Ultrasonic Coating of Poly(D,L-lactic acid)/Poly(lactic-co-glycolic acid) Electrospun Fibers with ZnO Nanoparticles to Increase Angiogenesis in the CAM Assay.

Critical-size bone defects necessitate bone void fillers that should be integrated well and be easily vascularized. One viable option is to use a biocompatible synthetic polymer and sonocoat it with zinc oxide (ZnO) nanoparticles (NPs). However, the ideal NP concentration and size must be assessed because a high dose of ZnO NPs may be toxic. Electrospun PDLLA/PLGA scaffolds were produced with different concentrations (0.5 or 1.0 s of sonocoating) and sizes of ZnO NPs (25 nm and 70 nm). They were characterized by SEM, EDX, ICP-OES, and the water contact angle. Vascularization and integration into the surrounding tissue were assessed with the CAM assay in the living chicken embryo. SEM, EDX, and ICP-OES confirmed the presence of ZnO NPs on polymer fibers. Sonocoated ZnO NPs lowered the WCA compared with the control. Smaller NPs were more pro-angiogenic exhibiting a higher vessel density than the larger NPs. At a lower concentration, less but larger vessels were visible in an environment with a lower cell density. Hence, the favored combination of smaller ZnO NPs at a lower concentration sonocoated on PDLLA/PLGA electrospun meshes leads to an advanced state of tissue integration and vascularization, providing a valuable synthetic bone graft to be used in clinics in the future.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Chemical composition and anti-angiogenic activity of the essential oil from Blumea eriantha D.C.

Blumea eriantha D.C is a weed from Asteraceae family and is reported to have anticancer activity. The essential oil from the aerial parts was extracted by steam distillation method with the yield of 0.36%. Through GC-MS analysis of the oil, seventeen compounds could be identified by comparing with linear retention indices with the library. Out of the seventeen compounds ß-Caryophylline oxide was isolated by column chromatography with gradient elution and the structure was determined through FT-IR, MS, 1HNMR, 13 C NMR and DEPT. The oil was evaluated for its effect on angiogenesis using Chorioallantoic Membrane Assay (CAM Assay). The concentration dependent antiangiogenic effect was observed with IC 50 value of 19.28 ppm.

Phytochemical evaluation, embryotoxic, and teratogenic effects of Buwakan (Decalobanthus peltatus (L.) A.R.Simões & Staples) leaf extracts on duck embryo.

Decalobanthus peltatus is a woody vine that is commonly utilized in traditional Southeast Asian medicinal preparations. Despite the documented therapeutic uses of D. peltatus, there is hardly any information regarding its toxic effects on its consumers. In this study, crude leaf extracts (aqueous, methanol, ethyl acetate, and hexane) from D. peltatus were prepared and evaluated for their embryotoxicity and teratogenic effects. Phytochemical screening of bioactive compounds from the plants showed the presence of alkaloids, flavonoids, saponins, steroids, and tannins. In addition, investigations on the toxicity of the crude leaf extracts were determined using brine shrimp lethality assay, in which the LC50 was calculated. Results showed that the ethyl acetate leaf extract was the most toxic among the crude leaf extracts, with an LC50 of 14.54 ppm. Based on this result, ethyl acetate leaf extract was treated on duck embryos, and the alteration of vascular branching patterns in the chorioallantoic membrane was quantified. Gross morphological and histological analysis of the skin tissues from the treated duck embryos were also examined. We found significant reduction of primary and tertiary vessel diameters in the duck embryos treated with ethyl acetate leaf extracts in both concentrations compared to the control group. Treated duck embryos exhibited gross malformations, growth retardation, and hemorrhages on the external body surfaces at 1000 ppm. Histopathological analysis of the skin tissues from the 14-day-old treated duck embryos showed a reduced number of feather follicles compared to the control group. These results suggest that D. peltatus crude leaf extracts present risks when taken in significant dosages and comprehensive toxicity testing on therapeutic herbs should be performed to ensure their safety on the consumers.

Role of Protein Tyrosine Phosphatase Receptor Type E (PTPRE) in Chemoresistant Retinoblastoma.

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma.

Pro-angiogenic and antibacterial copper containing nanoparticles in PLGA/amorphous calcium phosphate bone nanocomposites.

Large bone defects after trauma demand for adequate bone substitutes. Bone void fillers should be antibacterial and pro-angiogenic. One viable option is the use of composite materials like the combination of PLGA and amorphous calcium phosphate (aCaP). Copper stimulates angiogenesis and has antibacterial qualities. Either copper oxide (CuO) nanoparticles (NPs) were therefore added to PLGA/aCaP/CuO in different concentrations (1, 5 and 10 w/w %) or copper-doped tricalcium phosphate NPs (TCP with 2% of copper) were electrospun into PLGA/CuTCP nanocomposites. Bi-layered nanocomposites of PLGA/aCaP with different copper NPs (CuO or TCP) and a second layer of pristine PLGA were fabricated. Two clinical bacterial isolates (Staphylococcus aureus and Staphylococcus epidermidis) were used to assess antibacterial properties of the copper-containing materials. For angiogenesis, the chorioallantoic membrane (CAM) assay of the chicken embryo was performed. The higher the CuO content, the higher were the antibacterial properties, with 10 % CuO reducing bacterial adhesion most effectively. Vessel and cell densities were highest in the 5 % CuO containing scaffolds, while tissue integration was more pronounced at lower CuO content. The PLGA/aCaP/CuO (1 % CuO) behaved similar like PLGA/CuTCP in all angiogenic and antibacterial readouts, based on the same copper fraction. We conclude that CuO NPs or CuTCP NPs are useful components to increase angiogenic properties of nanocomposites and at the same time exhibiting antibacterial characteristics.

Organic-inorganic biohybrid films from wool-keratin/jellyfish-collagen/silica/boron via sol-gel reactions for soft tissue engineering applications.

Therapeutic angiogenesis is pivotal in creating effective tissue-engineered constructs that deliver nutrients and oxygen to surrounding cells. Hence, biomaterials that promote angiogenesis can enhance the efficacy of various medical treatments, encompassing tissue engineering, wound healing, and drug delivery systems. Considering these, we propose a rapid method for producing composite silicon-boron-wool keratin/jellyfish collagen (Si-B-WK/JFC) inorganic-organic biohybrid films using sol-gel reactions. In this approach, reactive tetraethyl orthosilicate and boric acid (pKa ⩾ 9.24) were used as silicon and boron sources, respectively, and a solid-state gel was formed through the condensation reaction of these reactive groups with the keratin/collagen mixture. Once the resulting gel was thoroughly suspended in water, the films were prepared by a casting/solvent evaporation methodology. The fabricated hybrid films were characterized structurally and mechanically. In addition, angiogenic characteristics were determined by the in ovo chick chorioallantoic membrane assay, which revealed an increased vascular network within the Si-B-WK/JFC biohybrid films. In conclusion, it is believed that Si-B-WK/JFC biohybrid films with mechanical and pro-angiogenic properties have the potential to be possessed in soft tissue engineering applications, especially wound healing.

Development and optimization of Clitoria teratea synthesized silver nanoparticles and its application to nanogel systems for wound healing.

OBJECTIVE: The present research deals with sequential optimization strategy based on Central Composite Design to optimize the process variables for efficient production of Clitoria teratea (CLT) synthesized silver nanoparticles (AgNPs) using biological synthesis. METHODS: Two substantial factors influencing the dependent variables viz UV-visible absorbance, particle size, zeta potential and polydispersity index (PDI) were identified as NaOH concentration, RH concentration, temperature as independent variables. In-vitro and ex-vivo studies of prepared CLT-AgNPs gel and marketed gel were carried out using dialysis membrane and egg membrane, respectively. In addition, antimicrobial study was also performed on the bacterial strains. RESULTS: The particles size (114 nm), PDI (0.45), and zeta potential (-29.5 mV) of optimized formulation were found, respectively. In-vitro profile of AgNPs from prepared CLT-AgNPs gel was noted (95.6%) in 8 h. It was found that the prepared CLT-AgNPs gel stimulates fibroblast and agranulocytosis development resulting better and timely wound healing. CONCLUSIONS: The prepared CLT-AgNPs gel can be as a potential substitute in the management and treatment of acute and chronic wounds.

Platelet-rich fibrin for rehydration and pre-vascularization of an acellular, collagen membrane of porcine origin.

OBJECTIVES: Pre-vascularization of the collagen membranes with autologous platelet concentrates is a standard procedure in oral and maxillofacial surgery. This study analyzed the possible interaction of an acellular collagen membrane of porcine origin (NM) with platelet-rich fibrin (PRF) regarding its rehydration protocol with differences in pH values and effect on angiogenesis. MATERIALS AND METHODS: NM was analyzed alone and combined with solid PRF by plotting or co-culturing with injectable PRF. Different media (venous blood, buffer solution with a fixed pH value of 7, saline solution, and injectable PRF) were used to analyze the influence on pH value during rehydration. Chorion allantois membrane assay (CAM) was applied to check pro-angiogenic effects after 24, 48, and 72 h, followed by immunohistochemical analysis. RESULTS: Rehydration in injectable PRF showed acidity over time (p < 0.05). A definite pro-angiogenic effect of NM alone was found regarding neo-vessel formation supported by the respective light microscopically analysis without significant differences to PRF alone (p > 0.005). This pro-angiogenic effect could not be exaggerated when NM was combined with liquid/solid PRF (each p > 0.005). CONCLUSIONS: Rehydration with liquid PRF of the collagen membrane results in acidity compared to a saline solution or patient's blood. The significant pro-angiogenic potential of the membrane alone resulted in enhanced neo-vessel formation that could not be optimized with the addition of PRF. CLINICAL RELEVANCE STATEMENT: Using injectable PRF for rehydration protocol of the collagen membrane leads to acidosis that can ultimately optimize wound healing. Differences in the physio-mechanical interplay of collagen matrices and autologous platelet concentrates must result in clinical algorithms if pre-vascularization can maximize outcomes.

Apoptosis-inducing, anti-angiogenic and anti-migratory effects of a dinuclear Pd(II) complex on breast cancer: A promising novel compound.

Because of the high mortality and morbidity rate of breast cancer, successful management of the disease requires synthesis of novel compounds. To this end, ongoing attempts to create new candidates include synthesis of multinuclear metal complexes. The high DNA binding affinity and cytotoxic activity of these complexes makes them promising as breast cancer treatments. This study investigated anti-growth/cytotoxic effect of the dinuclear Pd(II) complex on breast cancer cell lines (MCF-7, MDA-MB-231) using various methods of staining, flow cytometry, and immunoblotting. The study conducted colony formation, invasion, and migration assays were to assess the effect of the complex on metastasis. Increased caspase-3/7 levels and positive annexin V staining were observed in both cell lines, proving apoptosis. Altered TNFR1 and TRADD expression with caspase-8 cleavage followed by BCL-2 inactivation with loss of mitochondrial membrane potential confirmed the presence of apoptosis in MCF-7 and MDA-MB-231, regardless of p53 expression status. The results implied anti-migration properties. Finally, the study used the CAM assay to assess antiangiogenic properties and showed that the complex inhibited angiogenesis. The study concluded the dinuclear Pd(II) complex warrants further in vivo experiments to show its potential in the treatment of breast cancer.

Combination of Phenethyl Isothiocyanate and Dasatinib Inhibits Hepatocellular Carcinoma Metastatic Potential through FAK/STAT3/Cadherin Signalling and Reduction of VEGF Secretion.

Cancerous cells are characterised by their ability to invade, metastasise, and induce angiogenesis. Tumour cells use various molecules that can be targeted to reverse these processes. Dasatinib, a potent Src inhibitor, has shown promising results in treating hepatocellular carcinoma (HCC) in vitro and in vivo. However, its effectiveness is limited by focal adhesion kinase (FAK) activation. Isothiocyanates, on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effect of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac membrane (YSM) assays to evaluate the effect of the drug combination on HCC metastatic potential and angiogenesis in vitro and in vivo. The results showed that the combination inhibited the adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Additionally, the combination reduced angiogenesis in vitro, diminishing the growth of vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin expression and reduced VEGF secretion, reducing HCC metastatic potential. Therefore, a combination of PEITC and dasatinib could be a potential therapeutic strategy for the treatment of HCC.

Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumorigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumorigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin ß-3, its binding partner fibronectin-1 and matrix metalloproteinase enzymes, promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a chicken embryo xenograft model for in vivo validation, we show that CA-induced (+CA) MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional oncogenic alterations) is sufficient to confer early pro-tumorigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

Co-delivery of beta-caryophyllene and indomethacin in the oily core of nanoemulsions potentiates the anti-inflammatory effect in LPS-stimulated macrophage model.

The potentiation of pharmacological effects can be achieved through several strategies, such as the association of substances and delivery in nanostructured systems. In practice, potentiation can be measured by the law of mass action and joint evaluation of the combination index (CI) and dose-response curves. In this context, this study aimed to evaluate the anti-inflammatory effect of the association of ß-caryophyllene and indomethacin in the free form and delivered in nanoemulsions using the in vitro model of LPS-stimulated murine macrophage. The results indicated potentiation of the anti-inflammatory effect of nanoemulsified substances compared to free substances, as well as synergistic action between the sesquiterpene and the selected NSAID. In comparison, the association of ß-caryophyllene and indomethacin in the free form inhibited the production of nitric oxide by 50% at 48.60 µg/mL (CI = 0.21), while the nanoemulsified association of these substances resulted in an IC50 of 1.45 µg/mL (CI = 0.14). In parallel, cytotoxicity assays on HaCaT and MRC-5 cell lines demonstrated the safety of IC50-equivalent concentrations of the anti-inflammatory action, and no irritating effects on the chorioallantoic membrane of embryonated eggs were observed (HET-CAM assay). The results suggest that ß-caryophyllene may be an alternative to replace an inert oily core in nanoemulsion systems when anti-inflammatory effects are desirable.

F3 peptide functionalized liquid crystalline nanoparticles for delivering Salinomycin against breast cancer.

Salinomycin (Sal) is a potent veterinary antibiotic known to offer significant toxicity to the variety of neoplastic cells. Its therapeutic utility is limited due to its higher lipophilicity (logP 7.5) and poor hydrophilicity. Liquid crystalline nanoparticles (LCNPs) known to offer a suitable delivery platform for these kinds of drugs. The overexpressed nucleolin receptor on the cell surface and cytoplasm, could be selected as a target in cancer therapy. The present study involves the development and characterization of the F3 peptide functionalized LCNPs for delivering Sal (F3-Sal-NPs) for selectively targeting to the nucleolin receptor. The optimized LCNPs were characterized for particle size, zeta potential, surface morphology, drug release kinetics and stability. The LCNPs have a structure similar to nematic phases. In vitro drug release studies revealed sustained drug release characteristics (89.5 ± 1.5% at 120 h) with F3-Sal-NPs. The cytotoxicity results demonstrated that F3-Sal-NPs were 4.8, 2.6 and 5.5 folds more effective than naïve drug in MDA-MB-468, MDA-MB-231 and MCF-7 cells, respectively and the cell cycle was arrested in the S and G2/M phases. The expression of the gene responsible for the stemness (CD44 gene), apoptosis (BAX/Bcl-2 ration) and angiogenesis (LCN-2) was reduced by F3-Sal-NPs treatment. Ex vivo hemolytic toxicity was reduced (6.5 ± 1.5%) and the pharmacokinetics and bioavailability of Sal was improved with F3-Sal-NPs. The in vivo antitumor efficacy was tested in EAC bearing mice, where F3-Sal-NPs significantly reduced the tumor growth by 2.8-fold compared to pure Sal and induced necrosis of tumor cells. The results clearly demonstrate the outstanding performance of F3 peptide functionalized LCNPs for delivering Sal against breast cancer.

A new 3D-printed polylactic acid-bioglass composite for bone tissue engineering induces angiogenesis in vitro and in ovo.

Large bone defects such as those that occur after trauma or resections due to cancer still are a challenge for surgeons. Main challenge in this area is to find a suitable alternative to the gold-standard therapy, which is highly risky, and a promising option is to use biomaterials manufactured by 3D printing. In former studies, we demonstrated that the combination of polylactic acid (PLA) and bioglass (BG) resulted in a stable 3D-printable material, and porous and finely structured scaffolds were printed. These scaffolds exhibited osteogenic and anti-inflammatory properties. This 3D-printed material fulfills most of the requirements described in the diamond concept of bone healing. However, the question remains as to whether it also meets the requirements concerning angiogenesis. Therefore, the aim of this study was to analyze the effects of the 3D-printed PLA-BG composite material on angiogenesis. In vitro analyses with human umbilical vein endothelial cells (HUVECs) showed a positive effect of increasing BG content on viability and gene expression of endothelial markers. This positive effect was confirmed by an enhanced vascular formation analyzed by Matrigel assay and chicken chorioallantoic membrane (CAM) assay. In this work, we demonstrated the angiogenic efficiency of a 3D-printed PLA-BG composite material. Recalling the osteogenic potential of this material demonstrated in former work, we manufactured a mechanically stable, 3D-printable, osteogenic and angiogenic material, which could be used for bone tissue engineering.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Background: Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. Methods: A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Results: Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Conclusions: Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Development of PCL PolyHIPE Substrates for 3D Breast Cancer Cell Culture.

Cancer is a becoming a huge social and economic burden on society, becoming one of the most significant barriers to life expectancy in the 21st century. In particular, breast cancer is one of the leading causes of death for women. One of the most significant difficulties to finding efficient therapies for specific cancers, such as breast cancer, is the efficiency and ease of drug development and testing. Tissue-engineered (TE) in vitro models are rapidly developing as an alternative to animal testing for pharmaceuticals. Additionally, porosity included within these structures overcomes the diffusional mass transfer limit whilst enabling cell infiltration and integration with surrounding tissue. Within this study, we investigated the use of high-molecular-weight polycaprolactone methacrylate (PCL-M) polymerised high-internal-phase emulsions (polyHIPEs) as a scaffold to support 3D breast cancer (MDA-MB-231) cell culture. We assessed the porosity, interconnectivity, and morphology of the polyHIPEs when varying mixing speed during formation of the emulsion, successfully demonstrating the tunability of these polyHIPEs. An ex ovo chick chorioallantoic membrane assay identified the scaffolds as bioinert, with biocompatible properties within a vascularised tissue. Furthermore, in vitro assessment of cell attachment and proliferation showed promising potential for the use of PCL polyHIPEs to support cell growth. Our results demonstrate that PCL polyHIPEs are a promising material to support cancer cell growth with tuneable porosity and interconnectivity for the fabrication of perfusable 3D cancer models.

Toxicological Analysis by Assessment of Vascularization and Cell Viability Using the Chicken's Chorioallantoic Membrane (CAM Assay).

The chorioallantoic membrane (CAM) assay is an increasingly popular method using a hen's egg as an experimental organism. Animal models have been established in scientific research for centuries. Yet, awareness of animal welfare in society rises, and the transferability of findings obtained in rodent models to human physiology is challenged. Thus, using fertilized eggs as an alternative platform for animal experimentation might be a promising alternative. The CAM assay is utilized for toxicological analysis by determination of CAM irritation as well as analysis of organ damage and ultimately death of the embryo. Furthermore the CAM provides a micromilieu suited for the implantation of xenografts. Xenogene tissues and tumors grow on the CAM due to a lack of rejection by the immune system and a dense vascular network providing oxygen and nutrients. Multiple analytical methods including in vivo microscopy and various imaging techniques are applicable to this model. Additionally, ethical aspects, a comparatively low financial burden, and low bureaucratic hurdles legitimize the CAM assay.We here describe an in ovo model utilized for xenotransplantation of a human tumor. The model can be used to evaluate the efficacy as well as the toxicity of different therapeutic agents after intravascular injection. Additionally, we present the evaluation of vascularization and viability by intravital microscopy, ultrasonography, and immunohistochemistry.

Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis ΔsigF Mutant.

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.

Reassignment of NMR spectroscopic data of oleana-9(11),12-diene-3ß-ol isolated from Jeffreycia zeylanica and its wound healing potential in terms of cell migration and proangiogenic activity1.

Jeffreycia zeylanica (Asteraceae), a plant endemic to Sri Lanka, is used for the treatment of wounds. The scratch wound assay (SWA) guided fractionation of hexanes extract of J. zeylanica led to the isolation of oleana-9(11),12-diene-3ß-ol (1) which showed enhanced cell migration in SWA and significant proangiogenic response in chorioallantoic membrane (CAM) assay. Since the reported 1H NMR assignments of 1 were incomplete, and some 13C NMR assignments were inconsistent with our observations, reassignment of NMR spectroscopic data of 1 was carried out. Herein we report unambiguous assignment of NMR data of 1 based on 1D and 2D NMR spectra. This is the first report of 1 in J. zeylanica.