Cell/cell and cell/ECM interaction at the nano-scale for orthopaedic tissue engineering

PhD ThesisTissue engineering involves the use of cells, growth factors/cytokines, and scaffolds to regenerate damaged tissue. The choice of cells, scaffold and their delivery is crucial to the successful outcome of the treatment and this is particularly the case in bone and cartilage where the repair tissue has to recreate a structural hierarchy to restore long lasting function. One approach to deliver high numbers of cells to a defect site is as cell aggregates or spheroids. Experiments are described here that aim to understand how mesenchymal stem cells (MSCs) and osteoblastic cells behave in a cell aggregate and how this could be refined by the inclusion a self -assembling hydrogel to influence cellcell and cell-extracellular matrix (ECM) interactions. Forming cellular spheroids in vitro can be achieved using various methods, including hanging drop, static suspension culture, encapsulation/entrapment, and low adherence multi-well. Extensive analysis identified that the most efficient and reproducible method for the formation of spheroids using rat osteoblasts and human MSCs was through their culture at a specific concentration in polyHEMA coated plates. Both viability and ability of cells to differentiate was investigated. The MTT assay was used to assess cells viability while their ability to differentiate was assessed by measuring alkaline phosphatase activity as well as measuring gene osteogenic markers expression via qRT-PCR. Analysis of cell differentiation under these conditions revealed that alkaline phosphatase activity appeared more elevated in 2D cultures compared to 3D. However, it was noted that there were contrasting results between the two types of cells with expression of osteogenic genes higher when MSCs were grown in osteogenic media while with calvarial significant expression was also observed when grown in normal media. Because of the distinct regulatory cues given by cell-cell contact in the spheroid, analysis was performed for connexin (Cx)-43, a gap junction protein and members of the ephrin/Eph family. Cx-43 iv was immunolocalised to gap junction structures in cells after osteogenic treatment on a flat substrate but this was more difficult to assess in the 3D spheroids. Analysis of transcript patterns reflected the increased abundance of Cx-43 in cells treated with osteogenic supplements and parallel changes in expression of Ephrin B1 and Ephrin B2. Experiments were also performed including a Puramatrix hydrogel nanofibers scaffold that could encase the cells in an ECM-like environment, provide mechanical support and protect them and manipulate cell-cell interactions. The results obtained in this study concluded that calvaria cells viability and hence proliferation increased when grown embedded within 0.25% Puramtrix while mesenchymal stem cells increased when embedded in 0.5% Puramatrix. Similarly, alkaline phosphatase activity was higher in cells embedded within 0.25% Puramatrix while mesenchymal stem cells favoured 0.5%. On the other hand, osteogenic gene expression of both cells was enhanced with the use of Puramatrix scaffold

Antiproliferative effect of the Red Sea cone snail, Conus geographus

Purpose: To investigate the antiproliferative effect of the Red Sea cone snail, Conus geographus, against 4 MCF-7 (breast), MDA-MB-231 (epithelial human breast), HepG2 (hepatocellular) and SKOV-3 (ovarian) cancer cell lines. Methods: Extraction of Red Sea cone snail sample with a mixture of CH2Cl2 and CH3OH (1:1, v/v) yielded 0.55 g of a green viscous material. The cytotoxic effects of the organic extract against the cancer cell lines were determined using cell proliferation (MTT) assay, and the half-maximal concentration (IC50) values measured. The effect of the crude extract on the cell cycle of the HepG-2 was determined by flow cytometry. Results: The extract produced significant inhibitory effects against SKOV-3, MDA-MB-231, MCF-7 and HepG2, with IC50 values of 22.7 ± 2.2, 68.7 ± 6.2, 47 ± 4.2 and 19 ± 2.1 μg/mL, respectively. Cell cycle analysis revealed that the extract enhanced accumulation of HepG2 cells in the Go/G1 phase, at a level of 23.4 and 24.1 % at IC50 (19 μg/mL) and ½ IC50 (9.5 μg/mL), respectively, when compared to the untreated cells. Conclusion: These results indicate that C. geographus extract exhibits potent cytotoxic effect against HepG2 cells via a mechanism involving G0/G1 cell cycle arrest. Thus, C. geographus is a potential source of a new anti-cancer agent. Keywords: Conus geographus, Marine invertebrate, HepG2, Antiproliferatio

<i>Alcyonium</i> Octocorals: Potential Source of Diverse Bioactive Terpenoids

Alcyonium corals are benthic animals, which live in different climatic areas, including temperate, Antarctic and sub-Antarctic waters. They were found to produce different chemical substances with molecular diversity and unique architectures. These metabolites embrace several terpenoidal classes with different functionalities. This wide array of structures supports the productivity of genus Alcyonium. Yet, majority of the reported compounds are still biologically unscreened and require substantial efforts to explore their importance. This review is an entryway to push forward the bio-investigation of this genus. It covers the era from the beginning of reporting metabolites from Alcyonium up to March 2019. Ninety-two metabolites are presented; forty-two sesquiterpenes, twenty-five diterpenes and twenty-five steroids have been reported from sixteen species

Alcyonacea: A Potential Source for Production of Nitrogen-Containing Metabolites

Alcyonacea (soft corals and gorgonia) are well known for their production of a wide array of unprecedented architecture of bioactive metabolites. This diversity of compounds reported from Alcyonacea confirms its productivity as a source of drug leads and, consequently, indicates requirement of further chemo-biological investigation. This review can be considered a roadmap to investigate the Alcyonacea, particularly those produce nitrogen-containing metabolites. It covers the era from the beginning of marine nitrogen-containing terpenoids isolation from Alcyonacea up to December 2018. One hundred twenty-one compounds with nitrogenous moiety are published from fifteen genera. Their prominent biological activity is evident in their antiproliferative effect, which makes them interesting as potential leads for antitumor agents. For instance, eleutherobin and sarcodictyins are in preclinical or clinical stages

Exosomal miRNAs as a Promising Source of Biomarkers in Colorectal Cancer Progression

Colorectal cancer (CRC) is the third most common type of cancer worldwide amongst males and females. CRC treatment is multidisciplinary, often including surgery, chemotherapy, and radiotherapy. Early diagnosis of CRC can lead to treatment initiation at an earlier stage. Blood biomarkers are currently used to detect CRC, but because of their low sensitivity and specificity, they are considered inadequate diagnostic tools and are used mainly for following up patients for recurrence. It is necessary to detect novel, noninvasive, specific, and sensitive biomarkers for the screening and diagnosis of CRC at earlier stages. The tumor microenvironment (TME) has an essential role in tumorigenesis; for example, extracellular vesicles (EVs) such as exosomes can play a crucial role in communication between cancer cells and different components of TME, thereby inducing tumor progression. The importance of miRNAs that are sorted into exosomes has recently attracted scientists’ attention. Some unique sequences of miRNAs are favorably packaged into exosomes, and it has been illustrated that particular miRNAs can be directed into exosomes by special mechanisms that occur inside the cells. This review illustrates and discusses the sorted and transported exosomal miRNAs in the CRC microenvironment and their impact on CRC progression as well as their potential use as biomarkers

Urtica pilulifera leaves extract mitigates cadmium induced hepatotoxicity via modulation of antioxidants, inflammatory markers and Nrf-2 signaling in mice

Introduction: Cadmium (Cd) is a harmful heavy metal that results in many toxic issues. Urtica pilulifera showed potential pharmaceutical applications. This study investigated the possible ameliorative mechanism of Urtica pilulifera leaves extract (UPLE) against hepatotoxicity induced by cadmium chloride (CdCl2) in mice.Methods:In vitro phytochemical screening and the metal-chelating activity of UPLE were ascertained. Four groups of forty male mice were used (n = 10) as follows; Group 1 (G1) was a negative control. G2 was injected i.p., with UPLE (100 mg/kg b. wt) daily. G3 was injected i.p., with Cd (5 mg/kg b. wt) daily. G4 was injected with Cd as in G3 and with UPLE as in G2. On day 11, the body weight changes were evaluated, blood, and serum samples were collected for hematological and biochemical assessments. Liver tissues were used for biochemical, molecular, and histopathological investigations.Results: The results showed that UPLE contains promising secondary metabolites that considerably lessen the negative effects of Cd on liver. Furthermore, UPLE inhibited oxidative stress and inflammation; restored antioxidant molecules; and promoted nuclear-related factor-2 (Nrf-2) expression. Also, UPLE improved the histopathological alterations induced by Cd.Discussion: This study explored the beneficial role of UPLE treatment in Cd-induced liver injury through enhancing Nrf-2 signaling and antioxidant enzyme gene expression in the liver of mice. Therefore, UPLE could have valuable implications against hepatotoxicity induced by environmental cadmium exposure. Which can be used as a chelating agent against Cd

Euryops arabicus Promotes Healing of Excised Wounds in Rat Skin: Emphasis on Its Collagen-Enhancing, Antioxidant, and Anti-Inflammatory Activities

Euryops arabicus Steud (E. arabicus) belongs to the family Asteraceae. It has several uses in folk medicine in the Arabian Peninsula. The current study aimed at evaluating the wound healing properties of the E. arabicus extract in rats. Primarily, E. arabicus successfully accelerated cell migration in vitro and it also showed no signs of dermal toxicity. Topical application of E. arabicus extract (5% or 20%) expedited healing of excised skin in rats. Histological examinations indicated that E. arabicus shortened epithelization period, stimulated fibroblast activity, and increased collagen deposition in wound tissues. The plant extract exerted antioxidant activity as evidenced by inhibition of GSH depletion and MDA accumulation and enhanced mRNA expression of Sod1 in wound tissues collected at the end of the experiment. Further, E. arabicus inhibited the rise of TNF-α and IL-1β in the skin wound region. The anti-inflammatory was confirmed by the observed down regulation of Ptgs2, Nos2, IL-6, and NF-κB mRNA expression. In addition, the extract enhanced the expression of TGF-β1 and HIF-1α in wounded skin tissues as indicated immunohistochemically. Conclusively, E. arabicus expedites excision wound healing in rats. Collagen-enhancing, anti-inflammatory, and antioxidant properties mediate the observed wound healing activity. These findings might contribute to our understanding of the ethnobotanical use of E. arabicus in wounds

Monoterpene Indole Alkaloids from the Aerial Parts of <i>Rhazya stricta</i> Induce Cytotoxicity and Apoptosis in Human Adenocarcinoma Cells

Chromatographic investigation of the aerial parts of the Rhazya stricta (Apocynaceae) resulted in the isolation of two new monoterpene indole alkaloids, 6-nor-antirhine-N1-methyl (1) and razyamide (2), along with six known compounds, eburenine (3), epi-rhazyaminine (4), rhazizine (5), 20-epi-sitsirikine (6), antirhine (7), and 16-epi-stemmadenine-N-oxide (8). The chemical structures were established by various spectroscopic experiments. Compounds 1–8 exhibited cytotoxic effects against three cancer cells with IC50 values ranging between 5.1 ± 0.10 and 93.2 ± 9.73 µM against MCF-7; 5.1 ± 0.28 and 290.2 ± 7.50 µM against HepG2, and 3.1 ± 0.17 and 55.7 ± 4.29 µM against HeLa cells. Compound 2 showed the most potent cytotoxic effect against all cancer cell lines (MCF-7, HepG2 and HeLa with IC50 values = 5.1 ± 0.10, 5.1 ± 0.28, and 3.1 ± 0.17 µM, respectively). Furthermore, compound 2 revealed a significant increase in the apoptotic cell population of MCF-7, HepG2, and HeLa cells, with 31.4 ± 0.2%, 29.2 ± 0.5%, and 34.9 ± 0.6%, respectively. Compound 2 decreased the percentage of the phagocytic pathway on HepG2 cells by 15.0 ± 0.1%. These findings can explain the antiproliferative effect of compound 2