4.5.1. Clinical Evaluation

Surgical wounds were evaluated regarding the integrity of the sutures and the presence of secretion. Physical examination quantified the heart rate (HR), respiratory rate (RR), rectal temperature (RT) as well as the intestinal motility, hydration status, apparent mucous color, and capillary filling time. Physical examination was performed before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation and then weekly up to 8 weeks, fortnightly up to 38 weeks, and 57 weeks. The same evaluator obtained all the measurements.

## 4.5.2. Plasma Fibrinogen (PF)

To determine PF concentration, the chronometric technique described by Clauss (1957) [37] was used. Blood samples (3.6 mL) were collected in tubes containing 3.2% sodium citrate. Plasma was separated and frozen at −80 ◦C. After thawing, 200 μL aliquots of the samples were diluted in a buffer solution containing sodium barbital at a 1:10 ratio. Subsequently, 100 μL of thrombin (Fibrinogênio hemostasis, Labtest diagnóstica, Brazil) was added. Clot formation time was determined at 37 ◦C using a coagulometer (COAG 1000; Wama Diagnóstica, Brazil), which automatically converted the time obtained into fibrinogen concentration (mg/dL−1). PF was determined before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation and again at 2 and 3 weeks.

## 4.5.3. Mechanical Nociceptive Threshold (MNT)

A commercial set of Von Frey Filaments (VFF) were used to assess the skin MNT (Touch-TestTM Sensory Evaluators, Stoelting Company, Wood Dale, IL, USA). Six filaments of sizes 5.07 to 6.65 were used, which represent applied forces ranging from 11.8 to 446.7 g, respectively. The filaments were applied perpendicularly to the horse's skin until the nylon thread started to bend. Four applications, separated approximately 1 cm from each other, were performed around the implantation site, at intervals of 3 s. Initially, the thinnest filament was used and, when no aversive response was observed, the next filament was used until the animal showed an aversive reaction or the largest filament was used. An aversive reaction was defined as a movement of the tail, ears, or head, kicking, or stepping to the side. Simple motion reflexes upon first touching the filament on the skin were not accepted as an aversion response, and in these cases, the test was repeated after 10 s. Evaluations were performed before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation, then weekly until 8 weeks, and fortnightly until 38 weeks. The same operator performed all measurements, and these were made with the

horse in a quadrupedal position in an area without movement restrictions. The values obtained were converted into force (g) according to the table provided by the manufacturer.

## 4.5.4. Collection of Biopsy Specimens

The implants were removed along with surrounding tissue fragments. Two implants were removed after 24 weeks of implantation (PLA24 and PLA24F), one implant was removed at 28 weeks (PLA28), one implant at 34 weeks (PLA34), one at 38 weeks (PLA38), and the final implant was removed at 57 weeks (PLA57). As in the implantation procedure, the animal was properly sedated, and local infiltrative anesthesia was performed before the surgical removal of the fragment. One of the fragments removed after 24 weeks of implantation (PLA24F) was fixed in 10% buffered formalin for 24 h. The other implants and surrounding tissue fragments were fixed in 3.5% glutaraldehyde solution with PBS (pH 7.2) for 24–48 h and subsequently subjected to three washes using 5% glucose in 0.1 m sodium cacodylate buffer solution for 5 min. As we did not know whether fixation in glutaraldehyde would interfere with the histological evaluation and because this horse was used in the pilot study by Carvalho et al., 2020 [22], at 24 weeks two polymers were removed, one of which was fixed in formalin, and the other one in glutaraldehyde. After the slides were evaluated by an experienced pathologist, who confirmed that fixation in glutaraldehyde did not interfere with the histological quality, we proceeded to prepare the samples fixed in glutaraldehyde for SEM analysis. All materials were cut into two fragments, one for histopathological analysis and one for scanning electron microscopy (SEM), except for the PLA24F material, which was submitted to histopathological analysis only. Materials were stored in 70% alcohol until further use.
