, and, possibly, in the maintenance of its conformational stability. The biological function of b-Lg is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. b-Lg is also one of major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. Despite the frequency of use of heating, the effects of heating b-Lg and its denaturation on its recognition by IgE of patients are not fully described. Binding of IgE from patients with cow milk allergy to native b-Lg and to heated b-Lg in the temperature range 65-95C was measured by ELISA. Since b-Lg can be subjected to modifications during heating by the existing reducing sugars in the milk through Maillard reaction, binding of IgE of patients to glycated b-Lg was also measured. Loss of tertiary and secondary structures of b-Lg by heating above 75C resulted in a decrease in its recognition by IgE, whose reactivities differ between the patients. The intensity of the decrease of IgE binding was also observed in case of intensively glycated b-Lg. The expression in the yeast Pichia pastoris of a mutant bovine b-Lg, in which C121 was changed into Ser (C121S) was accomplished during this study. The C121S mutation blocks completely the irreversible aggregation induced by heat treatment. Binding of IgE from patients with cow milk allergy to native b-Lg, wild-type b-Lg and C121S mutant b-Lg was aslo measured by ELISA. b-Lg has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5-hydroxytryptamine, 5-HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In the last part of this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA-5HT), with b-Lg was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with b-Lg forming equimolar complexes. The binding constant for the serotonin/b-Lg interaction is between 105 and 106 M-1, while for the AA-5HT/b-Lg complex it is between 104 and 105 M-1 as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25 % EtOH). The interactions between serotonin/b-Lg and AA-5HT/b-Lg may compete with self-association (micellization) of both the ligand and the protein. According to far-and near-UV CD results, these ligands have no apparent influence on b-Lg secondary structure, however they partially destabilize its tertiary structure. Their binding by b-Lg may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk-fed animals.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Bienzymatic modification of polymeric membranes to mitigate biofouling

© 2019 Elsevier B.V. Staphylococcus aureus and Staphylococcus epidermidis are considered as major human pathogens and their resistance to antibiotic treatment and host defense systems can be increased due to the formation of biofilms. The biofilm-associated biofouling of industrial surfaces, particularly membranes, remains a serious concern that challenges investigators to develop practical solutions for the reduction of their impact. The present study developed antibacterial membrane surfaces that can mitigate biofilm formation. α-Amylase and lysozyme, as antibacterial enzymes, were covalently immobilized on polydopamine/cyanuric chloride functionalized polyethersulfone (PES) membranes to form biocompatible antibacterial surfaces. Several methods including SEM, AFM, Bradford, water contact angle goniometry, and surface free energy measurement techniques have been used to demonstrate the attachment of enzymes onto PES membranes by changing the physicochemical properties of the surface. The two enzymatic systems alter the membrane surface chemistry by rendering lower free surface energy and higher hydrophilicity, which leads to the creation of a layer of hydration energy barrier preventing microorganisms from being anchored on the surface. Those microorganisms that managed to overcome the energy barrier and get attached to the surface are attached by the enzymes' bond cleavage functionality. This multilevel defense system protects the membrane against any biofilm formation. The results of microtiter test and flow cytometry assay indicated that α-amylase/lysozyme mixture treated membrane samples came with more than 87% removal of biofilms. The results of the biofouling experiment in a dead-end cell demonstrated that the modified membrane surface had only a slightly impaired water flow compared to an unmodified membrane, which was due to the removal of biofilms by the enzymes’ activity. The results also showed that the modification of membranes with antibacterial enzymes could create a new biotechnological horizon to prevent biofilm formation

Domino P-µMB: A New Approach for the Sequential Immobilization of Enzymes Using Polydopamine/Polyethyleneimine Chemistry and Microfabrication

© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Developing a facile approach for the manipulation of the direction and order of the enzymatic reactions via sequential immobilization on inexpensive substrates is a continuous demand. Herein, a new methodology is introduced that allows making a desired enzymatic reaction pathway on a paper-based microfluidic-membrane based biosensor (P-µMB). Although the method is universal, here, as a proof-of-concept, the sequential immobilization of α-amylase, glucose oxidase (GOx) and horseradish peroxidase (HRP) is presented for fabricating a P-µMB. To this end, hydrophilic polydopamine/polyethyleneimine patterns are created on the hydrophobic polypropylene membrane using 3D printing and a polydimethylsiloxane (PDMS) mold, and a coating layer of silver nanoparticles (AgNPs) is used to modify the patterns. The enzymes are then individually immobilized on the desired locations with another set of PDMS molds. It is observed that AgNPs P-µMB in the sequential immobilization system has stable activity at various temperature and pH regimes, high selectivity toward starch, wide-range linear sensitivity, and a limit of detection of 0.002% w/w starch. A smartphone camera is used for the quantitative analysis of the analyte with the mean gray intensity as the analytical parameter. This developed system provides a platform for further sequential immobilization of other types of biological elements

New generation of drug delivery systems based on ginsenoside Rh2-, Lysine- and Arginine-treated highly porous graphene for improving anticancer activity

Abstract In this study, Rh2–treated graphene oxide (GO-Rh2), lysine-treated highly porous graphene (Gr-Lys), arginine-treated Gr (Gr-Arg), Rh2–treated Gr-Lys (Gr-Lys-Rh2) and Rh2–treated Gr-Arg (Gr-Arg-Rh2) were synthesized. MTT assay was used for evaluation of cytotoxicity of samples on ovarian cancer (OVCAR3), breast cancer (MDA-MB), Human melanoma (A375) and human mesenchymal stem cells (MSCs) cell lines. The percentage of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The hemolysis and blood coagulation activity of nanostructures were performed. Interestingly, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2 were more active against cancer cell lines in comparison with their cytotoxic activity against normal cell lines (MSCs) with IC50 values higher than 100 μg/ml. The results of TUNEL assay indicates a significant increase in the rates of TUNEL positive cells by increasing the concentrations of nanomaterials. Results were also shown that aggregation and changes of RBCs morphology were occurred in the presence of GO, GO-Rh2, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2. Note that all the samples had effect on blood coagulation system, especially on PTT. All nanostrucure act as antitumor drug so that binding of drugs to a nostructures is irresolvable and the whole structure enter to the cell as a drug

Surface modification of polypropylene membrane for the removal of iodine using polydopamine chemistry.

The development of stable and effective iodine removal systems would be highly desirable in addressing environmental issues relevant to water contamination. In the present research, a novel iodine adsorbent was synthesized by self-polymerization of dopamine (PDA) onto inert polypropylene (PP) membrane. This PP/PDA membrane was thoroughly characterized and its susrface propeties was analyzed by various analytical techniques indcluding field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH), contact angle, and surface free energy measurement. The PP/PDA membranes were subsequently used for batchwise removal of iodine at different temperatures (25-70 °C), pH (2-7), and surface areas (1-10 cm2) to understand the underlying adsorption phenomena and to estimate the membrane capacity for iodine uptake. The increase in temperature and pH both led to higher adsorption of iodine. The present approach showed a removal efficiency of over 75% for iodine using 10 cm2 PP/PDA membrane (18.87 m2 g-1) within 2 h at moderate temperatures (∼50 °C) and pH > 4, about 15 fold compared to the PP control membrane. The adsorption kinetics and isotherms were well fitted to the pseudo-second-order kinetic and Langmuir isotherm models (R2 > 0.99). This adsorbent can be recycled and reused at least six times with stable iodine adsorption. These findings were attributed to the homogenous monolayer adsorption of the iodide on the surface due to the presence of catechol and amine groups in the PP/PDA membrane. This study proposes an efficient adsorbent for iodine removal

In vitroandin vivostudy of hazardous effects of Ag nanoparticles and Arginine-treated multi walled carbon nanotubes on blood cells: Application in hemodialysis membranes

One of the novel applications of the nanostructures is the modification and development of membranes for hemocompatibility of hemodialysis. The toxicity and hemocompatibility of Ag nanoparticles and arginine-treated multiwalled carbon nanotubes (MWNT-Arg) and possibility of their application in membrane technology are investigated here. MWNT-Arg is prepared by amidation reactions, followed by characterization by FTIR spectroscopy, Raman spectroscopy, and thermogravimetric analysis. The results showed a good hemocompatibility and the hemolytic rates in the presence of both MWNT-Arg and Ag nanoparticles. The hemolytic rate of Ag nanoparticles was lower than that of MWNT-Arg. In vivo study revealed that Ag nanoparticle and MWNT-Arg decreased Hematocrit and mean number of red blood cells (RBC) statistically at concentration of 100 μg mL-1. The mean decrease of RBC and Hematocrit for Ag nanoparticles (18% for Hematocrit and 5.8 × 1,000,000/μL) was more than MWNT-Arg (20% for Hematocrit and 6 × 1000000/μL). In addition, MWNT-Arg and Ag nanoparticles had a direct influence on the White Blood Cell (WBC) drop. Regarding both nanostructures, although the number of WBC increased in initial concentration, it decreased significantly at the concentration of 100 μg mL-1. It is worth mentioning that the toxicity of Ag nanoparticle on WBC was higher than that of MWNT-Arg. Because of potent antimicrobial activity and relative hemocompatibility, MWNT-Arg could be considered as a new candidate for biomedical applications in the future especially for hemodialysis membranes