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Authors: J. D. Cabral, E. Murray, P. Turner, E. Hewitt, A. Ali, M. McConnell

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Worldwide demand for organ replacement and tissue regeneration is progressively increasing. Three-dimensional (3D) bioprinting, where a physical construct is produced using computer-aided design, is a promising tool to advance the tissue engineering and regenerative medicine fields. In this paper we describe two different approaches to developing 3D bioprinted constructs for use in tissue regeneration. Bioink development is critical in achieving the 3D biofabrication of functional, regenerative tissues. Hydrogels, cross-linked macromolecules that absorb large amounts of water, have received widespread interest as bioinks due to their relevant soft tissue mechanics, biocompatibility, and tunability. In turn, not only is bioink optimisation crucial, but the creation of vascularized tissues remains a key challenge for the successful fabrication of thicker, more clinically relevant bioengineered tissues. Among the various methodologies, cell-laden hydrogels are regarded as a favorable approach; and when combined with novel core/shell 3D bioprinting technology, an innovative strategy towards creating new vessel-like structures. In this work, we investigate this cell-based approach by using human umbilical endothelial cells (HUVECs) entrapped in a viscoelastic chitosan/dextran (CD)-based core hydrogel, printed simulataneously along with a gelatin methacrylate (GelMA) shell. We have expanded beyond our previously reported FDA approved, commercialised, post-surgical CD hydrogel, Chitogel®, by functionalizing it with cell adhesion and proteolytic peptides in order to promote bone marrow-derived mesenchymal stem cell (immortalized BMSC cell line, hTERT) and HUVECs growth. The biocompatibility and biodegradability of these cell lines in a 3D bioprinted construct is demonstrated. Our studies show that particular peptide combinations crosslinked within the CD hydrogel was found to increase in vitro growth of BMSCs and HUVECs by more than two-fold. These gels were then used as a core bioink combined with the more mechanically robust, UV irradiated GelMA shell bioink, to create 3D regenerative, vessel-like scaffolds with high print fidelity. As well, microporous MEW scaffolds made from milk proteins blended with PCL were found to show promising bioactivity, exhibiting a significant increase in keratinocyte (HaCaTs) and fibroblast (normal human dermal fibroblasts, NhDFs) cell migration and proliferation when compared to PCL only scaffolds. In conclusion, our studies indicate that a peptide functionalized CD hydrogel bioink reinforced with a GelMA shell is biocompatible, biodegradable, and an appropriate cell delivery vehicle in the creation of regenerative 3D constructs. In addition, a novel 3D printing technique, melt electrowriting (MEW), which allows fabrication of micrometer fibre meshes, was used to 3D print polycaprolactone (PCL) and bioactive milk protein, lactorferrin (LF) and whey protein (WP), blended scaffolds for potential skin regeneration applications. MEW milk protein/PCL scaffolds exhibited high porosity characteristics, low overall biodegradation, and rapid protein release. Human fibroblasts and keratinocyte cells were seeded on to the scaffolds. Scaffolds containing high concentrations of LF and combined proteins (LF+WP) showed improved cell viability over time as compared to PCL only scaffolds. This research highlights two scaffolds made using two different 3D printing techniques using a combination of both natural and synthetic biomaterial components in order to create regenerative constructs as potential chronic wound treatments.

Keywords: biomaterials, hydrogels, regenerative medicine, 3D bioprinting

Procedia PDF Downloads 244

Authors: F. Varga, Z. Liber, J. Jakše, A. Turudić, Z. Šatović, I. Radosavljević, N. Jeran, M. Grdiša

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Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.; Asteraceae), a source of the commercially dominant plant insecticide pyrethrin, is a species endemic to the eastern Adriatic. Genetic diversity of T. cinerariifolium was previously studied using amplified fragment length polymorphism (AFLP) markers. However, microsatellite markers (single sequence repeats - SSRs) are more informative because they are codominant, highly polymorphic, locus-specific, and more reproducible, and thus are most often used to assess the genetic diversity of plant species. Dalmatian pyrethrum is an outcrossing diploid (2n = 18) whose large genome size and high repeatability have prevented the success of the traditional approach to SSR markers development. The advent of next-generation sequencing combined with the specifically developed method recently enabled the development of, to the author's best knowledge, the first set of SSRs for genomic characterization of Dalmatian pyrethrum, which is essential from the perspective of plant genetic resources conservation. To evaluate the effectiveness of the developed SSR markers in genetic differentiation of Dalmatian pyrethrum populations, a preliminary genetic diversity study was conducted on 30 individuals from three geographically distinct natural populations in Croatia (northern Adriatic island of Mali Lošinj, southern Adriatic island of Čiovo, and Mount Biokovo) based on 12 SSR loci. Analysis of molecular variance (AMOVA) by randomization test with 10,000 permutations was performed in Arlequin 3.5. The average number of alleles per locus, observed and expected heterozygosity, probability of deviations from Hardy-Weinberg equilibrium, and inbreeding coefficient was calculated using GENEPOP 4.4. Genetic distance based on the proportion of common alleles (DPSA) was calculated using MICROSAT. Cluster analysis using the neighbor-joining method with 1,000 bootstraps was performed with PHYLIP to generate a dendrogram. The results of the AMOVA analysis showed that the total SSR diversity was 23% within and 77% between the three populations. A slight deviation from Hardy-Weinberg equilibrium was observed in the Mali Lošinj population. Allele richness ranged from 2.92 to 3.92, with the highest number of private alleles observed in the Mali Lošinj population (17). The average observed DPSA between 30 individuals was 0.557. The highest DPSA (0.875) was observed between several pairs of Dalmatian pyrethrum individuals from the Mali Lošinj and Mt. Biokovo populations, and the lowest between two individuals from the Čiovo population. Neighbor-joining trees, based on DPSA, grouped individuals into clusters according to their population affiliation. The separation of Mt. Biokovo clade was supported (bootstrap value 58%), which is consistent with the previous study on AFLP markers, where isolated populations from Mt. Biokovo differed from the rest of the populations. The developed SSR markers are an effective tool for assessing the genetic diversity and structure of natural Dalmatian pyrethrum populations. These preliminary results are encouraging for a future comprehensive study with a larger sample size across the species' range. Combined with the biochemical data, these highly informative markers could help identify potential genotypes of interest for future development of breeding lines and cultivars that are both resistant to environmental stress and high in pyrethrins. Acknowledgment: This work has been supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) insecticidal potential’- (PyrDiv) (IP-06-2016-9034) and by project KK.01.1.1.01.0005, Biodiversity and Molecular Plant Breeding, at the Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Zagreb, Croatia.

Keywords: Asteraceae, genetic diversity, genomic SSRs, NGS, pyrethrum, Tanacetum cinerariifolium

Procedia PDF Downloads 91

Authors: Jolene M. Helena, Annie M. Joubert, Peace Mabeta, Magdalena Coetzee, Roy Lakier, Anne E. Mercier

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Targeting the distant tumour and its microenvironment whilst preserving bone density is important in improving the outcomes of patients with bone metastases. 2-Ethyl-3-O-sulphamoyl-estra1,3,5(10)16-tetraene (ESE-16) is an in-silico-designed 2- methoxyestradiol analogue which aimed at enhancing the parent compound’s cytotoxicity and providing a more favourable pharmacokinetic profile. In this study, the potential radiosensitization effects of ESE-16 were investigated in an in vitro bone metastasis model consisting of murine pre-osteoblastic (MC3T3-E1) and pre-osteoclastic (RAW 264.7) bone cells, metastatic prostate (DU 145) and breast (MDA-MB-231) cancer cells, as well as human umbilical vein endothelial cells (HUVECs). Cytotoxicity studies were conducted on all cell lines via spectrophotometric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. The experimental set-up consisted of flow cytometric analysis of cell cycle progression and apoptosis detection (Annexin V-fluorescein isothiocyanate) to determine the lowest ESE-16 and radiation doses to induce apoptosis and significantly reduce cell viability. Subsequent experiments entailed a 24-hour low-dose ESE-16-exposure followed by a single dose of radiation. Termination proceeded 2, 24 or 48 hours thereafter. The effect of the combination treatment was investigated on osteoclasts via tartrate-resistant acid phosphatase (TRAP) activity- and actin ring formation assays. Tumour cell experiments included investigation of mitotic indices via haematoxylin and eosin staining; pro-apoptotic signalling via spectrophotometric quantification of caspase 3; deoxyribonucleic acid (DNA) damage via micronuclei analysis and histone H2A.X phosphorylation (γ-H2A.X); and Western blot analyses of bone morphogenetic protein-7 and matrix metalloproteinase-9. HUVEC experiments included flow cytometric quantification of cell cycle progression and free radical production; fluorescent examination of cytoskeletal morphology; invasion and migration studies on an xCELLigence platform; and Western blot analyses of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor receptor 1 and 2. Tumour cells yielded half-maximal growth inhibitory concentration (GI50) values in the nanomolar range. ESE-16 concentrations of 235 nM (DU 145) and 176 nM (MDA-MB-231) and a radiation dose of 4 Gy were found to be significant in cell cycle and apoptosis experiments. Bone and endothelial cells were exposed to the same doses as DU 145 cells. Cytotoxicity studies on bone cells reported that RAW 264.7 cells were more sensitive to the combination treatment than MC3T3-E1 cells. Mature osteoclasts were more sensitive than pre-osteoclasts with respect to TRAP activity. However, actin ring morphology was retained. The mitotic arrest was evident in tumour and endothelial cells in the mitotic index and cell cycle experiments. Increased caspase 3 activity and superoxide production indicated pro-apoptotic signalling in tumour and endothelial cells. Increased micronuclei numbers and γ-H2A.X foci indicated increased DNA damage in tumour cells. Compromised actin and tubulin morphologies and decreased invasion and migration were observed in endothelial cells. Western blot analyses revealed reduced metastatic and angiogenic signalling. ESE-16-induced radiosensitization inhibits metastatic signalling and tumour cell survival whilst preferentially preserving bone cells. This low-dose combination treatment strategy may promote the quality of life of patients with metastatic bone disease. Future studies will include 3-dimensional in-vitro and murine in-vivo models.

Keywords: angiogenesis, apoptosis, bone metastasis, cancer, cell migration, cytoskeleton, DNA damage, ESE-16, radiosensitization.

Procedia PDF Downloads 133

Authors: Essam Gooda, Chadi Abdallah, Hamdi Seif, Safaa Baydoun, Rouya Hdeib, Hilal Obeid

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Floods are common natural disaster occurring in semi-arid regions in Lebanon. This results in damage to human life and deterioration of environment. Despite their destructive nature and their immense impact on the socio-economy of the region, flash floods have not received adequate attention from policy and decision makers. This is mainly because of poor understanding of the processes involved and measures needed to manage the problem. The current understanding of flash floods remains at the level of general concepts; most policy makers have yet to recognize that flash floods are distinctly different from normal riverine floods in term of causes, propagation, intensity, impacts, predictability, and management. Flash floods are generally not investigated as a separate class of event but are rather reported as part of the overall seasonal flood situation. As a result, Lebanon generally lacks policies, strategies, and plans relating specifically to flash floods. Main objective of this research is to improve flash flood prediction by providing new knowledge and better understanding of the hydrological processes governing flash floods in the East Catchments of El Assi River. This includes developing rainstorm time distribution curves that are unique for this type of study region; analyzing, investigating, and developing a relationship between arid watershed characteristics (including urbanization) and nearby villages flow flood frequency in Ras Baalbeck and Fekha. This paper discusses different levels of integration approach¬es between GIS and hydrological models (HEC-HMS & HEC-RAS) and presents a case study, in which all the tasks of creating model input, editing data, running the model, and displaying output results. The study area corresponds to the East Basin (Ras Baalbeck & Fakeha), comprising nearly 350 km2 and situated in the Bekaa Valley of Lebanon. The case study presented in this paper has a database which is derived from Lebanese Army topographic maps for this region. Using ArcMap to digitizing the contour lines, streams & other features from the topographic maps. The digital elevation model grid (DEM) is derived for the study area. The next steps in this research are to incorporate rainfall time series data from Arseal, Fekha and Deir El Ahmar stations to build a hydrologic data model within a GIS environment and to combine ArcGIS/ArcMap, HEC-HMS & HEC-RAS models, in order to produce a spatial-temporal model for floodplain analysis at a regional scale. In this study, HEC-HMS and SCS methods were chosen to build the hydrologic model of the watershed. The model then calibrated using flood event that occurred between 7th & 9th of May 2014 which considered exceptionally extreme because of the length of time the flows lasted (15 hours) and the fact that it covered both the watershed of Aarsal and Ras Baalbeck. The strongest reported flood in recent times lasted for only 7 hours covering only one watershed. The calibrated hydrologic model is then used to build the hydraulic model & assessing of flood hazards maps for the region. HEC-RAS Model is used in this issue & field trips were done for the catchments in order to calibrated both Hydrologic and Hydraulic models. The presented models are a kind of flexible procedures for an ungaged watershed. For some storm events it delivers good results, while for others, no parameter vectors can be found. In order to have a general methodology based on these ideas, further calibration and compromising of results on the dependence of many flood events parameters and catchment properties is required.

Keywords: flood risk management, flash flood, semi arid region, El Assi River, hazard maps

Procedia PDF Downloads 460