In ovo assessment of the angiogenic response of silk/elastin bioprinted scaffolds

Intervertebral disc (IVD) degeneration generally associated with low back pain (LBP) affects million of people worldwide and presents a huge socioeconomic impact. Despite the large number of pre-clinical and clinical studies that focus on developing a novel therapeutic for IVD disorders, there is a clear need for alternative treatments to the conventional surgical invasive methods, that address repair/regeneration of this critical tissue1. The IVD is an avascular and alymphatic structure composed of a centrally situated gelatinous tissue, the nucleus pulposus (NP) and an external surrounding tissue, the annulus fibrosus fibrous (AF), which differs substantially from the inner part, being more fibrocartilaginous and vascularized on the periphery. A patient-specific approach based on a 3D bioprinted silk/elastin construct has been proposed by our group2 as a promising candidate for IVD tissue engineering. Silk fibroin scaffolds already proved their potential for AF tissue engineering applications, whereas blending with elastin can enhance the biological performance of the scaffold by mimicking the lamellar oriented structure of AF and creating a more favorable environment for ECM deposition.
Considering the avascular nature of native tissue, it is crucial that the constructs mimic/reproduce the vascular organization of the IVD. In this work, the angiogenic response of bioprinted silk/elastin scaffolds was investigated using a classical in vivo model for angiogenesis: the chick embryo chorioallantoic membrane assay. The CAM model has been effectively applied in tissue engineering for the last 40 years as a refined model for animal research. This is an accessible, simple and accepted system to examine the angiogenic response and the material-tissue biocompatibility through a minimally invasive approach3. The evaluation of the angiogenic response of the bioprinted scaffolds was performed by counting the number of blood vessels converging toward the implanted materials. Histological characterization (H&E staining) and immunohistochemical analysis of endothelial cells of chick origin (SNA-lectin) were also performed on excised sections of CAM.