Expression of integrin alphav beta3 in the quail embryo chorioallantoic membrane in response to ionic dissolution products

L. A. HARO DURAND; G.E. VARGAS; R. VERA MESONES; M. ROMERO; J.M. PORTO LÓPEZ; A.R. BOCCACCINI; M.P. ZAGO; A. GORUSTOVICH.

Centro Binacional (Argentina–Italia) de Criobiología Clínica y Aplicada , Facultad de Ciencias Bioquímicas y Farmacéuticas,Rosario, Sta Fe

Workshop; First Workshop on Artificial Organs, Biomaterials and Tissue Engineering.; 2009

Universidad Nacional de Rosario y Sociedad Latinoamericana de Biomateriales, Ingeniería de Tejidos y Órganos Artificiales

 A number of in-vitro and in-vivo studies have highlighted the angiogenic potential of bioactive glasses (BGs), in particular the composition 45S5 Bioglass®. However, the cellular and molecular mechanisms by which BGs affects angiogenesis remain to be elucidated. The aim of the present study was to determine whether the ionic dissolution products of melt-derived BGs affect the expression of integrin avb3, a marker of angiogenic vascular tissue, employing the quail embryo chorioallantoic membrane (CAM) as the experimental model of angiogenesis. Fertilized eggs of Japanese quail (Coturnix coturnix japonica) were incubated in-ovo at 37ºC under ambient atmosphere, cracked at embryonic day 3 (E3) into 10-cm2 wells of tissue culture polystyrene,  and cultured further ex-ovo at 37ºC. The extract containing the dissolution products of BGs was prepared by soaking 1% w/v 45S5 BG particles (< 5 µm) of composition (in wt.%): 45% SiO2, 24.5% Na2O, 24.5% CaO and 6% P2O5 or 45S5 BG containing B2O3 (2 % wt) (labeled 45S5.2B) in Hank’s balanced salt solution (HBSS, Gibco, pH 7.4) in an orbital shaker at 37ºC for 72 h. The elementary content of calcium (Ca), silicon (Si), boron (B), phosphorus (P) and sodium (Na) in the filtered extracts were determined by ICP analysis. At E7, 0.5 mL prewarmed BG-conditioned HBSS solutions were applied dropwise to the surface of each CAM. HBSS without added BG-ionic dissolution products served as control. The embryos were incubated further at 37ºC for 24 h. An enzyme-linked immunoadsorbent assay (ELISA) was performed to measure the endogenous levels of avb3 in the CAM after treatment. Briefly, CAMs were homogenized in RIPA buffer, and 2-4 µg of total protein as determined by the Bradford protein assay was coated in triplicate in each well of a 96-well plastic dish. The plates were dried at 37ºC, blocked in PBS/5%BSA, incubated with an anti-avb3 primary monoclonal antibody (LM609, 1µg/mL), washed with ELISA wash buffer, incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody, and developed in o-phenylenediamide. Plates were read in an ELISA scanner at 492 nm. The avb3 levels remained unchanged in CAMs exposed to a single application of control solution (HBSS) and /or BG-conditioned HBSS solutions for 24 h. The avb3 levels were not significantly increased above the baseline level detectable at developmental embryonic day 8 (E8) in untreated CAMs. The ionic dissolution products from BGs 45S5 and/or 45S5.2B used in the present work did not produce an angiogenic response, as determined by ELISA assay for avb3 expression.