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Introduction: Among scaffold preparation approaches, biofabrication can provide tridimensional scaffolds with precise and defined architectures that can promote tissue integration and neoangiogenesis after implantation (1). Therefore, the evaluation of tissue ingrowth and vascularization of 3D printed scaffolds is a crucial step in establishing a functional scaffold that could direct the successful tissue regeneration (2). The hen chorioallantoic membrane (CAM) has been proposed as a biorelevant alternative to animal studies for the assessment of implantable scaffolds in vivo. However, current approaches to quantify integration and vascularization, such as histology, lack of spatial resolution. As an alternative, microCT can be helpful to study the extent of tissue integration and vessel architecture within porous implants (3). In this work, we developed a simple and non-destructive method to evaluate tissue ingrowth and neoangiogenesis in 3D printed scaffolds using a CAM model, and further validated in two distinct porous architectures.
Methodology: PLA scaffolds (10 mm in diameter, 4 mm height) with 500 µm pore size designed with/without open lateral porosity were prepared by 3D printing. Sterilized scaffolds were placed on the CAM of fertilized eggs previously incubated for 7 days (Coren, Spain) and incubated at 37 °C for 5 or 7 days. In order to quantify the scaffold integration and vascularization, the CAM tissue surrounding the scaffolds (25 mm in diameter) was fixed in paraformaldehyde, extracted using a scalpel, and immersed in lugol 0.1% for 4 h. Then, samples were washed in PBS, water excess removed and then, scanned at 5 µm voxel resolution using a Skyscan 1272 microCT (Bruker, Belgium). Tissue ingrowth was obtained from the structural changes of structure volume before and after incubation. Finally, the structure and porosity of the reconstructed samples and the new vessels developed within the structure of the scaffolds were analyzed using CTAn software (Bruker, Belgium). Histological analysis was carried out in paraffin-embedded samples after microCT analysis to further quantify new tissue ingrowth and vessel formation and dimensions.
Results: scaffolds designed with open lateral porosity showed a significant higher integration with the CAM since tissue migrated towards the inner regions of the scaffolds not only from the underneath but also from the side surface of the scaffolds. The incubation with lugol allowed for the precise visualization of vessels and new tissue growing within the scaffolds due to the distinct contrast-enhanced radiopacity of vessels, tissue and scaffold components, as validated by histological and histomorphometrical analysis.
Conclusion: The developed methodology enabled the precise evaluation of tissue integration and vascularization in an embryonic in vivo model. As a non-destructive technique, the developed microCT method is a robust approach for initial in vivo screening of novel porous biomaterials, while complying with the principles of 3Rs.
References:
1. Agarwal, R. and Garcia, A.J. Adv Drug Deliv Rev 94, 53-62 (2015).
2. Velasco, M.A. et al., BioMed Res Int, 729076 (2015)
3. Moreno-Jimenez, I. et al., Tissue Eng Part C Methods 23, 938-952 (2017)
Acknowledgements:
This research was funded by Xunta de Galicia (ED481D-2021-014).
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