Skyscan 1076 scanner

The optical images of the hydrogel before and after gelatin formation were recorded with a digital camera (Canon EOS 550D, Japan). The sample porosity was measured according to the Archimedes' principle by the water-displacement technique. Porosity was further detected by microcomputed tomography (micro-CT) (voltage: 102 kV, current: 100 mA) (SkyScan 1076 scanner, Bruker micro-CT NV, Kontich, Belgium) and NRecon software (version 1.6.6; Bruker micro-CT). To quantify the average pore size and distribution, the sample microstructure was observed under a JEOL JSM-6700F scanning electron microscope (SEM), and images (n = 10) were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA).

To investigate the dbPT distribution in 3D-printed GEL-dbPTs scaffolds, μCT analysis was performed. The tomograms of the scaffolds were recorded on a Skyscan 1076 scanner (Bruker, Kontich, Belgium), applying a source voltage of 37 ​kV and a source current of 228 ​mA. To reduce beam hardening artifacts, a 0.025 ​mm titanium filter was used. The scan resolution was set to 9 ​μm per voxel. For noise reduction, an average of 4 frames was recorded every 0.3°. The scans were reconstructed applying the cone-beam algorithm in the NRecon software package (Bruker, Kontich, Belgium). The datasets were segmented globally, and the lower gray threshold was set to 155 and the upper gray threshold to 255 to account for the denser dbPTs. A 3D analysis of the segmented datasets was performed to determine the particle number and particle size distribution. For the segmentation and 3D analysis, the software CT analyser (Bruker, Kontich, Belgium) was used. High-resolution 3D renderings were created using CTVox software (Bruker, Kontich, Belgium).

Cylindrical 3D printed Ti6Al4V porous scaffolds, which was 6 mm diameter and 8 mm height, with pre-designed pore sizes about 600 μm and porosity about 70 % were manufactured layer-by-layer by an EBM system (Arcam Q10, Sweden). Briefly, the 3D model date was designed and imported into the UG NX6.0 system (Unigraphics Solutions, US). Medical-graded Ti6Al4V powder with an average diameter of 45–105 μm were used as basic material. The maximum scanning speed of electron beam was 8000 m s^− 1 and the accuracy of printing is ± 0.4 mm. Finally, all samples were cleaned in Powder Recovery System and washed in acetone, ethanol, and distilled water with ultrasonic machine in turn for 30 min, respectively, before cell experiments and animal procedures [29 ].
To demonstrate whether the parameters of the prepared scaffolds, such as pore size and porosity, are the same as those of the pre-designed model, we performed characterization tests on the scaffolds. The porosity of prepared porous scaffolds was measured by a SkyScan 1076 scanner Microcomputed Tomography (Micro-CT, Bruker, Kontich, Belgium). In addition, in order to detect the average pore diameter of these Ti6Al4V scaffolds, the microstructure of samples was photographed by a SIGMA500 scanning electron microscope (SEM, ZEISS, Oberkochen, Germany), and pictures were quantitative analysis by Image J software (NIH, Bethesda, MD, USA).