Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 830

Search results for: in vivo biocompatibility

830 The Biocompatibility and Osteogenic Potential of Experimental Calcium Silicate Based Root Canal Sealer, Capseal

Authors: Seok Woo Chang


Aim: Capseal I and Capseal II are calcium silicate and calcium phosphate based experimental root canal sealer. The aim of this study was to evaluate the biocompatibility and mineralization potential of Capseal I and Capseal II. Materials and Methods: The biocompatibility and mineralization-related gene expression (alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN)) of Capseal I and Capseal II were compared using methylthiazol tetrazolium assay and reverse transcription-polymerization chain reaction analysis, respectively. The results were analyzed by Kruskal-Wallis test. P-value of < 0.05 was considered significant. Result: Both Capseal I and Capseal II were favorable in biocompatibility and influenced the messenger RNA expression of ALP and BSP. Conclusion: Within the limitation of this study, Capseal is biocompatible and have mineralization promoting potential, and thus could be a promising root canal sealer.

Keywords: biocompatibility, mineralization-related gene expression, Capseal I, Capseal II

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829 Bio-Functional Polymeric Protein Based Materials Utilized for Soft Tissue Engineering Application

Authors: Er-Yuan Chuang


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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828 Cryotopic Macroporous Polymeric Matrices for Regenerative Medicine and Tissue Engineering Applications

Authors: Archana Sharma, Vijayashree Nayak, Ashok Kumar


Three-dimensional matrices were fabricated from blend of natural-natural polymers like carrageenan-gelatin and synthetic -natural polymers such as PEG- gelatin (PEG of different molecular weights (2,000 and 6,000) using two different crosslinkers; glutaraldehyde and EDC-NHS by cryogelation technique. Blends represented a feasible approach to design 3-D scaffolds with controllable mechanical, physical and biochemical properties without compromising biocompatibility and biodegradability. These matrices possessed interconnected porous structure, good mechanical strength, biodegradable nature, constant swelling kinetics, ability to withstand high temperature and visco-elastic behavior. Hemocompatibility of cryogel matrices was determined by coagulation assays and hemolytic activity assay which demonstrated that these cryogels have negligible effects on coagulation time and have excellent blood compatibility. In vitro biocompatibility (cell-matrix interaction) inferred good cell adhesion, proliferation, and secretion of ECM on matrices. These matrices provide a microenvironment for the growth, proliferation, differentiation and secretion of ECM of different cell types such as IMR-32, C2C12, Cos-7, rat bone marrow derived MSCs and human bone marrow MSCs. Hoechst 33342 and PI staining also confirmed that the cells were uniformly distributed, adhered and proliferated properly on the cryogel matrix. An ideal scaffold used for tissue engineering application should allow the cells to adhere, proliferate and maintain their functionality. Neurotransmitter analysis has been done which indicated that IMR-32 cells adhered, proliferated and secreted neurotransmitters when they interacted with these matrices which showed restoration of their functionality. The cell-matrix interaction up to molecular level was also evaluated so to check genotoxicity and protein expression profile which indicated that these cryogel matrices are non-genotoxic and maintained biofunctionality of cells growing on these matrices. All these cryogels, when implanted subcutaneously in balb/c mice, showed no adverse systemic or local toxicity effects at implantation site. There was no significant increase in inflammatory cell count has otherwise been observed after scaffold implantation. These cryogels are supermacroporous and this porous structure allows cell infiltration and proliferation of host cells. This showed the integration and presence of infiltrated cells into the cryogel implants. Histological analysis confirmed that the implanted cryogels do not have any adverse effect in spite of host immune system recognition at the site of implantation, on its surrounding tissues and other vital host organs. In vivo biocompatibility study after in vitro biocompatibility analysis has also concluded that these synthesized cryogels act as important biological substitutes, more adaptable and appropriate for transplantation. Thus, these cryogels showed their potential for soft tissue engineering applications.

Keywords: cryogelation, hemocompatibility, in vitro biocompatibility, in vivo biocompatibility, soft tissue engineering applications

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827 Crosslinked Porous 3-Dimensional Cellulose Nanofibers/Gelatin Based Biocomposite Aerogels for Tissue Engineering Application

Authors: Ali Mirtaghavi, Andy Baldwin, Rajendarn Muthuraj, Jack Luo


Recent advances in biomaterials have led to utilizing biopolymers to develop 3D scaffolds in tissue regeneration. One of the major challenges of designing biomaterials for 3D scaffolds is to mimic the building blocks similar to the extracellular matrix (ECM) of the native tissues. Biopolymer based aerogels obtained by freeze-drying have shown to provide structural similarities to the ECM owing to their 3D format and a highly porous structure with interconnected pores, similar to the ECM. Gelatin (GEL) is known to be a promising biomaterial with inherent regenerative characteristics owing to its chemical similarities to the ECM in native tissue, biocompatibility abundance, cost-effectiveness and accessible functional groups, which makes it facile for chemical modifications with other biomaterials to form biocomposites. Despite such advantages, gelatin offers poor mechanical properties, sensitive enzymatic degradation and high viscosity at room temperature which limits its application and encourages its use to develop biocomposites. Hydrophilic biomass-based cellulose nanofibrous (CNF) has been explored to use as suspension for biocomposite aerogels for the development of 3D porous structures with excellent mechanical properties, biocompatibility and slow enzymatic degradation. In this work, CNF biocomposite aerogels with various ratios of CNF:GEL) (90:10, 70:30 and 50:50) were prepared by freeze-drying technique, and their properties were investigated in terms of physicochemical, mechanical and biological characteristics. Epichlorohydrin (EPH) was used to investigate the effect of chemical crosslinking on the molecular interaction of CNF: GEL, and its effects on physicochemical, mechanical and biological properties of the biocomposite aerogels. Ultimately, chemical crosslinking helped to improve the mechanical resilience of the resulting aerogels. Amongst all the CNF-GEL composites, the crosslinked CNF: GEL (70:30) biocomposite was found to be favourable for cell attachment and viability. It possessed highly porous structure (porosity of ~93%) with pore sizes ranging from 16-110 µm, adequate mechanical properties (compression modulus of ~47 kPa) and optimal biocompatibility both in-vitro and in-vivo, as well as controlled enzymatic biodegradation, high water penetration, which could be considered a suitable option for wound healing application. In-vivo experiments showed improvement on inflammation and foreign giant body cell reaction for the crosslinked CNF: GEL (70:30) compared to the other samples. This could be due to the superior interaction of CNF with gelatin through chemical crosslinking, resulting in more optimal in-vivo improvement. In-vitro cell culture investigation on human dermal fibroblasts showed satisfactory 3D cell attachment over time. Overall, it has been observed that the developed CNF: GEL aerogel can be considered as a potential scaffold for soft tissue regeneration application.

Keywords: 3D scaffolds, aerogels, Biocomposites , tissue engineering

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826 Biocompatibility assessment of different origin Barrier Membranes for Guided Bone Regeneration

Authors: Antonio Munar-Frau, Sascha Klismoch, Manfred Schmolz, Federico Hernandez-Alfaro, Jordi Caballe-Serrano


Introduction: Biocompatibility of biomaterials has been proposed as one of the main criteria for treatment success. For guided bone regeneration (GBR), barrier membranes present a conflict given the number of origins and modifications of these materials. The biologic response to biomaterials is orchestrated by a series of events leading to the integration or rejection of the biomaterial, posing questions such as if a longer occlusive property may trigger an inflammatory reaction. Whole blood cultures are a solution to study the immune response to drugs or biomaterials during the first 24-48 hours. The aim of this study is to determine the early immune response of different origins and chemical modifications of barrier membranes. Materials & Methods: 5 different widely used barrier membranes were included in this study: Acellular dermal matrix (AlloDerm, LifeCell®), Porcine Peritoneum (BioGide, Geistlich Pharma®), Porcine Pericardium (Jason, Botiss Biomaterials GmbH®), Porcine Cross-linked collagen (Ossix Plus, Datum Dental®) and d-PTFE (Cytoplast TXT, Osteogenics Biomedical®). Blood samples were extracted from 3 different healthy donors and incubated with the different samples of barrier membranes for 24 hours. After the incubation time, serum samples were obtained and analyzed by means of biocompatibility assays taking into account 42 markers. Results: In an early stage of the inflammatory response, the Acellular dermal matrix, porcine peritoneum and porcine cross-linked collagen expressed similar patterns of cytokine expression with a great manifestation of ENA 78. Porcine pericardium and d-PTFE presented similar cytokine activation, especially for MMP-3 and MMP-9, although other cytokines were highlighted with lower expression. For the later immune response, Porcine peritoneum and acellular dermal matrix MCP-1 and IL-15 were evident. Porcine pericardium, porcine cross-linked collagen and d-PTFE presented a high expression of IL-16 and lower manifestation of other cytokines. Different behaviors depending on an earlier or later stage of the inflammation process were observed. Barrier membrane inflammatory expression does not only differ depending on the origin, variables such as treatment of the collagen and polymers may also have a great impact on the cytokine expression of the studied barrier membranes during inflammation. Conclusions: Surface treatment and modifications might affect the biocompatibility of the membranes, as different cytokine expressions were evidently depending on the origin of the biomaterial. This study is only a brushstroke regarding the biocompatibility of materials, as it is one of the pioneer studies for ex vivo barrier membranes assays. Studies regarding surface modification are needed in order to clarify mystifications of barrier membrane science.

Keywords: biomaterials, bone regeneration, biocompatibility, inflammation

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825 Biocompatibility and Sensing Ability of Highly Luminescent Synthesized Core-Shell Quantum Dots

Authors: Mohan Singh Mehata, R. K. Ratnesh


CdSe, CdSe/ZnS, and CdSe/CdS core-shell quantum dots (QDs) of 3-4 nm were developed by using chemical route and following successive ion layer adsorption and reaction (SILAR) methods. The prepared QDs have been examined by using X-ray diffraction, high-resolution electron microscopy and optical spectroscopy. The photoluminescence (PL) quantum yield (QY) of core-shell QDs increases with respect to the core, indicating that the radiative rate increases by the formation of shell around core, as evident by the measurement of PL lifetime. Further, the PL of bovine serum albumin is quenched strongly by the presence of core-shall QDs and follow the Stern-Volmer (S-V) relation, whereas the lifetime does not follow the S-V relation, demonstrating that the observed quenching is predominantly static in nature. Among all the QDs, the CdSe/ZnS QDs shows the least cytotoxicity hence most biocompatibility.

Keywords: biocompatibility, core-shell quantum dots, photoluminescence and lifetime, sensing ability

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824 A Systematic Review Examining the Experimental methodology behind in vivo testing of hiatus hernia and Diaphragmatic Hernia Mesh

Authors: Whitehead-Clarke T., Beynon V., Banks J., Karanjia R., Mudera V., Windsor A., Kureshi A.


Introduction: Mesh implants are regularly used to help repair both hiatus hernias (HH) and diaphragmatic hernias (DH). In vivo studies are used to test not only mesh safety but increasingly comparative efficacy. Our work examines the field of in vivo mesh testing for HH and DH models to establish current practices and standards. Method: This systematic review was registered with PROSPERO. Medline and Embase databases were searched for relevant in vivo studies. 44 articles were identified and underwent abstract review, where 22 were excluded. 4 further studies were excluded after full text review – leaving 18 to undergo data extraction. Results: Of 18 studies identified, 9 used an in vivo HH model and 9 a DH model. 5 studies undertook mechanical testing on tissue samples – all uniaxial in nature. Testing strip widths ranged from 1-20mm (median 3mm). Testing speeds varied from 1.5-60mm/minute. Upon histology, the most commonly assessed structural and cellular factors were neovascularization and macrophages, respectively (n=9 each). Structural analysis was mostly qualitative, where cellular analysis was equally likely to be quantitative. 11 studies assessed adhesion formation, of which 8 used one of four scoring systems. 8 studies measured mesh shrinkage. Discussion: In vivo studies assessing mesh for HH and DH repair are uncommon. Within this relatively young field, we encourage surgical and materials testing institutions to discuss its standardisation.

Keywords: hiatus, diaphragmatic, hernia, mesh, materials testing, in vivo

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823 Biological Evaluation of Some Modern Titanium Alloys for Dental Implants

Authors: Roxana Maria Angelescu, Raluca Ion, Anişoara Cîmpean, Doina Răducanu, Mariana Lucia Angelescu


In an attempt to find titanium alloys that fulfill the requirements for mechanical and biological compatibility, laboratory and material related tests were performed during the years, as well as preclinical and clinical trials. The multidisciplinary scientific research facilitates the global evaluation of biocompatibility and osseointegration regarding the dental implant alloys. The aim of this study was to determine the in vitro biocompatibility of three modern titanium alloys: Ti-31.7Nb-6.21Zr-1.4Fe-0.16O (wt%), Ti-36.5Nb-4.5Zr-3Ta-0.16O (wt%) and Ti-20Nb-5Ta (wt%), in order to establish whether the use of these titanium alloys can have any toxic or injurious effects on biological systems. The commonly used Ti-6Al-4V alloy was investigated as a reference material. The behavior of MC3T3-E1 pre-osteoblasts on all these four metallic surfaces was evaluated. The tests of immunofluorescence, cytotoxicity and cellular proliferation lead to the conclusion that the newly-developed titanium alloys elicit a good cellular response in terms of cellular survival, adhesion, morphology and proliferative potential as well.

Keywords: biocompatibility tests, dental implants, titanium alloys, biomedical engineering

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822 Stimuli-Responsive Zwitterionic Dressings for Chronic Wounds Management

Authors: Konstans Ruseva, Kristina Ivanova, Katerina Todorova, Margarita Gabrashanska, Tzanko Tzanov, Elena Vassileva


Zwitterionic polymers (ZP) are well-known with their ultralow biofouling. They are successfully competing with poly(ethylene glycols) (PEG), which are considered as the “golden standard” in this respect. These unique properties are attributed to their strong hydration capacity, defined by the dipole-dipole interactions, arising between the ZP pendant groups as well as to the dipoles interaction with water molecules. Beside, ZP are highly resistant to bacterial adhesion thus ensuring an excellent anti-biofilm formation ability. Moreover, ZP are able to respond upon external stimuli such as temperature, pH, salt concentration changes which in combination with their anti-biofouling effect render this type of polymers as materials with a high potential in biomedical applications. The present work is focused on the development of zwitterionic hydrogels for efficient treatment of highly exudating and hard-to-heal chronic wounds. To this purpose, two types of ZP networks with different crosslinking degree were synthesized - polysulfobetaine (PSB) and polycarboxybetaine (PCB) ones. They were characterized in terms of their physico-mechanical properties, e.g. microhardness, swelling ability, smart behaviour. Furthermore, the potential of ZP networks to resist biofilm formation towards Staphylococcus aureus and Escherichia coli was studied. Their ability to reduce the high levels of myeloperoxidase and metalloproteinase, two enzymes that are part of the chronic wounds enviroenment, was revealed. Moreover, the in vitro cytotoxic assessment of PSB and PCB networks along with their in vivo performance in rats was also studied to reveal their high biocompatibility.

Keywords: absorption properties, biocompatibility, enzymatic inhibition activity, wound healing, zwitterionic polymers

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821 A Novel Peptide Showing Universal Effect against Multiple Viruses in Vitro and in Vivo

Authors: Hanjun Zhao, Ke Zhang, Bojian Zheng


Background: So far, there is no universal antiviral agent which can inhibit multiple viral infections. More and more drug-resistant viral strains emerge after the antiviral drug application for treatment. Defensins are the front line of host innate immunity and have broad spectrum antibacterial and antiviral effects. However, there is limited data to show if these defensins have good antiviral activity in vivo and what the antiviral mechanism is. Subjects: To investigate a peptide with widespread antivirus activity in vitro and in vivo and illustrate the antiviral mechanism. Methods: Antiviral peptide library designed from mouse beta defensins was synthesized by the company. Recombinant beta defensin was obtained from E. coli. Antiviral activity in vitro was assayed by plaque assay, qPCR. Antiviral activity in vivo was detected by animal challenge with 2009 pandemic H1N1 influenza A virus. The antiviral mechanism was assayed by western blot, ELISA, and qPCR. Conclusions: We identify a new peptide which has widespread effects against multiple viruses (H1N1, H5N1, H7N9, MERS-CoV) in vitro and has efficient antivirus activity in vivo. This peptide inhibits viral entry into target cells and subsequently blocks viral replication. The in vivo study of the antiviral peptide against other viral infections and the investigation of its more detail antiviral mechanism are ongoing.

Keywords: antiviral peptide, defensin, Influenza A virus, mechanism

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820 Antimicrobial Evaluation of Polyphenon 60 and Ciprofloxacin Loaded Nano Emulsion against Uropathogenic Escherichia coli Bacteria and Its in vivo Analysis

Authors: Atinderpal Kaur, Shweta Dang


Our aim is to develop a nanoemulsion-based delivery system containing polyphenon 60 (P60) and ciprofloxacin (Cipro) for intravaginal delivery to treat urinary tract infection. In the present study Polyphenon 60 (P60) and ciprofloxacin (Cipro) were loaded in a single nano emulsion (NE) system via ultra-sonication technique and characterized for particle size, in vitro release and antibacterial efficacy against Bcl-2 level Escherichia coli bacteria. To determine in vivo pharmacokinetic parameters and intravaginal transportation of NE, gamma scintigraphy and biodistribution study was conducted by radiolabelling NE with technetium pertechnetate (99mTc). The preliminary antibacterial investigation showed synergy between these compounds with FICindex of 0.42. The developed formulation showed zeta potential +55.3 and particle size of 151.7 nm, with PDI of 0.196. The in vitro release percentage of P60 at the end of 7th hours was 94.8 ± 0.9 % whereas the release for Cipro was 75.1± 0.15 % in simulated vaginal media. MBC was identified and the findings demonstrated that in both ESBL (Extended Spectrum β- lactamase) and MBL (Metallo β- lactamase) cultures the P60+Cipro NE showed inhibition of growth of all the isolates at 2 mg/ml dilutions. The percentage per gram of radiolabelled drug was found (3.50±0.26) and (3.81±0.30) in kidney and urinary bladder, respectively at 3 h. From the findings, it was concluded that the developed P60+Cipro NE was transported efficiently throughout the target organs, had long duration of action and high biocompatibility via intravaginal administration as compared to oral administration.

Keywords: ciprofloxacin, gamma scintigraphy, intravaginal drug delivery, Polyphenon 60

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819 A Comparative Evaluation of Antioxidant Activity of in vivo and in vitro Raised Holarrhena antidysenterica Linn.

Authors: Gayatri Nahak, Satyajit Kanungo, Rajani Kanta Sahu


Holarrhena antidysenterica Linn. (Apocynaceae) is a typical Indian medicinal plant popularly known as “Indrajav”. Traditionally the plant has been considered a popular remedy for the treatment of dysentery, diarrhea, intestinal worms and the seeds of this plant are also used as an anti-diabetic remedy. In the present study axillary shoot multiplication, callus induction and shoot regeneration from callus culture were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. Then in vivo and in vitro grown healthy plants were selected for study of antioxidant activity through DPPH and OH methods. Significantly higher antioxidant activity and phenol contents were observed in vitro raised plant in comparison to in vivo plants. The findings indicated the greater amount of phenolic compounds leads to more potent radical scavenging effect as shown in in vitro raised plant in comparison to in vivo plants which showed the ability to utilize tissue culture techniques towards development of desired bioactive metabolites from in vitro culture as an alternative way to avoid using endangered plants in pharmaceutical purposes.

Keywords: Holarrhena antidysenterica, in vitro, in vivo, antioxidant activity

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818 Vascularized Adipose Tissue Engineering by Using Adipose ECM/Fibroin Hydrogel

Authors: Alisan Kayabolen, Dilek Keskin, Ferit Avcu, Andac Aykan, Fatih Zor, Aysen Tezcaner


Adipose tissue engineering is a promising field for regeneration of soft tissue defects. However, only very thin implants can be used in vivo since vascularization is still a problem for thick implants. Another problem is finding a biocompatible scaffold with good mechanical properties. In this study, the aim is to develop a thick vascularized adipose tissue that will integrate with the host, and perform its in vitro and in vivo characterizations. For this purpose, a hydrogel of decellularized adipose tissue (DAT) and fibroin was produced, and both endothelial cells and adipocytes that were differentiated from adipose derived stem cells were encapsulated in this hydrogel. Mixing DAT with fibroin allowed rapid gel formation by vortexing. It also provided to adjust mechanical strength by changing fibroin to DAT ratio. Based on compression tests, gels of DAT/fibroin ratio with similar mechanical properties to adipose tissue was selected for cell culture experiments. In vitro characterizations showed that DAT is not cytotoxic; on the contrary, it has many natural ECM components which provide biocompatibility and bioactivity. Subcutaneous implantation of hydrogels resulted with no immunogenic reaction or infection. Moreover, localized empty hydrogels gelled successfully around host vessel with required shape. Implantations of cell encapsulated hydrogels and histological analyses are under study. It is expected that endothelial cells inside the hydrogel will form a capillary network and they will bind to the host vessel passing through hydrogel.

Keywords: adipose tissue engineering, decellularization, encapsulation, hydrogel, vascularization

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817 Nano-Particle of π-Conjugated Polymer for Near-Infrared Bio-Imaging

Authors: Hiroyuki Aoki


Molecular imaging has attracted much attention recently, which visualizes biological molecules, cells, tissue, and so on. Among various in vivo imaging techniques, the fluorescence imaging method has been widely employed as a useful modality for small animals in pre-clinical researches. However, the higher signal intensity is needed for highly sensitive in vivo imaging. The objective of the current study is the development of a fluorescent imaging agent with high brightness for the tumor imaging of a mouse. The strategy to enhance the fluorescence signal of a bio-imaging agent is the increase of the absorption of the excitation light and the fluorescence conversion efficiency. We developed a nano-particle fluorescence imaging agent consisting of a π-conjugated polymer emitting a fluorescence signal in a near infrared region. A large absorption coefficient and high emission intensity at a near infrared optical window for biological tissue enabled highly sensitive in vivo imaging with a tumor-targeting ability by an EPR (enhanced permeation and retention) effect. The signal intensity from the π-conjugated fluorescence imaging agent is larger by two orders of magnitude compared to a quantum dot, which has been known as the brightest imaging agent. The π-conjugated polymer nano-particle would be a promising candidate in the in vivo imaging of small animals.

Keywords: fluorescence, conjugated polymer, in vivo imaging, nano-particle, near-infrared

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816 Development of Polymer Nano-Particles as in vivo Imaging Agents for Photo-Acoustic Imaging

Authors: Hiroyuki Aoki


Molecular imaging has attracted much attention to visualize a tumor site in a living body on the basis of biological functions. A fluorescence in vivo imaging technique has been widely employed as a useful modality for small animals in pre-clinical researches. However, it is difficult to observe a site deep inside a body because of a short penetration depth of light. A photo-acoustic effect is a generation of a sound wave following light absorption. Because the sound wave is less susceptible to the absorption of tissues, an in vivo imaging method based on the photoacoustic effect can observe deep inside a living body. The current study developed an in vivo imaging agent for a photoacoustic imaging method. Nano-particles of poly(lactic acid) including indocyanine dye were developed as bio-compatible imaging agent with strong light absorption. A tumor site inside a mouse body was successfully observed in a photo-acoustic image. A photo-acoustic imaging with polymer nano-particle agent would be a powerful method to visualize a tumor.

Keywords: nano-particle, photo-acoustic effect, polymer, dye, in vivo imaging

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815 Formulation Development and Characterization of Oligonucleotide Containing Chitosan Nanoparticles

Authors: Gyati Shilakari Asthana, Abhay Asthana


Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1 and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

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814 The Effect of Surface Modified Nano-Hydroxyapatite Incorporation into Polymethylmethacrylate Cement on Biocompatibility and Mechanical Properties

Authors: Yu-Shan Wu, Po-Liang Lai, I-Ming Chu


Poly(methylmethacrylate)(PMMA) is the most frequently used bone void filler for vertebral augmentation in osteoporotic fracture. PMMA bone cement not only exhibits strong mechanical properties but also can fabricate according to the shape of bone defect. However, the adhesion between the PMMA-based cement and the adjacent bone is usually weak and as PMMA bone cement is inherently bioinert. The combination of bioceramics and polymers as composites may increase cell adhesion and improve biocompatibility. The nano-hydroxyapatite(HAP) not only plays a significant role in maintaining the properties of the natural bone but also offers a favorable environment for osteoconduction, protein adhesion, and osteoblast proliferation. However, defects and cracks can form at the polymer/ceramics interface, resulting in uneven distribution of stress and subsequent inferior mechanical strength. Surface-modified HAP nano-crystals were prepared by chemically grafting poly(ε-caprolactone)(PCL) on surface-modified nano-HAP surface to increase the affinity of polymer/ceramic phases .Thus, incorporation of surface-modified nano-hydroxyapatite (EC-HAP) may not only improve the interfacial adhesion between cement and bone and between nanoparticles and cement, but also increase biocompatibility. In this research, PMMA mixing with 0, 5, 10, 15, 20, 25 and 30 wt% EC-HAP were examined. MC3T3-E1 cells were used for the biological evaluation of the response to the cements in vitro. Morphology was observed using scanning electron microscopy (SEM). Mechanical properties of HAP/PMMA and EC-HAP/PMMA cement were investigated by compression test. Surface wettability of the cements was measured by contact angles.

Keywords: bone cement, biocompatibility, nano-hydroxyapatite, polycaprolactone, PMMA, surface grafting

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813 Comparative Study of Antioxidant Activity in in vivo and in vitro Samples of Purple Greater Yam (Dioscorea alata L).

Authors: Sakinah Abdullah, Rosna Mat Taha


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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812 Self-Organized TiO₂–Nb₂O₅–ZrO₂ Nanotubes on β-Ti Alloy by Anodization

Authors: Muhammad Qadir, Yuncang Li, Cuie Wen


Surface properties such as topography and physicochemistry of metallic implants determine the cell behavior. The surface of titanium (Ti)-based implant can be modified to enhance the bioactivity and biocompatibility. In this study, a self-organized titania–niobium pentoxide–zirconia (TiO₂–Nb₂O₅–ZrO₂) nanotubular layer on β phase Ti35Zr28Nb alloy was fabricated via electrochemical anodization. Energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and water contact angle measurement techniques were used to investigate the nanotubes dimensions (i.e., the inner and outer diameters, and wall thicknesses), microstructural features and evolution of the hydrophilic properties. The in vitro biocompatibility of the TiO₂–Nb₂O₅–ZrO₂ nanotubes (NTs) was assessed by using osteoblast cells (SaOS2). Influence of anodization parameters on the morphology of TiO₂–Nb₂O₅–ZrO₂ NTs has been studied. The results indicated that the average inner diameter, outer diameter and the wall thickness of the TiO₂–Nb₂O₅–ZrO₂ NTs were ranged from 25–70 nm, 45–90 nm and 5–13 nm, respectively, and were directly influenced by the applied voltage during anodization. The average inner and outer diameters of NTs increased with increasing applied voltage, and the length of NTs increased with increasing anodization time and water content of the electrolyte. In addition, the size distribution of the NTs noticeably affected the hydrophilic properties and enhanced the biocompatibility as compared with the uncoated substrate. The results of this study could be considered for developing nano-scale coatings for a wide range of biomedical applications.

Keywords: Titanium alloy, TiO₂–Nb₂O₅–ZrO₂ nanotubes, anodization, surface wettability, biocompatibility

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811 Preparation and Characterization of Chitosan Nanoparticles for Delivery of Oligonucleotides

Authors: Gyati Shilakari Asthana, Abhay Asthana, Dharm Veer Kohli, Suresh Prasad Vyas


Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self-assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1, and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, the particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

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810 Identification and Characterization of in Vivo, in Vitro and Reactive Metabolites of Zorifertinib Using Liquid Chromatography Lon Trap Mass Spectrometry

Authors: Adnan A. Kadi, Nasser S. Al-Shakliah, Haitham Al-Rabiah


Zorifertinib is a novel, potent, oral, a small molecule used to treat non-small cell lung cancer (NSCLC). zorifertinib is an Epidermal Growth Factor Receptor (EGFR) inhibitor and has good blood–brain barrier permeability for (NSCLC) patients with EGFR mutations. zorifertinibis currently at phase II/III clinical trials. The current research reports the characterization and identification of in vitro, in vivo and reactive intermediates of zorifertinib. Prediction of susceptible sites of metabolism and reactivity pathways (cyanide and GSH) of zorifertinib were performed by the Xenosite web predictor tool. In-vitro metabolites of zorifertinib were performed by incubation with rat liver microsomes (RLMs) and isolated perfused rat liver hepatocytes. Extraction of zorifertinib and it's in vitro metabolites from the incubation mixtures were done by protein precipitation. In vivo metabolism was done by giving a single oral dose of zorifertinib(10 mg/Kg) to Sprague Dawely rats in metabolic cages by using oral gavage. Urine was gathered and filtered at specific time intervals (0, 6, 12, 18, 24, 48, 72,96and 120 hr) from zorifertinib dosing. A similar volume of ACN was added to each collected urine sample. Both layers (organic and aqueous) were injected into liquid chromatography ion trap mass spectrometry(LC-IT-MS) to detect vivozorifertinib metabolites. N-methyl piperizine ring and quinazoline group of zorifertinib undergoe metabolism forming iminium and electro deficient conjugated system respectively, which are very reactive toward nucleophilic macromolecules. Incubation of zorifertinib with RLMs in the presence of 1.0 mM KCN and 1.0 Mm glutathione were made to check reactive metabolites as it is often responsible for toxicities associated with this drug. For in vitro metabolites there were nine in vitro phase I metabolites, four in vitro phase II metabolites, eleven reactive metabolites(three cyano adducts, five GSH conjugates metabolites, and three methoxy metabolites of zorifertinib were detected by LC-IT-MS. For in vivo metabolites, there were eight in vivo phase I, tenin vivo phase II metabolitesofzorifertinib were detected by LC-IT-MS. In vitro and in vivo phase I metabolic pathways wereN- demthylation, O-demethylation, hydroxylation, reduction, defluorination, and dechlorination. In vivo phase II metabolic reaction was direct conjugation of zorifertinib with glucuronic acid and sulphate.

Keywords: in vivo metabolites, in vitro metabolites, cyano adducts, GSH conjugate

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809 The Effect of Substitution of CaO/MgO and CaO/SrO on in vitro Bioactivity of Sol-Gel Derived Bioactive Glass

Authors: Zeinab Hajifathali, Moghan Amirhosseinian


This study had two main aims: firstly, to determine how the individual substitution of CaO/MgO and CaO/SrO can affect the in vitro bioactivity of sol-gel derived substituted 58S bioactive glass (BG) and secondly to introduce a composition in the 60SiO2–(36-x)CaO–4P2O5–(x)MgO and 60SiO2–(36-x)CaO–4P2O5–(x)SrO quaternary systems (where x= 0, 5, 10 mol.%) with enhanced biocompatibility, alkaline phosphatase (ALP) activity, and more efficient antibacterial activity against MRSA bacteria. Results showed that both magnesium-substituted bioactive glasses (M-BGs) and strontium- substituted bioactive glasses (S-BGs) retarded the Hydroxyapatite (HA) formation. Meanwhile, magnesium had more pronounced effect. The 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and ALP assays revealed that the presence of moderate amount (5 mol%) of Mg and Sr had a stimulating effect on increasing of both proliferation and differentiation of MC3T3-E1 cells. Live dead and Dapi/actin staining revealed both substitution of CaO/MgO and CaO/SrO resulted in more biocompatibility and stimulation potential of the MC3T3 cells compared with control. Taken together, among all of the synthesized magnesium substituted (MBGs) and strontium substituted (SBGs), the sample 58- BG with 5 mol% CaO/MgO substitution (BG-5M) was considered as a multifunctional biomaterial in bone tissue regeneration field with enhanced biocompatibility, ALP activity as well as the highest antibacterial efficiency against methicillin-resistant Staphylococcus aureus (MRSA) bacteria.

Keywords: apatite, alkaline earth, bioactivity, biomedical applications, Sol-gel

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808 Starch Incorporated Hydroxyapatite/Chitin Nanocomposite as a Novel Bone Construct

Authors: Reshma Jolly, Mohammad Shakir, Mohammad Shoeb Khan, Noor E. Iram


A nanocomposite system integrating hydroxyapatite, chitin and starch (n-HA/CT/ST) has been synthesized via co-precipitation approach at room temperature, addressing the issues of biocompatibility, mechanical strength and cytotoxicity required for Bone tissue engineering. The interactions, crystallite size and surface morphology against n-HA/CT (nano-hydroxyapatite/chitin) nanocomposite have been obtained by correlating and comparing the results of FTIR, SEM, TEM and XRD. The comparative study of the bioactivity of n-HA/CT and n-HA/CT/ST nanocomposites revealed that the incorporation of starch as templating agent improved these properties in n-HA/CT/ST nanocomposite. The rise in thermal stability in n-HA/CT/ST nanocomposite as compared to n-HA/CT has been observed by comparing the TGA results. The comparison of SEM images of both the scaffolds indicated that the addition of ST influenced the surface morphology of n-HA/CT scaffold which appeared to be rougher and porous. The MTT assay on murine fibroblast L929 cells and in-vitro bioactivity of n-HA/CT/ST matrix referred superior non-toxic property of n-HA/CT/ST nanocomposite and higher possibility of osteo-integration in-vivo, respectively.

Keywords: bioactive, chitin, hyroxyapatite, nanocomposite

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807 In vitro and invivo Antioxidant Studies of Grewia crenata Leaves Extract in Albino Rats

Authors: A. N.Ukwuani, A. K. Abdulfatah


G. crenata is used locally for the treatment of fractured bones, wound healing and inflammatory conditions. In vitro and in vivo antioxidant activity of hydromethanolic extracts of the leaves of G. crenata were assessed. The phytochemical analysis shows the presence of phenols, flavonoids, saponins, cardiac glycosides and tannins. An in vitro quantitative analysis of phenols, flavonoids and tannins respectively were (164±1.20, 199±0.88 and 88.67±0.88 mg/100g FW). In vivo studies of hydromethanolic extract demonstrated a dose dependent increase in hepatic superoxide dismutase (1.14±0.14, 2.13±0.11, 2.55±0.11 U/mg Protein) with improvement in hepatic glutathione (6.98±0.42, 8.91±0.37, 11.07±0.46 µM/mg Protein) and Catalase (4.47±0.05, 6.24±0.02, 7.17±0.04 U/mg Protein) and Total protein (6.18±0.08, 6.69±0.18, 7.27±0.16 mg/ml) respectively at 100-300mg/kg body weight Grewia crenata leaves when compared to the control and standard drug. It can be concluded from the present findings of that G. crenata leaves possess antioxidant potential.

Keywords: Grewia crenata, antioxidant, hydromethanolic extract, in vivo, in vitro

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806 Synthesis, Characterization and Bioactivity of Methotrexate Conjugated Fluorescent Carbon Nanoparticles in vitro Model System Using Human Lung Carcinoma Cell Lines

Authors: Abdul Matin, Muhammad Ajmal, Uzma Yunus, Noaman-ul Haq, Hafiz M. Shohaib, Ambreen G. Muazzam


Carbon nanoparticles (CNPs) have unique properties that are useful for the diagnosis and treatment of cancer due to their precise properties like small size (ideal for delivery within the body) stability in solvent and tunable surface chemistry for targeted delivery. Here, highly fluorescent, monodispersed and water-soluble CNPs were synthesized directly from a suitable carbohydrate source (glucose and sucrose) by one-step acid assisted ultrasonic treatment at 35 KHz for 4 hours. This method is green, simple, rapid and economical and can be used for large scale production and applications. The average particle sizes of CNPs are less than 10nm and they emit bright and colorful green-blue fluorescence under the irradiation of UV-light at 365nm. The CNPs were characterized by scanning electron microscopy, fluorescent spectrophotometry, Fourier transform infrared spectrophotometry, ultraviolet-visible spectrophotometry and TGA analysis. Fluorescent CNPs were used as fluorescent probe and nano-carriers for anticancer drug. Functionalized CNPs (with ethylene diamine) were attached with anticancer drug-Methotrexate. In vitro bioactivity and biocompatibility of CNPs-drug conjugates was evaluated by LDH assay and Sulforhodamine B assay using human lung carcinoma cell lines (H157). Our results reveled that CNPs showed biocompatibility and CNPs-anticancer drug conjugates have shown potent cytotoxic effects and high antitumor activities in lung cancer cell lines. CNPs are proved to be excellent substitute for conventional drug delivery cargo systems and anticancer therapeutics in vitro. Our future studies will be more focused on using the same nanoparticles in vivo model system.

Keywords: carbon nanoparticles, carbon nanoparticles-methotrexate conjugates, human lung carcinoma cell lines, lactate dehydrogenase, methotrexate

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805 Self-Carried Theranostic Nanoparticles for in vitro and in vivo Cancer Therapy with Real-Time Monitoring of Drug Release

Authors: Jinfeng Zhang, Chun-Sing Lee


The use of different nanocarriers for delivering hydrophobic pharmaceutical agents to tumor sites has garnered major attention. Despite the merits of these nanocarriers, further studies are needed for improving their drug loading capacities (typically less than 10%) and reducing their potential systemic toxicity. So development of alternative self-carried nanodrug delivery strategies without using any inert carriers is highly desirable. In this study, we developed a self-carried theranostic curcumin (Cur) nanodrug for highly effective cancer therapy in vitro and in vivo with real-time monitoring of drug release. With a biocompatible C18PMH-PEG functionalization, the Cur nanoparticles (NPs) showed excellent dispersibility and outstanding stability in physiological environment, with drug loading capacity higher than 78 wt.%. Both confocal microscopy and flow cytometry confirmed the cellular fluorescent “OFF-ON” activation and real-time monitoring of Cur molecule release, showing its potential for cancer diagnosis. In vitro and in vivo experiments clearly show that therapeutic efficacy of the PEGylated Cur NPs is much better than that of free Cur. This self-carried theranostic strategy with real-time monitoring of drug release may open a new way for simultaneous cancer therapy and diagnosis.

Keywords: drug delivery, in vitro and in vivo cancer therapy, real-time monitoring, self-carried

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804 Biocompatibility and Electrochemical Assessment of Biomedical Ti-24Nb-4Zr-8Sn Produced by Spark Plasma Sintering

Authors: Jerman Madonsela, Wallace Matizamhuka, Akiko Yamamoto, Ronald Machaka, Brendon Shongwe


In this study, biocompatibility evaluation of nanostructured near beta Ti-24Nb-4Zr-8Sn (Ti2448) alloy with non-toxic elements produced utilizing Spark plasma sintering (SPS) of very fine microsized powders attained through mechanical alloying was performed. The results were compared with pure titanium and Ti-6Al-4V (Ti64) alloy. Cell proliferation test was performed using murine osteoblastic cells, MC3T3-E1 at two cell densities; 400 and 4000 cells/mL for 7 days incubation. Pure titanium took a lead under both conditions suggesting that the presence of other oxide layers influence cell proliferation. No significant difference in cell proliferation was observed between Ti64 and Ti2448. Potentiodynamic measurement in Hanks, 0.9% NaCl and cell culture medium showed no distinct difference on the anodic polarization curves of the three alloys, indicating that the same anodic reaction occurred on their surface but with different rates. However, Ti2448 showed better corrosion resistance in cell culture medium with a slightly lower corrosion rate of 2.96 nA/cm2 compared to 4.86 nA/cm2 and 5.62 nA/cm2 of Ti and Ti64 respectively. Ti2448 adsorbed less protein as compared to Ti and Ti64 though no notable difference in surface wettability was observed.

Keywords: biocompatibility, osteoblast, corrosion, surface wettability, protein adsorption

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803 Hepatoprotective Evaluation of Potent Antioxidant Fraction from Urtica dioica L.: In vitro and In vivo Studies

Authors: Bhuwan C. Joshi, Atish Prakash, Ajudhia N. Kalia


Ethnopharmacological relevance: The plant Urtica dioica L. (Urticaceae) is used in various diseases including hepatic ailments. Traditionally, the leaves and roots of the plant are used in jaundice. Objective: The aim of the present work was to evaluate hepatoprotective potential of potent antioxidant from Urtica dioica L. against CCl4 induced hepatotoxicity in-vitro and in-vivo model. Materials and methods: Antioxidant activity of hydro alcoholic extract and its fractions petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butanol fraction (NBF) and aqueous fraction (AF) were determined by DPPH radicals scavenging assay. Fractions were subjected to in-vitro cell line study. Further, the most potent fraction (EAF) was subjected to in-vivo study. The in-vivo hepatoprotective active fraction was chromatographed on silica column to isolate the bioactive constituent(s). Structure elucidation was done by using various spectrophotometric techniques like UV, IR, 1H NMR, 13C NMR and MS spectroscopy. Results and conclusion: The ethyl acetate fraction (EAF) of Urtica. dioica L. possessed the potent antioxidant activity viz. DPPH (IC50 78.99 ± 0.17 µg/ml). The in-vitro cell line study showed EAF prevented the cell damage. The EAF significantly attenuated the increased liver enzymes activities in serum and tissue. Column chromatography of most potent antioxidant fraction (EAF) leads to the isolation of 4-hydroxy-3-methoxy cinnamic acid which is responsible for its hepatoprotective potential. Hence, the present study suggests that EAF has significant antioxidant and hepatoprotective potential on CCl4 induced hepatotoxicity in-vitro and in-vivo.

Keywords: Urtica dioica L., antioxidant, HepG2 cell line, hepatoprotective

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802 Chemical and Physical Properties and Biocompatibility of Ti–6Al–4V Produced by Electron Beam Rapid Manufacturing and Selective Laser Melting for Biomedical Applications

Authors: Bing–Jing Zhao, Chang-Kui Liu, Hong Wang, Min Hu


Electron beam rapid manufacturing (EBRM) or Selective laser melting is an additive manufacturing process that uses 3D CAD data as a digital information source and energy in the form of a high-power laser beam or electron beam to create three-dimensional metal parts by fusing fine metallic powders together.Object:The present study was conducted to evaluate the mechanical properties ,the phase transformation,the corrosivity and the biocompatibility of Ti-6Al-4V by EBRM,SLM and forging technique.Method: Ti-6Al-4V alloy standard test pieces were manufactured by EBRM, SLM and forging technique according to AMS4999,GB/T228 and ISO 10993.The mechanical properties were analyzed by universal test machine. The phase transformation was analyzed by X-ray diffraction and scanning electron microscopy. The corrosivity was analyzed by electrochemical method. The biocompatibility was analyzed by co-culturing with mesenchymal stem cell and analyzed by scanning electron microscopy (SEM) and alkaline phosphatase assay (ALP) to evaluate cell adhesion and differentiation, respectively. Results: The mechanical properties, the phase transformation, the corrosivity and the biocompatibility of Ti-6Al-4V by EBRM、SLM were similar to forging and meet the mechanical property requirements of AMS4999 standard. a­phase microstructure for the EBM production contrast to the a’­phase microstructure of the SLM product. Mesenchymal stem cell adhesion and differentiation were well. Conclusion: The property of the Ti-6Al-4V alloy manufactured by EBRM and SLM technique can meet the medical standard from this study. But some further study should be proceeded in order to applying well in clinical practice.

Keywords: 3D printing, Electron Beam Rapid Manufacturing (EBRM), Selective Laser Melting (SLM), Computer Aided Design (CAD)

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801 Thermosensitive Hydrogel Development for Its Possible Application in Cardiac Cell Therapy

Authors: Lina Paola Orozco Marin, Yuliet Montoya Osorio, John Bustamante Osorno


Ischemic events can culminate in acute myocardial infarction by irreversible cardiac lesions that cannot be restored due to the limited regenerative capacity of the heart. Cell therapy seeks to replace these injured or necrotic cells by transplanting healthy and functional cells. The therapeutic alternatives proposed by tissue engineering and cardiovascular regenerative medicine are the use of biomaterials to mimic the native extracellular medium, which is full of proteins, proteoglycans, and glycoproteins. The selected biomaterials must provide structural support to the encapsulated cells to avoid their migration and death in the host tissue. In this context, the present research work focused on developing a natural thermosensitive hydrogel, its physical and chemical characterization, and the determination of its biocompatibility in vitro. The hydrogel was developed by mixing hydrolyzed bovine and porcine collagen at 2% w/v, chitosan at 2.5% w/v, and beta-glycerolphosphate at 8.5% w/w and 10.5% w/w in magnetic stirring at 4°C. Once obtained, the thermosensitivity and gelation time were determined, incubating the samples at 37°C and evaluating them through the inverted tube method. The morphological characterization of the hydrogels was carried out through scanning electron microscopy. Chemical characterization was carried out employing infrared spectroscopy. The biocompatibility was determined using the MTT cytotoxicity test according to the ISO 10993-5 standard for the hydrogel’s precursors using the fetal human ventricular cardiomyocytes cell line RL-14. The RL-14 cells were also seeded on the top of the hydrogels, and the supernatants were subculture at different periods to their observation under a bright field microscope. Four types of thermosensitive hydrogels were obtained, which differ in their composition and concentration, called A1 (chitosan/bovine collagen/beta-glycerolphosphate 8.5%w/w), A2 (chitosan/porcine collagen/beta-glycerolphosphate 8.5%), B1 (chitosan/bovine collagen/beta-glycerolphosphate 10.5%) and B2 (chitosan/porcine collagen/beta-glycerolphosphate 10.5%). A1 and A2 had a gelation time of 40 minutes, and B1 and B2 had a gelation time of 30 minutes at 37°C. Electron micrographs revealed a three-dimensional internal structure with interconnected pores for the four types of hydrogels. This facilitates the exchange of nutrients, oxygen, and the exit of metabolites, allowing to preserve a microenvironment suitable for cell proliferation. In the infrared spectra, it was possible to observe the interaction that occurs between the amides of polymeric compounds with the phosphate groups of beta-glycerolphosphate. Finally, the biocompatibility tests indicated that cells in contact with the hydrogel or with each of its precursors are not affected in their proliferation capacity for a period of 16 days. These results show the potential of the hydrogel to increase the cell survival rate in the cardiac cell therapies under investigation. Moreover, the results lay the foundations for its characterization and biological evaluation in both in vitro and in vivo models.

Keywords: cardiac cell therapy, cardiac ischemia, natural polymers, thermosensitive hydrogel

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