| Biocompatibility evaluation of nickel-titanium shape memory metal alloy: | ||
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The animals in all studies were checked weekly for any abnormalities and to ensure that the wounds had healed. The dissected samples were first observed visually. After removal, the implants were studied for possible marks of macroscopic corrosion.
The morphology and histology of the Goldner-Trichrome and Haematoxylin-Eosin stained sections were examined under a light microscope (magnification 12.8 – 320x in the studies II and III, 32-480x in study IV). Lamellar bone structures were studied in polarized light.
Analysis of metal ion concentrations in cell culture media (Study I): Media collected every second day were stored in plastic tubes and kept at -20 ºC until analysis. The media were collected from three flasks containing the same type of test material and mixed in a single tube. Corrosion analysis was done on this sample. Before the sample was analyzed, the media were burnt with acid. The nickel and titanium concentrations were assessed from the NiTi media. Nickel was determined from the stainless steel and titanium from the titanium flask media.
The analyses were performed using a graphite furnace atomic absorption spectrophotometry (GFAAS, Perkin Elmer SIMAA 6000, Rautaruukki Ltd., Raahe, Finland). Atomic absorption is a technique based on the unique spectrum of each element. For every element analyzed, characteristic wavelengths are generated in a discharge lamp (hollow cathode lamp) and then absorbed by a cloud or vapor of that element. The amount of absorption is proportional to the concentration of the element vaporized into the light beam.
Analysis of corrosion products in various organs (Study IV): The concentrations of nickel, chromium and iron in various organs were assessed at 26 and 60 weeks, to find out whether there had been release or accumulation of ions from the test nails. The samples were first freeze-dried for seven days. After that, they were weighed accurately (approximately to 0.1g) in Teflon decomposition vessels. Two milliliters of ultrapure nitric acid and two milliliters of hydrogen peroxide (pro analysis) were added. The samples were decomposed in a microwave oven (Milestone mls 1200) and diluted in 10 ml of pure water. Cr and Ni were determined by graphite furnace atomic absorption spectrometry (GFAAS) (Perkin-Elmer Zeeman/3030) and Fe by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (PU 7000). The concentrations were given as dry weights.
Study II: The thickness of the reactive and fibrous encapsule membranes around the implants were determined with a CCD camera-based digital image analysis system (MCID/M1, Imaging Research Inc., Canada). The system consisted of a microscope (Nikon Optiphot II, Japan), a videocamera (Dage MTI 72E, USA) and a personal computer with a digitizer (FG-100 AT; Imaging technology, USA). After several pilot stainings (Giemsa, Herovici, Van Gieson, Toluidine blue, Kossa), normal Haematoxylin-Eosin staining was chosen for subsequent use, since it gave the best distinction in the analysis of black-and-white video images. In order to randomize the points of measurement, we used an image overlay meshwork added to each picture. The screen area corresponded to 2.2 mm and the mesh size was 260 x 260 7um. The capsular thickness was determined in the orthogonal direction of each intersection point of horizontal and vertical mesh lines coinciding with the boundary between the capsule and the hole left by the implant. The capsule thickness was expressed as a mean value of 5-18 (average 12) hits. Two sections taken from different midsagittal areas of each sample were measured. There were a few ragged views, in which case no measurement was made. About 2000 single measurements were made during this study.
Study III: The real-color CCD camera-based digital image analysis system (MCID/M4, Imaging Research Inc., Canada) was used in bone histomorphometry. The system consisted of a microscope (Nikon Optiphot II, Japan), a videocamera (Sony DXC 930P, Japan) and a personal computer with a digitizer (Matrox Image 640 with CLD color board, Imaging Technology, USA). Goldner-Trichrome and Haematoxylin-Eosin stainings were used, since they gave the best distinction in the analysis of color video images. The measurement area of each slice was standard, corresponding to a screen of 2.2 mm2. The mid-point of the bone cortex was adjusted to the middle of the screen, just under the cross-sectional radius of the implant (in the paraffin-embedded slices to the hole left by the implant). Whenever possible, the histomorphometric terminology recommended by Parfitt et al. (1987) was used.
The mean cortical width (Ct.Wi) was measured automatically from the given area as the median internal distance perpendicular to the maximum curved chord. The bone area (B.Ar) was measured in a defined area, excluding the trabecular spaces. The erosion area (E.Ar) of resorption pits was measured after reconstruction of the bone surface. The active erosion surface (erosion surface covered by osteoclasts and mononuclear cells) was not determined distinctly, but the active erosion surface perimeter was measured as a fraction of the total bone surface perimeter (E.Pm/B.Pm). The area of new woven bone (N.Wo.B) was measured like B.Ar. Two sections taken from different mid-sagittal areas of each sample were measured.
Study IV: Medial radiographs of the operated femurs were obtained postoperatively at 1 week and along the killing schedule. The live animals were filmed under anesthesia. The maximum length (mm) and width of the callus area were measured from plain radiographs using a CCD camera (Dage MTI 72E), a light table and a PC with MCID/M4 software (Imaging Research Inc., St Catharines, Canada). The union of the osteotomy was judged visually as a consensus of two surgeons. Completely healed bone union with uniform, unbroken callus and no visible osteotomy line was classified as a ”good” outcome. Marked but not uniformly overstepping callus formation with a blur osteotomy line was classified as a ”satisfactory” outcome. Non-union with a clearly visible osteotomy line and no overstepping callus at the osteotomy site was classified as a ”poor” healing outcome.
Study IV: The structure of the callus and the changes in both total (BMD) and cortical bone mineral density (CtBMD) (mg/ccm) were assessed using a pQCT system (Stratec XCT 960A with software v. 5.20, Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany). An attenuation threshold of 0.700 cm-1 and a voxel size of 0.092 X 0.092 x 1.25 mm3 were used. Nine sagittal cuts with a slice thickness of 1 mm were obtained from the operated femur. Three of these were in the callus area, three in the diaphyseal nail area outside the callus, and three in the area of proximal intact femur. Three cuts were performed on non-operated femurs corresponding to the osteotomy sites as controls.
Field emission scanning electron microscopy (FESEM) (Jeol JSM-6300F, Japan Electronoptics LTD, Japan) with a digital image processor (SemAfore pro-S, J. Rimppi Inc., Finland) was used.
In study III, FESEM was used to obtain high-quality magnifications (ad 170,000x) from the cell-implant interface. Direct cell contact or other signs of close attachment between cells and different materials were observed. The tissue-implant adhesion morphology at micro- and nanoscales was analyzed and compared with qualitative criteria.
In study IV: FESEM was used to evaluate the marks of corrosion on the surface of the retrieved nails. The differences between the implants and the influence of time were analyzed and compared with the qualitative criteria.
The values were expressed as mean ± standard deviation (SD). The data were analyzed using unpaired two-sample t-test in study I and one-way analysis of variance (ANOVA) with t-test in the study III. In study IV, the data on bone unions were analyzed using chi-square test and the trace metals using t-test. The differences were considered significant at a probability level of 95% (P < 0.05). All statistical analyses were performed with commercially available software.