Pre-screening the intrinsic angiogenic capacity of biomaterials in an optimised ex ovo chorioallantoic membrane model

Abstract

Biomaterial development for clinical applications is currently on the rise. This necessitates adequate in vitro testing, where the structure and composition of biomaterials must be specifically tailored to withstand in situ repair and regeneration responses for a successful clinical outcome. The chorioallantoic membrane of chicken embryos has been previously used to study angiogenesis, a prerequisite for most tissue repair and regeneration. In this study, we report an optimised ex ovo method using a glass-cling film set-up that yields increased embryo survival rates and has an improved protocol for harvesting biomaterials. Furthermore, we used this method to examine the intrinsic angiogenic capacity of a variety of biomaterials categorised as natural, synthetic, natural/synthetic and natural/natural composites with varying porosities. We detected significant differences in biomaterials’ angiogenesis with natural polymers and polymers with a high overall porosity showing a greater vascularisation compared to synthetic polymers. Therefore, our proposed ex ovo chorioallantoic membrane method can be effectively used to pre-screen biomaterials intended for clinical application.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

“…This cling film method provides survival rates of over 60%. 72 It has been noted that embryos die due to the hard, flat material of the Petri dish, the increased surface tension and/or poor egg cracking technique, leading to rupture of the yolk membrane. 125 Therefore, alternative ex ovo methods have been explored, such as application of a cup-CAM method where the egg resides within the bottom of an ice-cream cup to ensure that the embryo is not stretched, leading to 85%-90% survival rates.…”

“…Eggshell dust can prove to be an irritant for the CAM; 72 however, if the hard shell is removed, leaving the white inner shell membrane intact, as in our approach, then the dust can be removed before the CAM is exposed. Critically, for in ovo incubation, antibiotic or antimycotic 72 solutions are not necessary compared with ex ovo methods and eggs can be incubated 'end on' or 'side on' as described in articles and reviews. 51,122…”

“…Selection of areas at random on the CAM is employed when a diffuse substance has been applied to the CAM. The blood vessel length, width 84 and branching 72,110 are able to be measured. 84 However, this method is subjective and, typically considers angiogenesis in direct contact with the implanted material or on the surface.…”

“…To euthanise the embryos, freezing at ED 12 has been reported, but is likely to cause distress for the chick. 72 Similarly, carbon dioxide gas is unreliable and difficult to ‘dose’; while using a fixative on the CAM is considered painful 71 and not permitted under ASPA. Others have published alternative approaches – Cirligeriu et al 56 describe applying 10% buffered formalin for 30 min to the CAM, on ED 13, which appears counter to EU guidance; however, the method of euthanasia is not described.…”

“…Materials derived from natural polymers, with an enhanced textured surface are indicated as more angiogenic 72 in contrast to smooth, synthetic inert materials. 112 However, it should be noted that a material which is angiogenic may not give the optimal response if the porosity is sub-optimal.…”