Search results for: in vitro biocompatibility

Authors: M. Szechynska-Hebda, M. Sobczak, E. Rozanska, J. Troczynska, Z. Banaszak, N. Hordyńska, M. Dyda, M. Wedzony

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Chloroplasts, as essential organelles for photosynthesis, play a critical role in plant development. However, disturbances in the proper functioning of chloroplasts, in the extreme case manifesting as albinism of tissues and whole plants, are a phenomenon often occurring in conditions deviating from natural (e.g., in vitro cultures applied in breeding programs). Using whole-transcriptome analysis (RNA-Seq) together with light, fluorescent and electron microscopy, it was shown, that development of chloroplasts and formation of green or albino plants in the androgenesis process are genotype-dependent; however, they could be modulated by sub-optimal temperature treatment. The reprogramming of the microspore development from gametophytic to sporophytic, and then regeneration of green plant can be positively regulated by cold stress (4 ⁰C). A high temperature stress (32 ⁰C) can induce androgenesis, but it is a factor negatively influencing green plant regeneration (promoting albinism). A similar effect on microspores, androgenesis, and subsequent chloroplast formation, is elicited as a result of postponing the date of spike collection from spring to summer in field conditions (natural temperature rise). It is determined in both environmental or genotypic manner. The delay of the sowing date (environmental effect) or growing of late genotypes (genotypic effect) result in spike maturation at higher temperatures and significantly enhance albino plant formation in androgenesis process. Such a temperature system (4 ⁰C vs. 32 ⁰C) was used to study the chloroplast biogenesis process in wheat and triticale. It was shown, that efficiency of physiological processes differentiates microspore development during cold reprograming in genotypes susceptible and resistant to androgenesis. Moreover, a great variation in developmental stages of the microspores in one anther is observed for susceptible genotypes. Microspores that are more physiologically active under cold conditions can activate signaling pathways and processes, which provide an appropriate supply of metabolites to cell compartments. This, in turn, fully correlates with the genotype-dependent efficiency of chloroplast formation (or different types of plastid) at particular steps of androgenesis. The effect obtained after applying a high temperature stress is different. High temperature causes a significant acceleration of microspore development and less variation in developmental stages at the end of the treatment. Therefore, the developmental diversity of the microspores in one anther seems to be a critical factor for subsequent cell and chloroplast differentiation. The work was financed by Ministry of Agriculture and Rural Development within Program: 'Biological Progress in Plant Production', project no HOR.hn.802.15.2018

Keywords: androgenesis, chloroplast biogenesis, temperature stress, wheat

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Authors: Kumaran Letchmanan, Shou-Cang Shen, Wai Kiong Ng

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Postoperative implant-associated infections in soft tissues and bones remain a serious complication in orthopaedic surgery, which leads to impaired healing, re-implantation, prolong hospital stay and increase cost. Drug-loaded implants with sustained release of antibiotics at the local site are current research interest to reduce the risk of post-operative infections and osteomyelitis, thus, minimize the need for follow-up care and increase patient comfort. However, the improved drug release of the drug-loaded bone cements is usually accompanied by a loss in mechanical strength, which is critical for weight-bearing bone cement. Recently, more attempts have been undertaken to develop techniques to enhance the antibiotic elution as well as preserve the mechanical properties of the bone cements. The present study investigates the potential influence of addition of mesoporous silica nanoparticles (MSN) on the in vitro drug release kinetics of gentamicin (GTMC), along with the mechanical properties of bone cements. Simplex P was formulated with MSN and loaded with GTMC by direct impregnation. Meanwhile, Simplex P with water soluble poragen (xylitol) and high loading of GTMC as well as commercial bone cement CMW Smartset GHV were used as controls. MSN-formulated bone cements are able to increase the drug release of GTMC by 3-fold with a cumulative release of more than 46% as compared with other control groups. Furthermore, a sustained release could be achieved for two months. The loaded nano-sized MSN with uniform pore channels significantly build up an effective nano-network path in the bone cement facilitates the diffusion and extended release of GTMC. Compared with formulations using xylitol and high GTMC loading, incorporation of MSN shows no detrimental effect on biomechanical properties of the bone cements as no significant changes in the mechanical properties as compared with original bone cement. After drug release for two months, the bending modulus of MSN-formulated bone cements is 4.49 ± 0.75 GPa and the compression strength is 92.7 ± 2.1 MPa (similar to the compression strength of Simplex-P: 93.0 ± 1.2 MPa). The unaffected mechanical properties of MSN-formulated bone cements was due to the unchanged microstructures of bone cement, whereby more than 98% of MSN remains in the matrix and supports the bone cement structures. In contrast, the large portions of extra voids can be observed for the formulations using xylitol and high drug loading after the drug release study, thus caused compressive strength below the ASTM F541 and ISO 5833 minimum of 70 MPa. These results demonstrate the potential applicability of MSN-functionalized poly(methyl methacrylate)-based bone cement as a highly efficient, sustained and local drug delivery system with good mechanical properties.

Keywords: antibiotics, biomechanical properties, bone cement, sustained release

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Authors: Victoria Molina, Wendy Franco, Sergio Benavides, José M. Troncoso, Ricardo Luna, Jose R. PéRez-Correa

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Emamectin Benzoate (EB) is a crystal antiparasitic that belongs to the avermectin family. It is one of the most common treatments used in Chile to control Caligus rogercresseyi in Atlantic salmon. However, the sea lice acquired resistance to EB when it is exposed at sublethal EB doses. The low solubility rate of EB and its degradation at the acidic pH in the fish digestive tract are the causes of the slow absorption of EB in the intestine. To protect EB from degradation and enhance its absorption, specific microencapsulation technologies must be developed. Amorphous Solid Dispersion techniques such as Spray Drying (SD) and Ionic Gelation (IG) seem adequate for this purpose. Recently, Soluplus® (SOL) has been used to increase the solubility rate of several drugs with similar characteristics than EB. In addition, alginate (ALG) is a widely used polymer in IG for biomedical applications. Regardless of the encapsulation technique, the quality of the obtained microparticles is evaluated with the following responses, yield (Y%), encapsulation efficiency (EE%) and loading capacity (LC%). In addition, it is important to know the percentage of EB released from the microparticles in gastric (GD%) and intestinal (ID%) digestions. In this work, we microencapsulated EB with SOL (EB-SD) and with ALG (EB-IG) using SD and IG, respectively. Quality microencapsulation responses and in vitro gastric and intestinal digestions at pH 3.35 and 7.8, respectively, were obtained. A central composite design was used to find the optimum microencapsulation variables (amount of EB, amount of polymer and feed flow). In each formulation, the behavior of these variables was predicted with statistical models. Then, the response surface methodology was used to find the best combination of the factors that allowed a lower EB release in gastric conditions, while permitting a major release at intestinal digestion. Two approaches were used to determine this. The desirability approach (DA) and multi-objective optimization (MOO) with multi-criteria decision making (MCDM). Both microencapsulation techniques allowed to maintain the integrity of EB in acid pH, given the small amount of EB released in gastric medium, while EB-IG microparticles showed greater EB release at intestinal digestion. For EB-SD, optimal conditions obtained with MOO plus MCDM yielded a good compromise among the microencapsulation responses. In addition, using these conditions, it is possible to reduce microparticles costs due to the reduction of 60% of BE regard the optimal BE proposed by (DA). For EB-GI, the optimization techniques used (DA and MOO) yielded solutions with different advantages and limitations. Applying DA costs can be reduced 21%, while Y, GD and ID showed 9.5%, 84.8% and 2.6% lower values than the best condition. In turn, MOO yielded better microencapsulation responses, but at a higher cost. Overall, EB-SD with operating conditions selected by MOO seems the best option, since a good compromise between costs and encapsulation responses was obtained.

Keywords: microencapsulation, multiple decision-making criteria, multi-objective optimization, Soluplus®

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Authors: Syed Asif Hassan, Tabrej Khan

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Tuberculosis (TB) caused by bacillus Mycobacterium tuberculosis (Mtb) continues to take a disturbing toll on human life and healthcare facility worldwide. The global burden of TB remains enormous. The alarming rise of multi-drug resistant strains of Mycobacterium tuberculosis calls for an increase in research efforts towards the development of new target specific therapeutics against diverse strains of M. tuberculosis. Therefore, the discovery of new molecular scaffolds targeting new drug sites should be a priority for a workable plan for fighting resistance in Mycobacterium tuberculosis (Mtb). Mtb non-acylated lipoprotein LprG (Rv1411c) has a Toll-like receptor 2 (TLR2) agonist actions that depend on its association with triacylated glycolipids binding specifically with the hydrophobic pocket of Mtb LprG lipoprotein. The detection of a glycolipid carrier function has important implications for the role of LprG in Mycobacterial physiology and virulence. Therefore, considering the pivotal role of glycolipids in mycobacterial physiology and host-pathogen interactions, designing competitive antagonist (chemotherapeutics) ligands that competitively bind to glycolipid binding domain in LprG lipoprotein, will lead to inhibition of tuberculosis infection in humans. In this study, a unified approach involving ligand-based virtual screening protocol USRCAT (Ultra Shape Recognition) software and molecular docking studies using Auto Dock Vina 1.1.2 using the X-ray crystal structure of Mtb LprG protein was implemented. The docking results were further confirmed by DSX (DrugScore eXtented), a robust program to evaluate the binding energy of ligands bound to the Ligand binding domain of the Mtb LprG lipoprotein. The ligand, which has the higher hypothetical affinity, also has greater negative value. Based on the USRCAT, Lipinski’s values and molecular docking results, [(2R)-2,3-di(hexadecanoyl oxy)propyl][(2S,3S,5S,6R)-3,4,5-trihydroxy-2,6-bis[[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6 (hydroxymethyl)tetrahydropyran-2-yl]oxy]cyclohexyl] phosphate (XPX) was confirmed as a promising drug-like lead compound (antagonist) binding specifically to the hydrophobic domain of LprG protein with affinity greater than that of PIM2 (agonist of LprG protein) with a free binding energy of -9.98e+006 Kcal/mol and binding affinity of -132 Kcal/mol, respectively. A further, in vitro assay of this compound is required to establish its potency in inhibiting molecular evasion mechanism of MTB within the infected host macrophages. These results will certainly be helpful in future anti-TB drug discovery efforts against Multidrug-Resistance Tuberculosis (MDR-TB).

Keywords: antagonist, agonist, binding affinity, chemotherapeutics, drug-like, multi drug resistance tuberculosis (MDR-TB), RV1411c protein, toll-like receptor (TLR2)

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Authors: Urvi Panwar, Kanchan Mishra, Kanjaksha Ghosh, ShankerLal Kothari

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Background: Day-by-day the increasing prevalence of neurodegenerative diseases have become a global issue to manage them by medical sciences. The adult neural stem cells are rare and require an invasive and painful procedure to obtain it from central nervous system. Mesenchymal stem cell (MSCs) therapies have shown remarkable application in treatment of various cell injuries and cell loss. MSCs can be derived from various sources like adult tissues, human bone marrow, umbilical cord blood and cord tissue. MSCs have similar proliferation and differentiation capability, but the human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are proved to be more beneficial with respect to cell procurement, differentiation to other cells, preservation, and transplantation. Material and method: Human umbilical cord is easily obtainable and non-controversial comparative to bone marrow and other adult tissues. The umbilical cord can be collected after delivery of baby, and its tissue can be cultured using explant culture method. Cell culture medium such as DMEMF12+10% FBS and DMEMF12+Neural growth factors (bFGF, human noggin, B27) with antibiotics (Streptomycin/Gentamycin) were used to culture and differentiate mesenchymal stem cells into neural cells, respectively. The characterisations of MSCs were done with Flow Cytometer for surface markers CD90, CD73 and CD105 and colony forming unit assay. The differentiated various neural cells will be characterised by fluorescence markers for neurons, astrocytes, and oligodendrocytes; quantitative PCR for genes Nestin and NeuroD1 and Western blotting technique for gap43 protein. Result and discussion: The high quality and number of MSCs were isolated from human umbilical cord via explant culture method. The obtained MSCs were differentiated into neural cells like neurons, astrocytes and oligodendrocytes. The differentiated neural cells can be used to treat neural injuries and neural cell loss by delivering cells by non-invasive administration via cerebrospinal fluid (CSF) or blood. Moreover, the MSCs can also be directly delivered to different injured sites where they differentiate into neural cells. Therefore, human umbilical cord is demonstrated to be an inexpensive and easily available source for MSCs. Moreover, the hUCMSCs can be a potential source for neural cell therapies and neural cell regeneration for neural cell injuries and neural cell loss. This new way of research will be helpful to treat and manage neural cell damages and neurodegenerative diseases like Alzheimer and Parkinson. Still the study has a long way to go but it is a promising approach for many neural disorders for which at present no satisfactory management is available.

Keywords: bone marrow, cell therapy, explant culture method, flow cytometer, human umbilical cord, mesenchymal stem cells, neurodegenerative diseases, neuroprotective, regeneration

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Authors: Simon Grossemy, Peggy P. Y. Chan, Pauline M. Doran

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Nerve tissue engineering is the main field of research aimed at finding an alternative to autografts as a treatment for nerve injuries. Scaffolds are used as a support to enhance nerve regeneration. In order to successfully design novel scaffolds and in vitro cell culture systems, a deep understanding of the factors affecting nerve regeneration processes is needed. Physical and biological parameters associated with the culture environment have been identified as potentially influential in nerve cell differentiation, including electrical stimulation, exposure to extracellular-matrix (ECM) proteins, dynamic medium conditions and co-culture with glial cells. The mechanisms involved in driving the cell to differentiation in the presence of these factors are poorly understood; the complexity of each of them raises the possibility that they may strongly influence each other. Some questions that arise in investigating nerve regeneration include: What are the best protein coatings to promote neural cell attachment? Is the scaffold design suitable for providing all the required factors combined? What is the influence of dynamic stimulation on cell viability and differentiation? In order to study these effects, scaffolds adaptable to bioreactor culture conditions were designed to allow electrical stimulation of cells exposed to ECM proteins, all within a dynamic medium environment. Gold coatings were used to make the surface of viscose rayon microfiber scaffolds (VRMS) conductive, and poly-L-lysine (PLL) and laminin (LN) surface coatings were used to mimic the ECM environment and allow the attachment of rat PC12 neural cells. The robustness of the coatings was analyzed by surface resistivity measurements, scanning electron microscope (SEM) observation and immunocytochemistry. Cell attachment to protein coatings of PLL, LN and PLL+LN was studied using DNA quantification with Hoechst. The double coating of PLL+LN was selected based on high levels of PC12 cell attachment and the reported advantages of laminin for neural differentiation. The underlying gold coatings were shown to be biocompatible using cell proliferation and live/dead staining assays. Coatings exhibiting stable properties over time under dynamic fluid conditions were developed; indeed, cell attachment and the conductive power of the scaffolds were maintained over 2 weeks of bioreactor operation. These scaffolds are promising research tools for understanding complex neural cell behavior. They have been used to investigate major factors in the physical culture environment that affect nerve cell viability and differentiation, including electrical stimulation, bioreactor hydrodynamic conditions, and combinations of these parameters. The cell and tissue differentiation response was evaluated using DNA quantification, immunocytochemistry, RT-qPCR and functional analyses.

Keywords: bioreactor, electrical stimulation, nerve differentiation, PC12 cells, scaffold

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Authors: Mumuni Sumaila, Viness Pillay, Yahya E. Choonara, Pradeep Kumar, Pierre P. Kondiah

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Tuberculosis (TB) remains a serious challenge to public health globally, despite every effort put together to curb the disease. Current TB therapeutics available have proven to be inefficient due to a multitude of drawbacks that range from serious adverse effects/drug toxicity to inconsistent bioavailability, which ultimately contributes to the emergence of drug-resistant TB. An effective ‘cargo’ system designed to cleverly deliver therapeutic doses of anti-TB drugs to infection sites and in a sustained-release manner may provide a better therapeutic choice towards winning the war against TB. In the current study, we investigated mannose-functionalized lipopolysaccharide hybrid nanoparticles for safety and efficacy towards macrophage-targeted simultaneous delivery of the two first-line anti-TB drugs, rifampicin (RF) and isoniazid (IS). RF-IS-loaded lipopolysaccharide hybrid nanoparticles were fabricated using the solvent injection technique (SIT), incorporating soy lecithin (SL) and low molecular weight chitosan (CS) as the lipid and polysaccharide components, respectively. Surface-functionalized nanoparticles were obtained through the reaction of the aldehyde group of mannose with free amine functionality present at the surface of the nanoparticles. The functionalized nanocarriers were spherical with average particle size and surface charge of 107.83 nm and +21.77 mV, respectively, and entrapment efficiencies (EE) were 53.52% and 69.80% for RF and IS, respectively. FTIR spectrum revealed high-intensity bands between 1663 cm⁻¹ and 1408 cm⁻¹ wavenumbers (absent in non-functionalized nanoparticles), which could be attributed to the C=N stretching vibration produced by the formation of Schiff’s base (–N=CH–) during the mannosylation reaction. In vitro release studies showed a sustained-release profile for RF and IS, with less than half of the total payload released over a 48-hour period. The nanocarriers were biocompatible and safe, with more than 80% cell viability achieved when incubated with RAW 264.7 cells at concentrations 30 to 500 μg/mL over a 24-hour period. Cellular uptake studies (after a 24-hour incubation period with the murine macrophage cells, RAW 264.7) revealed a 13- and a 9-fold increase in intracellular accumulation of RF and IS, respectively, when compared with the unformulated RF+IS solution. A 6- and a 3-fold increase in intracellular accumulation of RF and IS, respectively, were observed when compared with the non-functionalized nanoparticles. Furthermore, fluorescent microscopy images showed nanoparticle internalization and accumulation within the RAW 264.7 cells, which was more significant in the mannose-functionalized system compared to the non-functionalized nanoparticles. The overall results suggested that the fabricated mannose-functionalized lipopolysaccharide nanoparticles are a safe and promising platform for macrophage-targeted delivery of anti-TB therapeutics. However, in vivo pharmacokinetic/pharmacodynamics studies are required to further substantiate the therapeutic efficacy of the nanosystem.

Keywords: anti-tuberculosis therapeutics, hybrid nanosystem, lipopolysaccharide nanoparticles, macrophage-targeted delivery

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Authors: Iman Zafarparandeh, Muzammil Mumtaz, Paniz Taherzadeh, Deniz Erbulut

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The instability in the atlantoaxial joint may occur due to cervical surgery, congenital anomalies, and trauma. There are different types of fixation techniques proposed for restoring the stability and preventing harmful neurological deterioration. Application of the screw constructs has become a popular alternative to the older techniques for stabilizing the joint. The main difference between the various screw constructs is the type of the screw which can be lateral mass screw, pedicle screw, transarticular screw, and translaminar screw. The aim of this paper is to study the effect of three popular screw constructs fixation techniques on the biomechanics of the atlantoaxial joint using the finite element (FE) method. A three-dimensional FE model of the upper cervical spine including the skull, C1 and C2 vertebrae, and groups of the existing ligaments were developed. The accurate geometry of the model was obtained from the CT data of a 35-year old male. Three screw constructs were designed to compare; Magerl transarticular screw (TA-Screw), Goel-Harms lateral mass screw and pedicle screw (LM-Screw and Pedicle-Screw), and Wright lateral mass screw and translaminar screw (LM-Screw and TL-Screw). Pure moments were applied to the model in the three main planes; flexion (Flex), extension (Ext), axial rotation (AR) and lateral bending (LB). The range of motion (ROM) of C0-C1 and C1-C2 segments for the implanted FE models are compared to the intact FE model and the in vitro study of Panjabi (1988). The Magerl technique showed less effect on the ROM of C0-C1 than the other two techniques in sagittal plane. In lateral bending and axial rotation, the Goel-Harms and Wright techniques showed less effect on the ROM of C0-C1 than the Magerl technique. The Magerl technique has the highest fusion rate as 99% in all loading directions for the C1-C2 segment. The Wright technique has the lowest fusion rate in LB as 79%. The three techniques resulted in the same fusion rate in extension loading as 99%. The maximum stress for the Magerl technique is the lowest in all load direction compared to other two techniques. The maximum stress in all direction was 234 Mpa and occurred in flexion with the Wright technique. The maximum stress for the Goel-Harms and Wright techniques occurred in lateral mass screw. The ROM obtained from the FE results support this idea that the fusion rate of the Magerl is more than 99%. Moreover, the maximum stress occurred in each screw constructs proves the less failure possibility for the Magerl technique. Another advantage of the Magerl technique is the less number of components compared to other techniques using screw constructs. Despite the benefits of the Magerl technique, there are drawbacks to using this method such as reduction of the C1 and C2 before screw placement. Therefore, other fixation methods such as Goel-Harms and Wright techniques find the solution for the drawbacks of the Magerl technique by adding screws separately to C1 and C2. The FE model implanted with the Wright technique showed the highest maximum stress almost in all load direction.

Keywords: cervical spine, finite element model, atlantoaxial, fixation technique

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Authors: Niharika Kaushal, Minni Singh

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Fruit crops are widely cultivated throughout the World, with citrus being one of the most common. Mandarins, oranges, grapefruits, lemons, and limes are among the most frequently grown varieties. Citrus cultivars are industrially processed into juice, resulting in approx. 25-40% by wt. of biomass in the form of peels and seeds, generally considered as waste. In consequence, a significant amount of this nutraceutical-enriched biomass goes to waste, which, if utilized wisely, could revolutionize the functional food industry, as this biomass possesses a wide range of bioactive compounds, mainly within the class of polyphenols and terpenoids, making them an abundant source of functional bioactive. Mandarin is a potential source of bioflavonoids with putative antioxidative properties, and its potential application for developing value-added products is obvious. In this study, ‘kinnow’ mandarin (Citrus nobilis X Citrus deliciosa) biomass was studied for its flavonoid profile. For this, dried and pulverized peels were subjected to green and sustainable extraction techniques, namely, supercritical fluid extraction carried out under conditions pressure: 330 bar, temperature: 40 ̊ C and co-solvent: 10% ethanol. The obtained extract was observed to contain 47.3±1.06 mg/ml rutin equivalents as total flavonoids. Mass spectral analysis revealed the prevalence of polymethoxyflavones (PMFs), chiefly tangeretin and nobiletin. Furthermore, the antioxidant potential was analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which was estimated to be at an IC₅₀ of 0.55μg/ml. The pre-systemic metabolism of flavonoids limits their functionality, as was observed in this study through in vitro gastrointestinal studies where nearly 50.0% of the flavonoids were degraded within 2 hours of gastric exposure. We proposed nanoencapsulation as a means to overcome this problem, and flavonoids-laden polylactic-co-glycolic acid (PLGA) nano encapsulates were bioengineered using solvent evaporation method, and these were furnished to a particle size between 200-250nm, which exhibited protection of flavonoids in the gastric environment, allowing only 20% to be released in 2h. A further step involved impregnating the nano encapsulates within alginate hydrogels which were fabricated by ionic cross-linking, which would act as delivery vehicles within the gastrointestinal (GI) tract. As a result, 100% protection was achieved from the pre-systemic release of bioflavonoids. These alginate hydrogels had key significant features, i.e., less porosity of nearly 20.0%, and Cryo-SEM (Cryo-scanning electron microscopy) images of the composite corroborate the packing ability of the alginate hydrogel. As a result of this work, it is concluded that the waste can be used to develop functional biomaterials while retaining the functionality of the bioactive itself.

Keywords: bioflavonoids, gastrointestinal, hydrogels, mandarins

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Authors: Ziyad S. Haidar

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Background: Saliva plays a major role in maintaining oral, dental, and general health and well-being; where it normally bathes the oral cavity acting as a clearing agent. This becomes more apparent when the amount and quality of saliva are significantly reduced due to medications, salivary gland neoplasms, disorders such as Sjögren’s syndrome, and especially ionizing radiation therapy for tumors of the head and neck, the 5th most common malignancy worldwide, during which the salivary glands are included within the radiation field/zone. Clinically, patients affected by salivary gland dysfunction often opt to terminate their radiotherapy course prematurely as they become malnourished and experience a significant decrease in their QoL. Accordingly, the formulation of a radio-protection/-prevention modality and development of an alternative Rx to restore damaged salivary gland tissue is eagerly awaited and highly desirable. Objectives: Assess the pre-clinical radio-protective effect and reparative/regenerative potential of layer-by-layer self-assembled lipid-polymer-based core-shell nanocapsules designed and fine-tuned for the sequential (ordered) release of dual cytokines, following a single local administration (direct injection) into a murine sub-mandibular salivary gland model of irradiation. Methods: The formulated core-shell nanocapsules were characterized by physical-chemical-mechanically pre-/post-loading with the drugs, followed by optimizing the pharmaco-kinetic profile. Then, nanosuspensions were administered directly into the salivary glands, 24hrs pre-irradiation (PBS, un-loaded nanocapsules, and individual and combined vehicle-free cytokines were injected into the control glands for an in-depth comparative analysis). External irradiation at an elevated dose of 18Gy was exposed to the head-and-neck region of C57BL/6 mice. Salivary flow rate (un-stimulated) and salivary protein content/excretion were regularly assessed using an enzyme-linked immunosorbent assay (3-month period). Histological and histomorphometric evaluation and apoptosis/proliferation analysis followed by local versus systemic bio-distribution and immuno-histochemical assays were then performed on all harvested major organs (at the distinct experimental end-points). Results: Monodisperse, stable, and cytocompatible nanocapsules capable of maintaining the bioactivity of the encapsulant within the different compartments with the core and shell and with controlled/customizable pharmaco-kinetics, resulted, as is illustrated in the graphical abstract (Figure) below. The experimental animals demonstrated a significant increase in salivary flow rates when compared to the controls. Herein, salivary protein content was comparable to the pre-irradiation (baseline) level. Histomorphometry further confirmed the biocompatibility and localization of the nanocapsules, in vivo, into the site of injection. Acinar cells showed fewer vacuoles and nuclear aberration in the experimental group, while the amount of mucin was higher in controls. Overall, fewer apoptotic activities were detected by a Terminal deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay and proliferative rates were similar to the controls, suggesting an interesting reparative and regenerative potential of irradiation-damaged/-dysfunctional salivary glands. The Figure below exemplifies some of these findings. Conclusions: Biocompatible, reproducible, and customizable self-assembling layer-by-layer core-shell delivery system is formulated and presented. Our findings suggest that localized sequential bioactive delivery of dual cytokines (in specific dose and order) can prevent irradiation-induced damage via reducing apoptosis and also has the potential to promote in situ proliferation of salivary gland cells; maxSALIVA is scalable (Good Manufacturing Practice or GMP production for human clinical trials) and patent-pending.

Keywords: cancer, head and neck, oncology, drug development, drug delivery systems, nanotechnology, nanoncology

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Authors: Antonio J. Melendez-Martinez

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Beyond their role as natural colorants, some carotenoids are provitamins A and may be involved in health-promoting biological actions and contribute to reducing the risk of developing non-communicable diseases, including several types of cancer, cardiovascular disease, eye conditions, skin disorders or metabolic disorders. Given the versatility of carotenoids, the COST-funded European network to advance carotenoid research and applications in agro-food and health (EUROCAROTEN) is aimed at promoting health through the diet and increasing well-being by means. Stakeholders from 38 countries participate in this network, and one of its main objectives is to promote research on little-studied carotenoids. In this contribution, recent advances of our research group and collaborators in the study of two such understudied carotenoids, namely phytoene and phytofluene, the colorless carotenoids, are outlined. The study of these carotenoids is important as they have been largely neglected despite they are present in our diets, fluids, and tissues, and evidence is accumulating that they may be involved in health-promoting actions. More specifically, studies on their levels in diverse tomato and orange varieties were carried out as well as on their potential bioavailability from different dietary sources. Furthermore, the potential effect of these carotenoids on an animal model subjected to oxidative stress was evaluated. The tomatoes were grown in research greenhouses, and some of them were subjected to regulated deficit irrigation, a sustainable agronomic practice. The citrus samples were obtained from an experimental field. The levels of carotenoids were assessed using HPLC according to routine methodologies followed in our lab. Regarding the potential bioavailability (bioaccessibility) studies, different products containing colorless carotenoids, like fruits, juices, were subjected to simulated in vitro digestions, and their incorporation into mixed micelles was assessed. The effect of the carotenoids on oxidative stress was evaluated on the Caenorhabditis elegans model. For that purpose, the worms were subjected to oxidative stress by means of a hydrogen peroxide challenge. In relation to the presence of colorless carotenoids in tomatoes and orange varieties, it was observed that they are widespread in such products and that there are mutants with very high quantities of them, for instance, the Cara Cara or Pinalate mutant oranges. The studies on their bioaccessibility revealed that, in general, phytoene and phytofluene are more bioaccessible than other common dietary carotenoids, probably due to their distinctive chemical structure. About the in vivo antioxidant capacity of phytoene and phytofluene, it was observed that they both exerted antioxidant effects at certain doses. In conclusion, evidence on the importance of phytoene and phytofluene as dietary easily bioavailable and antioxidant carotenoids has been obtained in recent studies from our group, which can be important shortly to innovate in health-promotion through the development of functional foods and related products.

Keywords: carotenoids, health, functional foods, nutrition, phytoene, phytofluene

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Authors: Delphine Morales, Florian Lombart, Agathe Truchot, Pauline Maire, Pascale Vigneron, Antoine Galmiche, Catherine Lok, Muriel Vayssade

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Targeted therapy molecules are used as a first-line treatment for metastatic melanoma with B-Raf mutation. Nevertheless, these molecules can cause side effects to patients and are efficient on 50 to 60 % of them. Indeed, melanoma cell sensitivity to targeted therapy molecules is dependent on tumor microenvironment (cell-cell and cell-extracellular matrix interactions). To better unravel factors modulating cell sensitivity to B-Raf inhibitor, we have developed and compared several melanoma models: from metastatic melanoma cells cultured as monolayer (2D) to a co-culture in a 3D dermal equivalent. Cell response was studied in different melanoma cell lines such as SK-MEL-28 (mutant B-Raf (V600E), sensitive to Vemurafenib), SK-MEL-3 (mutant B-Raf (V600E), resistant to Vemurafenib) and a primary culture of dermal human fibroblasts (HDFn). Assays have initially been performed in a monolayer cell culture (2D), then a second time on a 3D dermal equivalent (dermal human fibroblasts embedded in a collagen gel). All cell lines were treated with Vemurafenib (a B-Raf inhibitor) for 48 hours at various concentrations. Cell sensitivity to treatment was assessed under various aspects: Cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK signaling pathway analysis (Western-Blotting), Apoptosis (TUNEL), Cytokine release (IL-6, IL-1α, HGF, TGF-β, TNF-α) upon Vemurafenib treatment (ELISA) and histology for 3D models. In 2D configuration, the inhibitory effect of Vemurafenib on cell proliferation was confirmed on SK-MEL-28 cells (IC50=0.5 µM), and not on the SK-MEL-3 cell line. No apoptotic signal was detected in SK-MEL-28-treated cells, suggesting a cytostatic effect of the Vemurafenib rather than a cytotoxic one. The inhibition of SK-MEL-28 cell proliferation upon treatment was correlated with a strong expression decrease of phosphorylated proteins involved in the MAPK pathway (ERK, MEK, and AKT/PKB). Vemurafenib (from 5 µM to 10 µM) also slowed down HDFn proliferation, whatever cell culture configuration (monolayer or 3D dermal equivalent). SK-MEL-28 cells cultured in the dermal equivalent were still sensitive to high Vemurafenib concentrations. To better characterize all cell population impacts (melanoma cells, dermal fibroblasts) on Vemurafenib efficacy, cytokine release is being studied in 2D and 3D models. We have successfully developed and validated a relevant 3D model, mimicking cutaneous metastatic melanoma and tumor microenvironment. This 3D melanoma model will become more complex by adding a third cell population, keratinocytes, allowing us to characterize the epidermis influence on the melanoma cell sensitivity to Vemurafenib. In the long run, the establishment of more relevant 3D melanoma models with patients’ cells might be useful for personalized therapy development. The authors would like to thank the Picardie region and the European Regional Development Fund (ERDF) 2014/2020 for the funding of this work and Oise committee of "La ligue contre le cancer".

Keywords: 3D human skin model, melanoma, tissue engineering, vemurafenib efficiency

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Authors: Z. Karahaliloğlu, E. Yalçın, M. Demirbilek, E.B. Denkbaş

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Critical-sized bone defects caused by trauma, bone diseases, prosthetic implant revision or tumor excision cannot be repaired by physiological regenerative processes. Current orthopedic applications for critical-sized bone defects are to use autologous bone grafts, bone allografts, or synthetic graft materials. However, these strategies are unable to solve completely the problem, and motivate the development of novel effective biological scaffolds for tissue engineering applications and regenerative medicine applications. In particular, scaffolds combined with a variety of bio-agents as fundamental tools emerge to provide the regeneration of damaged bone tissues due to their ability to promote cell growth and function. In this study, a magnetic silk fibroin (SF) hydrogel scaffold was prepared by electrogelation process of the concentrated Bombxy mori silk fibroin (8 %wt) aqueous solution. For enhancement of osteoblast-like cells (SaOS-2) growth and adhesion, basal fibroblast growth factor (bFGF) were conjugated physically to the HSA-coated magnetic nanoparticles (Fe3O4) and magnetic SF e-gel scaffolds were prepared by incorporation of Fe3O4, HSA (human serum albumin)=Fe3O4 and HSA=Fe3O4-bFGF nanoparticles. HSA=Fe3O4, HSA=Fe3O4-bFGF loaded and bare SF e-gels scaffolds were characterized using scanning electron microscopy (SEM.) For cell studies, human osteoblast-like cell line (SaOS-2) was used and an MTT assay was used to assess the cytotoxicity of magnetic silk fibroin e-gel scaffolds and cell density on these surfaces. For the evaluation osteogenic activation, ALP (alkaline phosphatase), the amount of mineralized calcium, total protein and collagen were studied. Fe3O4 nanoparticles were successfully synthesized and bFGF was conjugated to HSA=Fe3O4 nanoparticles with %97.5 of binding yield which has a particle size of 71.52±2.3 nm. Electron microscopy images of the prepared HSA and bFGF incorporated SF e-gel scaffolds showed a 3D porous morphology. In terms of water uptake results, bFGF conjugated HSA=Fe3O4 nanoparticles has the best water absorbability behavior among all groups. In the in-vitro cell culture studies realized using SaOS-2 cell line, the coating of Fe3O4 nanoparticles surface with a protein enhance the cell viability and HSA coating and bFGF conjugation, the both have an inductive effect in the cell proliferation. One of the markers of bone formation and osteoblast differentiation, according to the ALP activity and total protein results, HSA=Fe3O4-bFGF loaded SF e-gels had significantly enhanced ALP activity. Osteoblast cultured HSA=Fe3O4-bFGF loaded SF e-gels deposited more calcium compared with SF e-gel. The proposed magnetic scaffolds seem to be promising for bone tissue regeneration and used in future work for various applications.

Keywords: basic fibroblast growth factor (bFGF), e-gel, iron oxide nanoparticles, silk fibroin

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Authors: I. C. Orabueze, A. A. Amudalat, S. A. Adesegun, A. A. Usman

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Dental and associated oral diseases are increasingly affecting a considerable portion of the population and are considered some of the major causes of tooth loss, discomfort, mouth odor and loss of confidence. This study focused on the ethnobotanical survey of medicinal plants used in oral therapy and evaluation of the antimicrobial activities of methanolic extracts of two selected plants from the survey for their efficacy against dental microorganisms. The ethnobotanical survey was carried out in six herbal markets in Lagos State, Nigeria by oral interviewing and information obtained from an old family manually complied herbal medication book. Methanolic extracts of Olax subscorpioidea (stem bark) and Bridelia ferruginea (stem bark) were assayed for their antimicrobial activities against clinical oral isolates (Aspergillus fumigatus, Candida albicans, Streptococcus spp, Staphylococcus aureus, Lactobacillus acidophilus and Pseudomonas aeruginosa). In vitro microbial technique (agar well diffusion method and minimum inhibitory concentration (MIC) assay) were employed for the assay. Chlorhexidine gluconate was used as the reference drug for comparison with the extract results. And the preliminary phytochemical screening of the constituents of the plants were done. The ethnobotanical survey produced plants (28) of diverse family. Different parts of plants (seed, fruit, leaf, root, bark) were mentioned but 60% mentioned were either the stem or the bark. O. subscorpioidea showed considerable antifungal activity with zone of inhibition ranging from 2.650 – 2.000 cm against Aspergillus fumigatus but no such encouraging inhibitory activity was observed in the other assayed organisms. B. ferruginea showed antibacterial sensitivity against Streptococcus spp, Staphylococcus aureus, Lactobacillus acidophilus and Pseudomonas aeruginosa with zone of inhibitions ranging from 3.400 - 2.500, 2.250 - 1.600, 2.700 - 1.950, 2.225 – 1.525 cm respectively. The minimum inhibitory concentration of O. subscorpioidea against Aspergillus fumigatus was 51.2 mg ml-1 while that of B. ferruginea against Streptococcus spp was 0.1mg ml-1 and for Staphylococcus aureus, Lactobacillus acidophilus and Pseudomonas aeruginosa were 25.6 mg ml-1. A phytochemical analysis reveals the presence of alkaloids, saponins, cardiac glycoside, tannins, phenols and terpenoids in both plants, with steroids only in B. ferruginea. No toxicity was observed among mice given the two methanolic extracts (1000 mg Kg-1) after 21 days. The barks of both plants exhibited antimicrobial properties against periodontal diseases causing organisms assayed, thus up-holding their folkloric use in oral disorder management. Further research could be done viewing these extracts as combination therapy, checking for possible synergistic value in toothpaste and oral rinse formulations for reducing oral bacterial flora and fungi load.

Keywords: antimicrobial activities, Bridelia ferruginea, dental disinfection, methanolic extract, Olax subscorpioidea, ethnobotanical survey

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Authors: Dian Mayasari, Yosi Bayu Murti, Sylvia Utami Tunjung Pratiwi, Sudarsono

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Melastoma malabathricum L. is an Indo-Pacific herb that has been traditionally used to treat several ailments such as wounds, dysentery, diarrhea, toothache, and diabetes. This plant is common across tropical Indo-Pacific archipelagos and is tolerant of a range of soils, from low-lying areas subject to saltwater inundation to the salt-free conditions of mountain slopes. How the soil and environmental variation influences secondary metabolite production in the herb, and an understanding of the plant’s utility as traditional medicine, remain largely unknown and unexplored. The objective of this study is to evaluate the variability of the metabolic profiles of M. malabathricum L. across its geographic distribution. By employing high-performance liquid chromatography-diode array detector (HPLC-DAD), a highly established, simple, sensitive, and reliable method was employed for establishing the chemical fingerprints of 72 samples of M. malabathricum L. leaves from various geographical locations in Indonesia. Specimens collected from six terrestrial and archipelago regions of Indonesia were analyzed by HPLC to generate chromatogram peak profiles that could be compared across each region. Data corresponding to the common peak areas of HPLC chromatographic fingerprint were analyzed by hierarchical component analysis (HCA) and principal component analysis (PCA) to extract information on the most significant variables contributing to characterization and classification of analyzed samples data. Principal component values were identified as PC1 and PC2 with 41.14% and 19.32%, respectively. Based on variety and origin, the high-performance liquid chromatography method validated the chemical fingerprint results used to screen the in vitro antioxidant activity of M. malabathricum L. The result shows that the developed method has potential values for the quality of similar M. malabathrium L. samples. These findings provide a pathway for the development and utilization of references for the identification of M. malabathricum L. Our results indicate the importance of considering geographic distribution during field-collection efforts as they demonstrate regional metabolic variation in secondary metabolites of M. malabathricum L., as illustrated by HPLC chromatogram peaks and their antioxidant activities. The results also confirm the utility of this simple approach to a rapid evaluation of metabolic variation between plants and their potential ethnobotanical properties, potentially due to the environments from whence they were collected. This information will facilitate the optimization of growth conditions to suit particular medicinal qualities.

Keywords: fingerprint, high performance liquid chromatography, Melastoma malabathricum l., metabolic profiles, principal component analysis

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Authors: Lamzira Ebralidze, Aleksandre Tsertsvadze, Dali Berashvili, Aliosha Bakuridze

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Nowadays, there is an increasing demand for biologically active substances from vegetable, animal, and mineral resources. In terms of the use of natural compounds, pharmaceutical, cosmetic, and nutrition industry has big interest. The biggest drawback of conventional extraction methods is the need to use a large volume of organic extragents. The removal of the organic solvent is a multi-stage process. And their absolute removal cannot be achieved, and they still appear in the final product as impurities. A large amount of waste containing organic solvent damages not only human health but also has the harmful effects of the environment. Accordingly, researchers are focused on improving the extraction methods, which aims to minimize the use of organic solvents and energy sources, using alternate solvents and renewable raw materials. In this context, green extraction principles were formed. Green Extraction is a need of today’s environment. Green Extraction is the concept, and it totally corresponds to the challenges of the 21st century. The extraction of biologically active compounds based on green extraction principles is vital from the view of preservation and maintaining biodiversity. Novel technologies of green extraction are known, such as "cold methods" because during the extraction process, the temperature is relatively lower, and it doesn’t have a negative impact on the stability of plant compounds. Novel technologies provide great opportunities to reduce or replace the use of organic toxic solvents, the efficiency of the process, enhance excretion yield, and improve the quality of the final product. The objective of the research is the development of green technologies of flavonoids containing preparations. Methodology: At the first stage of the research, flavonoids containing preparations (Tincture Herba Leonuri, flamine, rutine) were prepared based on conventional extraction methods: maceration, bismaceration, percolation, repercolation. At the same time, the same preparations were prepared based on green technologies, microwave-assisted, UV extraction methods. Product quality characteristics were evaluated by pharmacopeia methods. At the next stage of the research technological - economic characteristics and cost efficiency of products prepared based on conventional and novel technologies were determined. For the extraction of flavonoids, water is used as extragent. Surface-active substances are used as co-solvent in order to reduce surface tension, which significantly increases the solubility of polyphenols in water. Different concentrations of water-glycerol mixture, cyclodextrin, ionic solvent were used for the extraction process. In vitro antioxidant activity will be studied by the spectrophotometric method, using DPPH (2,2-diphenyl-1- picrylhydrazyl) as an antioxidant assay. The advantage of green extraction methods is also the possibility of obtaining higher yield in case of low temperature, limitation extraction process of undesirable compounds. That is especially important for the extraction of thermosensitive compounds and maintaining their stability.

Keywords: extraction, green technologies, natural resources, flavonoids

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Authors: P.P. Rupasinghe, A.H. Farid

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The Aleutian mink disease virus (AMDV, Carnivore amdoparvovirus 1) causes persistent infection, plasmacytosis, and formation and deposition of immune complexes in various organs in adult mink, leading to glomerulonephritis, arteritis and sometimes death. The disease has no cure nor an effective vaccine, and identification and culling of mink positive for anti-AMDV antibodies have not been successful in controlling the infection in many countries. The failure to eradicate the virus from infected farms may be caused by keeping false-negative individuals on the farm, virus transmission from wild animals, or neighboring farms. The identification of sources of infection, which can be performed by comparing viral sequences, is important in the success of viral eradication programs. High mutation rates could cause inaccuracies when viral sequences are used to trace back an infection to its origin. There is no published information on the mutation rate of AMDV either in vivo or in vitro. The in vivo estimation is the most accurate method, but it is difficult to perform because of the inherent technical complexities, namely infecting live animals, the unknown numbers of viral generations (i.e., infection cycles), the removal of deleterious mutations over time and genetic drift. The objective of this study was to determine the mutation rate of AMDV on which no information was available. A homogenate was prepared from the spleen of one naturally infected American mink (Neovison vison) from Nova Scotia, Canada (parental template). The near full-length genome of this isolate (91.6%, 4,143 bp) was bidirectionally sequenced. A group of black mink was inoculated with this homogenate (descendant mink). Spleen sampled were collected from 10 descendant mink after 16 weeks post-inoculation (wpi) and from anther 10 mink after 176 wpi, and their near-full length genomes were bi-directionally sequenced. Sequences of these mink were compared with each other and with the sequence of the parental template. The number of nucleotide substitutions at 176 wpi was 3.1 times greater than that at 16 wpi (113 vs 36) whereas the estimates of mutation rate at 176 wpi was 3.1 times lower than that at 176 wpi (2.85×10-3 vs 9.13×10-4 substitutions/ site/ year), showing a decreasing trend in the mutation rate per unit of time. Although there is no report on in vivo estimate of the mutation rate of DNA viruses in animals using the same method which was used in the current study, these estimates are at the higher range of reported values for DNA viruses determined by various techniques. These high estimates are logical based on the wide range of diversity and pathogenicity of AMDV isolates. The results suggest that increases in the number of nucleotide substitutions over time and subsequent divergence make it difficult to accurately trace back AMDV isolates to their origin when several years elapsed between the two samplings.

Keywords: Aleutian mink disease virus, American mink, mutation rate, nucleotide substitution

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Authors: Tomasz Borowski, Dawid Sołoducha, Agata Markowska-Szczupak, Aneta Wesołowska, Marian Kordas, Rafał Rakoczy

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Essential oils (EOs) are fragrant volatile oils obtained from plants. These are used for cooking (for flavor and aroma), cleaning, beauty (e.g., rosemary essential oil is used to promote hair growth), health (e.g. thyme essential oil cures arthritis, normalizes blood pressure, reduces stress on the heart, cures chest infection and cough) and in the food industry as preservatives and antioxidants. Rosemary and thyme essential oils are considered the most eminent herbs based on their history and medicinal properties. They possess a wide range of activity against different types of bacteria and fungi compared with the other oils in both in vitro and in vivo studies. However, traditional uses of EOs are limited due to rosemary and thyme oils in high concentrations can be toxic. In light of the accessible data, the following hypothesis was put forward: Low frequency rotating magnetic field (RMF) increases the antimicrobial potential of EOs. The aim of this work was to investigate the antimicrobial activity of commercial Salvia Rosmarinus L. and Thymus vulgaris L. essential oil from Polish company Avicenna-Oil under Rotating Magnetic Field (RMF) at f = 25 Hz. The self-constructed reactor (MAP) was applied for this study. The chemical composition of oils was determined by gas chromatography coupled with mass spectrometry (GC-MS). Model bacteria Escherichia coli K12 (ATCC 25922) was used. Minimum inhibitory concentrations (MIC) against E. coli were determined for the essential oils. Tested oils in very small concentrations were prepared (from 1 to 3 drops of essential oils per 3 mL working suspensions). From the results of disc diffusion assay and MIC tests, it can be concluded that thyme oil had the highest antibacterial activity against E. coli. Moreover, the study indicates the exposition to the RMF, as compared to the unexposed controls causing an increase in the efficacy of antibacterial properties of tested oils. The extended radiation exposure to RMF at the frequency f= 25 Hz beyond 160 minutes resulted in a significant increase in antibacterial potential against E. coli. Bacteria were killed within 40 minutes in thyme oil in lower tested concentration (1 drop of essential oils per 3 mL working suspension). Rapid decrease (>3 log) of bacteria number was observed with rosemary oil within 100 minutes (in concentration 3 drops of essential oils per 3 mL working suspension). Thus, a method for improving the antimicrobial performance of essential oil in low concentrations was developed. However, it still remains to be investigated how bacteria get killed by the EOs treated by an electromagnetic field. The possible mechanisms relies on alteration in the permeability of ionic channels in ionic channels in the bacterial cell walls that transport in the cells was proposed. For further studies, it is proposed to examine other types of essential oils and other antibiotic-resistant bacteria (ARB), which are causing a serious concern throughout the world.

Keywords: rotating magnetic field, rosemary, thyme, essential oils, Escherichia coli

Procedia PDF Downloads 132

Authors: C. Watson, T. Glare, M. O'Callaghan, M. Hurst

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The endemic New Zealand grass grub (Costelytra giveni, Coleoptera: Scarabaeidae) is an economically significant grassland pest in New Zealand. Due to their impacts on production within the agricultural sector, one of New Zealand's primary industries, several methods are being used to either control or prevent the establishment of new grass grub populations in the pasture. One such method involves the use of a biopesticide based on the bacterium Serratia entomophila. This species is one of the causative agents of amber disease, a chronic disease of the larvae which results in death via septicaemia after approximately 2 to 3 months. The ability of S. entomophila to cause amber disease is dependant upon the presence of the amber disease associated plasmid (pADAP), which encodes for the key virulence determinants required for the establishment and maintenance of the disease. Following the collapse of grass grub populations within the soil, resulting from either natural population build-up or application of the bacteria, non-pathogenic plasmid-free Serratia strains begin to predominate within the soil. Whilst the interactions between S. entomophila and grass grub larvae are well studied, less information is known on the interactions between plasmid-bearing and plasmid-free strains, particularly the potential impact of these interactions upon the efficacy of an applied biopesticide. Using a range of constructed strains with antibiotic tags, in vitro (broth culture) and in vivo (soil and larvae) experiments were conducted using inoculants comprised of differing ratios of isogenic pathogenic and non-pathogenic Serratia strains, enabling the relative growth of pADAP+ and pADAP- strains under competition conditions to be assessed. In nutrient-rich, the non-pathogenic pADAP- strain outgrew the pathogenic pADAP+ strain by day 3 when inoculated in equal quantities, and by day 5 when applied as the minority inoculant, however, there was an overall gradual decline in the number of viable bacteria for both strains over a 7-day period. Similar results were obtained in additional experiments using the same strains and continuous broth cultures re-inoculated at 24-hour intervals, although in these cultures, the viable cell count did not diminish over the 7-day period. When the same ratios were assessed in soil microcosms with limited available nutrients, the strains remained relatively stable over a 2-month period. Additionally, in vivo grass grub co-infections assays using the same ratios of tagged Serratia strains revealed similar results to those observed in the soil, but there was also evidence of horizontal transfer of pADAP from the pathogenic to the non-pathogenic strain within the larval gut after a period of 4 days. Whilst the influence of competition is more apparent in broth cultures than within the soil or larvae, further testing is required to determine whether this competition between pathogenic and non-pathogenic Serratia strains has any influence on efficacy and disease progression, and how this may impact on the ability of S. entomophila to cause amber disease within grass grub larvae when applied as a biopesticide.

Keywords: biological control, entomopathogen, microbial ecology, New Zealand

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Authors: Malkhaz Jokhadze, Vakhtang Mshvildadze, Levan Makaradze, Ekaterine Mosidze, Salome Barbaqadze, Mariam Murtazashvili, Dali Berashvili, Koba sivsivadze, Lasha Bakuridze, Aliosha Bakuridze

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Essential oils are one of the most important groups of biologically active substances present in plants. Due to the chemical diversity of components, essential oils and their preparations have a wide spectrum of pharmacological action. They have bactericidal, antiviral, fungicidal, antiprotozoal, anti-inflammatory, spasmolytic, sedative and other activities. They are expectorant, spasmolytic, sedative, hypotensive, secretion enhancing, antioxidant remedies. Based on preliminary pharmacological studies, we have developed a formulation called “Phytobiotic” containing essential oils, a feed additive for poultry as an alternative to antibiotics. Phytobiotic is a water-soluble powder containing a composition of essential oils of thyme, clary, monarda and auxiliary substances: dry extract of liquorice and inhalation lactose. On this stage of research, the goal was to study the chemical composition of provided phytobiotic, identify the main substances and determine their quantity, investigate the biological activity of phytobiotic through in vitro and in vivo studies. Using gas chromatography-mass spectrometry, 38 components were identified in phytobiotic, representing acyclic-, monocyclic-, bicyclic-, and sesquiterpenes. Together with identification of main active substances, their quantitative content was determined, including acyclic terpene alcohol β-linalool, acyclic terpene ketone linalyl acetate, monocyclic terpenes: D-limonene and γ-terpinene, monocyclic aromatic terpene thymol. Provided phytobiotic has pronounced and at the same time broad spectrum of antibacterial activity. In the cell model, phytobiotic showed weak antioxidant activity, and it was stronger in the ORAC (chemical model) tests. Meanwhile anti-inflammatory activity was also observed. When fowls were supplied feed enriched with phytobiotic, it was observed that gained weight of the chickens in the experimental group exceeded the same data for the control group during the entire period of the experiment. The survival rate of broilers in the experimental group during the growth period was 98% compared to -94% in the control group. As a result of conducted researches probable four different mechanisms which are important for the action of phytobiotics were identified: sensory, metabolic, antioxidant and antibacterial action. General toxic, possible local irritant and allergenic effects of phytobiotic were also investigated. Performed assays proved that formulation is safe.

Keywords: clary, essential oils, monarda, poultry, phytobiotics, thyme

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Authors: Agnieszka Markowska-Radomska, Ewa Dluska

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Maintaining a bioactive substances dense diet is important for the elderly, especially to prevent diseases and to support healthy ageing. Adequate bioactive substances intake can reduce the risk of developing chronic diseases (e.g. cardiovascular, osteoporosis, neurodegenerative syndromes, diseases of the oral cavity, gastrointestinal (GI) disorders, diabetes, and cancer). This can be achieved by introducing a comprehensive supplementation of components necessary for the proper functioning of the ageing body. The paper proposes the multiple emulsions of the W1/O/W2 (water-in-oil-in-water) type as carriers for effective co-encapsulation and co-delivery of bioactive substances in supplementation of the elderly. Multiple emulsions are complex structured systems ("drops in drops"). The functional structure of the W1/O/W2 emulsion enables (i) incorporation of one or more bioactive components (lipophilic and hydrophilic); (ii) enhancement of stability and bioavailability of encapsulated substances; (iii) prevention of interactions between substances, as well as with the external environment, delivery to a specific location; and (iv) release in a controlled manner. The multiple emulsions were prepared by a one-step method in the Couette-Taylor flow (CTF) contactor in a continuous manner. In general, a two-step emulsification process is used to obtain multiple emulsions. The paper contains a proposal of emulsion functionalization by introducing pH-responsive biopolymer—carboxymethylcellulose sodium salt (CMC-Na) to the external phase, which made it possible to achieve a release of components controlled by the pH of the gastrointestinal environment. The membrane phase of emulsions was soybean oil. The W1/O/W2 emulsions were evaluated for their characteristics (drops size/drop size distribution, volume packing fraction), encapsulation efficiency and stability during storage (to 30 days) at 4ºC and 25ºC. Also, the in vitro multi-substance co-release process were investigated in a simulated gastrointestinal environment (different pH and composition of release medium). Three groups of stable multiple emulsions were obtained: emulsions I with co-encapsulated vitamins B12, B6 and resveratrol; emulsions II with vitamin A and β-carotene; and emulsions III with vitamins C, E and D3. The substances were encapsulated in the appropriate emulsion phases depending on the solubility. For all emulsions, high encapsulation efficience (over 95%) and high volume packing fraction of internal droplets (0.54-0.76) were reached. In addition, due to the presence of a polymer (CMC-Na) with adhesive properties, high encapsulation stability during emulsions storage were achieved. The co-release study of encapsulated bioactive substances confirmed the possibility to modify the release profiles. It was found that the releasing process can be controlled through the composition, structure, physicochemical parameters of emulsions and pH of the release medium. The results showed that the obtained multiple emulsions might be used as potential liquid complex carriers for controlled/modified/site-specific co-delivery of bioactive substances in dietary supplementation in the elderly.

Keywords: bioactive substance co-release, co-encapsulation, elderly supplementation, multiple emulsion

Procedia PDF Downloads 175

Authors: Aoxue Yang, Weili Tian, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Glucocorticoids (GCs) are used to treat GBM-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and BTSCs. Identifying markers specifically expressed by brain tumor stem cells (BTSCs) may enable specific therapies that spare their regular tissue-resident counterparts. By ribosome profiling analysis, we have identified that glycerol-3-phosphate dehydrogenase 1 (GPD1) is expressed by dormant BTSCs but not by NSCs. Through different stress-induced experiments in vitro, we found that only dexamethasone (DEXA) can significantly increase the expression of GPD1 in NSCs. Adversely, mifepristone (MIFE) which is classified as glucocorticoid receptors antagonists, could decrease GPD1 protein level and weaken the proliferation and stemness in BTSCs. Furthermore, DEXA can induce GPD1 expression in tumor-bearing mice brains and shorten animal survival, whereas MIFE has a distinct adverse effect that prolonged mice lifespan. Knocking out GR in NSC can block the upregulation of GPD1 inducing by DEXA, and we find the specific sequences on GPD1 promotor combined with GR, thus improving the efficiency of GPD1 transcription from CHIP-Seq. Moreover, GR and GPD1 are highly co-stained on GBM sections obtained from patients and mice. All these findings confirmed that GR could regulate GPD1 and loss of GPD1 Impairs Multiple Pathways Important for BTSCs Maintenance GPD1 is also a critical enzyme regulating glycolysis and lipid synthesis. We observed that DEXA and MIFE could change the metabolic profiles of BTSCs by regulating GPD1 to shift the transition of cell dormancy. Our transcriptome and lipidomics analysis demonstrated that cell cycle signaling and phosphoglycerides synthesis pathways contributed a lot to the inhibition of GPD1 caused by MIFE. In conclusion, our findings raise concern that treatment of GBM with GCs may compromise the efficacy of chemotherapy and contribute to BTSC dormancy. Inhibition of GR can dramatically reduce GPD1 and extend the survival duration of GBM-bearing mice. The molecular link between GPD1 and GR may give us an attractive therapeutic target for glioblastoma.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides

Procedia PDF Downloads 100

Authors: Federico Herrera, Valle Gomez García de Soria, Itxaso Portero Sainz, Carlos Fernández Arandojo, Mercedes Royg, Ana Marcos Jimenez, Anna Kreutzman, Cecilia MuñozCalleja

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Introduction: Graft-versus-host disease (GVHD) still remains the major complication associated with allogeneic stem cell transplantation (SCT). The pathogenesis involves migration of donor naïve T-cells into recipient secondary lymphoid organs. Two molecules are important in this process: CD62L and CCR7, which are characteristically expressed in naïve/central memory T-cells. With this background, we aimed to study the influence of CCR7 and CD62L on donor lymphocytes in the development and severity of GVHD. Material and methods: This single center study included 98 donor-recipient pairs. Samples were collected prospectively from the apheresis product and phenotyped by flow cytometry. CCR7 and CD62L expression in CD4+ and CD8+ T-cells were compared between patients who developed acute (n=40) or chronic GVHD (n=33) and those who did not (n=38). Results: The patients who developed acute GVHD were transplanted with a higher percentage of CCR7+CD4+ T-cells (p = 0.05) compared to the no GVHD group. These results were confirmed when these patients were divided in degrees according to the severity of the disease; the more severe disease, the higher percentage of CCR7+CD4+ T-cells. Conversely, chronic GVHD patients received a higher percentage of CCR7+CD8+ T-cells (p=0.02) in comparison to those who did not develop the complication. These data were also confirmed when patients were subdivided in degrees of the disease severity. A multivariable analysis confirmed that percentage of CCR7+CD4+ T-cells is a predictive factor of acute GVHD whereas the percentage of CCR7+CD8+ T-cells is a predictive factor of chronic GVHD. In vitro functional assays (migration and activation assays) supported the idea of CCR7+ T-cells were involved in the development of GVHD. As low levels of CD62L expression were detected in all apheresis products, we tested the hypothesis that CD62L was shed during apheresis procedure. Comparing CD62L surface levels in T-cells from the same donor immediately before collecting the apheresis product, and the final apheresis product we found that this process down-regulated CD62L in both CD4+ and CD8+ T cells (p=0.008). Interestingly, when CD62L levels were analysed in days 30 or 60 after engraftment, they recovered to baseline (p=0.008). However, to investigate the relation between CD62L expression and the development of GVHD in the recipient samples after the engraftment, no differences were observed comparing patients with GVHD to those who did not develop the disease. Discussion: Our prospective study indicates that the CCR7+ T-cells from the donor, which include naïve and central memory T-cells, contain the alloreactive cells with a high ability to mediate GVHD (in the case of both migration and activation). Therefore we suggest that the proportion and functional properties of CCR7+CD4+ and CCR7+CD8+ T-cells in the apheresis could act as a predictive biomarker to both acute and chronic GVHD respectively. Importantly, our study precludes that CD62L is lost in the apheresis and therefore it is not a reliable biomarker for the development of GVHD.

Keywords: CCR7, CD62L, GVHD, SCT

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Authors: Itzel Amaro-Estrada, Eduardo Vergara-Rivera, Virginia Juarez-Flores, Mayra Cobaxin-Cardenas, Rosa Estela Quiroz, Jesus F. Preciado, Sergio Rodriguez-Camarillo

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Bovine anaplasmosis is an infectious, tick-borne disease caused mainly by Anaplasma marginale; typical signs include anemia, fever, abortion, weight loss, decreased milk production, jaundice, and potentially death. Sick bovine can recover when antibiotics are administered; however, it usually remains as carrier for life, being a risk of infection for susceptible cattle. Anaplasma marginale is an obligate intracellular Gram-negative bacterium with genetic composition highly diverse among geographical isolates. There are currently no vaccines fully effective against bovine anaplasmosis; therefore, the economic losses due to disease are present. Vaccine formulation became a hard task for several pathogens as Anaplasma marginale, but peptide-based vaccines are an interesting proposal way to induce specific responses. Phage-displayed peptide libraries have been proved one of the most powerful technologies for identifying specific ligands. Screening of these peptides libraries is also a tool for studying interactions between proteins or peptides. Thus, it has allowed the identification of ligands recognized by polyclonal antiserums, and it has been successful for the identification of relevant epitopes in chronic diseases and toxicological conditions. Protective immune response to bovine anaplasmosis includes high levels of immunoglobulins subclass G2 (IgG2) but not subclass IgG1. Therefore, IgG2 from the serum of protected bovine can be useful to identify ligands, which can be part of an immunogen for cattle. In this work, phage display random peptide library Ph.D. ™ -12 was incubating with IgG2 or blood sera of immunized bovines against A. marginale as targets. After three rounds of biopanning, several candidates were selected for additional analysis. Subsequently, their reactivity with sera immunized against A. marginale, as well as with positive and negative sera to A. marginale was evaluated by immunoassays. A collection of recognized peptides tested by ELISA was generated. More than three hundred phage-peptides were separately evaluated against molecules which were used during panning. At least ten different peptides sequences were determined from their nucleotide composition. In this approach, three phage-peptides were selected by their binding and affinity properties. In the case of the development of vaccines or diagnostic reagents, it is important to evaluate the immunogenic and antigenic properties of the peptides. Immunogenic in vitro and in vivo behavior of peptides will be assayed as synthetic and as phage-peptide for to determinate their vaccine potential. Acknowledgment: This work was supported by grant SEP-CONACYT 252577 given to I. Amaro-Estrada.

Keywords: bovine anaplasmosis, peptides, phage display, veterinary vaccines

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Authors: Alba Barbara-i-Molinero, Rosalia Cascon-Pereira, Ana Beatriz Hernandez

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Students’ transitions from high school to the university have been challenged by the lack of continuity between both contexts. This mismatch directly affects students by generating feelings of anxiety and uncertainty, which increases the dropout rates and reduces students’ academic success. This discontinuity emanates because ‘transitions concern a restructuring of what the person does and who the person perceives him or herself to be’. Hence, identity becomes essential in these transitions. Generally, identity is the answer to questions such as who am I? or who are we? This is integrated by personal identity, and as many social identities as groups, the individual feels he/she is a part. A case in point to construct a social identity is the identification with a profession. For this reason, a way to lighten the generated tension during transitions is applying strategies orientated to enhance students’ professional identity in their point of entry to the higher education institution. That would create a sense of continuity between high school and higher education contexts, increasing their Professional Identity Strength. To develop the strategies oriented to enhance students Professional Identity, it is important to analyze what influences it. There exist several influencing factors that influence Professional Identity (e.g., professional status, the recommendation of family and peers, the academic environment, or the chosen bachelor degree). There is a gap in the literature analyzing the impact of these factors on more than one bachelor degree. In this regards, our study takes an additional step with the aim of evaluating the influence of several factors on Professional Identity using a cohort of university students from multiple degrees between the ages of 17-19 years. To do so, we used hierarchical regression analyses to assess the impact of the following factors: External Motivation Conditionals (EMC), Educational Experience Conditionals (EEC) and Personal Motivational Conditional (PMP). After conducting the analyses, we found that the assessed factors influenced students’ professional identity differently according to their bachelor degree and discipline. For example, PMC and EMC positively affected science students, while architecture, law and economics and engineering students were just influenced by PMC. Basing on that influences, we proposed three different strategies aimed to enhance students’ professional identity, in the short and long term. These strategies are: to enhance students’ professional identity before the incorporation to university through campuses and icebreaker activities; to apply recruitment strategies aimed to provide realistic information of the bachelor degree; and to incorporate different activities, such as in-vitro, in situ and self-directed activities aimed to enhance longitudinally students’ professional identity from the university. From these results, theoretical contributions and practical implications arise. First, we contribute to the literature by identifying which factors influence students from different bachelor degrees since there is still no evidence. And, second, using as a benchmark the obtained results, we contribute from a practical perspective, by proposing several alternative strategies to increase students’ professional identity strength aiming to lighten their transition from high school to higher education.

Keywords: professional identity, higher education, educational strategies , students

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Authors: Eun-Seop Kim, Eun-Ha Jang, Rana R. Kim, Sae-Byul Jang

Abstract:

Objective: Preterm labor is the most common antecedent of preterm birth(PTB), which is characterized by regular uterine contraction before 37 weeks of pregnancy and cervical change. In acute preterm labor, tocolytics are administered as the first-line medication to suppress uterine contractions but rarely delay pregnancy to 37 weeks of gestation. On the other hand, according to the Korean Traditional Medicine, PTB is caused by the deficiency of Qi and unnecessary energy in the body of the mother. The aim of this study was to demonstrate the benefit of Traditional Korean Medicine as an adjuvant therapy in management of early uterine contractions and the prevention of PTB. Methods: It is a case report of a 38-year-old woman (0-0-6-0) hospitalized for irregular uterine contractions and cervical change at 33+3/7 weeks of gestation. Past history includes chemical pregnancies achieved by Artificial Rroductive Technology(ART), one stillbirth (at 7 weeks) and a laparoscopic surgery for endometriosis. After seven trials of IVF and articificial insemination, she had succeeded in conception via in-vitro fertilization (IVF) with help of Traditional Korean Medicine (TKM) treatments. Due to irregular uterine contractions and cervical changes, 2 TKM were prescribed: Gami-Dangguisan, and Antae-eum, known to nourish blood and clear away heat. 120ml of Gami-Dangguisan was given twice a day monring and evening along with same amount of Antae-eum once a day from 31 August 2013 to 28 November 2013. Tocolytics (Ritodrine) was administered as a first aid for maintenance of pregnancy. Information regarding progress until the delivery was collected during the patient’s visit. Results: On admission, the cervix of 15mm in length and cervical os with 0.5cm-dilated were observed via ultrasonography. 50% cervical effacement was also detected in physical examination. Tocolysis had been temporarily maintained. As a supportive therapy, TKM herbal preparations(gami-dangguisan and Antae-eum) were concomitantly given. As of 34+2/7 weeks of gestation, however intermittent uterine contractions appeared (5-12min) on cardiotocography and vaginal bleeding was also smeared at 34+3/7 weeks. However, enhanced tocolytics and continuous administration of herbal medicine sustained the pregnancy to term. At 37+2/7 weeks, no sign of labor with restored cervical length was confirmed. The woman gave a term birth to a healthy infant via vaginal delivery at 39+3/7 gestational weeks. Conclusions: This is the first successful case report about a preter labor patient administered with conventional tocolytic agents as well as TKM herbal decoctions, delaying delivery to term. This case deserves attention considering it is rare to maintain gestation to term only with tocolytic intervention. Our report implies the potential of herbal medicine as an adjuvant therapy for preterm labor treatment. Further studies are needed to assess the safety and efficacy of TKM herbal medicine as a therapeutic alternative for curing preterm birth.

Keywords: preterm labor, traditional Korean medicine, herbal medicine, integrative treatment, complementary and alternative medicine

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Authors: Vanessa Roulet, Marc Turini, Juliane Hey, Stéphanie Bord-Romeu, Emilie Bonzom, Mahmoud Badawi, Mohammed-Amine Chakir, Valérie Simon, Vanessa Viotti, Jérémie Gautier, Françoise Le Boulaire, Catherine Coignard, Claire Vincent, Sandrine Greaume, Isabelle Voisin

Abstract:

Background: Beckman Coulter, Inc. has recently developed fully automated assays for the detection of HBsAg on a new immunoassay platform. The objective of this European multicenter study was to evaluate the performance of the ACCESS HBsAg and ACCESS HBsAg Confirmatory assays† on the recently CE-marked DxI 9000 ACCESS Immunoassay Analyzer. Methods: The clinical specificity of the ACCESS HBsAg and HBsAg Confirmatory assays was determined using HBsAg-negative samples from blood donors and hospitalized patients. The clinical sensitivity was determined using presumed HBsAg-positive samples. Sample HBsAg status was determined using a CE-marked HBsAg assay (Abbott ARCHITECT HBsAg Qualitative II, Roche Elecsys HBsAg II, or Abbott PRISM HBsAg assay) and a CE-marked HBsAg confirmatory assay (Abbott ARCHITECT HBsAg Qualitative II Confirmatory or Abbott PRISM HBsAg Confirmatory assay) according to manufacturer package inserts and pre-determined testing algorithms. False initial reactive rate was determined on fresh hospitalized patient samples. The sensitivity for the early detection of HBV infection was assessed internally on thirty (30) seroconversion panels. Results: Clinical specificity was 99.95% (95% CI, 99.86 – 99.99%) on 6047 blood donors and 99.71% (95%CI, 99.15 – 99.94%) on 1023 hospitalized patient samples. A total of six (6) samples were found false positive with the ACCESS HBsAg assay. None were confirmed for the presence of HBsAg with the ACCESS HBsAg Confirmatory assay. Clinical sensitivity on 455 HBsAg-positive samples was 100.00% (95% CI, 99.19 – 100.00%) for the ACCESS HBsAg assay alone and for the ACCESS HBsAg Confirmatory assay. The false initial reactive rate on 821 fresh hospitalized patient samples was 0.24% (95% CI, 0.03 – 0.87%). Results obtained on 30 seroconversion panels demonstrated that the ACCESS HBsAg assay had equivalent sensitivity performances compared to the Abbott ARCHITECT HBsAg Qualitative II assay with an average bleed difference since first reactive bleed of 0.13. All bleeds found reactive in ACCESS HBsAg assay were confirmed in ACCESS HBsAg Confirmatory assay. Conclusion: The newly developed ACCESS HBsAg and ACCESS HBsAg Confirmatory assays from Beckman Coulter have demonstrated high clinical sensitivity and specificity, equivalent to currently marketed HBsAg assays, as well as a low false initial reactive rate. †Pending achievement of CE compliance; not yet available for in vitro diagnostic use. 2023-11317 Beckman Coulter and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

Keywords: dxi 9000 access immunoassay analyzer, hbsag, hbv, hepatitis b surface antigen, hepatitis b virus, immunoassay

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Authors: Ling-Ling E, Lin Feng, Hong-Chen Liu, Dong-Sheng Wang, Zhanping Shi, Juncheng Wang, Wei Luo, Yan Lv

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The objective of this study was to compare the effects of the two calcium phosphate composite scaffolds on the attachment, proliferation and osteogenic differentiation of rabbit dental pulp stem cells (DPSCs). One nano-hydroxyapatite/collagen/poly (L-lactide) (nHAC/PLA), imitating the composition and the micro-structure characteristics of the natural bone, was made by Beijing Allgens Medical Science & Technology Co., Ltd. (China). The other beta-tricalcium phosphate (β-TCP), being fully interoperability globular pore structure, was provided by Shanghai Bio-lu Biomaterials Co, Ltd. (China). We compared the absorption water rate and the protein adsorption rate of two scaffolds and the characterization of DPSCs cultured on the culture plate and both scaffolds under osteogenic differentiation media (ODM) treatment. The constructs were then implanted subcutaneously into the back of severe combined immunodeficient (SCID) mice for 8 and 12 weeks to compare their bone formation capacity. The results showed that the ODM-treated DPSCs expressed osteocalcin (OCN), bone sialoprotein (BSP), type I collagen (COLI) and osteopontin (OPN) by immunofluorescence staining. Positive alkaline phosphatase (ALP) staining, calcium deposition and calcium nodules were also observed on the ODM-treated DPSCs. The nHAC/PLA had significantly higher absorption water rate and protein adsorption rate than ß-TCP. The initial attachment of DPSCs seeded onto nHAC/PLA was significantly higher than that onto ß-TCP; and the proliferation rate of the cells was significantly higher than that of ß-TCP on 1, 3 and 7 days of cell culture. DPSCs+ß-TCP had significantly higher ALP activity, calcium/phosphorus content and mineral formation than DPSCs+nHAC/PLA. When implanted into the back of SCID mice, nHAC/PLA alone had no new bone formation, newly formed mature bone and osteoid were only observed in β-TCP alone, DPSCs+nHAC/PLA and DPSCs+β-TCP, and this three groups displayed increased bone formation over the 12-week period. The percentage of total bone formation area had no difference between DPSCs+β-TCP and DPSCs+nHAC/PLA at each time point，but the percentage of mature bone formation area of DPSCs+β-TCP was significantly higher than that of DPSCs+nHAC/PLA. Our results demonstrated that the DPSCs on nHAC/PLA had a better proliferation and that the DPSCs on β-TCP had a more mineralization in vitro, much more newly formed mature bones in vivo were presented in DPSCs+β-TCP group. These findings have provided a further knowledge that scaffold architecture has a different influence on the attachment, proliferation and differentiation of cells. This study may provide insight into the clinical periodontal bone tissue repair with DPSCs+β-TCP construct.

Keywords: dental pulp stem cells, nano-hydroxyapatite/collagen/poly(L-lactide), beta-tricalcium phosphate, periodontal tissue engineering, bone regeneration

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Authors: N. Slepickova Kasalkova, P. Slepicka, L. Bacakova, V. Svorcik

Abstract:

Tissue engineering includes combination of materials and techniques used for the improvement, repair or replacement of the tissue. Scaffolds, permanent or temporally material, are used as support for the creation of the "new cell structures". For this important component (scaffold), a variety of materials can be used. The advantage of some polymeric materials is their cytocompatibility and possibility of biodegradation. Poly(L-lactic acid) (PLLA) is a biodegradable,  semi-crystalline thermoplastic polymer. PLLA can be fully degraded into H₂O and CO₂. In this experiment, the effect of the surface modification of biodegradable polymer (performed by plasma treatment) on the various cell types was studied. The surface parameters and changes of the physicochemical properties of modified PLLA substrates were studied by different methods. Surface wettability was determined by goniometry, surface morphology and roughness study were performed with atomic force microscopy and chemical composition was determined using photoelectron spectroscopy. The physicochemical properties were studied in relation to cytocompatibility of human osteoblast (MG 63 cells), rat vascular smooth muscle cells (VSMC), and human stem cells (ASC) of the adipose tissue in vitro. A fluorescence microscopy was chosen to study and compare cell-material interaction. Important parameters of the cytocompatibility like adhesion, proliferation, viability, shape, spreading of the cells were evaluated. It was found that the modification leads to the change of the surface wettability depending on the time of modification. Short time of exposition (10-120 s) can reduce the wettability of the aged samples, exposition longer than 150 s causes to increase of contact angle of the aged PLLA. The surface morphology is significantly influenced by duration of modification, too. The plasma treatment involves the formation of the crystallites, whose number increases with increasing time of modification. On the basis of physicochemical properties evaluation, the cells were cultivated on the selected samples. Cell-material interactions are strongly affected by material chemical structure and surface morphology. It was proved that the plasma treatment of PLLA has a positive effect on the adhesion, spreading, homogeneity of distribution and viability of all cultivated cells. This effect was even more apparent for the VSMCs and ASCs which homogeneously covered almost the whole surface of the substrate after 7 days of cultivation. The viability of these cells was high (more than 98% for VSMCs, 89-96% for ASCs). This experiment is one part of the basic research, which aims to easily create scaffolds for tissue engineering with subsequent use of stem cells and their subsequent "reorientation" towards the bone cells or smooth muscle cells.

Keywords: poly(L-lactic acid), plasma treatment, surface characterization, cytocompatibility, human osteoblast, rat vascular smooth muscle cells, human stem cells

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Authors: Djamila Chabane, Asma Rouane, Karim Arab

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Depending of the chemical substances responsible for the pharmacological effects, a future therapeutic drug might be produced by extraction from whole plants or by callus initiated from some parts. The optimized callus culture protocols now offer the possibility to use cell culture techniques for vegetative propagation and open minds for further studies on secondary metabolites and drug establishment. In Algerian traditional medicine, Pulicaria incisa (Asteraceae) is used in the treatment of daily troubles (stomachache, headhache., cold, sore throat and rheumatic arthralgia). Field findings revealed that many healers use some fresh parts (leaves, flowers) of this plant to treat skin wounds. This study aims to evaluate the healing efficiency of artisanal cream prepared from sticky mucilage isolated from calluses on dermal wounds of animal models. Callus cultures were initiated from reproductive explants (young inflorescences) excised from adult plants and transferred to a MS basal medium supplemented with growth regulators and maintained under dark for for months. Many calluses types were obtained with various color and aspect (friable, compact). Several subcultures of calli were performed to enhance the mucilage accumulation. After extraction, the mucilage extracts were tested on animal models as follows. The wound healing potential was studied by causing dermal wounds (1 cm diameter) at the dorsolumbar part of Rattus norvegicus; different samples of the cream were applied after hair removal on three rats each, including two controls (one treated by Vaseline and one without any treatment), two experimental groups (experimental group 1, treated with a reference ointment "Madecassol® and experimental group 2 treated by callus mucilage cream for a period of seventeen days. The evolution of the healing activity was estimated by calculating the percentage reduction of the area wounds treated by all compounds tested compared to the controls by using AutoCAD software. The percentage of healing effect of the cream prepared from callus mucilage was (99.79%) compared to that of Madecassol® (99.76%). For the treatment time, the significant healing activity was observed after 17 days compared to that of the reference pharmaceutical products without any wound infection. The healing effect of Madecassol® is more effective because it stimulates and regulates the production of collagen, a fibrous matrix essential for wound healing. Mucilage extracts also showed a high capacity to heal the skin without any infection. According to this pharmacological activity, we suggest to use calluses produced by in vitro culture to producing new compounds for the skin care and treatment.

Keywords: calluses, Pulicaria incisa, mucilage, Wounds

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