*2.5. Statistics*

Quantitative data are presented as mean ± standard deviation with variance analysis according to the Mann–Whitney U test. The Kruskal–Wallis test was used to compare among experimental groups using the PASW Statistics 18.0 software (SPSS Inc., Chicago, IL, USA). *p* < 0.05 was considered statistically significant.

### **3. Results and Discussion**

### *3.1. Evaluation of Filament Degradation in the Subcutaneous*

Figure 2a shows the volume changes of the eight different groups of filaments before and after 24 weeks implantation by micro-CT analysis. The volume change of filaments was used as an indicator of degradation. At 24 weeks, the volume change of groups (0, e) and (0, no e) was 1.54% ± 0.28% and 0.78% ± 0.24%, respectively. Groups (20, e) and (20, no e) was 10.16% ± 3.95% and 6.58% ± 1.04%, respectively. Groups (40, e) and (40, no e) was 10.33 ± 4.19 and 5.68 ± 3.17, respectively. Groups (60, e) and (60, no e) was 10.47 ± 3.59 and 6.80 ± 1.54, respectively. There was a significant difference in volume change between the (0, e) and (0, no e) groups, but there was no significant difference among the other groups because of relatively high standard deviations.

**Figure 2.** Volume change of filaments by micro-computer tomography (CT) and Equation (1). (**a**) Between E-beam and non-E-beam at 24 weeks (\* *p* < 0.05); (**b**,**<sup>c</sup>**) of 4 different tricalcium phosphate/polycaprolactone (TCP/PCL) ratio groups at 24 weeks (\* *p* < 0.05).

However, the volume changes of E-beam groups were greater than the non-E-beam groups in the same TCP:PCL ratio pair. The average difference between the E-beam and non-E-beam groups in each ratio was 0.76% (0% TCP), 3.30% (20% TCP), 4.65% (40% TCP), and 3.67% (60% TCP). The results sugges<sup>t</sup> that E-beam sterilization accelerated degradation, which is consistent with our in vitro degradation study [23] and other literature [19,20,23,26,27]. E-beam accelerates degradation because irradiation causes decreased crystallinity and shorter molecular chains of PCL due to chain scissions. The volume changes of pure PCL filaments are smaller than those of TCP/PCL composite filaments, suggesting that the addition of TCP also accelerated degradation. Compared to the slowly degradable hydrophobic PCL, the higher content of hydrophilic TCP ceramic particles increased water absorption from body fluids and accumulated more lipase enzymes onto the surface layer of the filaments [30,31], inducing hydrolysis and increasing PCL surface erosion [32]. It is worth noting that the increased hydrophilicity of the TCP/PCL surface likely facilitates cell adhesion, which also likely contributed to the increased degradation [32].

There were no significant differences in the degradation among the three different TCP content groups, in both E-beam and non-E-beam (Figure 2b,c). The results sugges<sup>t</sup> that the chemistry of composites plays a bigger role in biodegradation than the sterilization method. In particular, E-beam mainly affected the properties of PCL, not TCP. The concentration of TCP particles embedded into the PCL matrix was not sufficient to allow particles to be interconnected. The lack of interconnection then allows water to penetrate into the center of the composite [19].

In this experiment, live-CT was used to observe the volume change of filaments over time without euthanizing the rats. Using live-CT also reduces the number of rats needed for the study and discrepancies due to the individual characteristics of each rat. However, the 0% TCP group was difficult to scan with live-CT due to its low contrast and fine fiber diameter. The ability to scan with live-CT is affected by the signal-to-noise ratio, the phase difference, and the tissue around the artifact [33–36].

Table 2 lists the volume differences of samples that were estimated by Equation (3) by subtracting the volume before implantation by micro-CT from the volume at one day after implantation by live-CT. The average volumes by live-CT were slightly higher than those by micro-CT in all samples. This difference could be due to the filament swelling inside the live body at one day after implantation. A study found that swelling of PCL occurs rapidly within the first 24 h, with a difference of about 10–20% [37]. In another report, the rate of swelling varies with the amount of hydrophilic material mixed with PCL within 24 h, and the resulting volume increase is around 5–10% [38]. Our results in Table 2 are consistent with the values in these reports.


**Table 2.** Volume di fference between micro-CT and live-CT.

Figure 3A shows the volume change over time based on live-CT analysis and Equation (2). All TCP-containing groups exhibited a rapid volume reduction during the first four weeks of implantation. From 4 to 20 weeks, all groups showed a slow and gentle downward slope in volume change, and the E-beam groups resorbed more at every time point. It has been reported that the adsorption of bioactive proteins to the surface of biomaterials from serum and body fluid upon implantation a ffects the rate of degradation [39], because this influences the e ffects of cellular interactions on body fluids and active protein substances attached to implanted TCP/PCL specimens. The hydrolysis mechanism of PCL occurred simultaneously, which results in rapid degradation [40].

**Figure 3.** (**A**) Volume change at 1 day, 4, 12, and 20 weeks after implantation by live-CT and Equation (2). (**B**) Three-dimensional model of a sample 40% TCP filament, (**a**) E-beam and (**b**) non-E-beam by live-CT. (1–4) are filaments at 1 day, 4 weeks, 12 weeks, and 20 weeks, respectively. (5) is a superimposition between 1 day (red) and 20 weeks (yellow).

However, one weakness of this study was that we did not use live-CT to examine the volumes of filaments at 24 weeks after implantation, while they were still implanted into the rats; rather, we used micro-CT to examine the volumes of the filaments after explantation. Our decision was misled by our data comparison at day one listed in Table 2, in which the volume by live-CT one day after implantation was greater than the volume by micro-CT before implantation. It turns out that the volumes from micro-CT analysis of the explants after 24 weeks of implantation are actually larger than the values from live-CT analysis at 20 weeks of implantation. The lack of live-CT data at 24 weeks after implantation lost us the opportunity to continuously examine changes between 20 and 24 weeks. As such, we cannot compare the di fference between live-CT data before euthanization and micro-CT data after euthanization to further confirm the resolution of CT scanning.
