Search results for: ex vivo dentine model

Authors: M. Gowri, E. K. Girija, V. Ganesh

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Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate E. faecalis. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against E. faecalis. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on E. faecalis was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against E. faecalis. silver-curcumin nanoparticle exerted time kill effect. Further, SEM images of E. faecalis showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of E. faecalis and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Further, silver-curcumin nanoparticle was found to be hemocompatible, not cytotoxic to normal mammalian NIH 3T3 cells and non-mutagenic. Conclusion: The results of this study can pave the way for developing new antibacterial agents with well deciphered mechanisms of action and can be a promising antibacterial agent or medicament against root canal infection.

Keywords: ex vivo dentine model, inhibition of biofilm formation, root canal infection, silver-curcumin nanoparticle

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Authors: Behzad Babaei, B. Gangadhara Prusty

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The objective of this study is to assess the hypotheses that a restored tooth with a class II occlusal-distal (OD) cavity can be strengthened by designing an optimized cavity geometry, as well as selecting the composite restoration with optimized elastic moduli when there is a sharp de-bonded edge at the interface of the tooth and restoration. Methods: A scanned human maxillary molar tooth was segmented into dentine and enamel parts. The dentine and enamel profiles were extracted and imported into a finite element (FE) software. The enamel rod orientations were estimated virtually. Fifteen models for the restored tooth with different cavity occlusal depths (1.5, 2, and 2.5 mm) and internal cavity angles were generated. By using a semi-circular stone part, a 400 N load was applied to two contact points of the restored tooth model. The junctions between the enamel, dentine, and restoration were considered perfectly bonded. All parts in the model were considered homogeneous, isotropic, and elastic. The quadrilateral and triangular elements were employed in the models. A mesh convergence analysis was conducted to verify that the element numbers did not influence the simulation results. According to the criteria of a 5% error in the stress, we found that a total element number of over 14,000 elements resulted in the convergence of the stress. A Python script was employed to automatically assign 2-22 GPa moduli (with increments of 4 GPa) for the composite restorations, 18.6 GPa to the dentine, and two different elastic moduli to the enamel (72 GPa in the enamel rods’ direction and 63 GPa in perpendicular one). The linear, homogeneous, and elastic material models were considered for the dentine, enamel, and composite restorations. 108 FEA simulations were successively conducted. Results: The internal cavity angles (α) significantly altered the peak maximum principal stress at the interface of the enamel and restoration. The strongest structures against the contact loads were observed in the models with α = 100° and 105. Even when the enamel rods’ directional mechanical properties were disregarded, interestingly, the models with α = 100° and 105° exhibited the highest resistance against the mechanical loads. Regarding the effect of occlusal cavity depth, the models with 1.5 mm depth showed higher resistance to contact loads than the model with thicker cavities (2.0 and 2.5 mm). Moreover, the composite moduli in the range of 10-18 GPa alleviated the stress levels in the enamel. Significance: For the class II OD cavity models in this study, the optimal geometries, composite properties, and occlusal cavity depths were determined. Designing the cavities with α ≥100 ̊ was significantly effective in minimizing peak stress levels. The composite restoration with optimized properties reduced the stress concentrations on critical points of the models. Additionally, when more enamel was preserved, the sturdier enamel-restoration interface against the mechanical loads was observed.

Keywords: dental composite restoration, cavity geometry, finite element approach, maximum principal stress

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Authors: M. Gowri, E. K. Girija, V. Ganesh

Abstract:

Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate C. albicans. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against Candida albicans. Detailed molecular studies were carried out with silver-curcumin nanoparticle on C. albicans pathogenicity. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against C. albicans. Silver-curcumin nanoparticle exerted time kill effect and post antifungal effect. When used in combination with fluconazole or nystatin, silver-curcumin nanoparticle revealed a minimum inhibitory concentration (MIC) decrease for both drugs used. In-depth molecular studies with silver-curcumin nanoparticle on C. albicans showed that silver-curcumin nanoparticle inhibited yeast to hyphae (Y-H) conversion. Further, SEM images of C. albicans showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of C. albicans and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Conclusion: The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Keywords: C. albicans, ex vivo dentine model, inhibition of biofilm formation, root canal infection, yeast to hyphae conversion inhibition

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

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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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Authors: Zuliani Mahmood, Thirumulu Ponnuraj Kannan, Yean Yean Chan, Salahddin A. Al-Hudhairy

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Caries is the most common childhood disease which develops due to disturbances in the physiological equilibrium in the dental plaque resulting in demineralization of tooth structures. Plaque and dentine samples were collected from three different tooth surfaces representing caries progression (intact, over carious lesion and dentine) in children with early childhood caries (ECC, n=36). In caries free (CF) children, plaque samples were collected from sound tooth surfaces at baseline and after one year (n=12). The genomic DNA was extracted from all samples and subjected to 16S rRNA PCR amplification. The end products were cloned into pCR®2.1-TOPO® Vector. Five randomly selected positive clones collected from each surface were sent for sequencing. Identification of the bacterial clones was performed using BLAST against GenBank database. In the ECC group, the frequency of Lactobacillus sp. detected was significantly higher in the dentine surface (p = 0.031) than over the cavitated lesion. The highest frequency of bacteria detected in the intact surfaces was Fusobacterium nucleatum subsp. polymorphum (33.3%) while Streptococcus mutans was detected over the carious lesions and dentine surfaces at a frequency of 33.3% and 52.7% respectively. Fusobacterium nucleatum subsp. polymorphum was also found to be highest in the CF group (41.6%). Follow up at the end of one year showed that the frequency of Corynebacterium matruchotii detected was highest in those who remained caries free (16.6%), while Porphyromonas catoniae was highest in those who developed caries (25%). In conclusion, Streptococcus mutans and Porphyromonas catoniae are strongly associated with caries progression, while Lactobacillus sp. is restricted to deep carious lesions. Fusobacterium nucleatum subsp. polymorphum and Corynebacterium matruchotii may play a role in sustaining the healthy equilibrium in the dental plaque. These identified bacteria show promise as potential biomarkers in diagnosis which could help in the management of dental caries in children.

Keywords: early childhood caries, genotypic identification, oral bacteria, 16S rRNA

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Authors: Pompa Marcello, Mauro Capocelli, Vincenzo Piemonte

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This work focuses on a mathematical model able to describe the gastro-intestinal physiology and providing a rational tool for the design of an artificial gastro-intestinal system. This latter is mainly devoted to analyse the absorption and bioavailability of drugs and nutrients through in vitro tests in order to overcome (or, at least, to partially replace) in vivo trials. The provided model realizes a conjunction ring (with extended prediction capability) between in vivo tests and mechanical-laboratory models emulating the human body. On this basis, no empirical equations controlling the gastric emptying are implemented in this model as frequent in the cited literature and all the sub-unit and the related system of equations are physiologically based. More in detail, the model structure consists of six compartments (stomach, duodenum, jejunum, ileum, colon and blood) interconnected through pipes and valves. Paracetamol, Ketoprofen, Irbesartan and Ketoconazole are considered and analysed in this work as reference drugs. The mathematical model has been validated against in vivo literature data. Results obtained show a very good model reliability and highlight the possibility to realize tailored simulations for different couples patient-drug, including food adsorption dynamics.

Keywords: gastro-intestinal model, drugs bioavailability, paracetamol, ketoprofen

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

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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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Authors: Venoos Iman, Suzita Mohd Noor, Syam Mohan, Mohamad Ibrahim Noordin

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Girinimbine, a carbazole alkaloid was isolated from the stem bark and root of Murraya koenigii and its structure and purity was identified by HPLC and LC-MS. Here we report that Girinimbine strongly inhibit angiogenesis activity both in vitro and in vivo. MTT result showed that girinimbine inhibits cell proliferation of the HUVECS cell line in vitro. Result of endothelial cell invasion, migration, tube formation and wound healing assays also demonstrated significant time and does dependent inhibition by girinimbine. Moreover, girinibine mediates its anti-angiogenic activity through up- and down-regulation of angiogenic and anti-aniogenic proteins. Furthermore, anti-angiogenic potential of girinimbine was evidenced in vivo on zebrafish model. Girinimbine inhibited neo-vessels formation in zebrafish embryos during 24 hours exposure time. Together, these results demonstrated for the first time that girinimbine could effectively suppress angiogenesis and strongly suggest that it might be a novel angiogenesis inhibitor.

Keywords: anti-angiogenic, carbazole alkaloid, girinimbine, zebrafish

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Authors: Hanjun Zhao, Ke Zhang, Bojian Zheng

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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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Authors: Essia Sebai, Amel Abidi, Hayet benyeddem, Akkari Hafidh

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Herbal extracts are of particular interest to the drug industry; essential oil with significant anthelmintic activity has the potential to be used as an alternative to conventional chemical drugs. In the present study, we describe the chemical profile of Laurus nobilis essential oil (EO), the in vitro anthelmintic activity of laurel oil against Haemonchus contortus and its in vivo anthelmintic effect against the murine helminth parasite model Heligmosomoides polygyrus. The chromatographic profile of L. nobilis (EO) extracted from the leaves of L. nobilis has shown the presence of monoterpenes 1,8-cineol (Eucalyptol) (29.47%), D-Limonène (18.51%) and Linalool (10.84%) in high fractions. The in vitro anthelmintic potential was expressed by an ovicidal effect against H. contortus egg hatching with an inhibition value of 3.23 mg/mL and 87.5% of immobility of adult worms after 8 hours of exposure to 8 mg/mL of L. nobilis EO. Regarding the in vivo anthelmintic potential, L. nobilis (EO) at 2400 mg/kg completely eliminated the egg output of H. polygyrus after seven days of oral treatment, together with a 79.2% of reduction in total worm counts. Based on the obtained funding, L. nobilis EO showed promising in vitro and in vivo anthelmintic capacities against gastrointestinal parasites.

Keywords: lauris nobilis, anthelmintic, haemonchus, pylogyrus

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Authors: Nicoleta Simona Vedeanu, Rares Stiufiuc, Dana Alina Magdas

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A detailed knowledge of the teeth structure is mandatory to understand and explain the defects and the dental pathology, but especially to take a correct decision regarding dental prophylaxis and treatment. The present work is an alternative study to the traditional investigation methods used in dentistry, a study based on the use of modern, sensitive physical methods to investigate human enamel and dentin. For the present study, several teeth collected from patients of different ages were used for structural and dietary investigation. The samples were investigated by Raman spectroscopy for the molecular structure analysis of dentin and enamel, atomic force microscopy (AFM) to view the dental topography at the micrometric size and mass spectrometry for isotopic ratios as a fingerprint of patients’ personal diet. The obtained Raman spectra and their interpretation are in good correlation with the literature and may give medical information by comparing affected dental structures with healthy ones. AFM technique gave us the possibility to study in details the dentin and enamel surface to collect information about dental hardness or dental structural changes. δ¹³C values obtained for the studied samples can be classified in C4 category specific to young people and children diet (sweets, cereals, juices, pastry). The methods used in this attempt furnished important information about dentin and enamel structure and dietary habits and each of the three proposed methods can be extended at a larger level in the study of the teeth structure.

Keywords: AFM, dentine, enamel, Raman spectroscopy

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Authors: Huixin Wei, Shibin Wang, Linan Li, Lei Zhou, Xinhao Tu

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Facial skin is a biomedical material with complex mechanical properties of anisotropy, viscoelasticity, and hyperelasticity. The mechanical properties of facial skin are crucial for a number of applications including facial plastic surgery, animation, dermatology, cosmetic industry, and impact biomechanics. Skin is a complex multi-layered material which can be broadly divided into three main layers, the epidermis, the dermis, and the hypodermis. Collagen fibers account for 75% of the dry weight of dermal tissue, and it is these fibers which are responsible for the mechanical properties of skin. Many research on the anisotropic mechanical properties are mainly concentrated on in vitro, but there is a great difference between in vivo and in vitro for mechanical properties of the skin. In this study, we presented a method to measure the mechanical properties of facial skin in vivo. Digital image correlation (DIC) and indentation tests were used to obtain the experiment data, including the deformation of facial surface and indentation force-displacement curve. Then, the experiment was simulated using a finite element (FE) model. Application of Computed Tomography (CT) and reconstruction techniques obtained the real tissue geometry. A three-dimensional FE model of facial skin, including a bi-layer system, was obtained. As the epidermis is relatively thin, the epidermis and dermis were regarded as one layer and below it was hypodermis in this study. The upper layer was modeled as a Gasser-Ogden-Holzapfel (GOH) model to describe hyperelastic and anisotropic behaviors of the dermis. The under layer was modeled as a linear elastic model. In conclusion, the material properties of two-layer were determined by minimizing the error between the FE data and experimental data.

Keywords: facial skin, indentation test, finite element, digital image correlation, computed tomography

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Authors: Gayatri Nahak, Satyajit Kanungo, Rajani Kanta Sahu

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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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Authors: Hiroyuki Aoki

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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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Authors: Hiroyuki Aoki

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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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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: 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: Kun Chun, Jin-Chun Heo, Sang-Han Lee

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Asthma is a chronic airway inflammatory disease characterized by the infiltration of inflammatory cells and tissue remodeling. In this study, we examined the anti-asthmatic activity of a derivative of L-allo threonine by in vitro and in vivo anti-asthmatic assays. Ovalbumin (OVA)-induced C57BL/6 mice were used to analyze lung inflammation and cytokine expressions for exhibiting anti-atopic activity of the derivative. LX519290, a derivative of L-allo threonine, induced an increased IFN-γ and a decreased IL-10 mRNA level. This compound exhibited potent anti-asthmatic activity by decreasing immune cell infiltration in the lung, and IL-4 and IL-13 cytokine levels in the serum of OVA-induced mice. These results indicated that chronic airway injury was decreased by LX519290. We also assessed that LX519290 inhibits infiltration of immune cell, mucus release and cytokine expression in an in vivo model. Our results collectively suggest that the L-allo threonine is effective in alleviating asthmatic symptoms by treating inflammatory factors in the lung.

Keywords: asthma, L -allo threonine, LX519290, mice

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Authors: Rodolfo Alfonso, David Alonso, Albin Garcia, Jose Luis Alonso

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Recently a new kilovoltage radiotherapy unit model Xstrahl 200 - donated to the INOR´s Department of Radiotherapy (DR-INOR) in the framework of a IAEA's technical cooperation project- has been commissioned. This unit is able to treat shallow and low deep laying lesions, as it provides 8 discrete beam qualities, from 40 to 200 kV. As part of the patient-specific quality assurance program established at DR-INOR for external beam radiotherapy, it has been recommended to implement in vivo dose measurements (IVD), as they allow effectively discovering eventual errors or failures in the radiotherapy process. For that purpose a radio-photoluminescence (RPL) dosimetry system, model XXX, -also donated to DR-INOR by the same IAEA project- has been studied and commissioned. Main dosimetric parameters of the RPL system, such as reproducibility, linearity, and filed size influence were assessed. In a similar way, the response of radiochromic EBT3 type film was investigated for purposes of IVD. Both systems were calibrated in terms of entrance surface dose. Results of the dosimetric commissioning of RPL and EBT3 for IVD, and their pre-clinical implementation through end-to-end test cases are presented. The RPL dosimetry seems more recommendable for hyper-fractionated schemes with larger fields and curved patient contours, as those in chest wall irradiations, where the use of more than one dosimeter could be required. The radiochromic system involves smaller corrections with field size, but it sensibility is lower; hence it is more adequate for hypo-fractionated treatments with smaller fields.

Keywords: glass dosimetry, in vivo dosimetry, kilovotage radiotherapy, radiochromic dosimetry

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Authors: Jasbir Arora, Indu Talwar, Daisy Sahni, Vidya Rattan

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Introduction: Age estimation is a crucial step to build the identity of a person, both in case of deceased and alive. In adults, age can be estimated on the basis of six regressive (Attrition, Secondary dentine, Dentine transparency, Root resorption, Cementum apposition and Periodontal Disease) changes in teeth qualitatively using scoring system and quantitatively by micrometric method. The present research was designed to establish the reliability of qualitative (method 1) and quantitative (method 2) of age estimation among North Indians and to compare the efficacy of these two methods. Method: 250 single-rooted extracted teeth (18-75 yrs.) were collected from Department of Oral Health Sciences, PGIMER, Chandigarh. Before extraction, periodontal score of each tooth was noted. Labiolingual sections were prepared and examined under light microscope for regressive changes. Each parameter was scored using Gustafson’s 0-3 point score system (qualitative), and total score was calculated. For quantitative method, each regressive change was measured quantitatively in form of 18 micrometric parameters under microscope with the help of measuring eyepiece. Age was estimated using linear and multiple regression analysis in Gustafson’s method and Kedici’s method respectively. Estimated age was compared with actual age on the basis of absolute mean error. Results: In pooled data, by Gustafson’s method, significant correlation (r= 0.8) was observed between total score and actual age. Total score generated an absolute mean error of ±7.8 years. Whereas, for Kedici’s method, a value of correlation coefficient of r=0.5 (p<0.01) was observed between all the eighteen micrometric parameters and known age. Using multiple regression equation, age was estimated, and an absolute mean error of age was found to be ±12.18 years. Conclusion: Gustafson’s (qualitative) method was found to be a better predictor for age estimation among North Indians.

Keywords: forensic odontology, age estimation, North India, teeth

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

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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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Authors: Seham A. S. Mohamed, Sarah R. Baker, Christopher Deery, Mario V. Vettore

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Background: The link between oral health conditions and school performance and attendance remain unclear among Middle Eastern children. The association has been studied extensively in the Western region; however, several concerns have been raised regarding the reliability and validity of measures, low quality of studies, inadequate inclusion of potential confounders, and the lack of a conceptual framework. These limitations have meant that, to date, there has been no detailed understanding of the association or of the key social, clinical, behavioural and parental factors which may impact the association. Aim: To examine the association between oral health conditions and children’s school performance and attendance at Grade 2 in Muharraq city in the Kingdom of Bahrain using Heilmann et al.’s (2015) life course framework for oral health. Objectives: To (1) describe the prevalence of oral health conditions among 7-8 years old schoolchildren in the city of Muharraq; (2) analyse the social, biological, behavioural, and parental pathways that link early and current life exposures with children’s current oral health status; (3) examine the association between oral health conditions and school performance and attendance among schoolchildren; (4) explore the early and current life course social, biological, behavioural and parental factors associated with children’s school outcomes. Design: A time-ordered-cross-sectional study was conducted with 466 schoolchildren aged 7-8 years and their parents from Muharraq city in KoB. Data were collected through parents’ self-administered questionnaires, children’s face-face interviews, and dental clinical examinations. Outcome variables, including school performance and school attendance data, were obtained from the parents and school records. The data were analysed using structural equation modelling (SEM). Results: Dental caries, the consequence of dental caries (PUFA/pufa), and enamel developmental defects (EDD) prevalence were 93.4%, 25.7%, and 17.2%, respectively. The findings from the SEM showed that children born in families with high SES were less likely to suffer from dentine dental caries (β= -0.248) and more likely to earn high school performance (β= 0.136) at 7-8 years of age in Muharraq. From the current life course of children, the dental plaque was associated significantly and directly with enamel caries (β= 0.094), dentine caries (β= 0.364), treated teeth (filled or extracted because of dental caries) (β= 0.121), and indirectly associated with dental pain (β= 0.057). Further, dentine dental caries was associated significantly and directly with low school performance (β= -0.155). At the same time, the dental plaque was indirectly associated with low school performance via dental caries (β = −0.044). Conversely, treated teeth were associated directly with high school performance (β= 0.100). Notably, none of the OHCs, biological, SES, behavioural, or parental conditions was related to school attendance in children. Conclusion: The life course approach was adequate to examine the role of OHCs on children’s school performance and attendance. Birth and current (7-8-year-olds) social factors were significant predictors of poor OH and poor school performance.

Keywords: dental caries, life course, Bahrain, school outcomes

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Authors: Mahesh M. Patil, K. A. Anu-Appaiah

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Overweight and obesity is a serious medical problem, increasing in prevalence, and affecting millions worldwide. Investigators have been trying from decades to articulate the burden of obesity and related risk factors. To answer this problem, we suggest a new therapeutic anti-obesity compounds from Garcinia xanthochymus fruit. However, there is little published scientific information on non-hydroxycitric acid Garcinia species. Our findings include biochemical characterization of the fruit; in vivo toxicity and bio-efficacy study of G. xanthochymus in high fat diet wistar rat model. We observed that Garcinia pericarp is a rich source of organic acids, polyphenols, mono- (40.63%) and poly-unsaturated fatty acids (16.45%; omega-3: 10.02%). Toxicological studies have showed that Garcinia is safe and had no observed adverse effect level up to 400 mg/kg/day. Body weight and food intake was significantly (P<0.05) reduced in oral gavage treated rats (sonicated Garcinia powder) in 13 weeks. Subcutaneous fat was significantly (P<0.05) reduced in Garcinia treated rats. Hepatocytes significantly (p<0.05) overexpressed sterol regulatory element binding protein 2, liver X receptor- α, liver X receptor- β, lipoprotein lipase and monoacylglycerol lipase. Fatty acid binding protein 1 and peroxisome proliferator activated receptor- α were down regulated as assessed by real time qPCR. Currently our research is focused on the adipocyte obesity related gene expressions, effect of Garcinia on 3T3-adipocyte cell lines and high fat diet induced mice model. This in vivo pre-clinical data suggests that G. xanthochymus may have clinical utility for the treatment of obesity. However, further studies are required to establish its potency.

Keywords: Garcinia xanthochymus, anti-obesity, high fat diet, real time qPCR

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Authors: Rafay Ahmed, Condon Lau

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Bone healing is a complex and sequential process involving changes at the molecular level. Raman spectroscopy is a promising technique to study bone mineral and matrix environments simultaneously. In this study, subcritical calvarial defects are used to study bone composition during healing without discomposing the fracture. The model allowed to monitor the natural healing of bone avoiding mechanical harm to the callus. Calvarial defects were created using 1mm burr drill in the parietal bones of Sprague-Dawley rats (n=8) that served in vivo defects. After 7 days, their skulls were harvested after euthanizing. One additional defect per sample was created on the opposite parietal bone using same calvarial defect procedure to serve as control defect. Raman spectroscopy (785 nm) was established to investigate bone parameters of three different skull surfaces; in vivo defects, control defects and normal surface. Principal component analysis (PCA) was utilized for the data analysis and interpretation of Raman spectra and helped in the classification of groups. PCA was able to distinguish in vivo defects from normal surface and control defects. PC1 shows that the major variation at 958 cm⁻¹, which corresponds to ʋ1 phosphate mineral band. PC2 shows the major variation at 1448 cm⁻¹ which is the characteristic band of CH2 deformation and corresponds to collagens. Raman parameters, namely, mineral to matrix ratio and crystallinity was found significantly decreased in the in vivo defects compared to surface and controls. Scanning electron microscope and optical microscope images show the formation of newly generated matrix by means of bony bridges of collagens. Optical profiler shows that surface roughness increased by 30% from controls to in vivo defects after 7 days. These results agree with Raman assessment parameters and confirm the new collagen formation during healing.

Keywords: Raman spectroscopy, principal component analysis, calvarial defects, tissue characterization

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Authors: W. Mohammedsaeed, A. J. Mcbain, S. M. Cruickshank, C. A. O’Neill

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Many studies have demonstrated the importance of probiotics and their potential therapeutic effects within the gut. Recently, the possible therapeutic effects of probiotics in other tissues have also begun to be investigated. Comparatively few studies have evaluated the use of topical probiotics in relation to the skin. In this study, we have conducted preliminary investigations into whether a well-known probiotic, Lactobacillus rhamnosus GG (LGG), can increase the rate of re-epithelialization in a model wound. Full-thickness skin was obtained from individuals undergoing elective cosmetic surgery. This skin was wounded using excisional punch and cultured using a serum-free medium, either in the presence or absence of L. rhamnosus GG lysate. Histological staining of the sections was performed with Haematoxylin& Eosin E to quantify “epithelial tongue length”. This is the length of the new epithelial ‘tongue’ that grows and covers the exposed dermis at the inner wound edges. The length of the new epithelial ‘tongue’ was compared in untreated section and section treated with and L. rhamnosus GG made using108CFU/ml bacterial cells. L. rhamnosus GG lysate enhanced significantly the re-epithelialisation of treated wounds compared with that of untreated wounds (P=0.005, n=3). Tongue length, at day 1 was 7.55μm 0.15, at day 3 it was 18.5μm 0.25 and at day 7 was 22.9μm 0.35. These results can be compared with untreated cultures in which tongue length was 3.25μm 0.35, day 3 was 9.65μm 0.25 and day 7 was 13.5μm 0.15 post-wounding. In ex-vivo proliferation and migration cells were measured by determining the expression of nuclear proliferation marker Ki-67 and the expression of Phosphorylated cortactin respectively demonstrated that L. rhamnosus GG significantly increased NHEK proliferation and migration rates relative to controls. However, the dominant mechanism was migration because in ex-vivo skin treated with the L. rhamnosus GG up-regulated the gene expression of the chemokine receptor and ligands CXCR2 and CXCL2 comparing with controls (P=0.02, P=0.03 respectively, n=3). High levels of CXCL2/CXCL2 have already been implicated in multiple aspects of stimulation of wound healing through activation of keratinocyte migration. These data demonstrate that lysates from Lactobacillus rhamnosus GG increase re-epithelialization by stimulation of keratinocyte migration. The current study identifies the partial mechanism that contribute to stimulating the wound-healing process ex vivo in response to L. rhamnosus GG lysate is an increase in the production of CXCL2/ CXCR2 in ex vivo models. The use of probiotic lysates potentially offers new options to develop treatments that could improve wound healing.

Keywords: Lactobacillus rhamnosus GG, wounds, migration, lysate

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Authors: Solene Masloh, Anne Chevrel, Maxime Culot, Leonardo Scapozza, Magali Zeisser-Labouebe

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The limited stability of clinically proven therapeutic antibodies limits their administration by the parenteral route. However, oral administration remains the best alternative as it is the most convenient and less invasive one. Obtaining a targeted treatment based on biologics, which can be orally administered, would, therefore, be an ideal situation to improve patient adherence and compliance. Nevertheless, the delivery of macromolecules through the intestine remains challenging because of their sensitivity to the harsh conditions of the gastrointestinal tract and their low permeability across the intestinal mucosa. To address this challenge, this project aims to demonstrate that targeting receptor-mediated endocytosis followed by transcytosis could maximize the intestinal uptake and transport of large molecules, such as Nanofitins. These affinity proteins of 7 kDa with binding properties similar to antibodies have already demonstrated retained stability in the digestive tract and local efficiency. However, their size does not allow passive diffusion through the intestinal barrier. Nanofitins having a controlled affinity for membrane receptors involved in the transcytosis mechanism used naturally for the transport of large molecules in humans were generated. Proteins were expressed using ribosome display and selected based on affinity to the targeted receptor and other characteristics. Their uptake and transport ex vivo across viable porcine intestines were investigated using an Ussing chambers system. In this paper, we will report the results achieved while addressing the different challenges linked to this study. To validate the ex vivo model, first, we proved the presence of the receptors targeted in humans on the porcine intestine. Then, after the identification of an optimal way of detection of Nanofitins, transport experiments were performed on porcine intestines with viability followed during the time of the experiment. The results, showing that the physiological process of transcytosis is capable of being triggered by the binding of Nanofitins on their target, will be reported here. In conclusion, the results show that Nanofitins can be transported across the intestinal barrier by triggering the receptor-mediated transcytosis and that the ex vivo model is an interesting technique to assess biologics absorption through the intestine.

Keywords: ex-vivo, Nanofitins, oral administration, transcytosis

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

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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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Authors: Jinfeng Zhang, Chun-Sing Lee

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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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Authors: Zakaria Baka, Halima Alem

Abstract:

Cancer is a major worldwide health problem that has caused around ten million deaths in 2020. In addition, efforts to develop new anti-cancer drugs still face a high failure rate. This is partly due to the lack of preclinical models that recapitulate in-vivo drug responses. Indeed conventional cell culture approach (known as 2D cell culture) is far from reproducing the complex, dynamic and three-dimensional environment of tumors. To set up more in-vivo-like cancer models, 3D bioprinting seems to be a promising technology due to its ability to achieve 3D scaffolds containing different cell types with controlled distribution and precise architecture. Moreover, the introduction of microfluidic technology makes it possible to simulate in-vivo dynamic conditions through the so-called “cancer-on-chip” platforms. Whereas several cancer types have been modeled through the cancer-on-chip approach, such as lung cancer and breast cancer, only a few works describing ovarian cancer models have been described. The aim of this work is to combine 3D bioprinting and microfluidic technics with setting up a 3D dynamic model of ovarian cancer. In the first phase, alginate-gelatin hydrogel containing SKOV3 cells was used to achieve tumor-like structures through an extrusion-based bioprinter. The desired form of the tumor-like mass was first designed on 3D CAD software. The hydrogel composition was then optimized for ensuring good and reproducible printability. Cell viability in the bioprinted structures was assessed using Live/Dead assay and WST1 assay. In the second phase, these bioprinted structures will be included in a microfluidic device that allows simultaneous testing of different drug concentrations. This microfluidic dispositive was first designed through computational fluid dynamics (CFD) simulations for fixing its precise dimensions. It was then be manufactured through a molding method based on a 3D printed template. To confirm the results of CFD simulations, doxorubicin (DOX) solutions were perfused through the dispositive and DOX concentration in each culture chamber was determined. Once completely characterized, this model will be used to assess the efficacy of anti-cancer nanoparticles developed in the Jean Lamour institute.

Keywords: 3D bioprinting, ovarian cancer, cancer-on-chip models, microfluidic techniques

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Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

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