The fields of regenerative medicine and tissue engineering offer significant promise to address the urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases, and tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies need to be assessed before clinical application, often necessitating animal research as a prerequisite. The growing number of newly developed potential treatments, together with the ethical concerns involved in the application of in vivo studies, requires the implementation of alternative models to facilitate such screening of new treatments. The present review examines the current in vitro and in vivo models of preclinical research with particular emphasis on the chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative. Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a noninnervated rapidly growing vascular bed, which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis. Moreover, the application of imaging techniques (magnetic resonance imaging, microcomputed tomography, fluorescence labeling for tracking) will be discussed for the evaluation of biomaterials cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to facilitate the adoption of this technique across laboratories and the regenerative medicine community, and thus aid the reduction, replacement, and refinement of animal experiments in research.

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BackgroundA dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation.MethodsEarly osteogenic activities of the co-cultures (±VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31.ResultsALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive  cells in the mid-diaphyseal region of the femurs.ConclusionThese studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0270-3) contains supplementary material, which is available to authorized users.

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal–endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal–endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.—Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.