The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering

Abstract

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

Remodelling of human bone on the chorioallantoic membrane of the chicken egg:De novobone formation and resorption

“…In agreement with our previous study (Moreno‐Jiménez et al, ), we demonstrated that CAM‐implantation of human viable bone cylinders resulted in a significant increase in bone volume (9.79% ± 2.24 SD ; p <0.001). The pre‐existing mineralised tissue of the bone cylinder prevented the quantification or visualisation of the newly formed bone alone.…”

“…Using our previously published protocol (Moreno‐Jiménez et al, ), we implanted freshly isolated human bone cylinders on the CAM and were able to measure increases in bone volume following incubation. CAM‐implanted bone cylinders displayed a significant increase (9.79% ± 2.24 SD ; n = 6–8; p 0.001) compared with in vitro ‐cultured bone cylinders (1.60% ± 1.06 SD) and control (−0.19% ± 0.33 SD ; bone cylinders maintained at 4 °C) from the same donor.…”

“…Our previous studies demonstrated that human (HLA+) cells remain viable following in vivo culture on CAM (Moreno‐Jiménez et al, ). However, the current results indicated that RUNX2+/SOX9+ cell condensations colocalised with GFP expression, only present on the avian cells.…”

“…These cell condensations also costained with GFP, confirming their avian origin. We formerly described the formation of these cell condensations in association with collagen type II deposition from the CAM, again coexpressing with SOX9 and GFP next to the human bone tissue (Moreno‐Jiménez et al, ). Early studies from Hancox, showed formation of similar cell condensations following engraftment of chick embryo calvaria fragments on CAM.…”

“…In contrast to the human bone tissue cultured in vitro, CAM‐implanted bone cylinders showed the presence of avian blood vessels, condensations of progenitor (SOX9+) cells, extracellular matrix deposition, and significant increase in bone volume (Moreno‐Jiménez et al, ). Moreover, human bone autologous cells remained viable following CAM implantation (Moreno‐Jiménez et al, ). However, in the previous study, we were unable to identify the mechanisms involved in the bone formation.…”