Chorioallantoic Membrane Assay Research Articles

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

Polytetrafluoroethylene (PTFE) Microplastics Affect Angiogenesis and Central Nervous System (CNS) Development of Duck Embryo

Prolonged exposure to teratogens is known to cause neural tube defects (NTDs), a severe malformation of the central nervous system (CNS) that significantly contributes to global infant mortality. In recent years, exposure to nanoplastics (NPs) has been linked to faulty neural crest closure and altered neurulation by altering cellular adhesion molecules and accumulation of plastic particles in the neural tube leading to NTDs. However, research on the influence of various types of microplastics (MPs) on malformations of the CNS are still limited. In this study, we investigated whether MPs of polytetrafluoroethylene (PTFE)—a type of plastic commonly used as non-stick coatings of cooking utensils can affect angiogenesis and CNS development using ducks as model organisms. PTFE MPs were administered on Day 3 of duck embryo development at varying concentrations (0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, and 5 mg/ml), and angiogenesis was evaluated using a chorioallantoic membrane (CAM) assay. Gross morphology and histology of the spinal column and brain were analyzed on Days 8 and 18, respectively. FTIR confirmed PTFE's structure, while SEM and DLS analyses showed particle sizes between 300 nm and 5 μm, classifying them as MPs. High concentrations (5 mg/ml) of PTFE MPs treated on duck embryos resulted in a 35% mortality rate and reduced vascular density, suggesting anti-angiogenic effects. Brain and spinal abnormalities, such as encephalomalacia and spinal cord discontinuities were observed in the PTFE-treated embryos. Based on these results, PTFE is an anti-angiogenic and teratogenic agent affecting the development of duck embryos.

Synergistic integration of plant derived galactomannan and MXene to produce multifunctional nanocomposites with antibacterial and osteogenic properties

This study proposes to combine plant-derived galactomannan and MXene to form an organic-inorganic hydrogel network with integrative antibacterial and osteogenic properties. Oxidized galactomannan (OxGa) was blended with N-succinyl chitosan (NSC) and varying concentrations of Mxene, owing to its high reactivity and biocompatibility. The results indicated that Mxene-loaded OxGa/NSC scaffolds exhibited biomimetic topography, adequate mechanical features and excellent physicochemical properties with strong antibacterial properties against pathogenic microbes. The cell culture experiments showed dose-dependent cytotoxic behaviour. An optimized MXene content of ≤ 4 mg/ml revealed no cytotoxicity and augmented the proliferation of osteoblast cells. Similar results were obtained in gene expression analysis where ALP, COL1A1, RUNX2, and SOX2 genes showed up-regulation. In addition, the chorioallantoic membrane (CAM) assay also illustrated augmentation of angiogenesis without any toxicity. The overall results highlight the dual performance of OxGa/NSC-MXene scaffolds to exhibit anti-infection with simultaneous osteogenic properties, which is purely determined by the concentration of MXene. Therefore, regulating MXene concentration can serve as a chemical switch in imparting adequate antibacterial functions with the ability to allow new osseous generation. Our findings on the newly designed Mxene-loaded OxGa/NSC scaffolds can be a promising biomaterial for bone-related infections with a concurrent ability to promote osteogenesis.

Highly porous sildenafil loaded polylactic acid/polyvinylpyrrolidone based 3D printed scaffold containing forsterite nanoparticles for craniofacial reconstruction

Tissue engineering has emerged as a promising substitute for traditional tissue repair methods. Nowadays, advancements in 3D printing technology have enabled the fabrication of customized scaffolds to support tissue regeneration. In the present study, a polylactic acid-polyvinylpyrrolidone 3D-printed scaffold containing 10 % forsterite was fabricated. Subsequently, lyophilized fucoidan microstructures loaded with sildenafil were filled the channels of this 3D-printed scaffold. The fabricated scaffold loaded with sildenafil was thoroughly characterized, revealing that 97.46 % of the loaded sildenafil was released in a sustained manner over 28 days. Furthermore, the biocompatibility of MG63 was evaluated through cell viability and adhesion tests. The findings indicated a direct and favorable influence on cell behavior. Based on the chicken chorioallantoic membrane assay, the fabricated scaffold significantly increases angiogenesis due to the sustained release of sildenafil. Moreover, in-vivo studies conducted on a rat model demonstrated that the 3D-printed scaffold was able to stimulate and accelerate the repair of calvarial defects within 8 weeks, and the amount of new bone tissue formation was significantly higher than that of other experimental groups. Based on the comprehensive in-vitro and in-vivo assessments, the scaffold with a macro- and microporous structure combined with the ability to release sildenafil is suggested as a potential candidate for repairing bone tissue, especially in the context of skull defects.

Exosomal Delivery of miR-155 Inhibitor can Suppress Migration, Invasion, and Angiogenesis Via PTEN and DUSP14 in Triple-negative Breast Cancer.

Triple-Negative Breast Cancer (TNBC) is the most common type of breast cancer (BC). In order to develop effective treatments for TNBC, it is vital to identify potential therapeutic targets. Angiogenesis stimulates tumor growth and metastasis in TNBC, and miR-155 plays a crucial role in this process. The exosome is a nano-sized vesicle that carries many cargoes, including miRNAs. The present study investigated the effect of exosomal delivery of miR-155 antagomir on tumor migration, invasion, and angiogenesis in TNBC. From MDA-MB-231 cells, exosomes were extracted, characterized, and loaded with miR-155 antagomir using electroporation. The expression of miR-155 and its target genes, including PTEN and DUSP14, was analyzed using RTqPCR. The wound-healing and transwell assays were used to measure cell migration and invasion. Furthermore, angiogenesis was evaluated by tube formation and chorioallantoic membrane (CAM) assays. The results indicated that exosomal delivery of miR-155 antagomir to HUVEC cells significantly suppressed miR-155 expression while upregulating PTEN and DUSP14. The tube formation properties of HUVEC cells were also significantly reduced following treatment with exosomes containing miR-155 antagomirs, and these results were confirmed using CAM assay. The migration and invasion of MDA-MB-231 cells were significantly reduced after treatment with miR-155 antagomir-loaded exosomes. It was found that miR-155 antagomir delivery using exosomes can inhibit migration, invasion, and angiogenesis viaPTEN and DUSP14 in TNBC.

Cyclophilin A knockdown inhibits the proliferation and metastatic ability of AGS gastric cancer stem cells by downregulating CD147/STAT3/AKT/ERK and epithelial‑mesenchymal transition.

Gastric cancer stem cells (GCSCs) contribute to the challenging aspects of gastric cancer, such as progression, metastasis, treatment resistance and recurrence. Inhibitors targeting cyclophilin A (CypA) have shown potential in curtailing GCSC growth. Building upon this, the current study delved deeper into understanding the functional role of CypA in controlling the proliferation and metastatic capabilities of GCSCs, employing CypA‑specific small interfering RNA. The results revealed that knockdown of CypA led to significant suppression of the growth and tumorsphere‑forming capacity of GCSCs derived from AGS cells. This effect was mediated by arresting the cell cycle at the G0/G1 and S phases, and promoting apoptosis. Furthermore, silencing of CypA exerted inhibitory effects on the migration and invasion of AGS GCSCs by modulating the process of epithelial‑mesenchymal transition. Notably, the observed antiproliferative and antimetastatic effects of CypA knockdown were associated with the downregulation of critical regulators of gastric cancer stemness, such as CD44, CD133, aldehyde dehydrogenase 1 family member A1, NANOG, OCT4 and SOX2. This regulation occurred through inactivation of the CD147/STAT3/AKT/ERK signaling pathway. Additionally, CypA knockdown effectively curbed in vivo tumor growth of AGS GCSCs in a chorioallantoic membrane assay using chick embryos. These findings underscore the critical role of CypA in promoting the proliferation and metastasis of GCSCs, highlighting its potential as an effective therapeutic target for eradicating GCSCs and improving gastric cancer treatment outcomes.

Implementing Chicken Chorioallantoic Membrane (CAM) Assays for Validating Biomaterials in Tissue Engineering: Rationale and Methods.

Tissue engineering is a promising approach for generating or repairing living tissues. The development of innovative biomaterials for tissue engineering has the potential to address the unmet clinical needs in certain applications. However, before these biomaterials can be used in clinical settings, they must undergo preclinical testing to ensure safety and performance. The chicken chorioallantoic membrane (CAM) assay is a preferred screening tool for studying biocompatibility, angiogenesis, and inflammation induced by biomaterials owing to ethical and economic considerations. This CAM-based platform increased the throughput of biomaterial testing for tissue engineering before invivo testing. In this paper, we discuss the advantages of the CAM model. We also provided a step-by-step guide for implementing the CAM model in a research laboratory, along with tips and tricks for successfully running CAM assays. Finally, we present examples of biomaterials screened using CAM assays. CAM assay is a powerful invivo model for assessing the angiogenic potential of tissue-engineered scaffolds. This guide provides a framework for conducting the assay, but specific experimental conditions should be optimized based on the scaffold material and the research question.

Considerations of growth factor and material use in bone tissue engineering using biodegradable scaffolds in vitro and in vivo

Bone tissue engineering aims to harness materials to develop functional bone tissue to heal ‘critical-sized’ bone defects. This study examined a robust, coated poly(caprolactone) trimethacrylate (PCL-TMA) 3D-printable scaffold designed to augment bone formation. Following optimisation of the coatings, three bioactive coatings were examined, i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA), fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) applied sequentially (PEA/FN/BMP-2) and iii) both ELP and PEA/FN/BMP-2 coatings applied concurrently. The scaffold material was robust and showed biodegradability. The coatings demonstrated a significant (p < 0.05) osteogenic response in vitro in alkaline phosphatase gene upregulation and alkaline phosphatase production. The PCL-TMA scaffold and coatings supported angiogenesis and displayed excellent biocompatibility following evaluation on the chorioallantoic membrane assay. No significant (p < 0.05) heterotopic bone formed on the scaffolds within a murine subcutaneous implantation model, compared to the positive control of BMP-2 loaded collagen sponge following examination by micro-computed tomography or histology. The current studies demonstrate a range of innovative coated scaffold constructs with in vitro efficacy and clearly illustrate the importance of an appropriate in vivo environment to validate in vitro functionality prior to scale up and preclinical application.

A Facile Strategy for Preparing Flexible and Porous Hydrogel-Based Scaffolds from Silk Sericin/Wool Keratin by In Situ Bubble-Forming for Muscle Tissue Engineering Applications.

In the present study, it is aimed to fabricate a novel silk sericin (SS)/wool keratin (WK) hydrogel-based scaffolds using an in situ bubble-forming strategy containing an N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling reaction. During the rapid gelation process, CO2 bubbles are released by activating the carboxyl groups in sericin with EDC and NHS, entrapped within the gel, creating a porous cross-linked structure. With this approach, five different hydrogels (S2K1, S4K2, S2K4, S6K3, and S3K6) are constructed to investigate the impact of varying sericin and keratin ratios. Analyses reveal that more sericin in the proteinaceous mixture reinforced the hydrogel network. Additionally, the hydrogels' pore size distribution, swelling ratio, wettability, and in vitro biodegradation rate, which are crucial for the applications of biomaterials, are evaluated. Moreover, biocompatibility and proangiogenic properties are analyzed using an in-ovo chorioallantoic membrane assay. The findings suggest that the S4K2 hydrogel exhibited the most promising characteristics, featuring an adequately flexible and highly porous structure. The results obtained by in vitro assessments demonstrate the potential of S4K2 hydrogel in muscle tissue engineering. However, further work is necessary to improve hydrogels with an aligned structure to meet the features that can fully replace muscle tissue for volumetric muscle loss regeneration.

Therapeutic potency of marine collagen/pectin scaffolds - Fabrication, characterization and evaluation

Skin is an important vital organ that must be given proper care and protection from external damage and harmful microbes. If injured, it must be treated with an ideal wound dressing material with potent hemostatic and non-toxic properties. In the present study, fish collagen (FC) was extracted from the fins and tails of Black pomfret (Parastromateus niger). The isolated fish collagen was homogenized with pectin (P) and freeze dried to obtain fish collagen/pectin (FC/P) scaffolds. Scanning electron microscopic analysis showed the porous nature of scaffolds with intermittent holes. UV–Visible and Fourier infrared spectroscopic analyses demonstrated the physicochemical properties of FC/P scaffolds. Hemolytic assay performed using human blood demonstrated the percentage of hemolysis as 0.5 %. In vitro blood clotting assay carried out to determine the hemostatic behaviour displayed the formation of blood clot within 60 s in the presence of FC/P scaffolds. 95 % of cells were viable with the highest concentration of FC/P scaffold used for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Scratch wound assay demonstrated complete closure of wound in FC/P scaffold treated cells after 48 h of treatment. Chick embryo chorioallantoic membrane (CAM) assay showed the development of new blood vessels within 6 h of incubation with the FC/P scaffolds, thereby proving their angiogenic potency. These results indicate the potential use of FC/P scaffolds as effective biomaterials for tissue regenerative applications.

Highly porous polycaprolactone microspheres for skeletal repair promote a mature bone cell phenotype in vitro.

Improving our ability to treat skeletal defects is a critical medical challenge that necessitates the development of new biomaterials. One promising approach involves the use of degradable polymer microparticles with an interconnected internal porosity. Here, we employed a double emulsion to generate such round microparticles (also known as microspheres) from a polycaprolactone-based polymerised high internal phase emulsion (polyHIPE). These microspheres effectively supported the growth of mesenchymal progenitors over a 30-day period, and when maintained in osteogenic media, cells deposited a bone-like extracellular matrix, as determined by histological staining for calcium and collagen. Interestingly, cells with an osteocyte-like morphology were observed within the core of the microspheres indicating the role of a physical environment comparable to native bone for this phenotype to occur. At later timepoints, these cultures had significantly increased mRNA expression of the osteocyte-specific markers dentin matrix phosphoprotein-1 (Dmp-1) and sclerostin, with sclerostin also observed at the protein level. Cells pre-cultured on porous microspheres exhibited enhanced survival rates compared to those pre-cultured on non-porous counterparts when injected. Cells precultured on both porous and non-porous microspheres promoted angiogenesis in a chorioallantoic membrane (CAM) assay. In summary, the polycaprolactone polyHIPE microspheres developed in this study exhibit significant promise as an alternative to traditional synthetic bone graft substitutes, offering a conducive environment for cell growth and differentiation, with the potential for better clinical outcomes in bone repair and regeneration.

Hyaluronic acid-functionalized carboxymethyl dextran-coated melatonin nanoconjugates for targeted etoposide delivery in metastatic colon cancer: Extensive in-vitro investigation in HCT116 cell lines, antimicrobial efficacy, and anti-angiogenic potential in chick chorioallantoic membrane (CAM) assay

Managing advanced colon cancer is challenging, requiring targeted therapies. This study presents a novel nanoconjugate system, HA-CMD@ETP-MLT-NCs, designed to deliver etoposide (ETP) specifically to colon cancer cells. The system consists of Hyaluronic Acid (HA)-Functionalized Carboxymethyl Dextran (CMD) coated with Melatonin (MLT). The nanoconjugates showed good stability, with a zeta potential of −29.90 mV and a particle size of 199.1 nm. They achieved an 80.3 % yield and a high drug entrapment efficiency of 93.4 %. In vitro release studies demonstrated pH-dependent drug release, with 73.4 % released at pH 5.5 (tumour-like environment) and 42.6 % at pH 7.4 (normal tissue) over 24 h. The nanoconjugates improved cellular uptake, induced apoptosis, and reduced reactive oxygen species (ROS) in HCT116 colon cancer cells. Flow cytometry showed a significant decrease in ROS levels, and lipid peroxidation inhibition increased to 56.67 %. These findings suggest that HA-CMD@ETP-MLT-NCs enhance etoposide delivery and reduce side effects. Further in vivo studies and clinical trials are needed to confirm its therapeutic potential.

Chitosan/Fibroin Biopolymer-Based Hydrogels for Potential Angiogenesis in Developing Chicks and Accelerated Wound Healing in Mice.

Potential therapies for wound management remain one of the most challenging affairs to date. Biopolymer hydrogels possess inherent properties that facilitate the healing of damaged tissue by creating a supportive and hydrated environment. Chitosan/fibroin hydrogels were formulated with poly (vinyl pyrrolidone) and cross-linked using 3-aminopropyl (diethoxy) methylsilane (APDEMS) for the aforementioned function. The hydrogels were characterized through Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy, and their swelling response was observed using a variety of solvents. Additionally, hydrogels were investigated for biomedical applications. As the amount of fibroin added to the hydrogels increased, the swelling ratio decreased. The analysis of chorioallantoic membrane (CAM) assay revealed that higher concentrations of fibroin in the hydrogel were directly correlated with increased angiogenesis. The intragroup comparison showed that the vascular number in the CPF5 group was significantly increased (p ≤ 0.05) compared to other hydrogel groups. The wound healing efficiency of the prepared hydrogels showed that the rate of wound reduction (99.06%) was remarkably (p ≤ 0.05) high in the hydrogel group with a greater fibroin content against control (67.03%). Histological findings of wounded tissues corroborate the abovementioned results, showing dense fibrous connective tissues in the fibroin group compared to the control. The results of this work provide thorough preclinical evidence that chitosan-fibroin biopolymers are involved in enhanced angiogenesis in growing chicks and speed up wound healing in mice without any obvious toxicity.

Targeting Bone Tumours with 45S5 Bioactive Glass.

Despite advances in treatment modalities, bone tumour therapies still face significant challenges. Severe side effects of conventional approaches, such as chemo- and radiation therapy, result in poor survival rates and high tumour recurrence rates, which are the most common issues that need to be improved upon. The aim of this study was to evaluate the therapeutic properties of 45S5 bioactive glass (BG) for targeting bone tumours. The viability of the cells derived from osteosarcoma, chondrosarcoma, and giant cell tumours was significantly reduced in the presence of 45S5-BG. In contrast, the viability of non-malignant osteoblast-like cells, chondrocytes, and bone marrow-derived stromal cells was not or only slightly affected. While alterations to the particle surface induced by heat treatment, acid etching, or incubation in a simulated body fluid had only minor effects on cytotoxicity, reducing the particle size or sintering the material significantly improved the cytotoxic effect of 45S5-BG. Further, using a chicken chorioallantoic membrane assay, the co-transplantation of 45S5-BG resulted in a significant reduction in tumour formation in vivo. Given the known positive effects of BGs on bone regeneration, our findings suggest that 45S5-BG holds great potential for the development of new and effective bone tumour therapies, with minimal side effects on non-malignant cells and simultaneous contribution to bone healing.

Synthesis, pharmacological evaluation, and modeling of novel quaternary ammonium salts derived from β-carboline containing an imidazole moiety as angiogenesis inhibitors

In this study, a series of novel β-carboline condensed imidazolium derivatives (7a-7y) were designed and synthesized by incorporating imidazolium salt structures into β-carboline. The cytotoxicity of compounds 7a-7y was evaluated in various cancer cell lines, including lung cancer (A549), gastric cancer (BGC-823), mouse colon cancer (CT-26), liver cancer (Bel-7402), and breast cancer (MCF-7), using the MTT assay. Most compounds exhibited significant activity against one or more of the cancer cell lines. Notably, compounds 7 g, 7o, 7r, 7 s, 7u, 7v, 7x, and 7w showed the highest cytotoxic activity (IC50 < 2 μM) in the tested tumor cell lines. Compound 7x demonstrated cytotoxic activities of 1.3 ± 0.3 μM (for BGC-823), 2.4 ± 0.4 μM (against A549), 7.8 ± 0.9 μM (for Bel-7402), and 9.8 ± 1.4 μM (against CT-26). The chick chorioallantoic membrane assay revealed significant anti-angiogenic potential of compound 7x. Molecular imprinting studies suggested the anti-angiogenic effect of compound 7x might be attributed to inhibition of VEGFR2 kinase. Molecular docking and molecular dynamics further indicate that its activity may be primarily associated with the potential inhibition of VEGFR2. Our research outcomes have provided valuable lead compounds for the development of novel antitumor drugs and have offered beneficial insights for subsequent drug design and optimization.

Cryogenic 3D Printing of GelMA/Graphene Bioinks: Improved Mechanical Strength and Structural Properties for Tissue Engineering.

Tissue engineering aims to recreate natural cellular environments to facilitate tissue regeneration. Gelatin methacrylate (GelMA) is widely utilized for its biocompatibility, ability to support cell adhesion and proliferation, and adjustable mechanical characteristics. This study developed a GelMA and graphene bioink platform at concentrations of 1, 1.5, and 2 mg/mL to enhance scaffold properties for tissue engineering applications. Graphene was incorporated into GelMA matrices to improve mechanical strength and electrical conductivity of the bioinks. The compressive strength and thermal stability of the resulting GelMA/graphene scaffolds were assessed through DSC analysis and mechanical testing. Cytotoxicity assays were conducted to determine cell survival rates. Cryoprinting at -30°C was employed to preserve scaffold structure and function. The chorioallantoic membrane (CAM) assay was used to evaluate biocompatibility and angiogenic potential. The integration of graphene significantly amplified the compressive strength and thermal stability of GelMA scaffolds. Cytotoxicity assays indicated robust cell survival rates of 90%, confirming the biocompatibility of the developed materials. Cryoprinting effectively preserved scaffold integrity and functionality. The CAM assay validated the biocompatibility and angiogenic potential, demonstrating substantial vascularization upon scaffold implantation onto chick embryo CAM. Integrating graphene into GelMA hydrogels, coupled with low-temperature 3D printing, represents a potent strategy for enhancing scaffold fabrication. The resultant GelMA/graphene scaffolds exhibit superior mechanical properties, biocompatibility, and pro-vascularization capabilities, making them highly suitable for diverse tissue engineering and regenerative medicine applications.

Folate-Functionalized Chitosan-PLGA Nanoparticles: A Novel approach for targeted osthole delivery in pancreatic cancer

Efficient drug delivery systems targeting cancer cells are crucial for enhancing cancer therapy. In this study, we developed PLGA nanoparticles coated with folate-conjugated chitosan (osthole-PLGA-NPs/CS-FA) to deliver osthole to cancer cells and investigated its inhibitory and molecular signaling mechanisms in the PANC-1 pancreatic cancer cell line. Field emission scanning electron microscopy (FESEM) revealed that osthole-PLGA-NPs/CS-FA had a spherical structure with a uniform size distribution. Dynamic light scattering (DLS) analysis showed an average size of 171.76 nm, a dispersion index 0.26, and a surface charge of + 33.08 mV, indicating stability and uniform dispersion. Fourier-transform infrared (FTIR) spectrum analysis confirmed the successful incorporation of osthole into the PLGA nanoparticles, with an encapsulation efficiency of 93.12 %. These physicochemical properties suggest efficient cellular uptake and targeted delivery. The antioxidant potential of osthole-PLGA-NPs/CS-FA was evaluated using the ABTS assay, showing concentration-dependent inhibition of free radicals with an IC50 value of 172.95 μg/mL. The anticancer properties were assessed using the MTT assay, demonstrating a significant and concentration-dependent cytotoxic effect on PANC-1 cells (IC50 = 31.2 μg/mL) with minimal impact on normal human foreskin fibroblast (HFF) cells. DAPI staining and flow cytometry analyses confirmed a concentration-dependent increase in apoptosis in PANC-1 cells. The nanoparticles induced upregulation of Bax and downregulation of Bcl2, indicating activation of the intrinsic mitochondrial apoptotic pathway. The anti-angiogenic activity of osthole-PLGA-NPs/CS-FA was evaluated using the chick chorioallantoic membrane (CAM) assay. The results showed significant inhibition of angiogenesis in a concentration-dependent manner, starting at 40 μg/mL and increasing up to 120 μg/mL. In conclusion, osthole-PLGA-NPs/CS-FA nanoparticles exhibit promising potential for targeted pancreatic cancer therapy by enhancing cellular uptake, inducing apoptosis, and inhibiting angiogenesis.

3D printed polylactic acid/polyethylene glycol/bredigite nanocomposite scaffold enhances bone tissue regeneration via promoting osteogenesis and angiogenesis

Recently, the fabrication of personalized scaffolds with high accuracy has been developed through 3D printing technology. In the current study, polylactic acid/polyethylene glycol (PLA/PEG) composite scaffolds with varied weight percentages (0, 5, 10, 20 and 30 %) of bredigite nanoparticles (B) were fabricated using the 3D printing and then characterized through scanning electron microscopy and Fourier transform infra-red spectroscopy. The addition of B nanoparticles up to 20 wt% to PLA/PEG scaffold increased the compressive strength (from 7.59 to 13.84 MPa) and elastic modulus (from 142.42 to 268.33 MPa). The apatite formation ability as well as inorganic ion release in simulated body fluid were investigated for 28 days. The MG-63 cells viability and adhesion were enhanced by increasing the amount of B in the PLA/PEG scaffold and the osteogenic differentiation of the rat bone marrow mesenchymal stem cells was confirmed by alkaline phosphatase activity test and alizarin red staining. According to chorioallantoic membrane assay, the highest angiogenesis occurred around the PLA/PEG/B30 scaffold. In vivo experiments on a rat calvarial defect model demonstrated an almost complete recovery in the PLA/PEG/B30 group within 8 weeks. Based on the results, the PLA/PEG/B30 composite scaffold is proposed as an optimal scaffold to repair bone defects.

Critical appraisal of the chorioallantoic membrane model for studying angiogenesis in preclinical research.

Angiogenesis, the biological mechanism by which new blood vessels are generated from existing ones, plays a vital role in growth and development. Effective preclinical screening is necessary for the development of medications that may enhance or inhibit angiogenesis in the setting of different disorders. Traditional in vitro and, in vivo models of angiogenesis are laborious and time-consuming, necessitating advanced infrastructure for embryo culture. A challenge encountered by researchers studying angiogenesis is the lack of appropriate techniques to evaluate the impact of regulators on the angiogenic response. An ideal test should possess reliability, technical simplicity, easy quantifiability, and, most importantly, physiological relevance. The CAM model, leveraging the extraembryonic membrane of the chicken embryo, offers a unique combination of accessibility, low cost, and rapid development, making it an attractive option for angiogenesis assays. This review evaluates the strengths and limitations of the CAM model in the context of its anatomical and physiological properties, and its relevance to human pathophysiological conditions. Its abundant capillary network makes it a common choice for studying angiogenesis. The CAM assay serves as a substitute for animal models and offers a natural setting for developing blood vessels and the many elements involved in the intricate interaction with the host. Despite its advantages, the CAM model's limitations are notable. These include species-specific responses that may not always extrapolate to humans and the ethical considerations of using avian embryos. We discuss methodological adaptations that can mitigate some of these limitations and propose future directions to enhance the translational relevance of this model. This review underscores the CAM model's valuable role in angiogenesis research and aims to guide researchers in optimizing its use for more predictive and robust preclinical studies. The highly vascularized chorioallantoic membrane (CAM) of fertilized chicken eggs is a cost-effective and easily available method for screening angiogenesis, in comparison to other animal models.

Ex Ovo Chorioallantoic Membrane Assay as a Model of Bone Formation by Biomaterials.

Biomaterials play an increasingly critical role in bone tissue engineering. However, achieving effective clinical translation requires a careful choice of biomimetic materials and thorough assessment of their efficacy and safety. Existing in vitro and in vivo models have drawbacks including time and cost constraints, invasive procedures, and discordance between animal models and clinical outcomes. Therefore, there is a demand for an alternative model. We hypothesized that the chick embryo chorioallantoic membrane can serve as a bioreactor to evaluate the initial sign of bone formation on scaffolds. In parallel, we investigated the osteogenic potential of a previously fabricated fibrin-alginate-calcium phosphate biomaterial (FACaP). Blood vessels were observed to infiltrate the scaffolds with early signs of bone formation, confirmed via RUNX-2 and alpha smooth muscle actin markers. The scaffolds' chemical composition was evaluated by Fourier-transform infrared spectroscopy, and ion chromatography was used to assess calcium ion release. Finally, the topography was examined by atomic force microscopy. In conclusion, this system offers simple refinement for in vivo models in bone tissue engineering and highlights the great potential of FACaP as an angiogenic and osteogenic biomaterial for non-load-bearing applications.