**3. Discussion**

We previously studied the changes related to the safe biodegradation of the PLA polymer implanted subcutaneously in horses for 24 weeks [22]. Here we evaluated the changes incurred in a horse over 57 weeks.

We found that PLA implantation did not induce systemic inflammatory responses in the horse studied. HR values increased up to 144 h after implantation. This may have occurred because there was more frequent handling of the animal in this evaluation period, which probably triggered a mild anticipatory sympathetic response to general management. Indeed, HR decreased from the seventh day of implantation, remaining within normal values for equine species. Likewise, the concentrations of plasma fibrinogen, an acutephase protein commonly used to diagnose and monitor various inflammatory conditions in equine medicine [25], revealed that the injury caused by the skin incisions and the implantation of the polymers was not able to elicit a systemic inflammatory response.

On the other hand, MNT evaluation revealed the presence of a local inflammatory response, characterized by a decrease in nociception. Von Frey Filaments, used to assess the skin MNT, measures cutaneous hyperalgesia and allodynia, and usefully mimics clinical conditions that present increased cutaneous sensitivity [26]. In a study that evaluated the preemptive analgesic effect of epidural ketamine before performing skin incisions with sutures in horses, VFF were also able to quantify skin sensitivity [27].

Histopathological analysis revealed the formation of a fibrotic capsule delimiting the biomaterial from the initial stage of the inflammatory response, remaining present until 38 weeks post-implantation. Encapsulation of biomaterials occurs due to a chronic foreign body reaction [28]. In comparison with PLA24, the PLA38 presented a fibrotic capsule with multiple layers of fibroblasts and essential changes. Among these changes, we detected severe cell growth of fibrous tissue and polymer fragmentation, marked lymphohistioplasmocytic inflammation, marked phagocytosis of the material, and moderate angioplasia, which are characteristic alterations of a chronic inflammatory response [29].

Inflammation around the material, characterized by the presence mainly of lymphocytes and macrophages, accompanied the cell growth of the fibrous tissue amidst the biopolymers, which represented the local chronic inflammation and organization of this tissue. The presence of macrophages in the capsule and at the material's interface was observed in other studies, being related to phagocytosis and implant clearance [8,30]. Interestingly, accentuated phagocytosis was observed in both PLA24 and PLA38 samples. The intense phagocytosis observed in PLA38 was possibly related to an intense fragmentation and biodegradation of the biomaterial, which incited macrophages to phagocytose the resulting fragments for tissue cleaning. Fragmentation of the material leads to cell growth, and the resulting capsule insulated the material while some fibrous tissue infiltrated the polymer, thus promoting further fragmentation.

The proliferation of fibrous tissue around polymer fragments may be associated with the presence of pores and cracks in the materials, as observed by SEM. These pores promote cell growth as they provide a greater surface area [31]. Fifty-seven weeks after implantation, the polymer was no longer detected within the skin fragment. The assessment interval between 38 and 57 weeks is significant and relatively large, and it is not possible to determine what occurred in this period. However, a progression on polymer fragmentation associated with tissue clearance possibly happened to the point when the material was no longer detected. Thus, the fibrotic capsule was replaced by fibrovascular tissue rich in organized and well-differentiated collagen, similar to the collagen observed deep in the dermis. We also speculate that the intense phagocytosis observed in one of the polymers at 24 weeks following implantation is probably related to an initial foreign body response.

In addition to macrophages and multinucleated giant cells, lymphocytes were often present in the infiltrates around the implants. These findings are probably associated with a chronic inflammatory response at the injury site, which is usually characterized by the presence of macrophages, multinucleated giant cells, monocytes, and lymphocytes, in addition to fibrosis and angioplasia [32]. Furthermore, angioplasia was observed at all evaluated times, with mild intensity in PLA24 and moderate intensity in the other materials. The process of well-differentiated vascular proliferation is essential for tissue nutrition, maintenance of cell proliferation, and migration of inflammatory cells [8].

SEM data, obtained from the sample removed 24 weeks following implantation, revealed signs of biodegradation of the PLA. However, few pores were present with an median diameter of 0.477 μm. As post-implantation time progressed, we observed a gradual increase in the number of surface pores and a large variation in pore size. In vivo biodegradation of PLA depends on the intrinsic characteristics of the material, such as the D-PLA content, crystallinity, and molar mass and the recipient's conditions such as temperature, pH, and presence of cellular infiltrates [33]. The PLA used herein has a low content of D-PLA, which hampers its biodegradation. Indeed, chains with a predominance of L-PLA can organize into crystalline structures that are less susceptible to the permeation of water and extracellular enzyme infiltrates [34]. The biodegradation time established for the low content D-PLA implant used herein can be considered high when compared to that reported by Tschakaloff et al. (1994) [35], which showed that 70% of the mass of a L,D-PLA implant in rabbits biodegraded within 14 days.

Another important factor is the size of the PLA chains since only small molecules can enter cells and be used for cell metabolism. The average molar mass of the PLA used in this study is high (around 80,000 g.mol−1). The main in vivo biodegradation mechanism of PLA is the hydrolysis of ester groups, which leads to a slow reduction in chain size by chain scission to small oligomers or unitary lactic acid molecules. Once reduced to small lactic acid oligomers, they can be phagocytosed and take part in cellular metabolism [33].

The distribution of the histograms revealed that the biodegradation pattern of the polymer occurs with the emergence of small pores on the material's surface promoted by the hydrolysis and breakage of polymer chains by extracellular enzymes [33]. Small superficial pores allow the infiltration of water and extracellular enzymes into the polymeric bulk, thus promoting the appearance of more small pores inside the material. Eventually, the chains between the small pores are consumed, and the pores become larger. For this reason, the median pore size was very similar between samples with different post-implantation times, but the analysis of the micrographs revealed a higher number of pores as the post-implantation time progressed until the polymer was completely biodegraded.

The present in vivo study evaluating the long-term biocompatibility and biodegradability of PLA implants in a horse is innovative and sugges<sup>t</sup> that this biopolymer can be safely used in equine medicine. However, PLA has low flexibility and resistance, high apolarity, and low degradation rate, which are limiting properties for its use in human and veterinary medicine. Several approaches could be employed to overcome these limitations or to adjust the physical properties of the currently available PLA, such as the production of polymeric blends. Interestingly, although the PLA used herein was not of medical grade, it proved to be biocompatible and provided encouraging results that could pave the way for new low-cost biomaterials. In any case, future studies addressing the effectiveness of

PLA for further uses, such as drug development, tissue engineering, equine surgery, and general medicine are warranted.

## **4. Materials and Methods**

## *4.1. Ethics Statement*

The study followed the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation and was approved by the Ethics Committee on Animal Use of the CEUA–FCAV/Universidade Estadual Paulista (UNESP) under protocol n◦ 006548/17.
