Ct 80

Breeding of the C57BL/6 WISP-1 knock-out mice was approved by the local animal welfare committee (Az.: 84-02.04.2018.A284/LANUV, North Rhine-Westphalia, Germany) of the Leibniz Research Centre for the Working Environment and Human Factors (IfADo) and isolation of tibia was registered at the Tübingen district office (DE 08416113021/Az.: 32/9181.21/Mu approval at the 21 February 2018). Each of eight tibiae from wild-type C57BL/6 mice and C57BL/6 WISP-1 knock-out mice, with ages ranging from 8 to 14 weeks, were dissected and cleaned from any remaining muscles, fat, or tissue. In these animals, both the age difference and the knockout of WISP1 may affect bone strength [9 (link),10 (link)], which is ideal, as the FEM development and validation require different bone strength. Then, the bones were placed in a 2 mL reaction tube filled with water and scanned under a µCT (µCT 80, SCANCO MEDICAL) with an exposure time of 1000 ms. The rotation pattern was set as 360° of rotation with 360 steps [11 (link)]. The resulting scans were cropped and rotated so that all bones were in a straight position, based on a horizontal tibia plateau. Obtained µCT datasets were saved in the format of Digital Imaging and Communications in Medicine (DICOM). Dissected bones were stored at −80 °C until further use.

Femurs were isolated from 2- and 5-month-old mice. Fixed non-demineralized femurs and the femoral cancellous bones of the distal metaphysic and the middle shaft were scanned with micro-CT (µCT-80, Scanco Medical AG, Bassersdorf, Switzerland) as reported previously [25] (link). Images (IMAQ) were acquired at 70 kV and 113 mA. Two-dimensional images were used to generate three-dimensional reconstructions for 3D analysis. The analysis of the specimens involved the following bone measurements: trabecular and cortical bone volume fraction (Tb.BV/TV, Ct.BV/TV, %), trabecular number (Tb.N), trabecular and cortical thickness (Tb.Th, Ct.Th), trabecular separation (Tb.Sp), trabecular structure model index (Tb.SMI), trabecular and cortical bone mineral density (Tb.BMD, Ct. BMD) [26] (link).

High-resolution images of the real composite construct architectures were obtained through different stages of compressive deformation with the use of a micro-CT scanner (µCT 80, Scanco Medical AG, Switzerland). Pure scaffolds and reinforced gels with a fibre spacing of 200 and 800 µm were analysed at increasing compressive strain levels of 0, 15, 30 and 45%. The micro-CT device was equipped with a custom-made loading device. This loading system consists of a supporting tube driven by a bolt system and two spacers^{23 (link)} (Fig. 2C). When tightened, the system enabled the compression of the constructs to the required deformation level. A water-based contrast agent solution (Ioversol, Optiray 300 ^TM, Mallinckrodt Pharmaceuticals) was used in the reinforced constructs for staining the GelMA hydrogel. The acquisition parameters were set to a voltage of 70 kVp; an intensity of 114 µA and an integration time of 300 ms. After scanning, a Gauss filter was applied (sigma = 1, support = 0.8 voxel) and images were subsequently segmented. A global threshold of 24 per mile and 105–195 per mile were used for the pure scaffold and scaffold region on the reinforced hydrogel constructs.

High-resolution micro-CT images were used to assess new bone formation surrounded the implanted materials. The scanning was performed with X-ray tube set at 70 kVp and 114 µA (µCT80, Scanco Medical, Bassersdorf, Switzerland). Integration time (300 ms) was required for each sample and the region of interest had a resolution of 30 µm. The generated grey scale images were then reconstructed and analysed with the Scanco software. According to our previous work, a global threshold was adopted to differentiate the materials from bony tissue [27–29 (link)]. Three-dimensional (3D) images were extracted from the operated intervertebral lumbar region to visualize the connectivity between the implants and the new bone. Implant circumference and the length of all circumferential segments with direct contact between implant and bone were measured. Circumferential contact index (%) of the PMMA and PMMA-MC groups was calculated in accordance with literature [30 (link)].

After 56 days of in vitro corrosion, the implants were scanned using a µ-computer tomograph (µCT80; ScancoMedical, Zurich, Swiss; slice thickness: 20 µm; voltage: 70 kV; amperage: 114 µA; integration time: 400 ms). 3D images were computed (threshold: 108) and an assessment of the volume, density and the “true-3D-thickness” of the implants according to Huehnerschulte et al. [19 (link)] was performed.
Subsequently, the samples underwent three-point-bending testing as described in “Three-point bending test”.

Micro‐CT scans (µCT 80, Scanco Medical, Bruttisellen, Zurich, Switzerland) were performed on limbs harvested from the experimental and control groups. Specimens were scanned with a mean 20‐μm slice thickness under the conditions of 60 kV at 150 μA. HO formation was evaluated using reconstructed 3D images and ectopic bone volume was calculated.