**2. Results**

## *2.1. Clinical Evaluation*

Clinical evaluation was carried out during the experiment to verify the health evolution of the horse and/or the possible presence of systemic changes induced by the implantation of the biomaterials. In general, there was no discomfort associated with the implantation of the materials, and no behavioral and/or appetite changes were observed. Intestinal motility, hydration status, apparent mucous color, and capillary filling time were within the normal range for the species. RR and RT values were within reference values for adult horses throughout the implantation period (8–16 mpm; 37–38.3 ◦C; [23]), (Figure S1b,c). HR values increased at times up to 144 h after implantation; however, they returned to normal values for the equine species after 168 h (28–44 bpm; [23]) (Figure S1a).

## *2.2. Plasma Fibrinogen*

PF concentration was determined to check for systemic inflammatory processes due to the implantation of the biopolymers. PF values remained within the range of reference for the equine species (100–400 mg/dL; [24]) throughout the evaluation period (Figure S2).

## *2.3. Mechanical Nociceptive Threshold (MNT)*

Compared to baseline, MNT reduced 12 and 24 h (*F* = 9.431; *p* ≤ 0.001) after implantation. Forty-eight hours after implantation MNT was reversed (Figure 1).

**Figure 1.** Graphic representation of the means ± standard error of the mechanical nociceptive threshold (MNT) of a horse submitted to the implantation of six polymers of poly(lactic acid) (PLA). Means followed by the same letter do not differ by the Tukey's test (*p* < 0.05) in each moment.

## *2.4. Histopathological Analysis*

Histopathologic evaluation revealed fibrotic capsular formation involving the polymer at all implants surgical removal times (Figure 2), except at 57 weeks; however, over time, the capsules became more organized, as indicated by the progression in the scores of capsule characterizations (Table 1). We observed cellular growth, characterized as fragmentation of the biomaterial and the invasion of the fibrotic tissue, and the consequent surrounding of the evaluated fragments, which go<sup>t</sup> smaller at late implant removal times (Figure 3 and Table 1). We also detected lymphoplasmacytic and histiocytic inflammatory infiltrates associated with the fibrotic capsule, including epithelioid macrophages and giant multinucleated cells with intracytoplasmic polymer fragments that increased with time (Figure 4 and Table 1). These features revealed phagocytosis of the polymer, which was more evident at late implant removal times (Table 1). Further, the fibrotic capsule of all implants showed angioplasia (Figure 2). The sample removed after 57 weeks of implantation presented hemorrhage, some neutrophils, moderate angioplasia, and an abundant presence of collagen fibers. However, no polymer material or fibrotic capsule were identified, hampering the scoring of the fragmented tissue (Figure 5).



PLA24: 24 weeks following implantation; PLA24F: 24 weeks following implantation, formalin-fixed; PLA28: 28 weeks following implantation PLA34: 34 weeks following implantation; PLA38: 38 weeks following implantation; PLA57: 57 weeks following implantation. Ns: not scored.

**Figure 2.** Photomicrographs of implant polymer sites with the polymer (p) and fibrotic capsular formation (c) involving the material. (**A**) Fibrotic capsular formation at 24 weeks; (**B**) 28 weeks; (**C**) 34 weeks; and (**D**) 38 weeks. Hematoxylin and eosin stain, 100×. (A) Inlet: fibrotic capsular formation at 24 weeks with small vases proliferation (arrows), characterizing angioplasia. Hematoxylin and eosin stain, 400×.

**Figure 3.** Photomicrographs of implant polymer sites with the capsular formation (c), polymer (p) and cellular growth with fragmentation of the polymer (f). (**A**) Cellular growth and polymer fragments at 24 weeks; (**B**) 28 weeks; (**C**) 34 weeks; and (**D**) 38 weeks. Hematoxylin and eosin stain; 40× (**A**) and 100× (**B**–**D**).

**Figure 4.** Photomicrographs of implant polymer sites with the polymer (p), lymphoplasmacytic inflammatory infiltrate (arrowhead), and epithelioid macrophages and multinucleated giant cells, with intracytoplasmic polymer material, thus characterizing polymer phagocytosis (arrows). (**A**) Inflammatory infiltrate and polymer phagocytosis at 24 weeks; (**B**) 28 weeks; (**C**) 34 weeks; and (**D**) 38 weeks. Hematoxylin and eosin stain; 100×. Inlet (**A**,**D**): multinucleated giant cells with intracytoplasmic fragments of polymer material, characterizing phagocytosis (arrows). Inlet (**B**,**C**): lymphoplasmacytic inflammatory infiltrate (arrowhead). Inlet (**C**): epithelioid macrophages (arrow) delimiting a polymer fragment (f). Hematoxylin and eosin stain; 400×.

**Figure 5.** Photomicrographs of implant polymer site removed 57 weeks after implantation. No polymer was identified at this time (**A**) Site of implantation with abundant disorganized collagen fibers, no integral or fragmented polymer material or fibrotic capsule and intense diffuse hemorrhage. (**B**) Site of implantation evidencing the intense diffuse hemorrhage, characterized by many erythrocytes out of vessels. Hematoxylin and eosin; 100× (**A**) and 400× (**B**).

## *2.5. Scanning Electron Microscopy (SEM)*

Figure 6 reveals SEM micrographs of PLA surfaces implanted and exposed to biodegradation for 24 (Figure 6a), 28 (Figure 6b), 34 (Figure 6c), and 38 weeks (Figure 6d) and their respective pore size distribution histograms. The distribution of pore diameters was not normal. The median pore size is shown in Table 2. The data revealed surface irregularity

on the surface, porous morphological appearance, and the presence of cracks at all times, indicating a process of biodegradation on the material. Moreover, there was an increase in pore diameter at all times compared to the first moment (24 weeks) (Kruskal-Wallis, *H* = 423.113; *p* ≤ 0.001) (Table 2 and Figure 7).

**Figure 6.** SEM micrographs and pore size distribution histograms of the surface of PLA implanted subcutaneously in one horse. (**A**) Twenty-fourweeks following implantation; (**B**) 28 weeks following implantation; (**C**) 34 weeks following implantation; and (**D**) 38 weeks following implantation.


**Table 2.** Pore median diameter of PLA implanted subcutaneously in one horse and removed at different times.

PLA24: 24 weeks following implantation; PLA28: 28 weeks following implantation; PLA34: 34 weeks following implantation; PLA38: 38 weeks following implantation. \* Indicates larger pore diameter. IQR, interquartile range.

**Figure 7.** Medians and amplitude of pore size of PLA implanted subcutaneously in one horse. \* Indicates increase in pore size when compared to pore size at 24 weeks following implantation (Kruskal-Wallis, *H* = 423.113; *p* < 0.001).

Furthermore, over time, the polymer underwent degradation and changes in its morphology up to 57 weeks. Afterward it was no longer possible to detect the polymer within the skin fragment, thus impeding the removal of polymer fragments for analysis without the skin (Figure 8). We must recognize a limitation of our study. Owning to logistic restrictions, we have not been able to produce aa SEM image from a non-implanted PLA sample. However, this limitation does not compromise the study findings and more information on this non-implanted sample can be found in Carvalho et al., 2020 [22].

**Figure 8.** SEM micrographs of skin fragments with PLA implanted in one horse (**A**) 34 weeks following implantation; (**B**) 38 weeks following implantation; and (**C**) 57 weeks following implantation. Dotted red lines delimits the area of the implants.
