In the present study, macroscopic, histological, and molecular analyses were performed in vivo using a rabbit experimental OA model to assess the therapeutic effect of SVF and ADSCs on OA. Macroscopic and histological analysis revealed that the SVF group was superior to the lADSC group in inhibiting OA progression and synovitis and was almost equivalent to the hADSC group. In addition, the immunohistochemical staining and expression of collagen II and SOX9 was the highest in the hADSC and SVF groups, followed by the lADSC group, and was the lowest in the control group at 8 and 12 weeks post-OA onset. Sox9, a chondrogenic transcription factor, Sox-9, plays a crucial role in increasing chondrogenesis, particularly by activating the coexpression of collagen II [40, 41]. Taken together, the findings of the previous and the present studies suggest that intra-articularly injected SVF and ADSCs promote the secretion of anabolic factors in chondrocytes. In contrast, the immunohistochemical staining and mRNA expression of MMP-13 was low in the lADSC-, hADSC-, SVF and -injected knees compared to 4% RSA-injected knees; the suppression of its expression was higher in the SVF group than those in control and lADSC groups but was comparable to that of hADSC group. MMP-13 has been reported to be involved in cartilage damage in OA [42] and is used as an indicator of OA treatment efficacy in OA models in vivo [43]. In summary, in vivo, the SVF treatment showed significantly increased anabolic and reduced catabolic effects on the cartilage compared to the control and lADSC treatments; however, these effects of SVF treatment were comparable to those of hADSC.
Next, we explored the involvement of macrophage in the synovium in the in vivo experimental OA model to understand the underlying mechanism of the OA-alleviating effects of SVF and ADSCs on chondrocytes. Recent studies suggest that macrophages in the synovium play a key role in OA pathogenesis [20, 21, 44]. In vivo, M1 macrophages in the synovium aggravate experimental OA, while M2 macrophages reduce its progression[20, 45]. In particular, an increased M1/M2 ratio is associated with increased expression of inflammatory cytokines and has been shown to induce OA [46]. In this study, lADSCs, hADSCs and SVF treatments significantly ameliorated synovitis in the synovium and reduced the M1/M2 ratio at 8 weeks after OA onset compared to control treatment. Previous studies reported that ADSCs had an anti-inflammatory effect due to the M2 macrophage polarization [47, 48]. The translocation of intra-articularly administered SVF to the synovium was reported to increase the number of M2-like macrophages in the synovium in an OA mouse model. The co-culture of macrophages with SVF significantly increased the percentage of M2 macrophage-positive markers [49]. In the present study, the synovitis, M1/M2 macrophage ratio, and M2 macrophage polarization were significantly improved in the SVF group compared to those in the control and lADSC groups. Taken together, we inferred that immunomodulation via macrophage polarization to the synovium induced by intra-articular injections of SVF and ADSCs contributes to the attenuation of OA in rabbits. In addition, the present study also demonstrated that at 8 and 12 weeks post-OA onset, expression of TGF-β and IL-10, which are expected to be secreted by M2 macrophages, was significantly increased, while that of MMP-13, a catabolic factor, was significantly suppressed in the synovial cells of lADSC, hADSC and SVF groups compared to those in the synovial cells of the control group. These results suggest that the OA-alleviating effect of SVF is also attributed to the paracrine effect of M2 macrophages, which may improve synovial membrane and knee joint homeostasis and cartilage protection.
Furthermore, we performed in vitro experiments to confirm the findings of abovementioned in vivo experiments. Previous studies reporting the paracrine effects of SVF and ADSCs demonstrated an increase in chondrogenesis in the co-culture of chondrocytes with SVF and ADSCs [17, 50], confirming that SVF and ADSCs can promote the release of collagen II and SOX-9 expression in chondrocytes [16]. In the present study, the separated pellet co-culture showed a significantly larger pellet size in the SVF group than those in the control and lADSC groups. In addition, the levels of TGF-β and IL-10, the paracrine factors in ADSCs [51, 52], were higher in the SVF group than those in the lADSC group, as detected via ELISA using the culture medium at the first exchange. These results indicate that SVF paracrine factors may be secreted not only by ADSCs but also by other heterogeneous cells in SVF. Immunohistochemical staining also confirmed the anti-inflammatory phenotype of most macrophages in SVF by double staining with F4/80 and CD163. However, cells were not stained with F4/80 in the ADSC group, which could be because ADSCs are pure stem cells. Various authors have reported that macrophages comprise approximately 20% of the SVF [28]. Approximately 80% of these macrophages are also positive for F4/80, a pan-macrophage cell surface marker, and approximately 70% for the M2 anti-inflammatory marker [29, 30]. These results of the previous studies are similar to those observed in our study. M2 macrophages secrete TGF-β and IL-10, induce collagen II expression in chondrocytes, and decrease MMP-13 expression [53]. Additionally, TGF-β has been recently reported to contribute to chondrocyte regeneration partially through the Smad2/3 signaling pathway [54]. Macrophages that have acquired the M2 phenotype upregulate the expression of anti-inflammatory cytokines, including IL-10 and TGF-β, when co-cultured with MSCs and ADSCs [55]. In summary, a comparison of SVF and lADSCs suggests that the effect of SVF may be due to its heterogeneous cell composition, mainly M2 macrophages, which are responsible for the secretion of growth factors and cartilage protective cytokines such as TGF-β and IL-10, which may contribute to the therapeutic effect of SVF on chondrocytes.
Our study had several limitations. First, this study used SVF and ADSC in fixed doses by single administration; however, the optimal dose in the rabbit model has not yet been determined. Second, we used a rabbit OA model for this experiment. Rabbits are quadrupeds, and this OA model may not necessarily translate to a human model due to differences in postural and gait dynamics between rabbits and humans. Additionally, while the normal lifespan of a New Zealand rabbit is approximately 5 years, the New Zealand rabbits used in this study were only 6 months old; they may not correspond to middle-aged humans and their OA changes. Finally, histological and immunohistochemical evaluations in this study were performed primarily using semi-quantitative methods; therefore, more objective and accurate quantitative methods need to be investigated.