Search results for: edible tissue

Authors: Aleksandra BužArovska, Gordana Bogoeva Gaceva

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Biodegradable polymers have received significant scientific attention in tissue engineering (TE) application, in particular their composites consisting of inorganic nanoparticles. In the last 15 years, they are subject of intensive research by many groups, aiming to develop polymer scaffolds with defined biodegradability, porosity and adequate mechanical stability. The most important characteristic making these materials attractive for TE is their biodegradability, a process that could be time controlled and long enough to enable generation of a new tissue as a replacement for the degraded polymer scaffold. In this work poly(L-lactic acid) scaffolds, filled with TiO2 nanoparticles functionalized with oleic acid, have been prepared by thermally induced phase separation method (TIPS). The functionalization of TiO2 nanoparticles with oleic acid was performed in order to improve the nanoparticles dispersibility within the polymer matrix and at the same time to inhibit the cytotoxicity of the nanofiller. The oleic acid was chosen as amphiphilic molecule belonging to the fatty acid family because of its non-toxicity and possibility for mediation between the hydrophilic TiO2 nanoparticles and hydrophobic PLA matrix. The produced scaffolds were characterized with thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and mechanical compression measurements. The bioactivity for bone tissue engineering application was tested in supersaturated simulated body fluid. The degradation process was followed by Fourier transform infrared spectroscopy (FTIR). The results showed anisotropic morphology with elongated open pores (100 µm), high porosity (around 92%) and perfectly dispersed nanofiller. The compression moduli up to 10 MPa were identified independent on the nanofiller content. Functionalized TiO2 nanoparticles induced formation of hydroxyapatite clusters as much as unfunctionalized TiO2. The prepared scaffolds showed properties ideal for scaffold vascularization, cell attachment, growth and proliferation.

Keywords: biodegradation, bone tissue engineering, mineralization, PLA scaffolds

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Authors: Yamina Boukari, Nashiru Billa, Andrew Morris, Stephen Doughty, Kevin Shakesheff

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Poly (lactic-co-glycolic) acid (PLGA) is a synthetic polymer that can be used in bone tissue engineering with the aim of creating a scaffold in order to support the growth of cells. The formation of microspheres from this polymer is an attractive strategy that would allow for the development of an injectable system, hence avoiding invasive surgical procedures. The aim of this study was to develop a microsphere delivery system for use as an injectable scaffold in bone tissue engineering and evaluate various formulation parameters on its properties. Porous and lysozyme-containing PLGA microspheres were prepared using the double emulsion solvent evaporation method from various molecular weights (MW). Scaffolds were formed by sintering to contain 1 -3mg of lysozyme per gram of scaffold. The mechanical and physical properties of the scaffolds were assessed along with the release of lysozyme, which was used as a model protein. The MW of PLGA was found to have an influence on microsphere size during fabrication, with increased MW leading to an increased microsphere diameter. An inversely proportional relationship was displayed between PLGA MW and mechanical strength of formed scaffolds across loadings for low, intermediate and high MW respectively. Lysozyme release from both microspheres and formed scaffolds showed an initial burst release phase, with both microspheres and scaffolds fabricated using high MW PLGA showing the lowest protein release. Following the initial burst phase, the profiles for each MW followed a similar slow release over 30 days. Overall, the results of this study demonstrate that lysozyme can be successfully incorporated into porous PLGA scaffolds and released over 30 days in vitro, and that varying the MW of the PLGA can be used as a method of altering the physical properties of the resulting scaffolds.

Keywords: bone, microspheres, PLGA, tissue engineering

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Authors: Hayden R Schott, John M Felder III

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Introduction: Chronic lower extremity wounds in the diabetic and vasculopathic populations are associated with a high degree of morbidity.When wounds require more extensive treatment than can be offered by wound care centers, more aggressive solutions involve local tissue transfer and microsurgical free tissue transfer for achieving definitive soft tissue coverage. These procedures of autologous tissue transfer (ATT) offer resilient, soft tissue coverage of limb-threatening wounds and confer promising limb salvage rates. However, chronic osteomyelitis and recalcitrant soft tissue infections are common in severe diabetic foot wounds and serve to significantly complicate ATT procedures. Stimulan is a resorbable calcium sulfate antibiotic carrier. The use of stimulan antibiotic beads to treat chronic osteomyelitis is well established in the orthopedic and plastic surgery literature. In these procedures, the beads are placed beneath the skin flap to directly deliver antibiotics to the infection site. The purpose of this study was to quantify the success of Stimulan antibiotic beads in treating recalcitrant infections in patients with diabetic foot wounds receiving ATT. Methods: A retrospective review of clinical and demographic information was performed on patients who underwent ATT with the placement of Stimulan antibiotic beads for attempted limb salvage from 2018-21. Patients were analyzed for preoperative wound characteristics, demographics, infection recurrence, and adverse outcomes as a result of product use. The primary endpoint was 90 day infection recurrence, with secondary endpoints including 90 day complications. Outcomes were compared using basic statistics and Fisher’s exact tests. Results: In this time span, 14 patients were identified. At the time of surgery, all patients exhibited clinical signs of active infection, including positive cultures and erythema. 57% of patients (n=8) exhibited chronic osteomyelitis prior to surgery, and 71% (n=10) had exposed bone at the wound base. In 57% of patients (n=8), Stimulan beads were placed beneath a free tissue flap and beneath a pedicle tissue flap in 42% of patients (n=6). In all patients, Stimulan beads were only applied once. Recurrent infections were observed in 28% of patients (n=4) at 90 days post-op, and flap nonadherence was observed in 7% (n=1). These were the only Stimulan related complications observed. Ultimately, lower limb salvage was successful in 85% of patients (n=12). Notably, there was no significant association between the preoperative presence of osteomyelitis and recurrent infections. Conclusions: The use of Stimulanantiobiotic beads to treat recalcitrant infections in patients receiving definitive skin coverage of diabetic foot wounds does not appear to demonstrate unnecessary risk. Furthermore, the lack of significance between the preoperative presence of osteomyelitis and recurrent infections indicates the successful use of Stimulan to dampen infection in patients with osteomyelitis, as is consistent with the literature. Further research is needed to identify Stimulan as the significant contributor to infection treatment using future cohort and case control studies with more patients. Nonetheless, the use of Stimulan antibiotic beads in patients with diabetic foot wounds demonstrates successful infection suppression and maintenance of definitive soft tissue coverage.

Keywords: wound care, stimulan antibiotic beads, free tissue transfer, plastic surgery, wound, infection

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Authors: Rostika Flora, Muhammad Zulkarnain, Syokumawena

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Background: Aerobic physical exercises are recommended to keep body fit and healthy although physical exercises themselves can increase body metabolism and oxygen and can lead into tissue hypoxia. Oxygen pressure can serve as Vascular Endhothelial Growth Factor (VEGF) regulator. Hypoxia increases gene expression of VEGF through ascendant regulation of HIF-1. VEGF is involved in regulating angiogenesis process. Aerobic physical exercises can increase the concentration of VEGF in brain and enables angiogenesis process. We have investigated the influence of aerobic physical exercise to the VGEF concentration of wistar rat’s brain. Methods: This was experimental study using post test only control group design. Independent t-test was used as statistical test. The samples were twenty four wistar rat (Rattus Norvegicus) which were divided into four groups: group P1 (control group), group P2 (treatment group with once-a-week exercise), group P3 (treatment group with three time-a-week exercise), and group P4 (treatment group with seven time-a-week exercise). Group P2, P3, and P4 were treated with treadmil with speed of 20 m/minute for 30 minutes. The concentration of VEGF was determined by ELISA. Results: There was a significant increase of VEGF in treatment group compared with control one (<0.05). The maximum increase was found in group P2 (129.02±64.49) and the minimum increase was in group P4 (96.98±11.20). Conclusion: The frequency of aerobic physical exercises influenced the concentration of Vascular Endhothelial Growth Factor (VEGF) of brain tissue of Rattus Norvegicus.

Keywords: brain tissue, hypoxia, physical exercises, vascular endhothelial growth factor

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Authors: Z. Honarvar, M. Farhoodi, M. R. Khani, S. Shojaee-Aliabadi

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Recently, edible films and coating have attracted much attention in food industry due to their environmentally friendly nature and safety in direct contact with food. However edible films have relatively weak mechanical properties and high water vapor permeability. Therefore, the aim of the study was to develop bilayer carboxymethyl cellulose (CMC) coated polypropylene (PP) films to increase mechanical properties and water vapor resistance of each pure CMC or PP films. To modify the surface properties of PE for better attachment of CMC coating layer to PP the atmospheric cold plasma treatment was used. Then the PP surface changes were evaluated by contact angle, AFM, and ATR-FTIR. Furthermore, the physical, mechanical, optical and microstructure characteristics of plasma-treated and untreated films were analyzed. ATR-FTIR results showed that plasma treatment created oxygen-containing groups on PP surface leading to an increase in hydrophilic properties of PP surface. Moreover, a decrease in water contact angle (from 88.92° to 52.15°) and an increase of roughness were observed on PP film surface indicating good adhesion between hydrophilic CMC and hydrophobic PP. Furthermore, plasma pre-treatment improved the tensile strength of CMC coated-PP films from 58.19 to 61.82. Water vapor permeability of plasma treated bilayer film was lower in comparison with untreated film. Therefore, cold plasma treatment has potential to improve attachment of CMC coating to PP layer, leading to enhanced water barrier and mechanical properties of CMC coated polypropylene as food packaging in which also CMC is in contact with food.

Keywords: carboxymethyl cellulose film, cold plasma, Polypropylene, surface properties

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Authors: Muhammad Rafi Ullah Khan, Yaodong Guo, Vanee Chonhenchob, Jinjin Pei, Chongxing Huang

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The effect of sodium alginate (1%) based edible coating incorporated natural essential oils; thymol, carvone and carvacrol as antimicrobial agents at different concentrations (0.1, 0.5 and 1.0 %) on the quality changes of fresh-cut pineapple were investigated. Pineapple dipped in distilled water was served as control. After coating, fruit were sealed in a modified atmosphere package (MAP) using high permeable film; and stored at 5 °C. Gas composition in package headspace, color values (L*, a*, b*, C*), TSS, pH, ethanol, browning, and microbial decay were monitored during storage. Oxygen concentration continuously decreased while carbon dioxide concentration inside all packages continuously increased over time. Color parameters (L*, b*, c*) decreased and a* values increased during storage. All essential oils significantly (p ≤ 0.05) prevented microbial growth than control. A significantly higher (p ≤ 0.05) ethanol content was found in the control than in all other treatments. Visible microbial growth, high ethanol, and low color values limited the shelf life to 6 days in control as compared to 9 days in all other treatments. Among all essential oils, thymol at all concentrations maintained the overall quality of the pineapple and could potentially be used commercially in fresh fruit industries for longer storage.

Keywords: essential oils, antibrowning agents, antimicrobial agents, modified atmosphere packaging, microbial decay, pineapple

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Authors: Reyhane Hamed Kamran

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Early stage morphogenesis requires the execution of complex systems that direct the nearby conduct of gatherings of cells. The organization of such instruments has been, for the most part, deciphered through the recognizable proof of moderated groups of flagging pathways that spatially and transiently control cell conduct. In any case, how this data is handled to control cell shape and cell elements is an open territory of examination. The structure that rises up out of differing controls, for example, cell science, material science, and formative science, focuses to bond and cortical actin arranges as controllers of cell surface mechanics. In this specific circumstance, a scope of formative marvels can be clarified by the guideline of cell surface pressure.

Keywords: cell, tissue damage, morphogenesis, cell conduct

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Authors: Narin Salehiyan

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Early stage morphogenesis requires the execution of complex systems that direct the nearby conduct of gatherings of cells. The organization of such instruments has been, for the most part, deciphered through the recognizable proof of moderated groups of flagging pathways that spatially and transiently control cell conduct. In any case, how this data is handled to control cell shape and cell elements is an open territory of examination. The structure that rises up out of differing controls, for example, cell science, material science and formative science, focuses to bond and cortical actin arranges as controllers of cell surface mechanics. In this specific circumstance, a scope of formative marvels can be clarified by the guideline of cell surface pressure.

Keywords: cell, tissue damage, morphogenesis, cell conduct

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Authors: MyungGu Yeo, JongHan Ha, Gi-Hoon Yang, JaeYoon Lee, SeungHyun Ahn, Hyeongjin Lee, HoJun Jeon, YongBok Kim, Minseong Kim, GeunHyung Kim

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Tissue engineering is a rapidly growing interdisciplinary research area that may provide options for treating damaged tissues and organs. As a promising technique for regenerating various tissues, this technology requires biomedical scaffolds, which serve as an artificial extracellular matrix (ECM) to support neotissue growth. Electrospun micro/nanofibers have been used widely in tissue engineering because of their high surface-area-to-volume ratio and structural similarity to extracellular matrix. However, low mechanical sustainability, low 3D shape-ability, and low cell infiltration have been major limitations to their use. In this work, we propose new hybrid scaffolds interlayered with cell-laden electrospun micro/nano fibers and poly(caprolactone) microstructures. Also, we applied various concentrations of alginate and electric field strengths to determine optimal conditions for the cell-electrospinning process. The combination of cell-laden bioink (2 ⅹ 10^5 osteoblast-like MG63 cells/mL, 2 wt% alginate, 2 wt% poly(ethylene oxide), and 0.7 wt% lecithin) and a 0.16 kV/mm electric field showed the highest cell viability and fiber formation in this process. Using these conditions and PCL microstructures, we achieved mechanically stable hybrid scaffolds. In addition, the cells embedded in the fibrous structure were viable and proliferated. We suggest that the cell-embedded hybrid scaffolds fabricated using the cell-electrospinning process may be useful for various soft- and hard-tissue regeneration applications.

Keywords: bioink, cell-laden scaffold, micro/nanofibers, poly(caprolactone)

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Authors: P. Shrivastava, S. Vijayalakshmi, A. K. Singh, S. Dalai, R. Teotia, P. Sharma, J. Bellare

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This paper focuses on the synthesis and application of nanobioactive glass for bone regeneration studies. Nanobioactive glass has been synthesized by sol gel method having a combination of silicon, calcium and phosphorous in the molar ratio of 75:21:4. The prepared particles were analyzed for surface morphology by FEG SEM and FEG TEM. Physiochemical properties were investigated using ICP AES, FTIR spectroscopy and X-ray diffraction (XRD) techniques. To ascertain their use for therapeutic use, biocompatibility evaluation of the particles was done by performing soaking studies in SBF and in vitro cell culture studies on MG63 cell lines. Cell morphology was observed by FE SEM and phase contrast microscopy. Nanobioactive glasses (NBG) thus prepared were of 30-200 nm in size, which makes them suitable for nano-biomedical applications. The spherical shape of the particles imparts high surface to volume ratio, promoting fast growth of hydroxyapatite (HA), which is the mineral component of bone. As evaluated by in vitro cell culture studies the NBG was found to enhance the surface activation which enhances osteoblast adhesion. This is an essential parameter to improve bone tissue integration, thereby making nanobioactive glass therapeutically suitable for correcting bone defects.

Keywords: biocompatibility, bone tissue engineering, hydroxyapatite, nanobioactive glass

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Authors: Amir Davood Elmi

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From the earliest light microscope to the most innovative X-ray imaging techniques, all of them have refined and improved our knowledge about the organization and composition of living tissues. The old techniques are time consuming and ultimately destructive to the tissues under the examination. In recent few decades, thanks to the boost of technology, non-destructive visualization techniques, such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), selective plane illumination microscopy (SPIM), and optical projection tomography (OPT), have come to the forefront. Among these techniques, CT is excellent for mineralized tissues such as bone or dentine. In addition, CT it is faster than other aforementioned techniques and the sample remains intact. In this article, applications, advantages, and limitations of micro-CT is discussed, in addition to some information about micro-CT of soft tissue.

Keywords: Micro-CT, hard tissue, bone, attenuation coefficient, rapid prototyping

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Authors: Gileade P. Freitas, Helena B. Lopes, Alann T. P. Souza, Paula G. F. P. Oliveira, Adriana L. G. Almeida, Paulo G. Coelho, Marcio M. Beloti, Adalberto L. Rosa

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Bone tissue presents great capacity to regenerate when injured by trauma, infectious processes, or neoplasia. However, the extent of injury may exceed the inherent tissue regeneration capability demanding some kind of additional intervention. In this scenario, cell therapy has emerged as a promising alternative to treat challenging bone defects. This study aimed at evaluating the effect of local injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on bone regeneration of rat calvaria defects. BM-MSCs and AT-MSCs were isolated and characterized by expression of surface markers; cell viability was evaluated after injection through a 21G needle. Defects of 5 mm in diameter were created in calvaria and after two weeks a single injection of BM-MSCs, AT-MSCs or vehicle-PBS without cells (Control) was carried out. Cells were tracked by bioluminescence and at 4 weeks post-injection bone formation was evaluated by micro-computed tomography (μCT) and histology, nanoindentation, and through gene expression of bone remodeling markers. The data were evaluated by one-way analysis of variance (p≤0.05). BM-MSCs and AT-MSCs presented characteristics of mesenchymal stem cells, kept viability after passing through a 21G needle and remained in the defects until day 14. In general, injection of both BM-MSCs and AT-MSCs resulted in higher bone formation compared to Control. Additionally, this bone tissue displayed elastic modulus and hardness similar to the pristine calvaria bone. The expression of all evaluated genes involved in bone formation was upregulated in bone tissue formed by BM-MSCs compared to AT-MSCs while genes involved in bone resorption were upregulated in AT-MSCs-formed bone. We show that cell therapy based on the local injection of BM-MSCs or AT-MSCs is effective in delivering viable cells that displayed local engraftment and induced a significant improvement in bone healing. Despite differences in the molecular cues observed between BM-MSCs and AT-MSCs, both cells were capable of forming bone tissue at comparable amounts and properties. These findings may drive cell therapy approaches toward the complete bone regeneration of challenging sites.

Keywords: cell therapy, mesenchymal stem cells, bone repair, cell culture

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Authors: Yuri V. Plugatar, Vladimir P. Koba, Vladimir V. Papelbu, Vladimir N. Gerasimchuk, Tatjana M. Sakhno

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Using a supersonic shaft–wood tomography, the evaluation of a shaft-wood phyto-pathogenic damage level of Sequoiadendron giganteum (Lindl.) Buccholz was prosecuted. The digital bivariate reflections of the shaft tissue damage were obtained, the characteristics of comparative parameters of the wood-decay degree were given. The investigation results allowed to show up the role of some edaphic factors in their affection on a vital condition and the level of destructive processes while shaft tissue damaging of S.giganteum. It was pinned up that soil consolidation, and hydro-morphication equally make for a phyto-pathogenic damage of plants. While soil consolidation negative acting the shaft-wood damage is located in an underneath of a shaft. In the conditions of an enlarged hydro-morphication a tissue degradation runs less intensively, the destructive processes more active spread in a vertical section of a shaft. The use of a supersonic tomography method gives wide possibilities to diagnose a shaft-wood phyto-pathogenic damage.

Keywords: Sequoiadendron giganteum (Lindl.) Buccholz, supersonic tomography, diagnosis, phyto-pathogenic damage, a vital condition

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Authors: Er-Yuan Chuang

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Bio-mimetic matters have biological functionalities. This might be valuable in the development of versatile biomaterials. At biological fields, protein-based materials might be components to form a 3D network of extracellular biomolecules, containing growth factors. Also, the protein-based biomaterial provides biochemical and structural assistance of adjacent cells. In this study, we try to prepare protein based biomaterial, which was harvested from living animal. We analyzed it’s chemical, physical and biological property in vitro. Besides, in vivo bio-interaction of the prepared biomimetic matrix was tested in an animal model. The protein-based biomaterial has degradability and biocompatibility. This development could be used for tissue regenerations and be served as platform technologies.

Keywords: protein based, in vitro study, in vivo study, biomaterials

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Authors: Mahsa Ahmadi, Mehdi Mehdikhani-Nahrkhalaji, Jaleh Varshosaz, Shadi Farsaei

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Gelatin and hyaluronic acid are commonly used in skin tissue engineering scaffolds, but because of their low mechanical properties and high biodegradation rate, adding a synthetic polymer such as polycaprolactone could improve the scaffold properties. Therefore, we developed a gelatin-hyaluronic acid-polycaprolactone scaffold, containing 0.5 % atorvastatin loaded nanostructured lipid carriers (NLCs) for skin tissue engineering. The atorvastatin loaded NLCs solution was prepared by solvent evaporation method and freeze drying process. Synthesized atorvastatin loaded NLCs was added to the gelatin and hyaluronic acid solution, and a membrane was fabricated with solvent evaporation method. Thereafter it was coated by a thin layer of polycaprolactone via spine coating set. The resulting scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses. Moreover, mechanical properties, in vitro degradation in 7 days period, and in vitro drug release of scaffolds were also evaluated. SEM images showed the uniform distributed NLCs with an average size of 100 nm in the scaffold structure. Mechanical test indicated that the scaffold had a 70.08 Mpa tensile modulus which was twofold of tensile modulus of normal human skin. A Franz-cell diffusion test was performed to investigate the scaffold drug release in phosphate buffered saline (pH=7.4) medium. Results showed that 72% of atorvastatin was released during 5 days. In vitro degradation test demonstrated that the membrane was degradated approximately 97%. In conclusion, suitable physicochemical and biological properties of membrane indicated that the developed gelatin-hyaluronic acid-polycaprolactone nanocomposite scaffold containing 0.5 % atorvastatin loaded NLCs could be used as a good candidate for skin tissue engineering applications.

Keywords: atorvastatin, gelatin, hyaluronic acid, nano lipid carriers (NLCs), polycaprolactone, skin tissue engineering, solvent casting, solvent evaporation

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Authors: Tuhina-khatun, Mohamed Hanafi Musa, Mohd Rafii Yosup, Wong Mui Yun, Aktar-uz-Zaman, Mahbod Sahebi

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Suppression subtractive hybridization (SSH) method is valuable tool for identifying differentially regulated genes in disease specific or tissue specific genes important for cellular growth and differentiation. It is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. It is based primarily on a suppression polymerase chain reaction (PCR) technique and combines normalization and subtraction in a solitary procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs or genomic DNA fragments and simplifies analysis of the subtracted library. SSH technique is applicable to many comparative and functional genetic studies for the identification of disease, developmental, tissue specific, or other differentially expressed genes, as well as for the recovery of genomic DNA fragments distinguishing the samples under comparison.

Keywords: suppression subtractive hybridization, differentially expressed genes, disease specific genes, tissue specific genes

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Authors: Anita Conti, Riccardo Ossanna, Lindsey A. Quintero, Giamaica Conti, Andrea Sbarbati

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Ischemia/reperfusion (IR) injury induces muscle fiber atrophy and skeletal muscle fiber death with subsequently functionality loss. The heterogeneous pool of cells, especially mesenchymal stem cells, contained in the stromal vascular fraction (SVF) of adipose tissue could promote muscle fiber regeneration. To prevent SVF dispersion, it has been proposed the use of injectable biopolymers that work as cells carrier. A significant element of the extracellular matrix is hyaluronic acid (HA), which has been widely used in regenerative medicine as a cell scaffold given its biocompatibility, degradability, and the possibility of chemical functionalization. Connective tissue micro-fragments enriched with SVF obtained from mechanical disaggregation of adipose tissue were evaluated for IR muscle injury regeneration using low molecular weight HA as a scaffold. IR induction. Hindlimb ischemia was induced in 9 athymic nude mice through the clamping of the right quadriceps using a plastic band. Reperfusion was induced by cutting the plastic band after 3 hours of ischemic period. Contralateral (left) muscular tissue was used as healthy control. Treatment. Twenty-four hours after the IR induction, animals (n=3) were intramuscularly injected with 100 µl of SVF mixed with HA (SVF-HA). Animals treated with 100 µl of HA (n=3) and 100 µl saline solution (n=3) were used as control. Treatment monitoring. All animals were in vivo monitored by magnetic resonance imaging (MRI) at 5, 7, 14 and 18 days post-injury (dpi). High-resolution morphological T2 weighed, quantitative T2 map and Dynamic Contrast-Enhanced (DCE) images were acquired in order to assess the regenerative potential of SVF-HA treatment. Ex vivo evaluation. After 18 days from IR induction, animals were sacrificed, and the muscles were harvested for histological examination. At 5 dpi T2 high-resolution MR images clearly reveal the presence of an extensive edematous area due to IR damage for all groups identifiable as an increase of signal intensity (SI) of muscular and surrounding tissue. At 7 dpi, animals of the SVF-HA group showed a reduction of SI, and the T2relaxation time of muscle tissue of the HA-SVF group was 29±0.5ms, comparable with the T2relaxation time of contralateral muscular tissue (30±0.7ms). These suggest a reduction of edematous overflow and swelling. The T2relaxation time at 7dpi of HA and saline groups were 84±2ms and 90±5ms, respectively, which remained elevated during the rest of the study. The evaluation of vascular regeneration showed similar results. Indeed, DCE-MRI analysis revealed a complete recovery of muscular tissue perfusion after 14 dpi for the SVF-HA group, while for the saline and HA group, controls remained in a damaged state. Finally, the histological examination of SVF-HA treated animals exhibited well-defined and organized fibers morphology with a lateralized nucleus, similar to contralateral healthy muscular tissue. On the contrary, HA and saline-treated animals presented inflammatory infiltrates, with HA slightly improving the diameter of the fibers and less degenerated tissue. Our findings show that connective tissue micro-fragments enriched with SVF induce higher muscle homeostasis and perfusion restoration in contrast to control groups.

Keywords: ischemia/reperfusion injury, regenerative medicine, resonance imaging, stromal vascular fraction

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Authors: Lizeth Fuentes-Mera, Vanessa Perez-Silos, Nidia K. Moncada-Saucedo, Alejandro Garcia-Ruiz, Alberto Camacho, Jorge Lara-Arias, Ivan Marino-Martinez, Victor Romero-Diaz, Adolfo Soto-Dominguez, Humberto Rodriguez-Rocha, Hang Lin, Victor Pena-Martinez

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Biphasic scaffolds in cartilage tissue engineering have been designed to influence not only the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone to promote the implant integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a biphasic scaffold based on the assembly of a cartilage phase constituted by fibroin biofunctionalized with bovine cartilage matrix; cellularized with differentiated pre-chondrocytes from adipose tissue stem cells (autologous) and well attached to a bone phase (bone bovine decellularized) to mimic the structure of the nature of native tissue and to promote the cartilage regeneration in a model of joint damage in pigs. Biphasic scaffolds were assembled by fibroin crystallization with methanol. The histological and ultrastructural architectures were evaluated by optical and scanning electron microscopy respectively. Mechanical tests were conducted to evaluate Young's modulus of the implant. For the biological evaluation, pre-chondrocytes were loaded onto the scaffolds and cellular adhesion, proliferation, and gene expression analysis of cartilage extracellular matrix components was performed. The scaffolds that were cellularized and matured for 10 days were implanted into critical 3 mm in diameter and 9-mm in depth osteochondral defects in a porcine model (n=4). Three treatments were applied per knee: Group 1: monophasic cellular scaffold (MS) (single chondral phase), group 2: biphasic scaffold, cellularized only in the chondral phase (BS1), group 3: BS cellularized in both bone and chondral phases (BS2). Simultaneously, a control without treatment was evaluated. After 4 weeks of surgery, integration and regeneration tissues were analyzed by x-rays, histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular biphasic composites exhibited Young's modulus of 805.01 kPa similar to native cartilage (400-800 kPa). In vitro biological studies revealed the chondroinductive ability of the biphasic implant, evidenced by an increase in sulfated glycosaminoglycan (GAGs) and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, In group 1, the defects were not reconstructed. In group 2 and 3 a good integration of the implant with the surrounding tissue was observed. Defects in group 2 were fulfilled by hyaline cartilage and normal bone. Group 3 defects showed fibrous repair tissue. In conclusion; our findings demonstrated the efficacy of biphasic and bioactive scaffold based on silk fibroin, which entwined chondroinductive features and biomechanical capability with appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Keywords: biphasic scaffold, extracellular cartilage matrix, silk fibroin, osteochondral tissue engineering

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Authors: Alireza Barari, Maryam Firozi-Niyaki, Maryam Kamarlouei

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Background: Herbs have a strong anti-cancer effect. Also, exercise is one of several lifestyle factors known to lower the risk of developing cancer. The aim of this study was to investigate the effect of endurance training and taxol on cyclooxygenase-2 and prostaglandin E2 in the liver tissue of mice with cervical cancer. Materials and Methods: In this experimental study, 35 female C57 mice were randomly divided into 5 groups (n=7 in each group): control (healthy), control (cancer), complement (cancer), training-supplementary (cancer) and training (cancer). The implantation of cancerous tumors was performed under the skin of the upper pelvis. The training group completed the endurance training protocol, which included 3 sessions per week, 50 minutes per session, at a speed of 14-18 m/s for six weeks. A dose of 60 mg/kg/day of pure taxol was injected intra peritoneally. The dependent variables of this study were measured 24 hours after the last training session by ELISA. Results: The results showed that the use of taxol and endurance training reduced the levels of cyclooxygenase-2 and prostaglandin E2 in the liver tissues of C57 mice with cervical cancer. Conclusion: Induction of the cancerous tissue in mice with cervical cancer increases the levels of cyclooxygenase-2 and prostaglandin E2 and endurance training along with taxol may reduce these levels.

Keywords: cervical cancer, taxol, endurance training, cyclooxygenase-2, prostaglandin E2

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Authors: Pegah Ebrahimi, Farshad Oveissi, Iman Manavi-Tehrani, Sina Naficy, David F. Fletcher, Fariba Dehghani, David S. Winlaw

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Despite advances in modern surgery, congenital heart disease remains a medical challenge and a major cause of infant mortality. Cardiovascular prostheses are routinely used in surgical procedures to address congenital malformations, for example establishing a pathway from the right ventricle to the pulmonary arteries in pulmonary valvar atresia. Current off-the-shelf options including human and adult products have limited biocompatibility and durability, and their fixed size necessitates multiple subsequent operations to upsize the conduit to match with patients’ growth over their lifetime. Non-physiological blood flow is another major problem, reducing the longevity of these prostheses. These limitations call for better designs that take into account the hemodynamical and anatomical characteristics of different patients. We have integrated tissue engineering techniques with modern medical imaging and image processing tools along with mathematical modeling to optimize the design of cardiovascular grafts in a patient-specific manner. Computational Fluid Dynamics (CFD) analysis is done according to models constructed from each individual patient’s data. This allows for improved geometrical design and achieving better hemodynamic performance. Tissue engineering strives to provide a material that grows with the patient and mimic the durability and elasticity of the native tissue. Simulations also give insight on the performance of the tissues produced in our lab and reduce the need for costly and time-consuming methods of evaluation of the grafts. We are also developing a methodology for the fabrication of the optimized designs.

Keywords: computational fluid dynamics, cardiovascular grafts, design optimization, tissue engineering

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Authors: Matej Patzelt, Jan Dudak, Frantisek Krejci, Jan Zemlicka, Vladimir Musil, Jitka Riedlova, Viktor Sykora, Jana Mrzilkova, Petr Zach

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Introduction: Micro-CT is well used for examination of bone structures and teeth. On the other hand visualization of the soft tissues is still limited. The goal of our study was to create a new fixation method for soft tissue imaging in micro-CT. Methodology: We used organs of 18 mice - heart, lungs, kidneys, liver and brain, which we fixated in ethanol after meticulous preparation. We fixated organs in different concentrations of ethanol and for different period of time. We used three types of ethanol concentration - 97%, 50% and ascending ethanol concentration (25%, 50%, 75%, 97% each for 12 hours). Fixated organs were scanned after 72 hours, 168 hours and 336 hours period of fixation. We scanned all specimens in micro-CT MARS (Medipix All Resolution System). Results: Ethanol method provided contrast enhancement in all studied organs in all used types of fixation. Fixation in 97% ethanol provided very fast fixation and the contrast among the tissues was visible already after 72 hours of fixation. Fixation for the period of 168 and 336 hours gave better details, especially in lung tissue, where alveoli were visualized. On the other hand, this type of fixation caused organs to petrify. Fixation in 50% ethanol provided best results in 336 hours fixation, details were visualized better than in 97% ethanol and samples were not as hard as in fixation in 97% ethanol. Best results were obtained in fixation in ascending ethanol concentration. All organs were visualized in great details, best-visualized organ was heart, where trabeculae and valves were visible. In this type of fixation, organs stayed soft for whole time. Conclusion: New ethanol method is a great option for soft tissue fixation as well as the method for enhancing contrast among tissues in organs. The best results were obtained with fixation of the organs in ascending ethanol concentration, the best visualized organ was the heart.

Keywords: x-ray imaging, small animals, ethanol, ex-vivo

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Authors: Sakinah Abdullah, Rosna Mat Taha

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Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species such as singlet oxygen, superoxide, peroxyl radicals, and peroxynitrite which result in oxidative stress leading to cellular damage. Natural antioxidant are in high demand because of their potential in health promotion and disease prevention and their improved safety and consumer acceptability. Plants are rich sources of natural antioxidant. Dioscorea alata L. known as 'ubi badak' in Malaysia were well known for their antioxidant content, but this plant was seasonal. Thus, tissue culture technique was used to mass propagate this plant. In the present work, a comparative study between in vitro (from tissue culture) and in vivo (from intact plant) samples of Dioscorea alata L. for their antioxidant potential by 2,2-diphenil -1- picrylhydrazyl (DPPH) radical scavenging activity method and their total phenolic and flavonoid contents were carried out. All samples had better radical scavenging activity but in vivo samples had the strongest radical scavenging activity compared to in vitro samples. Furthermore, tubers from in vivo samples showed the greatest free radical scavenging effect and comparatively greater phenolic content than in vitro samples.

Keywords: Dioscorea alata, tissue culture, antioxidant, in vivo, in vitro, DPPH

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Authors: Dins Sumerags, Mara Pilmane, Vita Konopecka, Gunta Sumeraga

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Reinke’s oedema is a specific type of chronic laryngitis evolving only in smokers. Our study aimed to identify the presence and interaction of the immunohistochemical markers for inflammation [IL-1α] and [IL-10], proliferation [Ki-67] and immunoreactive innervation [PGP 9.5] in the laryngeal mucosa using biotin-streptavidin immunochemical staining method. The laryngeal tissue samples were taken from the vocal cord during the surgery of the Reinke’s oedema and compared to the control group from the tissue samples of the cadavers without any visual laryngeal disease. The study results confirm increased cellular proliferation and elevation of the inflammation markers in the laryngeal mucosa in the case of Reinke’s oedema by comparing with the control.

Keywords: reinke`s oedema, immunohistochemical markers, laryngeal mucosa, biotin-streptavidin

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Authors: P. Afsharian, S. Mousazadeh, M. Shahhoseini, R. Aflatoonian

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Introduction: Matrix MetalloProteinases (MMPs) degrade extracellular matrix components to provide normal remodeling and contribute to pathological tissue destruction and cell migration in endometriosis. It is accepted that MMPs are resistant to suppression by progesterone in endometriotic tissues. The physiological effects of progesterone are mediated by its two progesterone receptor (PGR) isoforms, namely PGR-A and PGR-B. The capacity of progesterone affect to gene expression is dependent on the PGR-A/PGR-B ratio. The imbalance ratio in endometriotic tissue may be an important mechanism to be resulted in Progesterone resistance and modify progesterone action via differential regulation of specific progesterone response genes and improve endometriosis disease. Material and methods: RNA was extracted from twenty ectopic (endometriotic) and eutopic (endometrial) tissue samples of women undergoing laparoscopy for endometriosis and 20 healthy fertile women at Royan Institute, Tehran, Iran. Analysis of PGR-A, PGR-B, MMP-2 and MMP-9 mRNA expression was performed using Real-time PCR in ectopic and eutopic tissues. Then, Statistical analysis was calculated according to the 2-ΔΔCT equation for all samples. Results: Quantitative RT–PCR analyses of PGR-A and PGR-B mRNA revealed that there were differences in both isoformes of PGRs mRNA expressions between ectopic and control eutopic tissues. We were able to demonstrate low expression levels of PGR-B isoforms in ectopic tissues. Although, PGR-A expression was significantly higher in the same ectopic samples compare to controls.This method permitted us to demonstrate significant overexpression of MMP-2 and MMP-9 in ectopic samples compared to control endometrial tissues, as well. Conclusions: Our data suggest that low expression levels of PGR-B and overexpression of PGR-A can alter PGR-A/PGR-B ratio in endometriotic ectopic tissues. Imbalance ratio of PGRs in endometriotic tissue may be able to consequence MMP-2 and MMP-9 overexpression which can be important in pathogenesis and treatment of disease.

Keywords: endometriosis, matrix metalloproteinases, progesterone receptor -A and -B, PGR-A/PGR-B ratio

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Authors: J. H. Bang, H. J. Baek, K. I. Kim, B. J. Lee, H. J. Jung, H. J. Jang, S. K. Jung

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Asthma is a chronic inflammatory lung disease characterized by airway hyper responsiveness (AHR), airway obstruction and airway wall remodeling responsible for significant morbidity and mortality worldwide. Genetic and environment factors may result in asthma, but there are no the exact causes of asthma. Chungpye-tang (CPT) has been prescribed as a representative aerosol agent for patients with dyspnea, cough and phlegm in the respiratory clinic at Kyung Hee Korean Medicine Hospital. This Korean herbal medicines have the effect of dispelling external pathogen and dampness pattern. CPT is composed of 4 species of herbal medicines. The 4 species of herbal medicines are Ephedrae herba, Pogostemonis(Agatachis) herba, Caryophylli flos and Zingiberis rhizoma crudus. CPT suppresses neutrophil infiltration and the production of pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. Moreover, the anti-inflammatory effects of CPT on a mouse model of Chronic Obstructive Pulmonary Disease (COPD) was proved. Activation of the NF-κB has been proven that it plays an important role in inflammation via inducing transcription of pro-inflammatory genes. Over-expression of NF-κB has been believed be related to many inflammatory diseases such as arthritis, gastritis, asthma and COPD. So we firstly hypothesize whether CPT has an anti-inflammatory effect on asthmatic human airway epithelial tissue via inhibiting NF-κB pathway. In this study, CPT was extracted with distilled water for 3 hours at 100°C. After process of filtration and evaporation, it was freeze dried. And asthmatic human lung tissues were provided by MatTek Corp. We investigated the precise mechanism of the anti-inflammatory effect of CPT by western blotting analysis. We observed whether the decoction extracts could reduce NF-κB activation, COX-2 protein expression and NF-κB-mediated pro-inflammatory cytokines such as TNF-α, eotaxin, IL-4, IL-9 and IL-13 in asthmatic human lung tissue. As results of this study, there was a trend toward decreased NF-κB expression in asthmatic human airway epithelial tissue. We found that the inhibition effects of CPT on COX-2 expression was not determined. IL-9 and IL-13 secretion was significantly reduced in the asthmatic human lung tissue treated with CPT. Overall, our results indicate that CPT has an anti-inflammatory effect through blocking the signaling pathway of NF-κB, thereby CPT may be a potential remedial agent for allergic asthma.

Keywords: Chungpye-tang, allergic asthma, asthmatic human airway epithelial tissue, nuclear factor kappa B (NF-κB) pathway, COX-2

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Authors: Nidhi Jindal, Ashok Chaudhury, Manisha Mangal

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Andrographis paniculata, (Burm f.) Wallich ex. Nees (Family Acanthaceae) popularly known as King of Bitters, is an important medicinal herb. It has an astonishingly wide range of medicinal properties such as anti-inflammatory,antidiarrhoeal, antiviral, antimalarial, hepatoprotective, cardiovascular, anticancer, and immunostimulatory activities. It is widely cultivated in southern Asia. Though propagation of this herb generally occurs through seeds, it has many germination problems which intrigued scientists to work out on the alternative techniques for its mass production. The potential of tissue culture techniques as an alternative tool for AP multiplication was found to be promising. However, the high mortality rate of explants caused by phenolic browning of explants is one of the difficulties reported. Low multiplication rates were reported in the proliferation phase, as well as cultures decline characterized by leaf fall and loss of overall vigor. In view of above problems, a study was undertaken to overcome seed dormancy to improve germination potential and to investigate further on the possible means for successful proliferation of cultures via preventive approaches to overcome failures caused by phenolic browning. Experiments were conducted to improve germination potential and among all the chemical and mechanical trials, scarification of seeds with sand paper proved to be the best method to enhance the germination potential (82.44%) within 7 days. Similarly, several pretreatments and media combinations were tried to overcome browning of explants leading to the conclusion that addition of 0.1% citric acid and 0.2% of ascorbic acid in the media followed by rapid sub culturing of explants controlled browning and decline of explants by 67.45%.

Keywords: plant tissue culture, empirical measure, germination, tissue culture

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Authors: Arun Jyothi Bheemagani, Chakrapani Pullagummi, Anupalli Roja Rani

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Exploration of the chemical constituents of the plants and pharmacological screening may provide us the basis for the development of novel agents. Plants have provided us some of the very important life saving drugs used in the modern medicine. The aim of our work was to screen the phytochemical constituents and antimicrobial and antioxidant activities of methanol extract of leaves of Annona cherimola Mill plant from Tirumala forest, Tirupathi. It was originally called Chirimuya by the Inca people who lived where it was growing in the Andes of South America, is an edible fruit-bearing species of the genus Annona from the family Annonaceae. Annona cherimola Mill is a multipurpose tree with edible fruits and is one of the sources of the medicinal products. The antibacterial activity was measured by agar well diffusion method; the diameter of the zone of bacterial growth inhibition was measured after incubation of plates. The inhibitory effect was studied against the pathogenic bacteria (Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus and Escherichia coli (E. coli). Antioxidant assays were also performed for the same extracts by spectrophotometric methods using known standard antioxidants as reference. The studied plant extracts were found to be very effective against the pathogenic microorganisms tested. The methanolic extract of Annona cherimola Mill from showed maximum activity against Escherichia coli and Staphylococcus aureus and the least concentration required showing the activity was 0.5mg/ml. Phytochemical screening of the plants revealed the presence of flavonoids, alkaloids, steroids and carbohydrates. Good presence of antioxidants was also found in the methanolic extracts.

Keywords: annona cherimola, phytochemicals, antioxidant and antibacterial activity, methanol extract

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Authors: Mesih Kocamuftuoglu, Gonca Ozan, Enver Ozan, Nalan Kaya, Sema Temizer Ozan

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Metabolic Syndrome, one of the common metabolic disorder, occurs with co-development of insulin resistance, obesity, dislipidemia and hypertension problems. Insulin resistance appears to play a pathogenic role in the metabolic syndrome. Also, there is a relationship between insulin resistance and infertility as known. Turmeric (Curcuma longa L.) a polyphenolic chemical is widely used for its coloring, flavoring, and medicinal properties, and exhibits a strong antioxidant activity. In this study, we assess the effects of turmeric on rat uterine tissue in metabolic syndrome model induced by high fructose diet. Thirty-two adult female Wistar rats weighing 220±20 g were randomly divided into four groups (n=8) as follows; control, fructose, turmeric, and fructose plus turmeric. Metabolic syndrome was induced by fructose solution 20% (w/v) in tap water, and turmeric (C.Longa) administered at the dose of 80 mg/kg body weight every other day by oral gavage. After the experimental period of 8 weeks, rats were decapitated, serum and uterine tissues were removed. Serum lipid profile, glucose, insülin levels were measured. Uterine tissues were fixed for histological analyzes. The uterine tissue sections were stained with hematoxylin-eosin (H & E) stain, then examined and photographed on a light microscope (Novel N-800Mx20). As a result, fructose consumption effected serum lipids, insulin levels, and insulin resistance significantly. Endometrium and myometrium layers were observed in normal structure in control group of uterine tissues. Perivascular edema, peri glandular fibrosis, and inflammatory cell increase were detected in fructose group. Sections of the fructose plus turmeric group showed a significant improvement in findings when compared to the fructose group. Turmeric group cell structures were observed similar with the control group. These results demonstrated that high-fructose consumption could change the structure of the uterine tissue. On the other hand, turmeric administration has beneficial effects on uterine tissue at that dose and duration when administered with fructose.

Keywords: metabolic syndrome, rat, turmeric, uterus

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Authors: E. Roshan Ara Begum, K. Bhavani, K. Subachitra, C. Kirthika, R. Shenbagarathai

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In recent years, there has been a growing concern for the production of chitosan blend nanofibrous scaffold for its favorable physicochemical properties which mimic the native extracellular matrix (ECM) both morphologically and chemically. Therefore, this study focused on production of β-chitosan(β-Cts) and Poly(vinyl alcohol)(PVA) blend nanofibrous scaffold by electrospinning. β-Cts was extracted from the squid pen waste of locally available squid variety Loligo duvauceli (Indian Squid). To the best of our knowledge, there are no reports on nanofibers preparation from the extracted β-Cts. Both the β-Cts and PVA polymers were mixed in two different proportions (30:70 and 40:60 respectively. The electrospun nanofibrous scaffolds were characterized by SEM, swelling property, in vitro enzymatic degradation, and hemo, biocompatibility properties. β-Cts/PVA nanofibers scaffolds had an average fiber diameter of 120 to 550nm.Among the two different β-Cts/PVA blends nanofibers the β-Cts/PVA (40:60) blend fibers demonstrated favourable tissue engineering properties. The β-Cts/PVA (40:60) blend nanofibers exhibited a swelling ratio of 36 ± 2.5% with mass loss percentage of 20 ± 2.71% after 4 weeks of degradation. It has exhibited good hemocompatible properties. HEK-293(Human Embryonic Kidney) cells lines were able to adhere and proliferate well in the β-Cts/PVA blends nanofibers. All these results indicated that electrospun β-Cts/PVA blends nanofibers are a suitable scaffold to be used for tissue engineering purposes.

Keywords: β-chitosan, electrospinning, nanofibers, poly(vinyl alcohol) (PVA)

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Authors: Krasimir I. Ivanov, Penka S. Zapryanova, Stefan V. Krustev, Violina R. Angelova

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Three commonly used digestion methods (dry ashing, acid digestion, and microwave digestion) in different variants were compared for digestion of tobacco leaves. Three main macroelements (K, Ca and Mg) were analysed using AAS Spectrometer Spectra АА 220, Varian, Australia. The accuracy and precision of the measurements were evaluated by using Polish reference material CTR-VTL-2 (Virginia tobacco leaves). To elucidate the problems with elemental recovery X-Ray and SEM–EDS analysis of all residues after digestion were performed. The X-ray investigation showed a formation of KClO₄ when HClO4 was used as a part of the acids mixture. The use of HF at Ca and Mg determination led to the formation of CaF₂ and MgF₂. The results were confirmed by energy dispersive X-ray microanalysis. SPSS program for Windows was used for statistical data processing.

Keywords: digestion methods, plant tissue, determination of macroelements, K, Ca, Mg

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