Effect of in ovo application of tetracalcium phosphate-containing polymeric scaffold on chicken embryo development

Effect of in ovo application of tetracalcium phosphate-containing polymeric scaffold on chicken embryo development
Atefeh Derakhshani,^1,* Saeed Hesaraki,² Nader Nezafati,³ Mahmoud Azami,⁴
1. Nanotechnology and advanced materials, Materials and energy research center, Karaj, Iran
2. Nanotechnology and advanced materials, Materials and energy research center, Karaj, Iran
3. Nanotechnology and advanced materials, Materials and energy research center, Karaj, Iran
4. Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
Introduction: Chick embryo chorioallantoic membrane (CAM) is well known as a powerful experimental tool for the study of normal and pathologic angiogenesis and chicken embryo development, due to its high vascular network. Additionally, CAM is an embryonic tissue lacking an immune system and having mesenchymal cells with stem-like potential able to differentiate depending on a speciﬁc microenvironment (Cirligeriu et al. 2018). Calcium (Ca) is an essential mineral which its vital roles (e.g. blood coagulation, bone formation, and nervous system) in the body are clear (Tuan et al. 1986). However, the injection of hydroxyapatite nanoparticles was not reflected in embryo growth and even slowed down bone development (Matuszewski et al. 2020). Biochemical approach is one of the Current techniques for enhancing the formation of vascular networks. This technique involves the incorporation of the small bioactive molecules or growth factors within scaffolds for promoting formation of the blood vessels which can prepare ideal nutrition for the embryo (Saik et al. 2011). Polymeric tissue-engineered scaffolds are the best candidate for delivery of molecules or other factors. The objective of the present study was to evaluate the effect of in ovo application of ion delivery from tetra-calcium phosphate-containing polymeric scaffold on chicken embryo development.
Methods: Fabrication of composite scaffold The preparation of TTCP is fixed to solid-state reaction of calcium carbonate and dicalcium phosphate anhydrate at high temperatures. Then 0.4-gram TTCP was incorporated in a hydroxyethyl cellulose/hyaluronic acid/gelatin scaffold which was previously fabricated by ( Derakhshani et al. 2020). CAM assay Fertilized eggs were purchased from a local company and the assay was performed as (Moreno et al. 2016) protocol.
Results: On day 8th, to study the chick embryo development, the eggs were evacuated. Fig.2 showed the scaffold treated chicken embryo(STCE). As shown in Fig. 2(a) the vitelline membrane covers almost the whole yolk. Eye pigmentation is readily visible. The beak’s upper and lower parts are differentiated (Fig. 2(b)), as well as the wings and legs. The neck stretches and the brain is completely settled in its cavity. Also, we can see the external auditory canal is opened. Additionally, the claws are formatted and the budding of first feather follicles are invisible (www.thepoultrysite.com). The last two phenomena belong to day 9th of the normal chick embryo development, but all of them happened on day 8th of STCE. The comparison of height (control=2.1 cm, STCE=3.3 cm) and weight (control=0.67 g, STCE=1.7 g) of control and STCE are depicted in Fig.3 and Table 1. The results indicated the presence of scaffold and released tetra-calcium phosphate ions (especially Ca, which is effective in cell proliferation) had a positive effect on chicken embryo development.
Conclusion: In this study, we prepared a polymeric scaffold that released inorganic ions such as calcium. The CAM assay indicated that the released ions positively affect chicken embryonic development and this approach is more effective than direct injection of a calcium phosphate member.
Keywords: tissue engineering, CAM assay, ion delivery, scaffold