Chorioallantoic Membrane Assay Research Articles

GELATIN AND VEGF INCORPORATION IN PVA-TYRAMINE HYDROGELS AS A STRATEGY TO ENHANCE VASCULAR INFILTRATION AND TREAT AVASCULAR NECROSIS

INTRODUCTIONStimulation of angiogenesis via the delivery of growth factors (GFs) like vascular endothelial growth factor (VEGF) is a promising strategy for the treatment of avascular necrosis (AVN). Tyraminated poly-vinyl-alcohol hydrogels (PVA-Tyr), which have the ability to covalently incorporate GFs, were proposed as a platform for the controlled delivery of therapeutic levels VEGF to the necrotic areas[1]. Nevertheless, PVA hydrophilicity and bioinertness limits its integration with the host tissues. The aim of this study was to investigated the effectiveness of incorporating gelatin, an FDA-approved, non-immunogeneic biomaterial with biological recognition sites, as a strategy to facilitate blood vessels invasion of PVA-Tyr hydrogels and to restore the vascular supply to necrotic tissues.METHODSProgressively higher gelatin concentrations (0.01–5wt%) were incorporated in the PVA-Tyr network. Hydrogel physico-chemical properties and endothelial cell attachment were evaluated. Afterwards, the capability of the released VEGF and gelatin to promote vascularization was evaluated via chorioallantoic membrane (CAM) assay. VEGF-loaded PVA-Tyr hydrogels with or without gelatin (n=7) were implanted in a subcutaneous mouse model for 3 weeks. Vascularization (CD31+ cells) and cell infiltration (H&E) were evaluated. Finally, AVN was induced in 6 weeks old male piglets as previously described [2]. A transphyseal hole (3mm) was drilled and PVA-Tyr hydrogels with 1% gelatin were delivered in the defects. Piglets were euthanized after 4 weeks and microCT analysis was performed.RESULTSThe incorporation of 1% gelatin significantly enhanced cell attachment without compromising hydrogels physical properties, degradation time, VEGF retention and release. Thus, this gelatin concentration was selected for further analysis. Additionally, the covalent incorporation of VEGF or gelatin to the PVA-Tyr network does not hamper their bioactivity, as both still promoted neo-angiogenesis in a CAM assay. Following subcutaneous implantation, the presence of gelatin did not increase the cellular infiltration in the PVA-Tyr hydrogels. Nevertheless, higher vascular infiltration was observed in the groups where either gelatin or VEGF were included. Additionally, preliminary microCT results indicated that the delivery of PVA-Tyr hydrogels containing 1% gelatin in an AVN model was effective in preventing the necrosis-associated resorption of the bone.DISCUSSION & CONCLUSIONSThese results indicated that the presence of either gelatin or VEGF was sufficient to promote vascular infiltration. Additionally, preliminary results suggested the suitability of the developed hydrogels to treat AVN.

Incorporation of Inclusion Complexes in the Dissolvable Microneedle Ocular Patch System for the Efficiency of Fluconazole in the Therapy of Fungal Keratitis.

Fluconazole (FNL) is one of the first-line treatments for fungal keratitis as it is an effective broad-spectrum antimicrobial commonly administered orally or topically. However, FNL has a very low water solubility, limiting its drug formulation, therapeutic application, and bioavailability through tissues. To overcome these limitations, this study aimed to develop FNL inclusion complexes (FNL-IC) with cyclodextrin (α-cyclodextrin, sulfobutylether-β-cyclodextrin, and hydroxypropyl-γ cyclodextrin) and incorporate it into a dissolvable microneedle (DMN) system to improve solubility and drug penetration. FNL-IC was evaluated for saturation solubility, Fourier transform infrared spectroscopy, differential scanning calorimetry, in vitro release, minimum inhibitory concentration, minimum fungicidal concentration, and time-killing assay. DMN-FNL-IC was evaluated for mechanical and insertion properties, surface pH, moisture absorption ability, water vapor transmission, and drug content recovery. Moreover, ocular kinetic, ex vivo antimicrobial, in vivo antifungal, and chorioallantoic membrane (HET-CAM) assays were conducted to assess the overall performance of the formulation. Mechanical strength and insertion properties revealed that DMN-FNL-IC has great mechanical and insertion properties. The in vitro release of FNL-IC was significantly improved, exhibiting a 9-fold increase compared to pure FNL. The ex vivo antifungal activity showed significant inhibition of Candida albicans from 6.54 to 0.73 log cfu/mL or 100-0.94%. In vivo numbers of colonies of 0.87 ± 0.13 log cfu/mL (F2), 4.76 ± 0.26 log cfu/mL (FNL eye drops), 3.89 ± 0.24 log cfu/mL (FNL ointments), and 8.04 ± 0.58 log cfu/mL (control) showed the effectiveness of DMN preparations against other standard commercial preparations. The HET-CAM assay showed that DMN-FNL-IC (F2) did not show any vascular damage. Finally, a combination of FNL-IC and DMN was developed appropriately for ocular delivery of FNL, which was safe and increased the effectiveness of treatments for fungal keratitis.

Gastric Inhibitory Polypeptide Receptor (GIPR) Overexpression Reduces the Tumorigenic Potential of Retinoblastoma Cells.

Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy.

Assessment of Antiangiogenic and Cytotoxic Effects of Moringa oleifera Silver Nanoparticles Using Cell Lines

Angiogenesis is the physiological process through which new blood vessels are developed from pre-existing vessels. It includes steps like migration, differentiation, and growth of endothelial cells. Furthermore, it plays a critical role in cancer formation and metastasis. Recently, Moringa oleifera (MO) has gained interest due to its properties in various fields, particularly in nanoparticle (NP) technology. This study aims to demonstrate the cytotoxic and antiangiogenic effects of MO silver nanoparticles (MO-AgNPs). Briefly, the cytotoxic effects of MO-AgNPs on Ea. hy 926 and HT 29 cell lines were observed in the Colony Formation Assay. The antiproliferative effects of MO-AgNPs were then further confirmed using MTT assay, in addition to morphological characterization, migration and closure percent calculation. To evaluate the antiangiogenic effect of MO, the chorioallantoic membrane (CAM) assay was done. Fertilized chicken eggs were divided into three groups: MO-AgNPs, MO ethanol extract and negative control. The results showed antiangiogenic effects were observed at 6-12 µg/mL concentration of MO-AgNPs. Whereas the plate efficiency was 45.7 % ± 1%. The surviving fraction at 12, 6, 3, and 1.5 μg/mL was 27.3± 0.5%, 30.1 ± 0.5 %, 41.8 ± 1.5%, and 69.8 ± 2.5 %, respectively. MO-AgNPs showed higher cytotoxic activity than MO ethanol extract on the Ea. hy926 cell line, where the IC50 of MO-AgNPs were 74, 35, and 12 µg/mL for 24, 48 and 72 hours, respectively. On the other hand, the IC50 of MO ethanol extract was above 200, 135 and 11 µg/mL for 24, 48 and 72 hours, respectively. Additionally, significant morphological changes were observed in Ea. hy cells. Whereas the closure percent average of Ea. hy cells at 1.5, 3, 6, and 12 µg/mL were 91.603, 88.507, 84.599, and 63.144%, respectively. Based on the results showed in this study, MO extract shows potential to be an effective anti-angiogenic agent and is a promising therapeutic option in cancer treatment.

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.

Development of eugenol loaded cellulose-chitosan based hydrogels; in-vitro and in-vivo evaluation for wound healing

Recent years have demonstrated that biopolymers can serve as effective woundhealing agents. We hereby report chitosan-oxidized cellulose (CS.OMCC) based hydrogels loaded with eugenol with distinctive antimicrobial, biocompatible, and angiogenic characteristics. CS.OMCC hydrogels in four different ratios were synthesized through Schiff base (-CN) between chitosan (CS) and oxidized microcrystalline cellulose (OMCC). The swelling and degradation study analyzed the maximum absorption strength and weight loss of 1165% and 56.8 %, respectively. Hydrogels were potent against E. coli and S. aureus. The zones of inhibition were dependent upon the chitosan and eugenol, where 30 % eugenol (Eu) loaded CS.OMCC 4:1 hydrogel revealed greater zones (17 mm). The hydrogels demonstrated favorable biocompatibility, adhesion, and migration potential of mouse fibroblast cells. Moreover, the in-vivo potential by Chorioallantoic membrane (CAM) assay displayed an increase in the number, length, and size of blood vessels, contributing superior angiogenic potential. Increased expression in the case of VEGF further supported the role of hydrogels in promoting angiogenesis. Our study presents eugenol loaded chitosan-oxidized cellulose hydrogels that presented a promising solution to target bacterial infection and promote angiogenesis, thereby promoting the wound healing process.

Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells

Background & aimsMetallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib.MethodsIntrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics.ResultsThe phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib.ConclusionshMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

Eriocitrin Inhibits Angiogenesis by Targeting VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways.

Eriocitrin, a flavanone found in peppermint and citrus fruits, is known to possess many physiological activities. However, the anti-angiogenic effects of eriocitrin are yet to be fully elucidated. Therefore, the objective of this research was to explore the anti-angiogenic effects of eriocitrin both in vitro and in vivo as well as its underlying mechanism. Anti-angiogenic effects of eriocitrin were evaluated utilizing in vitro models of angiogenesis, including inhibition of tube formation, and induction of apoptosis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay in chick embryos was also performed to evaluate the in vivo effects of eriocitrin on angiogenesis. Results showed significant eriocitrin effects on proliferation, tube formation, migration, and apoptosis in HUVECs. Furthermore, in vivo analysis revealed that eriocitrin significantly suppressed the formation of new blood vessels. In particular, it regulated MAPK/ERK signaling pathway and VEGFR2, inhibited the downstream PI3K/AKT/mTOR signaling pathway, and activated apoptosis signals such as caspase cascades. In HUVECs, the expression of matrix metalloproteinases (MMP-2 and MMP-9) exhibited an inhibitory effect on angiogenesis through the suppression of the signaling pathway. Therefore, eriocitrin presents potential for development into an antiangiogenic therapeutic agent.

The chicken chorioallantoic membrane assay revisited – A face-lifted approach for new perspectives in placenta research

The study of very early human placentation is largely limited due to ethical restrictions on the use of embryonic tissue and the fact that the placental anatomy of common laboratory animal models varies considerably from that of humans. In recent years several promising models, including trophoblast stem cell-derived organoids, have been developed that have also proven useful for the study of important trophoblast differentiation processes. However, the consideration of maternal blood flow in trophoblast invasion models currently appears to be limited to animal models. An almost forgotten model to study the invasive behavior of trophoblasts is to culture them in vitro on the chicken chorioallantoic membrane (CAM), showing an extraembryonic vascular network in its mesenchymal stroma that is continuously perfused by the chicken embryonic blood circulation.Here, we present an extension of the previously described ex ovo CAM assay and describe the use of cavity-bearing trophoblast spheroids obtained from the first trimester cell line ACH-3P. We demonstrate how spheroids penetrated the CAM and that erosion of CAM vessels by trophoblasts led to filling of the spheroid cavities with chicken blood, mimicking initial steps of intervillous space blood perfusion. Moreover, we prove that this model is useful for state-of-the-art techniques including immunofluorescence and in situ padlock probe hybridization, making it a versatile tool to study aspects of trophoblast invasion in presence of blood flow.

Perspectives on chick embryo models in developmental and reproductive toxicity screening

Teratology, the study of congenital anomalies and their causative factors intersects with developmental and reproductive toxicology, employing innovative methodologies. Evaluating the potential impacts of teratogens on fetal development and assessing human risk is an essential prerequisite in preclinical research. The chicken embryo model has emerged as a powerful tool for understanding human embryonic development due to its remarkable resemblance to humans. This model offers a unique platform for investigating the effects of substances on developing embryos, employing techniques such as ex ovo and in ovo assays, chorioallantoic membrane assays, and embryonic culture techniques. The advantages of chicken embryonic models include their accessibility, cost-effectiveness, and biological relevance to vertebrate development, enabling efficient screening of developmental toxicity. However, these models have limitations, such as the absence of a placenta and maternal metabolism, impacting the study of nutrient exchange and hormone regulation. Despite these limitations, understanding and mitigating the challenges posed by the absence of a placenta and maternal metabolism are critical for maximizing the utility of the chick embryo model in developmental toxicity testing. Indeed, the insights gained from utilizing these assays and their constraints can significantly contribute to our understanding of the developmental impacts of various agents. This review underscores the utilization of chicken embryonic models in developmental toxicity testing, highlighting their advantages and disadvantages by addressing the challenges posed by their physiological differences from mammalian systems.

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate.

Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

Preparation, Characterization, and Antiangiogenic Evaluation of a Novel 5-Fluorouracil Derivative Solid Lipid Nanoparticle with a Hen's Egg Chorioallantoic Membrane Assay and Wound Healing Response in HaCaT Keratinocytes.

5-Fluorouracil is a heterocyclic aromatic organic compound, and it is commonly used as a chemotherapeutic agent in many cancers. The present goal is to analyze and characterize the physicochemical and biological properties of a new therapeutic formulation of 5-FUD-Gal under simulated chronic wound and oxidative stress conditions. After synthesis of a new 5-fluorouracil derivative, preparation and characterization of the formulation were carried out. The antiangiogenic effect, wound healing, and oxidative stress responses were conducted with a HET-CAM assay and in vitro cell culture technique. The results initially demonstrated that 5-FUD-Gal synthesized by a series of reactions and the SLN formulation were prepared successfully. A strong cell protective effect above 98% cell viability was detected at 20 μM at 48 h. The wound closure of the HaCaT scratch assay was calculated to be 90.12 and 98.98% at 10 and 20 μM concentrations, respectively, at 48 h. Moreover, the strongest effect of 5-FUD-Gal-F was observed at 20 μM concentration on chicken embryos. This study provides novel insights that a new derivative of semisynthetic 5-FUD-Gal-F can be further evaluated as a therapeutic chemical compound in cancer disease.

Abstract 4018: An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is a revolutionary approach in cellular immunotherapy, where a patient’s T cells are genetically engineered to recognize and attack cancer cells. In the last decade, several CAR-T cell therapies have been approved by the FDA. However, this encouraging outcome was obtained only with certain blood cancers, like B cell leukemias, lymphomas, etc. Many research/clinical trials are now underway worldwide to extend the CAR T-cell therapy benefits to a large spectrum of cancers, using rodents. This model presents several drawbacks, such as the immunodeficiency of humanized models, ethical constraints, time, and cost. An alternative, pertinent, 3Rs-compliant in vivo model for developing CAR-T therapy is urgently needed. Xenografts on the chicken embryo’s ChorioAllantoic Membrane (CAM) have proven extremely valuable for in vivo cancerology studies. In this work, we evaluated in vivo the anti-tumor efficacy of CD19-targeting CAR-T cells on human lymphoma using Inovotion’s CAM assay. METHODS Human lymphoma Raji cells (ATCC) were grafted on the CAM on embryonic development day (EDD) 9. A 2nd generation of CD19-targeting CAR-T cells (CAR-T-(CD3/4-1BB)) developed by Allogenica (France) were co-injected at EDD9 at E/T (effector CAR-T cell vs target Raji cell) ratio 1:1. One group was injected with CAR-T (CD3/4-1BB) only once at EDD9 and a second group received a first injection of CAR-T (CD3/4-1BB) at EDD9 and a second injection at EDD13. Embryonic viability was checked daily. On EDD18. The efficacy of CAR-T therapy was evaluated basing on tumor weight, metastatic invasion in the lower CAM and CAR-T infiltration in tumors detected by qPCR. Activation of the chicken embryo immune system was detected as well. RESULTS The treatment with CAR-T-(CD3/4-1BB) cells was well tolerated in ovo, no significant embryo mortality/abnormality was observed. CAR-T-(CD3/4-1BB) cells prevented tumor formation (tumor weight was 88.67% to 90.62% lower than control, p < 0.001); and prevented metastasis formation in the lower CAM (86.88% to 81.43% lower than control, p < 0.001). CONCLUSION Different to in vitro models and immunodeficient murine models, chicken embryo possesses a progressively mature immune system. Our results show that CAR-T-(CD3/4-1BB) cells can prevent tumor formation and metastatic invasion when co-injected with tumor cells. Inovotion’s CAM assay is a viable alternative in vivo model for developing CAR-T cell therapy on a large spectrum of cancers. It is fully 3Rs-compliant. Citation Format: Yan Wang, Xavier Rousset, Chloé Prunier, Emilien Dosda, Alejandra Gutierrez-Guerrero, Pauline Abrial, Inna Menkova, Jean Viallet. An innovative in vivo model for CAR-T cell therapy development: Tolerability and efficacy evaluation of CD19-targeting CAR-T cells on human lymphoma using the chicken CAM assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4018.

Biomechanical analysis of the human derived soft tissue graft Epiflex for use in oral soft tissue augmentation

PurposeThis study aimed to investigate the biomechanical properties, cell migration, and revascularization of the acellular dermal matrix Epiflex. As a decellularized, freeze–dried human skin graft, Epiflex has broad applications in medical fields, particularly in implantology and dentistry. Understanding its biomechanical characteristics is crucial for its clinical adoption as a novel soft tissue graft option.MethodsEpiflex (n = 3) was evaluated in comparison to palatal tissue from body donors (n = 3). Key metrics, such as elongation and tear resistance, were quantified. Both graft types underwent histological analysis and scanning electron microscopy. Additionally, the healing properties of Epiflex were assessed using a Chorioallantoic Membrane (CAM) Assay.ResultsBiomechanically, Epiflex (mean = 116.01 N) demonstrated the ability to withstand greater forces (p = 0.013) than human palatal tissue (mean = 12.58 N). When comparing the elongation, no significant difference was measured (ASG mean = 9.93 mm, EF mean = 9.7 mm). Histologically, Epiflex exhibited a loosely connected network of collagen fibers with a dense upper layer. The CAM Assay indicated efficient revascularization.ConclusionEpiflex appears to be a viable option for soft tissue augmentation, particularly appealing to patient groups who avoid all or specific animal-derived products due to ethical or religious reasons.Graphical

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

Green Synthesis of Selenium Nanoparticles From Clove and Their Toxicity Effect and Anti-angiogenic, Antibacterial and Antioxidant Potential.

Introduction Nanoparticles, owing to their minuscule size, have become pivotal in diverse scientific endeavors, presenting unique characteristics with applications spanning medicine to environmental science. Selenium nanoparticles (SeNPs) exhibit potential in diverse biomedical uses. Aim This research investigates the potential anti-inflammatory and anticancer properties of SeNPs, which are synthesized using the green synthesis method. This eco-friendly approach aligns with sustainable practices and utilizes clove extract (Syzygium aromaticum). Materials and methods Clove extract facilitates SeNPsynthesis via sodium selenite reduction. The characterization methods comprised Fourier-transforminfrared (FTIR) spectroscopy, UV-VIS spectroscopy, and scanning electron microscopy (SEM). Assessments coveredantioxidant properties, chorioallantoic membrane assay (CAM) assay for antiangiogenic effects, toxicity evaluation, and antibacterial assays. Results Successful synthesis of SeNPswas verified by a UV-visible absorption peakat 256 nm and FTIR peaks around 3500-500 cm-1, and the spherical morphology was confirmed by SEM analysis with EDAX, which indicated the presence of SeNPs and their unique properties. Phytochemical substances are active chemicals that contribute to the properties of SeNPs. The SeNPs exhibited antioxidant activity with an IC50 value of 0.437 µg/mL and antibacterial properties against bacterial pathogenSalmonella species, with a zone of inhibition measuring 19 mm.The CAM assay demonstrated possible antiangiogenic actions, and toxicity testing on Artemia nauplii showed biocompatibility. Conclusion This study underscores the efficient synthesis of SeNPs using clove extract, emphasizing their potential applications. The notable properties of SeNPs emphasize their promise for diverse biomedical and environmental uses.

Pd(II) and Pt(II) saccharinate complexes with two phosphine derivatives: Synthesis, anticancer and antiangiogenic activities

As clinically used anticancer Pt(II) drugs have severe side effects, there is a growing interest for new metal complexes with great potential for cancer therapy. The current work aimed to prepare and characterize new Pd(II) and Pt(II) saccharinate (sac) complexes bearing pyridyl‐ and benzyldiphenylphosphines (PPh2Py and PPh2Bz, respectively), cis‐[Pd(sac)2(PPh2Py)2] (1), cis‐[PtCl(sac)(PPh2Py)2]·0.5DMF (2), cis‐[Pd(sac)2(PPh2Bz)2]·DMF (3) and trans‐[PtCl(sac)(PPh2Bz)2] (4) as promising anticancer and antiangiogenic drugs. The anticancer activity of the complexes was screened against seven cancer cell lines including HCT116 (colon), HepG2 (liver), MDA‐MB‐231 (breast), PANC‐1 (pancreatic), A549 (lung), C6 (glioma), DU145 (prostate) and normal human lung epithelial cells (BEAS‐2B). 1 and 2 did not show biological activity below 20 μM at 48 h, whereas 3 and 4 displayed significant cytotoxic effect on the cancer cells. 4 was the most potent complex (IC50 = 2.2–12.1 μM) and displayed much greater cytotoxicity than cisplatin in all the cancer cell lines. 4 caused apoptosis in HCT116 cells as evidenced by annexin V positivity and caspase 3/7 activity assays. Furthermore, the inhibition of antiapoptotic Bcl‐2 proteins by the complex suggested the intrinsic apoptosis. In addition, 4 greatly enhanced generation of intracellular reactive oxygen species (ROS) and consequently caused remarkable DNA double‐strand breaks in HCT116 cells. Moreover, the chick chorioallantoic membrane (CAM) assay was used to evaluate antiangiogenic potential of 4. The complex effectively inhibited angiogenesis at a dose of 50 ng, suggesting it as a promising multi‐targeted agent for antiangiogenic cancer treatment.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer

AimsExchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. Main methodsWe employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. Key findingsInhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. ConclusionsOur findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Analysis of Osteosarcoma Cell Lines and Patient Tissue Using a 3D In Vivo Tumor Model—Possible Effects of Punicalagin

Osteosarcomas are the most common primary malignant bone tumors and mostly affect children, adolescents, and young adults. Despite current treatment options such as surgery and polychemotherapy, the survival of patients with metastatic disease remains poor. In recent studies, punicalagin has reduced the cell viability, angiogenesis, and invasion in cell culture trials. The aim of this study was to examine the effects of punicalagin on osteosarcomas in a 3D in vivo tumor model. Human osteosarcoma biopsies and SaOs-2 and MG-63 cells, were grown in a 3D in vivo chorioallantoic membrane (CAM) model. After a cultivation period of up to 72 h, the tumors received daily treatment with punicalagin for 4 days. Weight measurements of the CAM tumors were performed, and laser speckle contrast imaging (LSCI) and a deep learning-based image analysis software (CAM Assay Application v.3.1.0) were used to measure angiogenesis. HE, Ki-67, and Caspase-3 staining was performed after explantation. The osteosarcoma cell lines SaOs-2 and MG-63 and osteosarcoma patient tissue displayed satisfactory growth patterns on the CAM. Treatment with punicalagin decreased tumor weight, proliferation, and tumor-induced angiogenesis, and the tumor tissue showed pro-apoptotic characteristics. These results provide a robust foundation for the implementation of further studies and show that punicalagin offers a promising supplementary treatment option for osteosarcoma patients. The 3D in vivo tumor model represents a beneficial model for the testing of anti-cancer therapies.

CRISPR/Cas9 screenings unearth protein arginine methyltransferase 7 as a novel essential gene in prostate cancer metastasis

Due to the limited effectiveness of current treatments, the survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) is significantly reduced. Consequently, it is imperative to identify novel therapeutic targets for managing these patients. Since the invasive ability of cells is crucial for establishing and maintaining metastasis, the aim of this study was to identify the essential regulators of invasive abilities of mCRPC cells by conducting two independent high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using siRNA technology, with protein arginine methyltransferase 7 (PRMT7) emerging as the most promising candidate. We demonstrated that its inhibition or depletion via genetic or pharmacological approaches significantly reduces invasive, migratory and proliferative abilities of mCRPC cells in vitro. Moreover, we confirmed that PRMT7 ablation reduces cell dissemination in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules by methylating various transcription factors, such as FoxK1, resulting in the loss of adhesion from the primary tumor and increased motility of mCRPC cells. Furthermore, PRMT7 higher expression correlates with tumor aggressivity and poor overall survival in prostate cancer patients. Thus, this study demonstrates that PRMT7 is a potential therapeutic target and potential biomarker for mPCa.