Grade-1 titanium soaked in a DMEM solution at 37°C.

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 (5M NaOH, 60°C, 24h) 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 number of previous studies focusing on the biomimetic coating of alkali-treated Ti6Al4V coupons in DMEM have actually 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, vitamins, 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 24h. The solutions and deposits of this study were analyzed by AAS, ICP-AES, FTIR, XRD, XPS, and surface profilometry.

The use of physiological solutions or media in calcium phosphate synthesis and processing.

This review examined the literature to spot uses, if any, of physiological solutions/media for the in situ synthesis of calcium phosphates (CaP) under processing conditions (i.e. temperature, pH, concentration of inorganic ions present in media) mimicking those prevalent in the human hard tissue environments. There happens to be a variety of aqueous solutions or media developed for different purposes; sometimes they have been named as physiological saline, isotonic solution, cell culture solution, metastable CaP solution, supersaturated calcification solution, simulated body fluid or even dialysate solution (for dialysis patients). Most of the time such solutions were not used as the aqueous medium to perform the biomimetic synthesis of calcium phosphates, and their use was usually limited to the in vitro testing of synthetic biomaterials. This review illustrates that only a limited number of research studies used physiological solutions or media such as Earle's balanced salt solution, Bachra et al. solutions or Tris-buffered simulated body fluid solution containing 27mM HCO3(-) for synthesizing CaP, and these studies have consistently reported the formation of X-ray-amorphous CaP nanopowders instead of Ap-CaP or stoichiometric hydroxyapatite (HA, Ca10(PO4)6(OH)2) at 37°C and pH 7.4. By relying on the published articles, this review highlights the significance of the use of aqueous solutions containing 0.8-1.5 mMMg(2+), 22-27mM HCO3(-), 142-145mM Na(+), 5-5.8mM K(+), 103-133mM Cl(-), 1.8-3.75mM Ca(2+), and 0.8-1.67mM HPO4(2-), which essentially mimic the composition and the overall ionic strength of the human extracellular fluid (ECF), in forming the nanospheres of X-ray-amorphous CaP.

Comparison of titanium soaked in 5 M NaOH or 5 M KOH solutions.

Commercially pure titanium plates/coupons and pure titanium powders were soaked for 24 h in 5 M NaOH and 5 M KOH solutions, under identical conditions, over the temperature range of 37° to 90 °C. Wettability of the surfaces of alkali-treated cpTi coupons was studied by using contact angle goniometry. cpTi coupons soaked in 5 M NaOH or 5 M KOH solutions were found to have hydrophilic surfaces. Hydrous alkali titanate nanofibers and nanotubes were identified with SEM/EDXS and grazing incidence XRD. Surface areas of Ti powders increased > 50­220 times, depending on the treatment, when soaked in the above solutions. A solution was developed to coat amorphous calcium phosphate, instead of hydroxyapatite, on Ti coupon surfaces. In vitro cell culture tests were performed with osteoblast-like cells on the alkali-treated samples.

X-ray-amorphous calcium phosphate (ACP) synthesis in a simple biomineralization medium.

An inorganic solution similar to the inorganic electrolyte compartment of the DMEM (Dulbecco's modified Eagle's medium) cell culture medium is developed. This biomineralization medium contains 44.05 mM HCO3 -, 126.86 mM Na+, 93.37 mM Cl-, 5.33 mM K+, 2.26 mM Ca2+, 0.905 mM H2PO4 -, and 0.81 mM Mg2+. Its Ca/P molar ratio is set to be identical to that of human blood plasma, i.e., 2.50. The medium is free of any Tris or Hepes but maintains a pH of 7.45 both at 37 and 65 °C. The first novelty of this solution is that it has the unique ability to homogeneously coat X-ray-amorphous calcium phosphate (ACP) on glass slides vertically immersed in it and kept at 37 °C for less than 48 h. The second innovative aspect of this solution is that it has the unprecedented ability to produce monodisperse ACP nanospheres with diameters less than 180 nm when simply heated at 65 °C for 1 h while being stirred. The third novelty of this solution is that it only forms ACP and it does not form apatite in stark contrast to many other synthetic calcification or biomineralization media known. Samples were characterized by X-ray diffraction, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, BET surface area, contact angle goniometry, field emission-scanning and transmission electron microscopy analyses.

Brushite (CaHPO4·2H2O) to octacalcium phosphate (Ca8(HPO4)2(PO4)4·5H2O) transformation in DMEM solutions at 36.5°C.

The purpose of this study was to investigate the transformation of brushite (dicalcium phosphate dihydrate, DCPD, CaHPO4·2H2O) powders at 36.5°C in DMEM (Dulbecco's Modified Eagle Medium) solutions. Two sets of brushite powders with different particle shapes were synthesized to use in the above DMEM study. The first of these brushite powders was prepared by using a method which consisted of stirring calcite (CaCO3) powders in a solution of ammonium dihydrogen phosphate (NH4H2PO4) from 6 to 60min at room temperature. These powders were found to consist of dumbbells of water lily-shaped crystals. The second one of the brushite powders had the common flat-plate morphology. Both powders were separately tested in DMEM-immersion experiments. Monetite (DCPA, CaHPO4) powders were synthesized with a unique water lily morphology by heating the water lily-shaped brushite crystals at 200°C for 2h. Brushite powders were found to transform into octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4·5H2O) upon soaking in DMEM (Dulbecco's Modified Eagle Medium) solutions at 36.5°C over a period of 24h to 1week. Brushite powders were known to transform into apatite when immersed in synthetic (simulated) body fluid (SBF) solutions. This study found that DMEM solutions are able to convert brushite into OCP, instead of apatite.

A new approach in biomimetic synthesis of calcium phosphate coatings using lactic acid-Na lactate buffered body fluid solution.

The main objective of this study was to investigate calcium phosphate (CaP) coatings on Ti6Al4V substrates by using the biomimetic technique. To this purpose, a new solution was developed to coat CaP on Ti6Al4V alloy substrates. The newly formulated body fluid (Lac-SBF) contained appropriate amounts of sodium lactate (NaL) and lactic acid (HL), as well as all the other ionic constituents of the human blood plasma. The inorganic ion concentrations of the Lac-SBF solutions were identical with those of human blood plasma. The new Lac-SBF solution of this study eliminated the need for using Tris/HCl or Hepes/NaOH buffers. Prior to coating, Ti6Al4V substrates were chemically treated in NaOH and/or NaOH+H(2)O(2) solutions as an alternative route and then heated at 600 degrees C for 1h in air. In the previous applications, the Cl(-) ion concentration was found to be higher than blood plasma 103mM, which exists in human blood plasma as a result of Tris/HCl which are used to prevent precipitation and to keep the pH level at certain values. In this study, instead of using Tris/HCl, HL/NaL which are generated by human body and do not show any toxic behavior, are used and Cl(-) concentration was kept at 103mM value for the first time. The prepared Lac-SBF was shown to have similar concentration to human blood plasma in terms of all inorganic ions for the first time. Solution properties were evaluated by using turbidimeter, pH meter and rheometer. The coatings were characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and a scratch tester. The obtained results are presented and discussed.

A new rhenanite (beta-NaCaPO(4)) and hydroxyapatite biphasic biomaterial for skeletal repair.

Biphasic beta-rhenanite (beta-NaCaPO(4))-hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2)) biomaterials were prepared by using a one-pot, solution-based synthesis procedure at the physiological pH of 7.4, followed by low-temperature (300-600 degrees C) calcination in air for 6 h. Calcination was for the sole purpose of crystallization. An aqueous solution of Ca(NO(3))(2). 4H(2)O was rapidly added to a solution of Na(2)HPO(4) and NaHCO(3), followed by immediate removal of gel-like, poorly-crystallized precursor precipitates from the mother liquors of pH 7.4. Freeze-dried precursors were found to be nanosize with an average particle size of 45 nm and a surface area of 128 m(2)/g. Upon calcination in air, precursor powders crystallized into biphasic (60% HA-40% rhenanite) biomaterials, while retaining their submicron particle sizes and high surface areas. beta-rhenanite is a high solubility sodium calcium phosphate phase. Samples were characterized by XRD, FTIR, SEM, TEM, ICP-AES, TG, DTA, DSC, and surface area measurements.

In vitro testing of calcium phosphate (HA, TCP, and biphasic HA-TCP) whiskers.

Calcium phosphate [single-phase hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)), single-phase tricalcium phosphate (beta-TCP, Ca(3)(PO(4))(2)), and biphasic HA-TCP] whiskers were formed by using a novel microwave-assisted molten salt mediated process. Aqueous solutions containing NaNO(3), HNO(3), Ca(NO(3))(2) x 4H(2)O, and KH(2)PO(4) (with or without urea) were used as starting reagents. These solutions were irradiated in a household microwave oven for 5 min. As-recovered precursors were then simply stirred in water at room temperature for 1 h to obtain the whiskers of the desired calcium phosphate (CaP) bioceramics. These whiskers were evaluated, respectively, in vitro by (1) soaking those in synthetic body fluid (SBF) solutions at 37 degrees C for one week, and (2) performing cell attachment and total protein assay tests on the neat whiskers by using a mouse osteoblast cell line (7F2). beta-TCP, HA, and HA-TCP biphasic whiskers were all found to possess apatite-inducing ability when soaked in SBF. SBF-soaked whiskers were found to have BET surface areas ranging from 45 to 112 m(2)/g. Although the osteoblast viability and protein concentrations were found to be the highest on the neat HA whiskers, cells were attached and proliferated on all the whiskers.