Author's personal copy Grade-1 titanium soaked in a DMEM solution at 37 °C A. Cuneyt Tas 1 Department of Materials Science and Engineering, University of Illinois, Urbana, IL 61801, USA abstract article info Article history: Received 12 July 2013 Received in revised form 27 October 2013 Accepted 28 November 2013 Available online 7 December 2013 Keywords: DMEM Amorphous Calcium phosphate Titanium DMEM (Dulbecco's modified Eagle medium) solutions are used in performing in vitro cell culture experiments to assess the cell biocompatibility of synthetic biomaterials. In this study, Hepes-buffered, phenol red- and sodium pyruvate-free DMEM solutions were used, for the first time as immersion media at 37 °C, to test alkali-treated (5 M NaOH, 60 °C, 24 h) grade-1 titanium substrates. Such DMEM solutions were found to deposit X-ray- amorphous calcium phosphate (ACP), in one or two weeks, on the soaked grade-1 Ti substrates. A limited num- ber of previous studies focusing on the biomimetic coating of alkali-treated Ti6Al4V coupons in DMEM have ac- tually used different DMEM solutions, which were not Hepes-buffered and containing phenol red and sodium pyruvate. The previous studies with such DMEM solutions reported the deposition of cryptocrystalline apatitic calcium phosphate (Ap-CaP) on Ti6Al4V substrates, but not ACP. An inorganic solution (free of amino acids, vita- mins, glucose, sodium pyruvate and phenol red), simulating the ion concentrations of the DMEM solutions, was also used for the first time in depositing ACP on grade-1 Ti substrates upon soaking at 37 °C for only 24 h. The solutions and deposits of this study were analyzed by AAS, ICP-AES, FTIR, XRD, XPS, and surface profilometry. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Alpha-minimum essential medium (α-MEM) and Dulbecco's modi- fied Eagle medium (DMEM) are solutions (media) which contain amino acids, vitamins, glucose and especially the inorganic salts at concentra- tions similar to those present in the whole mammalian serum. Both α-MEM and DMEM solutions, the preferred media to perform in vitro cell culture studies, originated from the pioneering work of Eagle [1,2], which were focused on developing synthetic media with components essential and sufficient for the survival and growth of a wide variety of animal cells. Eagle's original minimum essential medium (MEM) contained 13 amino acids, 8 vitamins, glucose and inorganic salts such as NaCl, KCl, CaCl 2 , MgCl 2 ·6H 2 O, NaH 2 PO 4 ·2H 2 O and NaHCO 3 [2]. Eagle's MEM solution had a Ca/P molar ratio of 1.64 and a HCO 3 - concen- tration of 23.8 mM. Dulbecco's modification to the Eagle medium consisted of adding 2% horse serum to it [3,4] resulting in an increase in the number of amino acids to 15. In a cell culture study directly comparing the α-MEM and DMEM solutions by using the human oste- oblastic bone marrow cells, Coelho et al. [5] reported that the cell prolif- eration was similar in cultures grown in the two media but ALP (alkaline phosphatase) activity and ability to form mineralized deposits were lower in DMEM cultures. DMEM, the physiological medium of interest in this study, can be obtained either as a powder or as a solution and there happens to be a number of variants of DMEM available [6], mainly in the forms containing high, low or no glucose at all, with or without glutamine, with or without Na-pyruvate, with or without phenol red, and with or without Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid). Therefore, it is important to specify the catalog number of the manufac- turer of the DMEM preferred in any study. The specific DMEM solution chosen for this study was previously used in testing the biomineraliza- tion of brushite powders at the human body temperature [7]. Most of the DMEM solutions produced today, in contrast to the original Eagle's MEM, have a Ca/P molar ratio of 1.99 and a HCO 3 - concentration of 44.05 mM. Blood plasma's Ca/P molar ratio and HCO 3 - concentration are 2.50 and 27 mM, respectively. Three differ- ent SBF (synthetic/simulated body fluid) solution formulations, which do not contain amino acids, vitamins and glucose, can match the Ca/P molar ratio (2.50) and the HCO 3 - concentration (27 mM) of human blood plasma [8–12], but cells cannot survive and grow in SBF solutions [13,14]. The direct comparison between DMEM and HCO 3 -deficient (i.e., 4.2 mM), Cl-rich (148 mM) SBF [15] solutions has been the sub- ject of a limited number of previous studies, in which bioglass [16,17] or calcium phosphate [18] samples have been soaked in both solutions, side-by-side, at 37 °C, followed by the microscopic examination of the spherulites (or globules) forming on the sample surfaces. These studies [16–18], by only reporting EDXS (energy-dispersive X-ray spectrosco- py) data, proved that the DMEM solution used was able to cover the bioglass, glass-ceramic, hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), β- and α- polymorphs of tricalcium phosphate (Ca 3 (PO 4 ) 2 ) surfaces with the spherulites of a calcium phosphate (CaP) phase just like the SBF solu- tions would do. Nevertheless, none of these reports [16–18] provided any X-ray diffraction (XRD) data to ascertain whether the CaP formed Materials Science and Engineering C 36 (2014) 84–94 E-mail address: c_tas@hotmail.com. URL: http://www.cuneyttas.com. 1 Tel.: +1 2173446708. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.045 Contents lists available at ScienceDirect Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec