Formation of hydroxyapatite on titanium implants in vivo precedes bone-formation during healing.

The bone material interface has been an area of intense study over many decades, where studies of the healing process ranging from simple mineral deposition in vitro to actual healing in vivo have given important clues to the importance of calcium minerals in the bone/implant interface. Here, the authors use a combination of in vitro cell culture methods and in vivo implantation to study how the role of the spontaneously formed hydroxyapatite layer on Ti-implants for the in vivo-healing into the bone tissue of rat tibia. Initial experiments were made in reduced systems by incubation of TiO in cell culture medium and analysis by time of flight secondary ion mass spectrometry (ToF-SIMS) and energy-dispersive x-ray spectroscopy followed by subsequent exposure of human embryological stem cells analyzed by von Kossa staining and environmental scanning electron microsopy.

Mg-corrosion, hydroxyapatite, and bone healing.

The different capacities of magnesium in the metallic form (Mg-metal) and magnesium oxide (MgO) to stimulate bone healing are possible clues in the search for products that may promote bone healing. Since both Mg-metal and MgO can be assumed to release comparable amounts of Mg ions during their reactions in the tissue where they have been implanted, it is of some importance to follow this process and analyze the resulting mineral formation in the tissue at the implantation site. Implants of MgO were inserted into rat tibia, and the bone healing was compared with sham-operated controls.

Mineralization at Titanium Surfaces is a Two-Step Process.

Mapping the initial reaction of implants with blood or cell culture medium is important for the understanding of the healing process in bone. In the present study, the formation of low crystalline carbonated hydroxyapatite (CHA) onto commercially pure titanium (Ti) implants from cell culture medium and blood, is described as an early event in bone healing at implants. The Ti-implants were incubated with cell culture medium (DMEM) or whole blood and the surface concentration of Ca, P and HA was analyzed by XPS, EDX and Tof-SIMS.

Increase of Compact Bone Thickness in Rat Tibia after Implanting MgO into the Bone Marrow Cavity.

The effect of implanting MgO paste into the bone marrow of rat tibia, was studied by light microscopy, time of flight-secondary ion mass spectrometry (ToF-SIMS), and environmental scanning electron microscopy (ESEM), and energy dispersive X-ray (EDX) analysis. After three weeks of implantation, the thickness of compact bone increased by 25% compared to sham-operated controls, while no effect was seen on the trabecular bone. In order to further elucidate the mechanism of the Mg-induced increase in bone mass, EDX and ToF-SIMS analysis of the bone samples was made at two weeks.

Analysis of As- and Hg-species in metal-resistant oral bacteria, by imaging ToF-SIMS.

Bacterial strains were isolated from the oral cavity of healthy volunteers and grown in the presence of Hg-ions (1-10 ppm) or arsenate ions at concentrations of 0.1-1.0%.

High-resolution, imaging TOF-SIMS: novel applications in medical research.

The use of high-resolution, imaging TOF-SIMS is described and examples are made to demonstrate the application of the method in medical research. Cytochemistry by TOF-SIMS is shown by localization of diacylglycerol (DG) in cryostat sections of hyaline cartilage and by localization of corticosterone in cryostat sections of the adrenal gland cortex. Quantitative measurements and comparison of groups is shown by comparing the lipid content of adipose tissue from two mouse strains, transgenic mouse expressing the FOXC2 gene and wild-type controls.

High-resolution imaging and proteomics of peptide fragments by TOF-SIMS.

Thyroglobulin is an iodinated glycoprotein (m.w. 660 kD) required for the storage and formation of thyroid hormone.

Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS).

The review describes methods available for analyzing mineralization of bone tissue in healing of fractures and implants in bone. The recent development of imaging MS, TOF-secondary ion MS (SIMS), enabling localization of hydroxyapatite (HA) in tissue samples will be presented in some detail. We strongly believe that imaging MS has the potential of becoming an important method for the studies of bone mineralization.

Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants.

Implant healing into bone tissue is a process where the mature bone grows towards and eventually fuses with the implant. In this study we investigated implant healing during 4 weeks with focus on the implant-tissue interface. Our main interest was to study the mineralization process around the implant.

High resolution imaging by organic secondary ion mass spectrometry.

Secondary-ion mass spectrometry (SIMS) is based on the acceleration of high-energy primary ions onto a target. Secondary electrons, neutrals and ions are emitted from the target, reflecting its chemical composition. This enables simultaneous analysis and localization of target molecules, giving valuable information that is difficult or impossible to obtain with other analytical methods.

Bioimaging TOF-SIMS: High resolution 3D imaging of single cells.

The distribution of phosphocholine ions (m/z 184, m/z 86), sodium ions, and potassium ions in thyroid tumor cells was analyzed by imaging TOF-SIMS. Repeated sputtering with a C(60) (+) source and subsequent analysis with a Bi(3) (+) gun produced a series of 138 images that were stacked to make a 3D display of the chemistry of cells. Phosphocholine was seen in the plasma membrane (m/z 184) and intracellular membranes (m/z 86).

Callus formation and remodeling at titanium implants.

Titanium is biocompatible with bone tissue, and during the healing process bone makes intimate contact with the implant surface. Although much is known about the long-term healing of implants, less is known about the callus formation at implants. In this study, the histology of bone healing was studied during the period between 4 and 14 days.

Imaging of lipids in human adipose tissue by cluster ion TOF-SIMS.

Biopsies of human subcutaneous adipose tissue were taken from healthy donors. Samples were high-pressure frozen, freeze-fractured, and freeze dried. Imaging mass spectrometry of samples was performed in a TOF-SIMS mass spectrometer equipped with a bismuth cluster ion source.

Localization of fatty acids with selective chain length by imaging time-of-flight secondary ion mass spectrometry.

Localization of fatty acids in biological tissues was made by using TOF-SIMS (time-of-flight secondary ion mass spectrometry). Two cell-types with a specific fatty acid distribution are shown. In rat cerebellum, different distribution patterns of stearic acid (C18:0), palmitic acid (C16:0), and oleic acid (C18:1) were found.

Analysis of bone minerals by time-of-flight secondary ion mass spectrometry: a comparative study using monoatomic and cluster ions sources.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an important tool for the analysis of bone minerals at implant surfaces. Most studies have been performed with monoatomic primary ion sources such as Ga(+) with poor secondary molecular ion production efficiency and only elemental distributions and minor fragments of bone minerals have been reported. By using cluster ion sources, such as Au(1-3) (+) and Bi(1-3) (+), identification of larger hydroxyapatite species at m/z 485, 541, 597 and 653, identified as Ca(5)P(3)O(12), Ca(6)P(3)O(13), Ca(7)P(3)O(14) and Ca(8)P(3)O(15), respectively, became possible.

Sulfatide with different fatty acids has unique distributions in cerebellum as imaged by time-of-flight secondary ion mass spectrometry (TOF-SIMS).

In order to elucidate the biological role of sulfatide it is important to define the cellular and subcellular distribution of its various molecular species (e.g. fatty acid chain length and hydroxylation).

Localization of lipids in the aortic wall with imaging TOF-SIMS.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of localization of lipids and inorganic ions in healthy rat aorta and human atherosclerotic plaque. Pieces of rat aorta were high pressure frozen, freeze-fractured and freeze dried. The samples were analyzed by imaging TOF-SIMS equipped with a Bi(1-7)(+)-source.

Distribution of cholesterol and galactosylceramide in rat cerebellar white matter.

White matter and the inner granular layer of rat cerebellum was analysed by imaging time-of-flight secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bi+ ion cluster gun. Samples were prepared by high pressure freezing, freeze-fracturing and freeze drying or by plunge freezing and cryostat sectioning. The identified and localized chemical species were: sodium, potassium, phosphocholine, cholesterol and galactosylceramide (GalC) with carbon chain lengths C18:0 (N-stearoyl-galactosylceramide) and C24:0 (N-lignoceroylgalactosylceramide) with CH24:0 (hydroxy-lignoceroylgalactosylceramide).

Imaging TOF-SIMS of rat kidney prepared by high-pressure freezing.

Phosphocholine, potassium ions, and sodium ions were localized in rat kidney with imaging TOF-SIMS. Tissue preparation was performed with high-pressure freezing, freeze-fracturing and freeze-drying. The distribution of sodium ions was visualized by imaging the signal at m/z 23 of positively charged secondary ions, and the distribution of potassium ions was visualized by imaging the signal at m/z 39.

Localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex with imaging TOF-SIMS equipped with a bismuth cluster ion source.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of co-localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex. Rat cerebellum was fixed, freeze-protected by sucrose, frozen and sectioned by cryoultramicrotomy and dried at room temperature. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi(1-7)+-source.

Bioimaging TOF-SIMS of tissues by gold ion bombardment of a silver-coated thin section.

The imaging time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) method was utilized to address the problem of cholesterol localization in rat tissues. Rat kidneys were fixed, cryoprotected by sucrose, frozen, sectioned by cryoultramicrotomy, and dried at room temperature. The samples were either covered with a thin silver layer or analyzed uncovered in an imaging TOF-SIMS instrument equipped with an Au1-3(+)-source.

Contact activation and down regulation of neutrophils by cellulose wound dressings can be prevented by radical scavengers.

Neutrophil interaction with wound dressing materials was studied. A meshed non-woven cellulose was chosen as model dressing. Neutrophils isolated from human blood was added to the cellulose, and the production of reactive oxygen species was measured by luminol-amplified chemiluminescence.

Bioimaging TOF-SIMS: localization of cholesterol in rat kidney sections.

Here, we show the localization of a whole organic molecule in biological tissue using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Rat kidneys were sectioned by cryoultramicrotomy and dried at room temperature. The samples were covered with a thin silver layer and analyzed in an imaging TOF-SIMS instrument equipped with a Ga(+)-source.

Implantation of hydrophilic and hydrophobic titanium discs in rat tibia: cellular reactions on the surfaces during the first 3 weeks in bone.

In a previous study, a method for evaluation of short-time (1-8 days) healing of titanium implants in rat tibiae was described (J. Biomed. Mater.

Thrombin, kallikrein and complement C5b-9 adsorption on hydrophilic and hydrophobic titanium and glass after short time exposure to whole blood.

Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5s and 24h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively.