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Pure Appl. Chem., 2011, Vol. 83, No. 11, pp. 2063-2069

Published online 2011-09-05

Automated high-throughput screening of carbon nanotube-based bio-nanocomposites for bone cement applications

Paula P. Gonçalves1*, Manoj K. Singh2*, Virgília S. Silva1, Filipa Marques1, Ana Marques1, Philip R. LeDuc3, José Grácio3*, Paula A. A. P. Marques3, Gil Gonçalves3 and António C. M. Sousa3

1 Departamento de Biologia and CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal
2 Nanotechnology Research Division, Center for Mechanical Technology and Automation, University of Aveiro, 3810-193 Aveiro, Portugal
3 Department of Mechanical Engineering, Carnegie Mellon University, 415 Scaife Hall, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA

Abstract: In this work we demonstrate the potential of using an automated cell viability analyzer for developing high-throughput screening of orthopedic bioactive materials. We used a biomaterial of carbon nanotubes (CNTs)-based composite integrated with hydroxyapatite/polymethyl methacrylate (HA/PMMA) with controlled physical and chemical properties to evaluate the usefulness of morphometric analysis in conjunction with trypan blue dye exclusion assays in MG63 cell cultures. The MG63 cell line, derived from human bone osteo-sarcoma, is often used as a model for studying osteoblast-like cellular response to bioactive materials for orthopedic surgery. The viability analyzer, Vi-CELLTM XR, Beckman Coulter, was used with trypan blue dye exclusion method in cell suspensions obtained after trypsinization along with determining the distribution plots of cell diameter and circularity, which are critical cellular characteristics. In addition, the activity of alkaline phosphatase (ALP), a typical representation of osteogenic activity of osteoblasts, was also measured spectro-photometrically using p-nitrophenol phosphate as the substrate. Comparative analysis of the frequency histogram of average cell diameter and circularity allowed for the analyses of significant alterations in cell morphology not only over time in control cultures (spherical vs. a flat morphology) but also with respect to PMMA and HA nanocomposites. After cell exposure to HA/PMMA/CNTs, a shift toward loss of cell circularity was observed. The appearances of more differentiated morphologic features were well correlated with the increase of secreted ALP activity. In conclusion, the evaluation of material-induced changes of cell morphology could represent a valuable prescreening test for bioactive properties.