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.

Department of Biology and Physiology University of Veterinary Medicine and Pharmacy in Kosice Kosice Slovakia

Department of Biomedical Research East Slovak Institute of Cardiovascular Diseases Inc Kosice Slovakia

Department of Morphological Disciplines University of Veterinary Medicine and Pharmacy in Kosice Kosice Slovakia

Department of Pharmacognosy and Botany Faculty of Pharmacy Comenius University in Bratislava Bratislava Slovakia

Department of Pharmacology Faculty of Medicine Pavol Jozef Safarik University in Kosice Kosice Slovakia

Institute of Materials Research The Slovak Academy of Sciences Kosice Slovakia

Prague Burn Centre 3rd Faculty of Medicine Charles University and University Hospital Prague Prague Czech Republic

University of Veterinary Medicine and Pharmacy in Kosice Komenskeho 73 Kosice 041 81 Slovakia

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