European Journal of Biomedical and Pharmaceutical Sciences

ANGIOGENIC PROPERTY OF COLLAGEN FROM THE EGGSHELL MEMBRANE OF CHICKEN USING EX-OVO CHORIO-ALLANTOIC MEMBRANE ASSAY OF DUCK EGGS

Aves Z. M. R., Chan C. G., Frias D. B., Laurio A.M.M.*, Mendoza M.A.J.A., Tabagan A.J.A.T. and Mylene Andal RPh, MSPharm

ABSTRACT

Ischemic heart disease remains one of the leading cause of death globally, and drug therapies, medical procedures and extracellular matrices such as heparin is used to promote formation of new blood vessels as the intervention for the disease. Angiogenesis is the process wherein new blood vessels form and grow on a pre-existing vasculature, wherein it is effective in treating or preventing ischemic heart disease. Collagen is abundant in the matrix of extracellular tissues of humans and plays a vital role in giving structural support and function on the growth and regeneration of tissues, which can be helpful in tissue engineering as its properties include high mechanical strength, low antigenicity, good biocompatibility and ability of being cross-linked that makes it an ideal biomaterial for such application. The purpose of this study is to utilize eggshell membrane, an industrial waste, to extract collagen and to determine its angiogenic property compared to standard drug, Heparin. Collagen was extracted from the separated membranes using acetic acid relative to membrane weight at a ratio of 1:4. The eggshell membrane collagen yield of 14.48%. At electron microscopic level, a distinct structure was observed having a spontaneously formed macro porous scaffolds similar to type 1 collagen. Fourier Transform Infrared Spectroscopy showed regions of amide A (3437.15), amide B (2929.87), amide I (1656.85), amide II (1558.48) and amide III (1247.94), which are essential functional groups for proteins and triple helical structure. Confirmation of purity was established by using High Performance Liquid Chromatography retention time comparison with the extracted collagen (1.803) and standard collagen (1.829). Analysis of Differential Scanning Calorimetry revealed that the thermal denaturation was about 106.85°C indicating high stability of structures. Chorio-allantoic membrane filter paper assay quantification was determined based on collateral counts. Concentration of 3mg/mL (p=0.169) and 10mg/mL(p=0.588) extracted collagen have no significant differences with the positive control, Heparin. At concentration of 5mg/mL (p=0.006) eggshell membrane collagen exhibited the most angiogenic activity greater than the positive control, Heparin. The results suggest that collagen of eggshell membrane could improve tissue perfusion reducing the occurrence of ischemic tissues.

Keywords: angiogenesis, egg shell, collagen, chorio-allantoic membrane, extracellular matrix (ECM), growth factor (GF).