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), being previously proven to be compatible with alginate capsules (20).
Recommended therapeutic cell concentration fluctuates around 10 to 30 million (26). However, free MSC injection can lead to cell dispersion in 7 days (17, 27–29). In order 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, bringing originality to the present study.
The maintenance of physical parameters after MSCs 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, absence of lameness has also been described after MSC injection (37).
Intraarticular injection of autologous, allogeneic or xenogeneic bone marrow MSCs can alone increase TNCC in horses (32, 37, 38). In our study, besides MSCs, the alginate and the experimental lesion contributed to the inflammation process. Although alginate itself is inert, the calcium used in the crosslinking process can exert an immunogenic effect (39). Another study using alginate capsules injected in the peritoneal cavity of mice also demonstrated a significant increase of inflammatory cells after 48, 96 and 168h (40). It is important to point out that the absence of a group with alginate alone (without MSCs), in order 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 throughout the time, so that at the end of the study the GB revealed the lowest cell count, which leads to the inference that MSCs exerted an immunomodulatory paracrine effect despite the initial inflammation. This effect is achieved by the release of cytokines and growth factors (41–43), added to the porous structure of alginate that allows oxygen, metabolite and nutrient diffusion (44), stimulating cell proliferation and survival (45). Similar immunomodulatory effect was observed in a previous study that used MSCs in alginate hydrogel to treat neuroinflammation, reinforcing that alginate can not only act as a delivery scaffold, but also enhance MSCs therapeutic effect (46).
Although MSC injection can have contributed 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 presented inflammation initially. While some studies show a decrease in neutrophil count after 7 to 9 days (33, 47), it remained high for 14 days in our study, corroborating other previous data (48).
Lymphocytes are cells from the adaptive immune system, attracted chemotactically by several cytokines (49). The increase in lymphocyte count at 168h only in the control group may have occurred due to the ability of MSCs to reduce lymphocyte activation (12, 50).
Macrophages have different subtypes with different functions. They can either polarize into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype, depending on the environment (51, 52). M2 macrophages release chondrogenic factors, including IL-10, IL-1Ra and TGF β (51, 52). Although specific labelling 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 GB indicate a tendency towards M2 polarization, since IL-10 is required for macrophage polarization (53). Interestingly, the control group also presented a correlation between MC and IL-10, which can indicate anti-inflammatory response against the inflammation. However, further analyses of cartilage scores revealed bad cartilage aspect and histologic architecture, highlighting the important role of MSCs in interacting, organizing and orchestrating the reparative process. The final result found in the control group substantiates the inference that most of synovial macrophages did not polarize into M2, given that a better outcome would be expected in a pro-resolutive (M2) scenario.
Increase in total protein (TP) up to 5g/dL has been demonstrated after allogeneic and xenogeneic MSC injections indicating inflammation (32), similar to the 24h time point in GB. The positive correlation between TP and macrophage count in the GB, with concomitant decrease of both variables, reinforces the hypothesis that MSCs acted modulating the inflammatory process.
Interferon 𝛾 increased at 24h in the GB, and at 24, 168 and 336 h in GA. Since IFN 𝛾 is related to M1 polarization of macrophages and increase in neutrophil and monocyte activity (54), the increase of its levels reflects the initial inflammatory process, where most macrophages generally adopt a M1 polarization (51, 52). Even with a strong positive correlation between IFN 𝛾 and TNF-α, a better outcome was noticed in both treated groups, which indicates that despite the pro-inflammatory commitment of these cytokines, the initial inflammatory process elicited a MSC anti-inflammatory response, corroborating previous findings (55).
The role of IL-6 in horses is not completely understood. It can be released in LPS-induced arthritis (56), acting as a pro-inflammatory cytokine through the delay in lymphocyte and neutrophil apoptosis and decrease in T-cell stimulation (56, 57). However, immunoregulatory properties have been also attributed to IL-6, demonstrating a dual role of this cytokine (58). Since the peak of IL-6 occurred at 96h in the GB, alongside with a decrease in neutrophil and total nucleated cell count and an increase in IL-10, we infer that IL-6 exerted an immunoregulatory effect in this case. The absence of simultaneous increase in IL-6, IL-1 and TNFα corroborates this affirmation, since in a pro-inflammatory scenario IL-6 is involved in chondral matrix degradation alongside IL-1 and TNFα (59–61).
In contrast, IL-10 was increased in the encapsulated GB. When properly stimulated, MSCs can release IL-10 and other anti-inflammatory molecules like IL-1ra, indoleamine 2,3-dioxygenase, TGF-β and PGE2 (10, 29, 30). Thus, it is possible that encapsulated MSCs released IL-10 in response to the initial inflammatory process.
The presence of a well-attached, white and firm cartilage-like tissue has been previously reported after PRP injection in both experimental and natural chondral lesions in horses (62–64). When PRP was associated with MSCs, a superiority in collagen type II deposition, macroscopic and histological appearance was reported (65). Better macro and microscopic aspects observed in the GB, with higher glycosaminoglycan deposition and better O’Driscoll scores, makes a great contrast with the GA and GC, which presented prevalence of fibrous tissue.
Taken together, data of this study indicate a better outcome in cartilage condition after injecting MSCs encapsulated in alginate, but the exact mechanisms that make this combination superior remain not completely elucidated. Besides providing a scaffold for MSCs tridimensional organization, alginate encapsulation is a cell delivery mechanism capable of improving cell therapeutic effects due to the maintenance of MSCs for a longer time at the site of injection (66–69), which can facilitate cell-to-cell interaction and consequent secretion of specific cytokines (70) and cell stimulation (29, 42, 71–73) that orientate the articular environment towards a pro-resolutive scenario. It is important to point out that the large synovial cavity allowed the injection of a high number of capsules. However, each case needs to be evaluated individually, since the number of capsules to be injected is directly related to the cell concentration.
A longer follow-up of the animals would have provided considerable data regarding collagen type II deposition, morphological and histological scores. Since each MSC source has its own particularities that may lead to different behaviors even facing the same conditions, the comparison of synovial membrane to other MSC sources would also contribute to the better understanding of the events associated with chondral healing in horses.