In recent years, the chorioallantoic membrane (CAM) has emerged as a crucial component of biocompatibility testing for biomaterials designed for regenerative strategies and tissue engineering applications. This study explores angiogenic potential of an innovative acellular and porous biopolymer scaffold, based on polyhydroxybutyrate and chitosan (PHB/CHIT), using the ex ovo quail CAM assay as an alternative to the conventional chick CAM test. On embryonic day 6 (ED6), we placed the tested biomaterials on the CAM alone or soaked them with various substances, including vascular endothelial growth factor (VEGF-A), saline, or the endogenous angiogenesis inhibitor Angiostatin. After 72 h (ED9), we analyzed blood vessels formation, a sign of ongoing angiogenesis, in the vicinity of the scaffold and within its pores. We employed marker for cell proliferation (PHH3), embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and endothelial cells (QH1) for morphological and histochemical analysis. Our findings demonstrated the robust angiogenic potential of the untreated scaffold without additional influence from the angiogenic factor VEGF-A. Furthermore, gene expression analysis revealed an upregulation of pro-angiogenic growth factors, including VEGF-A, ANG-2, and VE-Cadherin after 5 days of implantation, indicative of a pro-angiogenic microenvironment. These results underscore the inherent angiogenic potential of the PHB/CHIT composite. Additionally, monitoring of CAM microvilli growing to the scaffold provides a methodology for investigating the biocompatibility of materials using the ex ovo quail CAM assay as a suitable alternative model compared to the chicken CAM platform. This approach offers a rapid screening method for biomaterials in the field of tissue repair/regeneration and engineering.

• Klíčová slova
• Angiogenesis, Avian animal model, Bone regeneration, Chitosan, Polyhydroxybutyrate,
• MeSH
• biokompatibilní materiály * farmakologie   MeSH
• chitosan * farmakologie   MeSH
• chorioalantoická membrána * účinky léků   MeSH
• fyziologická neovaskularizace účinky léků   MeSH
• křepelky a křepelovití embryologie   MeSH
• testování materiálů MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biokompatibilní materiály * MeSH
• chitosan * MeSH

Birds, especially the chick and hen, have been important biomedical research models for centuries due to the accessibility of the avian embryo and the early discovery of avian viruses. Comprehension of avian tumor virology was a milestone in basic cancer research, as was that of non-viral genesis, as it enabled the discovery of oncogenes. Furthermore, studies on avian viruses provided initial insights into Kaposi's sarcoma and EBV-induced diseases. However, the role of birds in human carcinogenesis extends beyond the realm of virology research. Utilization of CAM, the chorioallantoic membrane, an easily accessible extraembryonic tissue with rich vasculature, has enabled studies on tumor-induced angiogenesis and metastasis and the efficient screening of potential anti-cancer compounds. Also, the chick embryo alone is an effective preclinical in vivo patient-derived xenograft model, which is important for the development of personalized therapies. Furthermore, adult birds may also closely resemble human oncogenesis, as evidenced by the laying hen, which is the only animal model of a spontaneous form of ovarian cancer. Avian models may create an interesting alternative compared with mammalian models, enabling the creation of a relatively cost-effective and easy-to-maintain platform to address key questions in cancer biology.

• Klíčová slova
• carcinogenesis, chorioallantoic membrane, hen model, oncoviruses, ovarian cancer,
• MeSH
• karcinogeneze * patologie   genetika   MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• nádory patologie   genetika   MeSH
• ptáci MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The combination of in ovo and ex ovo chorioallantoic membrane (CAM) assay provides an excellent platform which extends its relevance in studying carcinogenesis to the field of screening of anticancer activity of platinum nanoparticles (PtNPs) and further study of the amino acids' fluctuations in liver and brain. PtNPs are promising candidates for replacing cisplatin (CDDP); however, insufficient data of their antitumor efficiency and activity on the cancer-related amino acid metabolism are available, and the assessment of the in vivo performance has barely scratched the surface. Herein, we used CAM assay as in vivo model for screening of novel therapeutic modalities, and we conducted a comparative study of the effects of CDDP and polyvinylpyrrolidone coated PtNPs on MDA-MB-231 breast cancer xenograft. PtNPs showed a higher efficiency to inhibit the tumor growth and metastasis compared to CDDP. The amino acids profiling in the MDA-MB-231 ​cells revealed that the PtNPs had an overall depleting effect on the amino acids content. Noteworthy, more side effects to amino acid metabolism were deduced from the depletion of the amino acids in tumor, brain, and liver upon CDDP treatment. Different sets of enzymes of the tricarboxylic acid (TCA) cycle were targeted by PtNPs and CDDP, and while mRNA encoding multiple enzymes was downregulated by PtNPs, the treatment with CDDP affected only two TCA enzymes, indicating a different mechanism of action. Taken together, CAM assay represents and invaluable model, demonstrating the PtNPs capability of repressing angiogenesis, decrease amino acid contents and disrupt the TCA cycle.

• Klíčová slova
• Amino acids metabolism, Breast cancer, CAM assay, Cisplatin, Platinum nanoparticles, TCA cycle,
• Publikační typ
• časopisecké články MeSH

The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane widely used as an in vivo model to study angiogenesis and its inhibition in response to tissues, cells, or soluble factors. In recent years, the use of CAM has become an integral part of the biocompatibility testing process for developing biomaterials intended for regenerative strategies and tissue engineering applications. In this study, we used the chicken ex ovo CAM assay to investigate the angiogenic potential of innovative acellular biopolymer polyhydroxybutyrate/chitosan (PHB/CHIT) scaffold, which is intended for the treatment of hard tissue defects, depending on treatment with pro- and anti-angiogenic substances. On embryonic day (ED) 7, the experimental biomaterials were placed on the CAM alone or soaked in vascular endothelial growth factor (VEGF-A), saline solution (PHY), or tyrosine kinase inhibitor (SU5402). After 72 h, the formation of vessels was analyzed in the surrounding area of the scaffold and inside the pores of the implants, using markers of embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and macrophages (KUL-01). The morphological and histochemical analysis showed strong angiogenic potential of untreated scaffolds without additional effect of the angiogenic factor, VEGF-A. The lowest angiogenic potential was observed in scaffolds soaked with SU5402. Gene expression of pro-angiogenic growth factors, i.e., VEGF-A, ANG-2, and VE-CAD, was upregulated in untreated scaffolds after 72 h, indicating a pro-angiogenic environment. We concluded that the PHB/CHIT has a strong endogenous angiogenic potential and could be promising biomaterial for the treatment of hard tissue defects.

• Klíčová slova
• CAM assay, angiogenesis, biomaterial, bone tissue engineering, chitosan, polyhydroxybutyrate, regeneration,
• Publikační typ
• časopisecké články MeSH

PURPOSE: The chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential. METHODS: The Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed. RESULTS: Ex ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM. CONCLUSIONS: Taken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

• Klíčová slova
• CAM assay, drug testing, extracranial solid tumor, metastasis, neuroblastoma, preclinical trials,
• Publikační typ
• časopisecké články MeSH