Biomaterials need to be vigorously tested at every stage of preclinical development. As demand for in vivo culture environments continues to increase, traditional animal models are often technically complex, ethically undesirable, time-consuming, and resource intensive and thus present a barrier to high throughput screening. The chick chorioallantoic membrane (CAM) assay has long been used to study the effects of drugs on angiogenesis in vivo, providing researchers with a readily available, accessible, self-sustaining, and high throughput screen without requiring animal facilities. It has also been recognized as an in vivo assay to test initial tissue response to biomaterials; however it has not yet gained widespread acceptance. This could be due to lack of specific protocols on how to optimize this assay to specifically test biomaterials. Here we describe how the ex ovo (shell-less) CAM assay can be effectively used to study the angiogenic potential and initial tissue response to biomaterials. In comparison to alternative in vivo approaches, this technique provides additional advantages to the researcher as it allows better visualization of implanted biomaterials and the ability to implant several samples simultaneously enabling combinatorial biomaterial assays to be conducted.

Guided bone regeneration is a common dental implant treatment where a barrier membrane (BM) is used between epithelial tissue and bone or bone graft to prevent the invasion of the fast-proliferating epithelial cells into the defect site to be able to preserve a space for infiltration of slower-growing bone cells into the periodontal defect site. In this study, a bilayer polycaprolactone (PCL) BM was developed by combining electrospinning and emulsion templating techniques. First, a 250 µm thick polymerised high internal phase emulsion (polyHIPE) made of photocurable PCL was manufactured and treated with air plasma, which was shown to enhance the cellular infiltration. Then, four solvent compositions were investigated to find the best composition for electrospinning a nanofibrous PCL barrier layer on PCL polyHIPE. The biocompatibility and the barrier properties of the electrospun layer were demonstrated over four weeks in vitro by histological staining. Following in vitro assessment of cell viability and cell migration, cell infiltration and the potential of PCL polyHIPE for supporting blood vessel ingrowth were further investigated using an ex-ovo chick chorioallantoic membrane assay. Our results demonstrated that the nanofibrous PCL electrospun layer was capable of limiting cell infiltration for at least four weeks, while PCL polyHIPE supported cell infiltration, calcium and mineral deposition of bone cells, and blood vessel ingrowth through pores.

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Aim:In this study, we explored the angiogenic potential and proangiogenic concentration ranges of 2deoxy-D-ribose (2dDR) and -Estradiol (E2) in comparison with vascular endothelial growth factor (VEGF). 2dDR and E2 were then loaded into tissue engineering (TE) scaffolds to investigate their proangiogenic potential when released from fibres. Materials and Methods: Ex-ovo chick chorioallantoic membrane (CAM) assay was used to evaluate angiogenic activity of 2dDR and E2. Both factors were then introduced into scaffolds via electrospinning to assess their angiogenic potential when released from fibres. Results: Both factors were approximately 80% as potent as (VEGF) and showed a dose-dependent angiogenic response. The sustained release of both agents from the scaffolds stimulated neovascularisation over 7 days in the CAM assay. Conclusion: We conclude that both 2dDR and E2 provide attractive alternatives to VEGF for the functionalisation of TE scaffolds to promote angiogenesis in vivo. Graphical abstract: