A549 Lung Carcinoma Cells: Binary vs. Ternary Bioactive Gel-Glasses

The binary gel-glasses in the CaO-SiO 2 system are being studied for use as a bioceramic material and hence have to be compared to other materials that are similar in properties and are in use either for clinical or research purposes. In this paper, the att achment, proliferation and the morphology of human lung epithelial cells are investigated using substr ates of two compositions (one with and another without phosphate). The results of this study indicate that the ternary 58S bioactive gel-glass substrates offer favourable conditions for A549 cell adhesion and pr oliferation. Introduction Ternary bioactive gel-glasses in the CaO-SiO 2-P2O5 system have been widely studied since 1991 [1]. Both S70C30 and 58S gel-glass compositions are Class A bioactive [ 2,3] and have been compared to melt-derived 45S5 Bioglass®, which has been in clinical use for twenty year s. The aim of the investigation reported herein is to compare cell adhe sion and proliferation properties of binary and ternary substrates using human epithelial-li ke lung carcinoma cells (A549). Porous scaffolds of both compositions are being developed for a lung cell ba s d io-photonics toxicity detector system. Method Binary and ternary gel-glass monoliths (composition as given in Tabl e 1) were prepared using the sol-gel technique with tetraethyl orthosilicate, triethyl phosphate and calcium nitrate tetrahydrate as starting materials [4]. Physical characterization of porous subst rate consisted of pore size and textural analysis using N 2 sorption. Table 1. Nominal composition of the monoliths (mol %) SiO2 CaO P2O5 S70C30 70 30 58S 60 36 4 Human lung epithelial (A549) cells (purchased from ECACC, UK) were cultured in F12K medium supplemented with 10% foetal calf serum (FCS; Invitrogen) at 37°C in 5% CO2 and in humidified atmosphere. Cell seeding on S70C30 foams and monoliths was perfo rmed as previously described [5]. The cells were seeded at the same density (10 000 cell s / sample) on each sample, and incubated at 37°C in 5% CO 2 for various periods of time. The medium was changed twice a week. A549 adhesion was assessed using WST-1 test (Roche, Mannheim, Germany) . Two hours after cell seeding on materials or control plastic, non-adherent cells we re removed by gently washing twice in PBS. WST-1 substrate (tetrazolium salt) diluted to 10% in F12K medium was added to A549 culture and incubated at 37°C in 5% CO 2 atmosphere. After 3 hours, the absorbance (A) was measured at 450 nm using a MRX ELISA reader (Dynatech Laboratorie s). R sults are expressed in percentage of cell adhesion relative to cell adhesion onto cell cult ure plastic surface (positive Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 781-784 doi:10.4028/www.scientific.net/KEM.254-256.781 © 2004 Trans Tech Publications Ltd, Switzerland All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications Ltd, www.scientific.net. (Semanticscholar.org-13/03/20,19:47:20) control). Background absorbance was obtained by incubation of the WST-1 on substr ate in cell culture medium only or on material only. Cell proliferation was inves tigated after incubation for 2 hours, 1, 3, 7, 15 and 28 days before analysis. The same method was used to foll ow cell proliferation and the results are expressed as optical density unit s. All biological samples were repeated 4 times, and experiments were done 3 times. The U Mann-Whit ney test was applied to results. Values with p<0.05 were considered as significant (*) and p<0.01 is shown as (**). Cell morphology was studied using a JSM LV 5610 scanning electron micr oscopy. The substrates were gently washed in phosphate buffer after different incubation periods. They were fixed using 2.5% glutaraldehyde in 0.1M phosphate buffer for 40 minutes at 4°C and dehydrated through increasing concentrations of ethanol and hexamthyldisilasane (HM DS). The specimens were vacuum dried and sputter coated with gold. Image analysis was performed using 10kV accelerating voltage. Results N2 sorption analysis on the monoliths indicates that both substrates are m esoporous in nature (see Table 2) with interconnecting, non-perfect cylindrical pores of a narrow pore size dis tribut on. Table 2. Textural parameters S70C30 58S Surface Area 136 mg 151 mg Pore Volume 0.71 cmg 0.45 cmg Average Pore 210 Å 132 Å After 2 hours of cell incubation, about 70% of cell adhesion was obtained on bot h substrates. No significant differences were observed between cell adhesion to S70C30 and to 58S (Fig. 1). 0 20 40 60 80 100 120 control plastic 7030 monolith 58s monolith ce ll ad h es io n /c o n tr o l p la st ic