Several strategies have been developed to avoid chondral loss and OA development. Stem cells are an important treatment option due to their immunomodulatory properties (23, 24). Synovial membrane MSCs have demonstrated better chondrogenic differentiation (8, 25) and were previously proven to be compatible with alginate capsules (20).
The recommended therapeutic cell concentration fluctuates at approximately 10 to 30 million (26). However, free MSC injection can lead to cell dispersion in 7 days (17, 27–29). To reduce cell dispersion, previous studies used activated PRP as a cell scaffold, achieving significant results (30). Other studies using MSCs in scaffolds also demonstrated good therapeutic and differentiation potential in bone defects (31). However, to the authors’ knowledge, the use of MSCs encapsulated in sodium alginate to treat chondral lesions in horses has not been previously described.
The maintenance of physical parameters after MSC injection reinforces the biosafety of this treatment and corroborates previous studies (32, 33). Transitory lameness was described by some authors after MSC injection (32, 34). Similar findings were reported in LPS-induced synovitis treated with MSCs (35, 36). In contrast, the absence of lameness has also been described after MSC injection (37).
Intraarticular injection of autologous, allogeneic or xenogeneic bone marrow MSCs alone can increase TNCC in horses (32, 37, 38). In our study, in addition to MSCs, alginate and the experimental lesion could have contributed to the inflammation process. Although alginate itself is inert, the calcium used in the crosslinking process can exert an immunogenic effect (39). Additionally, alginate capsules injected in the peritoneal cavity of mice caused a significant increase in inflammatory cells after 48, 96 and 168 h (40). In this sense, the absence of a group with alginate alone (without MSCs) to evaluate the isolated effect of alginate capsules is a limiting factor of this study.
The insertion of MSCs in an alginate scaffold could have reduced their effect initially, leading to a higher inflammatory process. However, the inflammatory process decreased over time, such that at the end of the study, the encapsulated SMMSC group revealed the lowest cell count, which can indicate an immunomodulatory paracrine effect of MSCs despite the initial inflammation. This effect is achieved by the release of cytokines and growth factors (41–43) that can be added to the porous structure of alginate, allowing oxygen, metabolite and nutrient diffusion (44) and stimulating cell proliferation and survival (45). A similar immunomodulatory effect was observed in a previous study that used MSCs in alginate hydrogels to treat neuroinflammation, demonstrating that alginate can not only act as a delivery scaffold but also enhance the therapeutic effect of MSCs (46).
Although MSC injection can contribute to the increase in neutrophil count (47), we state that the experimental lesion was the main cause of the initial inflammatory process as all groups, including the control group, initially presented inflammation. While some studies showed a decrease in neutrophil count after 7 to 9 days (33, 48), it remained high for 14 days in our study, corroborating other previous data (49).
Lymphocytes are cells from the adaptive immune system that are attracted chemotactically by several cytokines (50). The increase in lymphocyte count (LC) at 168 h only in the control group might occurred due to the ability of MSCs to reduce lymphocyte activation (12, 51).
Macrophages have different subtypes with different functions. They can polarize into either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype depending on the environment (52, 53). M2 macrophages release chondrogenic factors, including IL-10, IL-1Ra and TGF β (52, 53). Although specific labeling for M1 and M2 was not performed, the positive correlation between macrophage count (MC) and IL-10 and the statistically higher IL-10 in the encapsulated SMMSC group indicate a tendency toward M2 polarization, as IL-10 is required for macrophage M2 polarization (54). Interestingly, the PBS Control group also presented a correlation between MCs and IL-10, which indicates an anti-inflammatory response against inflammation. However, as further analyses of cartilage scores revealed bad cartilage aspects and histologic architecture, the role of MSCs in interacting, organizing and orchestrating the reparative process was corroborated. The final result found in the PBS control group indicates that most of the synovial macrophages did not polarize into M2, as a better outcome would be expected in a pro-resolutive (M2) scenario.
An increase in total protein (TP) up to 5 g/dL has been demonstrated after allogeneic and xenogeneic MSC injections, indicating inflammation (32), similar to the 24 h time point in the encapsulated SMMSC group. The positive correlation between TP and macrophage count in the encapsulated SMMSC group, with a simultaneous decrease in both variables, suggests that MSCs modulated the inflammatory process.
Interferon 𝛾 increased only at 24 h in the encapsulated SMMSC group and at 24, 168 and 336 h in the free SMMSC group. As IFN 𝛾 is related to M1 polarization of macrophages and an increase in neutrophil and monocyte activity (55), the increase in its levels reflects the initial inflammatory process, where most macrophages generally adopt M1 polarization (52, 53). Even in the presence of a strong positive correlation between IFN 𝛾 and TNF-α, a better outcome was noticed in both treated groups, which shows that despite the proinflammatory commitment of these cytokines, the initial inflammatory process elicited an MSC anti-inflammatory response, corroborating previous findings (56).
The role of IL-6 in horses is not completely understood. It can be released in LPS-induced arthritis (57), acting as a proinflammatory cytokine through the delay in lymphocyte and neutrophil apoptosis and decrease in T-cell stimulation (58, 59). However, immunoregulatory properties have also been attributed to IL-6, demonstrating a dual role of this cytokine (60). As the peak of IL-6 occurred at 96 h in the encapsulated SMMSC group along with a decrease in neutrophil and total nucleated cell counts and an increase in IL-10, we assumed that IL-6 exerted an immunoregulatory effect in this case. The absence of simultaneous increases in IL-6, IL-1 and TNFα corroborates this affirmation because in a pro-inflammatory scenario, IL-6 is involved in chondral matrix degradation along with IL-1 and TNFα (61–63).
In contrast, IL-10 was increased in the encapsulated SMMSC group. When properly stimulated, MSCs can release IL-10 and other anti-inflammatory molecules, such as IL-1ra, indoleamine 2,3-dioxygenase (IDO), TGF-β and PGE2 (10, 29, 30). Thus, the release of IL-10 can have occurred in response to the initial inflammatory process.
The presence of well-attached, white and firm cartilage-like tissue has been previously reported after PRP injection in both experimental and natural chondral lesions in horses (64–66). When PRP was associated with MSCs, superior type II collagen deposition and macroscopic and histological appearance were reported (67). Better macro- and microscopic aspects were observed in the encapsulated SMMSC group, with higher glycosaminoglycan deposition and better O’Driscoll scores, showing great contrast with the free SMMSC and PBS control groups, which revealed the prevalence of fibrous tissue.
Taken together, the data of this study indicate a better outcome in cartilage condition after injecting MSCs encapsulated in alginate. In addition to providing a scaffold for MSCs tridimensional organization, alginate encapsulation is a cell delivery mechanism capable of improving therapeutic cellular effects due to the maintenance of MSCs for a longer time at the site of injection (68–71), which can facilitate cell-to-cell interaction and consequent secretion of specific cytokines (72) and cell stimulation (29, 42, 73–75) that direct the articular environment toward a pro-resolutive scenario. It is important to note that the large synovial cavity allowed the injection of a high number of capsules. However, each case needs to be evaluated individually because the number of capsules to be injected is directly related to the cell concentration. A longer follow-up period for the animals would have provided considerable data regarding type II collagen deposition and morphological and histological scores. As each MSC source has its own particularities that may lead to different behaviors, even when facing the same conditions, the comparison of the synovial membrane to other MSC sources would also contribute to a better understanding of the events associated with chondral healing in horses.