*3.2. Histological Examination*

Figure 4 shows histology images of sample implants surrounded by tissue. Fibrous encapsulation—a thick and homogeneous colonization by fibroblast cells—was found in all samples.

**Figure 4.** *Cont*.

**Figure 4.** Micrographs of subcutaneous tissue responses to filaments after 24 weeks of implantation. White arrows indicate fibroblasts; (\* with a white circle) indicates monocytes and macrophages; (\*\*) indicates protein and collagen; and (\*\*\*) indicates blood vessels. The micrographic images of each stained samples (10 × and 100 × magnification) are labelled with group code as follow; (**A**) and (**a**) are (0,e), (**B**) and (**b**)are (0,no e), (**C**) and(**c**) are (0,e), (**D**) and(**d**) are(0,no e), (**E**)and (**e**)are(0,e), (**F**)and (**f**)are (0,no e), (**G**) and (**g**)are (0,e),and (**H**) and (**h**)are(0,no e).

**60, no e**

Inflammatory macrophages (or monocytes) were found in the (0, e), (0, no e), (60, e), and (60, no e) groups. PCL degradation increases the acidity around the tissues, resulting in an inflammatory response [31]. Fibroblasts were at high-density and accompanied by collagen deposition. Blood vessel formation was also observed. A study of biodegradable polymers by Pêgo et al. shows that encapsulation of samples is mainly composed of macrophages, fibroblasts, and newly formed blood vessels due to immune reactions that are followed by inflammatory reactions, encapsulation characteristics commonly seen after implantation [41]. On the tissue surface of both the E-beam and non-E-beam 60% TCP groups, some multinucleated giant cells (MNGCs) were also observed (data not shown). When macrophages fail to remove foreign bodies due to the presence of slowly degradable PCL, they fuse to form MNGCs, and exhibit foreign body reactions during chronic inflammation [42]. The presence of MNGCs is closely related to the improvement of neovascularization, the degradation and uptake of the implanted biomaterial, and the encapsulation by the transplantation reaction [43,44]. MNGCs invade the implanted biomaterial and begin to destroy the original structure. Blood vessels and connective tissue grow into the biomaterial, leading to premature loss [45].

We understand that subcutaneous implantation may not reflect the degradation of orthotopic bone defect implantation. We used the filaments and subcutaneous implantation as a simple screening test before we test them in bone defects. In our other studies, we implanted 3D-printed TCP/PCL scaffolds of single formulation (20% TCP/80% PCL) into large bone defects in rabbit femoral heads [13,14] and rat femurs [46]. Both implantations showed that our scaffolds promoted bone ingrowth into the porous structure and retained structural integrity, suggesting excellent biocompatibility and osteointegration. The degradation rates of the macroporous 20% TCP/80% PCL scaffolds ranged from 10% to 25% at eight weeks after implantation in femoral head bone defects [13]. The slowest degradation rate in bone defects in rabbits was similar to those of subcutaneous implantation. The difference could be the result of higher surface areas in porous scaffolds versus rods and anatomical sites. We plan to further test E-beam sterilized 3D-printed scaffolds within bone defects in the future.
