2.1.2. E-Beam Specification

Half of all the samples were E-beam irradiated with a standard single dose of 25kGy (Steri-Tek, Fremont, CA, USA), in alignment with the ISO 11137-2:2006 norm. Steri-Tek uses two 10 MeV, 20 KW linear accelerators (Mevex, Stittsville, ON, Canada) to create a DualBeam™ processing method, which increases efficiency by administering uniform doses to products without having to rotate them. The Bruyas et al. study on E-beam and β-TCP/PCL scaffolds also used this E-beam specification [23]. This standard complies with the sterility assurance level (SAL) being less than 10−6. In other words, there can be at most one unsterile item for every one million objects, whether it be devices or sca ffolds, in order to qualify as sterile [22].

### *2.2. The Subcutaneous Implantation of Samples into Rats*

For this study, five Sprague Dawley rats (S.D Rat, Taconic Biosciences, Rensselaer, NY, USA) were grown in a pathogen-free environment for a period of 9 weeks. All experiments were conducted in accordance with animal testing ethics and were approved by Chonnam National University Institutional Animal Care and Use Committee (No. CNU IACUC-YB-2018-80). Specimens prepared for in vivo testing were classified as shown in Table 1, and a total of 40 specimens were prepared and divided into eight groups. The rats were anesthetized using 10 mg/kg of Xylazine (Rumpoon, Bayer, Leverkusen, Germany) and 20 mg/kg of Zoletil (Zolazepam + Tiletamine, Virbac, Carros, France) by intraperitoneal injection. To prevent bradycardia, 0.1 mg/kg of an anticholinergic drug (Atropine, JEIL Pharmaceutical, Seoul, Korea) was injected intramuscularly. Both the neck and hind limbs were shaved followed by iodine cure, ethanol (70% ethyl alcohol) disinfection, and incisions. Each filament from the experimental groups was implanted into the neck and hind limbs. Each rat was implanted with eight di fferent groups of cylindrical filaments that were placed in subcutaneous sacs internally and sutured (Vicryl-4.0, Johnson & Johnson Medical, New Brunswick, NJ, USA), with sutures at appropriate intervals to prevent movement of the samples. The transplanted samples were not in contact with each other (Figure 1).


**Table 1.** Classification codes for each group.

**Figure 1.** Schematic diagram of the filament implantation and experimental process.

Post-operatively, all the animals received 5 mg/kg of antibiotics (Enrofloxacin, Bayer Leverkusen, Germany) and 5 mg/kg of analgesic anti-inflammatory drugs (Ketoprofen, EagleVet, Seoul, Korea). After implantation, rats were subjected to in vivo live micro-computer tomography (CT) (live-CT) and micro-CT (Figure 1).

### *2.3. Micro-CT and In Vivo Live-CT*

Volumes of the samples before implantation and explants after animal euthanization were measured using micro-CT (SKYSCAN 1272, Bruker, Billerica, MA, USA) at 30 kV voltage, 150 μA current, and 10 μm pixel size. The scanned slices were reconstructed into DICOM files using the Cone Beam program (PerkinElmer, Waltham, MA, USA). After 24 weeks of implantation, the rats were sacrificed, and the implants were removed and subjected to micro-CT measurement under the same conditions as before implantation. The volume change of the specimens was calculated using Equation (1) and an image processing software (Mimics software, Materialize NV, Leuven, Belgium).

$$\text{Micro-CT volume change (\%)} = ((M\_{\Delta} - M\_0)/M\_0) \times 100,\tag{1}$$

where *M*0 is the micro-CT data of implants before implantation, and *M* is the micro-CT data of explants at 24 weeks after implantation.

The volume changes of the filaments implanted in vivo were measured using a live-CT device (Quantum GX2, PerkinElmer, Inc., USA). Exposure conditions were maintained at 90 kV voltage, 88 μA current, and 90 μm voxel size for 4 min. The volume of the samples was measured after 1 day of implantation and again at 4, 12, and 20 weeks after implantation. The volume change of the specimens was calculated by Equation (2) using an image processing software.

$$\text{Live-CT volume change } (\%) = ((L\_{\Delta} - L\_0) / L\_0) \times 100,\tag{2}$$

where *L*0 is the live-CT data at 1 day after implantation, and *L* is the data for each set period.

Equation (3) was used to compare the difference between micro-CT (*MCT*) volume before implantation and live-CT (*LCT*) volume after 1 day of implantation.

$$\text{Difference between } L\_{CT} \text{ and } M\_{CT} \text{ (\%)} \equiv ((L\_{CT} - M\_{CT})) \text{(} M\_{CT} \text{)} \times 100. \tag{3}$$
