Synchrotron radiation micro computed tomography

SRµCT imaging was performed at the P07 high-energy material science beamline [17], which is operated by Hereon at the PETRA III storage ring at the Deutsches Elektronen-Synchrotron in Hamburg, Germany. Samples were scanned at a photon energy of 60 keV with the use of a double crystal monochromator. Due to the large sample size, two ROIs in specific sections of the screw were imaged instead of the whole volume. The sample was imaged by rotating off-center ∼360° leading to a vertical and horizontal field of view of 2.88 mm and 12.56 mm, respectively. Projections were stitched prior to tomographic reconstruction using IDL (Harris Geospatial Solutions, Inc.). The effective pixel size was 1.06 µm, which was binned during reconstruction to a pixel size of 3.18 µm. Stitched tomograms were reconstructed using a reconstruction framework implemented in MATLAB [18, 19] and employing the ASTRA toolbox for tomographic back projection [20, 21]. The reconstructed data sets were filtered using an iterative nonlocal means filter [22].

The segmentation of the filtered data sets was performed in Avizo 2021.1 (FEI SAS, Thermo Scientific, France). The labels residual material (non-corroded alloy), degradation layer (corrosion products attached to the residual material), bone (mineralized tissue) and background (all remaining features not assigned to previously mentioned labels) were distinguished. The segmented data were used for quantitative analysis of parameters describing degradation of implants, osseointegration and bone regeneration over the healing process [13]. Prior to the analysis of VL and DR, the alignment of non-degraded screw and ROIs of degraded screw were necessary. The mid-resolution µCT scan of a non-degraded implant was used as a reference shape and it was registered and resampled on the degraded ones. With this operation, we ensure that compared volumes are from the same sections of the screw.

The quantitative parameters were VL (in mm³), DR (in mm/year), bone-to-implant contact (BIC) [%] and bone volume fraction (BV/TV) [%]. The volumes of non-degraded and degraded screws as well as the volume of bone and background in selected areas were calculated in software Fiji (ImageJ) [23] based on segmented labels.

VL and DR quantifications were quantified according to equation (1).

The BIC parameter was used for characterizing the osseointegration and as an indication for implant stability in the bone:

Both values were calculated with the use of a MATLAB R2018a (The MathWorks, Inc., USA) script which determined the contact voxels in the 3D volume between two different layers [24]—implant (degradation layer + residual metal or just material in case of Ti) and bone, and implant and background, respectively.

BV/TV is a parameter used for evaluating the bone formation surrounding an implant. A ROI was determined separately for each sample by enlarging the non-degraded, registered reference screw by 30 µm and 1 mm, respectively. The enlargement of 30 µm was selected to assess the effect of bone shrinkage during the embedding process on BIC; 30 µm were selected assuming a shrinkage to the extent of the average size of an osteoblast. The enlargement by 1 mm was performed to reduce the effect that the screw thread shape may have on bone formation. Therefore, 1 mm was selected, which is approximately double the size of the threads. Enlarging the non-degraded screws and quantifying the voxel numbers was performed in Fiji. The enlargement was based on applying a distance map to the non-degraded screw and selecting all voxels with a distance of ≤ 1 mm from the original screw surface.

Finally, BV/TV was determined as [25, 26]: