Use of Raman Microscopy and Multivariate Data Analysis to Observe the Biomimetic Growth of Carbonated Hydroxyapatite on Bioactive Glass Regina K. H. Seah, † Marc Garland, ‡ Joachim S. C. Loo,* ,† and Effendi Widjaja* ,‡ School of Materials Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, and Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (ASTAR), 1 Pesek Rd, Jurong Island, Singapore 627833 In the present contribution, the biomimetic growth of carbonated hydroxyapatite (HA) on bioactive glass were investigated by Raman microscopy. Bioactive glass samples were immersed in simulated body fluid (SBF) buffered solution at pH 7.40 up to 17 days at 37 °C. Raman microscopy mapping was performed on the bioglass samples immersed in SBF solution for different periods of time. The collected data was then analyzed using the band-target entropy minimization technique to extract the observable pure component Raman spectral information. In this study, the pure component Raman spectra of the precursor amorphous calcium phosphate, transient oc- tacalcium phosphate, and matured HA were all recovered. In addition, pure component Raman spectra of calcite, silica glass, and some organic impurities were also recovered. The resolved pure component spectra were fit to the normalized measured Raman data to provide the spatial distribution of these species on the sample sur- faces. The current results show that Raman microscopy and multivariate data analysis provide a sensitive and accurate tool to characterize the surface morphology, as well as to give more specific information on the chemical species present and the phase transformation of phos- phate species during the formation of HA on bioactive glass. In the past decade, there has been an increasing interest in the concept of biological fixation of prostheses, where the interface between implant and tissues develop a type of biological bond. 1 Bioactive glasses, ceramics, and composites have been developed as implants and put into clinical use as bone-regenerative material in dental and orthopedic applications. When these bioimplants are immersed in a simulated body fluid (SBF), a layer of bioactive coatings such as carbonated hydroxyapatite (HA) is formed on the surface that has chemical composition similar to the mineral phase of bones. 2 The first well-characterized bioactive glass is the melt-derived 45S5 Bioglass with the weight composition of 45% SiO 2 , 24.5% Na 2 O, 24.5% CaO, 4% P 2 O 5 developed by Hench et al. 3 This bioactive glass is considered as having the closest mechanical properties to cortical bone, as well as giving a quick biochemi- cal response when it is immersed in physiological fluid. 1,3,4 An extensive study on the mechanism of reactions occurring at the surface of this bioactive glass has been also carried out by Clark et al. 5 As reported, the formation of carbonated HA required the presence of hydrated silica, that is, Si-OH groups. The presence of the specific functional group Si-OH reacts with the OH - in the SBF to give Si-O - . The negatively charged Si-O - then attracts the positively charged calcium ions present in the body fluid, forming calcium silicate, Si-O-Ca + . The calcium ions attached to the Si-O - then further attract the phosphate ions, (HPO 4 2- ), forming an amorphous calcium phosphate, Ca x (PO 4 ) y , which is the precursor for the carbonated HA crystallites. Numerous in vitro spectroscopic studies have been performed to charac- terize and to understand the formation of HA on the surface of bioactive glasses. These included the use of spectroscopic techniques, such as Fourier Transform Infrared (FT-IR) Spectroscopy, 6-8 Raman, 8-11 X-ray Photoelectron Spectro- scopy, 12-15 and Nuclear Magnetic Resonance. 16-19 In a recent * To whom correspondence should be addressed. E-mail: joachimloo@ ntu.edu.sg (J.S.C.L.), effendi_widjaja@ices.a-star.edu.sg (E.W.). † Nanyang Technological University. ‡ Institute of Chemical and Engineering Sciences. (1) Jones, J. R.; Hench, L. L. Mater. Sci. 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