Bioreactor-Based Bone Tissue Engineering

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries

PCL-Coated Multi-Substituted Calcium Phosphate Bone Scaffolds with Enhanced Properties

[EN] Ionic substitutions within the hydroxyapatite lattice are a widely used approach to mimic the chemical composition of the bone mineral. In this work, Sr-substituted and Mg- and Sr-co-substituted calcium phosphate (CaP) scaffolds, with various levels of strontium and magnesium substitution, were prepared using the hydrothermal method at 200 degrees C. Calcium carbonate skeletons of cuttlefish bone, ammonium dihydrogenphosphate (NH4H2PO4), strontium nitrate (Sr(NO3)(2)), and magnesium perchlorate (Mg(ClO4)(2)) were used as reagents. Materials were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Whole powder pattern decomposition refinements of XRD data indicated that increased magnesium content in the Mg- and Sr-co-substituted scaffolds was related to an increased proportion of the whitlockite (WH) phase in the biphasic hydroxyapatite (HAp)/WH scaffolds. In addition, refinements indicate that Sr2+ ions have replaced Ca2+ sites in the WH phase. Furthermore, PCL-coated Mg-substituted and Sr- and Mg-co-substituted scaffolds, with the HAp:WH wt. ratio of 90:10 were prepared by vacuum impregnation. Results of compression tests showed a positive impact of the WH phase and PCL coating on the mechanical properties of scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on composite scaffolds in an osteogenic medium for 21 days. Immunohistochemical staining showed that Mg-Sr-CaP/PCL scaffold exhibited higher expression of collagen type I than the Mg-CaP/PCL scaffold, indicating the positive effect of Sr2+ ions on the differentiation of hMSCs, in concordance with histology results. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis confirmed an early stage of osteogenic differentiation.This research was funded by the Croatian Science Foundation (project IP-2014-09-3752) and the European Structural and Investments Funds (grant KK.01.1.1.07.0014.). The authors thank Inga Urli, Faculty of Science, University of Zagreb for providing Hek293 and hMSC cells. Compression experiments were carried out at the Centre for Biomaterials and Tissue Engineering (CBIT), Universitat Politecnica de Valencia, Valencia, Spain under the PID2019-106000RB-C21/AEI/10.13039/501100011033 project. The authors would like to thank Jorge Mas-Estelles for his generous assistance.Bauer, L.; Antunovic, M.; Gallego-Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2021). PCL-Coated Multi-Substituted Calcium Phosphate Bone Scaffolds with Enhanced Properties. Materials. 14(16):1-19. https://doi.org/10.3390/ma14164403S119141

Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold

[EN] Ionic substitutions are a promising strategy to enhance the biological performance of calcium phosphates (CaP) and composite materials for bone tissue engineering applications. However, systematic studies have not been performed on multi-substituted organic/inorganic scaffolds. In this work, highly porous composite scaffolds based on CaPs substituted with Sr2+, Mg2+, Zn2+ and SeO3 2¿ ions, and chitosan have been prepared by freezegelation technique. The scaffolds have shown highly porous structure, with very well interconnected pores and homogeneously dispersed CaPs, and high stability during 28 days in the degradation medium. Osteogenic potential of human mesenchymal stem cells seeded on scaffolds has been determined by histological, immunohistochemical and RT-qPCR analysis of cultured cells in static and dynamic conditions. Results indicated that ionic substitutions have a beneficial effect on cells and tissues. The scaffolds with multi-substituted CaPs have shown increased expression of osteogenesis related markers and increased phosphate deposits, compared to the scaffolds with non-substituted CaPs.The financial supports of the European Regional Development Fund (grant KK.01.1.1.07.0014) , the PID2019-106000RB-C21/AEI/10.13039/501100011033 project from the Spanish Research Agency, and the L'Oreal-UNESCO "For Women in Science" Foundation are gratefully acknowledged.Ressler, A.; Antunovic, M.; Teruel Biosca, L.; Ferrer GG; Babic, S.; Urlic, I.; Ivankovic, M.... (2022). Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold. Carbohydrate Polymers. 277:1-16. https://doi.org/10.1016/j.carbpol.2021.11888311627

Cytotoxic activity of novel palladium-based compounds on leukemia cell lines

Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)center dot 4H(2)O and [PdCl(terpy)](sac)center dot 2H(2)O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted

Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage

Malaria im Netz : Vergleich von Recherchestrategien und Retrievalergebnissen zum Themenfeld Malaria in proprietären Datenbanken und öffentlich zugänglichen Informationsquellen

Diese Diplomarbeit beschäftigt sich mit dem Themenfeld Malaria. Zu achtzehn aktuellen Fragen zur Malaria werden entsprechende elektronische Quellen vorgestellt, die der Beantwortung dieser Problemstellungen dienen. Des Weiteren werden drei Recherchequellen – die Suchmaschinen Google und Metager und die Datenbank LexisNexis – mit neun ausgesuchten Recherchefragen aus den zuvor bearbeiteten achtzehn Fragen auf ihre Trefferqualität getestet. Dieser Test besteht aus zwei Teilen: Zum einen werden die drei Recherchequellen mit einer Recherchestrategie getestet und deren Treffer beurteilt; zum anderen besteht der zweite Test aus einer einfachen Suchstrategie, wobei auf gleiche Recherchebedingungen geachtet wurde

Chapter Bioreactor-Based Bone Tissue Engineering

Omega-3 (ω-3) and omega-6 (ω-6) are polyunsaturated fatty acids (PUFAs) that play critical role in human health and have to be provided by food. In the brain, PUFAs are also precursors of endocannabinoids. The aim of this chapter is to review the existing literature on how dietary PUFAs impact on the endocannabinoid system in the brain and what are the consequences for brain function and dysfunction. In this chapter, we will first describe how PUFAs enter the brain, what are their metabolism processes and roles in brain function. We will describe the pathways from PUFAs to endocannabinoid production. Then, we will review the literature on how dietary ω-6/ω-3 ratio impacts the endocannabinoid system, in terms of endocannabinoid levels, proteins and endocannabinoid-dependent synaptic plasticity. In the next part, we will describe what we know about the interactions between PUFAs and endocannabinoids in neurological and neuropsychiatric disorders. Finally, we will conclude on the possible implications of the interactions between dietary PUFAs and endocannabinoids in the normal and pathological brain. In particular, we will discuss how dietary PUFAs, as homeostatic regulators of endocannabinoids, can constitute interesting therapeutic strategies for the prevention and/or treatment of neurological disorders with endocannabinoids impairment

Large Graphene Quantum Dots Alleviate Immune-Mediated Liver Damage

We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. <i>In vitro</i>, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect