Encapsulated Allogeneic Synovial Membrane Mesenchymal Stem Cells Provide Better Outcomes of Chondral Lesions in Horses

Vitor Hugo Santos FMVZ UNESP Botucatu https://orcid.org/0000-0001-5622-9152 João Pedro Hübbe Pfeifer UNESP FMB: Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Medicina Campus de Botucatu Gustavo dos Santos Rosa Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu Fernanda de Castro Stievani Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu Emanuel Vitor Pereira Apolonio Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu Mariana Correa Rossi Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu Leonel Vinicius Constantino UEL: Universidade Estadual de Londrina Thadeu Rodrigues de Melo UEL: Universidade Estadual de Londrina Carlos Eduardo Fonseca-Alves Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu Ana Liz Garcia Alves (  ana.liz@unesp.br ) Universidade Estadual Paulista Julio de Mesquita Filho Campus de Botucatu


Introduction
Osteoarthritis (OA) is the main cause of lameness in horses, leading to poor performance and important economic impacts (1). Even though its etiology is multifactorial, mechanical forces are the major causes of chondral lesions, generating chondrocyte injury and the release of proteases which result in chondral brillation (2,3). Cell-based therapies and tissue engineering can help to promote better chondral healing, reducing articular damage and pain (4).

Strategies combining mesenchymal stem cells (MSCs), biocompatible scaffolds and bioactive
components provide mechanical support and a cell source, contributing to joint repair (4,5). Although several cell sources have been used with positive results, synovial membrane-derived mesenchymal stem cells ( SM MSCs) present high proliferation capacity and chondrogenic differentiation (6). They can also be obtained through minimally invasive techniques (6-9). Treatment of OA using MSCs relies on its immunomodulatory and paracrine effects, decreasing lymphocyte activation and releasing several molecules (IL-10, IL 1ra, TGF-β and PGE2) (5, 10, 11) and growth factors (12)(13)(14)(15)(16)(17) involved in the articular repair process. Despite the noticeable bene t tissue engineering have brought to orthopedics, some undesired events remain, especially systemic dispersion (18) and decreased viability after MSC intraarticular injection (19). Thus, encapsulated stem cells can be used in joint injuries for protection and to maintain cell viability and stimulate chondrogenic differentiation (19).
The aim of this study was to evaluate the effect of SM MSCs encapsulated in alginate hydrogel on immunomodulation and healing of induced tibiotarsal lesions of horses.

Methods
Fifteen healthy geldings were used in this study, with ages between 3 and 8 years and a mean weight of 330 kg. The animals were divided into 3 groups of 5 animals each: SM MSCs alone (free SMMSC), encapsulated SM MSCs (encapsulated SMMSC) and the control (PBS control).
Cell culture and encapsulation All SM MSCs were previously characterized and stored in a biobank. Cells were cultured until reaching 1x10 7 SM MSCs in the third passage (P3) according to the technique described by (5,20) and divided into free and encapsulated groups. Cells from the encapsulated SM MSC group were resuspended in 1.5% (w/v) sodium alginate. The mixture was dripped in a gelling solution of 102 mM CaCl 2 using a 10-ml syringe and a 21-G needle in an infusion pump. The drops were maintained within the solution for 10 minutes for the crosslinking reaction and capsule formation. Capsules were washed three times in 0.15 M NaCl before the injection.
Cell viability was assessed by the trypan blue exclusion method in a Neubauer chamber after dissolving alginate hydrogel with sodium citrate 3 .

Arthroscopic procedure and treatments
Tibiotarsal joints were subjected to arthroscopy to induce chondral lesions, and treatment was performed at the moment of the surgical procedure. Thus, arthroscopy was set as the initial moment (0 h (21). In brief, a shaver was used to create lesions 15 mm in diameter ( Figure 1). Ferris-Smith forceps were used to remove the hyaline and calci ed cartilage without reaching subchondral bone (mean depth of 1.5-3.2 mm).
All treatments were performed at the initial time immediately after the induction of chondral lesions. The capsules were injected through the arthroscopic portal using a no. 20 Levine catheter. Free SM MSCs were diluted in 10 ml of PBS and injected. The control group received only 10 ml of PBS.

Clinical and laboratorial evaluations
Clinical parameters (physical examination and lameness score) were assessed before arthrocentesis by two evaluators in a blinded manner. Tibiotarsal arthrocentesis was performed at all time points. The synovial uid analysis included brinogen concentration and cytological evaluation (total nucleated cells and differential counting). Stored synovial uid was used for quanti cation of IL-1, IL-10, IL-6, INF-, TNF ∝ 4 , P substance 5 , serum amyloid A 6 , TGF-β 7 , IGF 8 and PGE2 9 by ELISA following the manufacturer's instructions. Absorbance was read at a wavelength of 450 nm.
A new arthroscopy was performed after 150 days to observe the macroscopic appearance of the chondral surface in situ and to harvest cartilage for histological analysis and immunohistochemistry for type II collagen. The cartilage aspect was graded blindly by 6 evaluators following the International Cartilage Repair Society (ICRS) score, which evaluates cartilage repair, integration of lesion edges, macroscopic appearance and general aspect of the repairing tissue.
Chondral tissue was harvested and cryopreserved using Tissue-Tek® O.C. T TM10 for histological evaluation (hematoxylin-eosin and toluidine blue staining) according to the O'Driscoll histological score. The evaluators were also uninformed of the experimental groups at the time of evaluation. Immunohistochemistry (IHC) was performed according to the technique described by (22) using the peroxidase and 3,3'-diaminobenzidine tetrahydrochloride (DAB) method. Antigen retrieval was performed with citrate buffer (pH 6) in a pressure cooker, 11 and the glass slides were put into the Dako Cytomation Autostainer 12 platform. Anti-type II collagen antibody was detected using a mouse secondary monoclonal antibody 13 at a 1/200 dilution. Immunologic staining was performed using the Histo ne 14 method, and the slides were counterstained with hematoxylin. The positive control was pulmonary tissue, and the negative control was made by not using primary antibody.
All samples were evaluated in bright eld microscopy and graded in a semiquantitative scoring system, where the absence of expression was graded as 0; from 1% to 25% of positive staining was graded as 1; from 26% to 50% of positive staining was graded as 2 (weak); from 51% to 75% of positive staining was graded as 3 (moderate); and a score of 4 was graded when more than 75% cells were stained (strong).

Statistical analysis
Normality tests were performed using the Kolmogorov-Smirnov test. In the absence of normality, the Kruskal-Wallis test was applied for comparisons between groups at the same time points, and the Friedman test was applied for comparisons between time points of each group. Tukey's test was used for comparisons between medians using Sigmastat 3.5 software when there was signi cance.
Correlations between treatments and cytokines are shown in a heatmap (pheatmap package) grouping data hierarchically by Euclidean distance and the Ward method. "R" software was used for Pearson's correlation coe cient.
Data regarding treatment response were analyzed by general linear model ANOVA (GLM) considering the evaluator and the experimental group as constants. Mean values of signi cant results were compared by Tukey's test.

Results
The mean count and viability of SM MSCs were 1.4x10 7  Macrophages increased signi cantly in all groups initially. However, the treated groups presented an earlier decrease illustrated by a signi cant difference between the control and treated groups at 96 h (P=0.025).
The total protein (TP) of both treated groups increased signi cantly at 24 h compared with the initial moment (P<0.05).
Values of interleukin-6 (IL-6) only showed signi cant differences at 168 h (P=0.030). The encapsulated SMMSC group presented higher values compared with the control group but did not differ from the free SM MSC group.
Individually, there was a peak of IL-6 in the free SM MSC group at 48 and 96 h (P=0.003) and only at 96 h in the encapsulated SM MSC group (P=0.004). The PBS control group did not present signi cant differences throughout the time points (P=0.218).
No differences were observed between groups or within the same group in SAA, P substance, PGE2, IL-1 , TNF , IGF 1 or TGF (P>0.05), even though the TGF-levels remained higher in the control group.
Higher levels of IL-10 at 24 h, 48 h and 96 h were found in the encapsulated SM MSC group compared with the initial moment (P=0.012), but not at 336 h.
The hierarchical grouping divided treatments into two groups: the control group and the treated group (which included both free and encapsulated cells). The variables were divided into four groups: 1: SAA and IGF; 2: TNCC, TP, IFN, TGF, TNF, IL-10, NC and IL-6; 3: MC, LC and IL-1; and 4: SP and PGE2. Encapsulated MSCs were related to higher SAA, IGF, TNCC, TP, IFN-, TGF , TNF , IL-10, neutrophil and IL-6 levels, whereas free MSCs led to higher IL-1, macrophage and lymphocyte counts, and the control group was associated with higher SP and PGE2 levels ( Figure 5).
The encapsulated SM MSC group presented a better macroscopic appearance similar to naive cartilage.
Conversely, chondral erosions and peripheral detachments were observed in the free SM MSC group.
However, no signs of synovitis, brillation or erosion were observed in either group (Figure 7). The PBS control group presented brosis with un lled, friable and eroded areas.
The Global Repair Evaluation (GRE) showed higher scores in the encapsulated SM MSC group than in the PBS control group (P=0.007). The results of all groups are presented in Table 1. Table 1 ANOVA for comparing the scores of the groups at T150 in the variable "Global Repair Evaluation" (GRE) (* there was a statistical difference between the groups). The histological analysis (HE and toluidine blue) revealed different degrees of brocartilage deposition.

Group
Overall, while the control group presented only brosis and brocartilage with no chondrocytes or extracellular glycosaminoglycan matrix, the encapsulated SM MSC and free SM MSC groups led to better tissue organization, visible chondrocytes and extracellular matrix deposition (Figure 8).
Immunohistochemistry for type II collagen did not reveal signi cant differences, even though encapsulated SM MSC and free SM MSC groups led to higher labeling scores (4 and 3, respectively) compared with the PBS control group (score of 2) (Figure 8).
O´Driscoll histological scoring of revealed signi cant differences between groups in structural integrity (P=0.028), chondrocyte grouping (P=0.013) and lateral integration of the tissue (P=0.031) ( Table 2).  (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 ndings 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 in ammation 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 signi cant increase in in ammatory 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 in ammatory process. However, the in ammatory 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 in ammation. This effect is achieved by the release of cytokines and growth factors (41)(42)(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 neuroin ammation, 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 in ammatory process as all groups, including the control group, initially presented in ammation. 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 proin ammatory (M1) or anti-in ammatory (M2) phenotype depending on the environment (52,53). M2 macrophages release chondrogenic factors, including IL-10, IL-1Ra and TGF β (52,53). Although speci c 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-in ammatory response against in ammation. 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 nal 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 proresolutive (M2) scenario.
An increase in total protein (TP) up to 5 g/dL has been demonstrated after allogeneic and xenogeneic MSC injections, indicating in ammation (32), similar to the 24 h time point in the encapsulated SM MSC 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 in ammatory process.
Interferon increased only at 24 h in the encapsulated SM MSC group and at 24, 168 and 336 h in the free SMMSC group. As IFN is related to M1 polarization of macrophages and an increase in neutrophil and monocyte activity (55), the increase in its levels re ects the initial in ammatory process, where most macrophages generally adopt M1 polarization (52,53). Even in the presence of a strong positive correlation between IFN and TNF-α, a better outcome was noticed in both treated groups, which shows that despite the proin ammatory commitment of these cytokines, the initial in ammatory process elicited an MSC anti-in ammatory response, corroborating previous ndings (56).
The role of IL-6 in horses is not completely understood. It can be released in LPS-induced arthritis (57), acting as a proin ammatory 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 SM MSC 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 a rmation because in a proin ammatory 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 SM MSC group. When properly stimulated, MSCs can release IL-10 and other anti-in ammatory 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 in ammatory process.
The presence of well-attached, white and rm 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 SM MSC group, with higher glycosaminoglycan deposition and better O'Driscoll scores, showing great contrast with the free SM MSC and PBS control groups, which revealed the prevalence of brous 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-tocell interaction and consequent secretion of speci c cytokines (72) and cell stimulation (29,42,(73)(74)(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.

Conclusion
Alginate capsules with MSCs elicited a marked initial in ammatory process. However, in ammation was modulated over time, and encapsulated MSCs produced better outcomes in terms of chondral aspect and composition. Some grade of in ammation is necessary and even desirable, which can be bene cial to the MSC response, releasing anti-in ammatory cytokines that guide the reparative process toward resolution. In addition to safety in application, the positive results observed after administration of encapsulated    Total nucleated cell, neutrophil, lymphocyte and macrophage counts (cells/µL) and total protein (g/dL) of synovial uid of the three groups. * indicates P<0.05.