The employment of ASCs in regenerative medicine is a rapidly growing area of research and some evidence of therapeutic success using these cells for osteochondral defect has been reported [39
]. Recently, this cell therapy is being used as a valid therapeutic tool also in the treatment of OA. Beneficial effects of ASCs were reported in the care of this pathology in some experimental animal models [25
]. However, most of these studies were limited to macroscopic and histological observations of articular cartilage after ASC treatment. No information was provided on the effects of these cells on catabolic and inflammatory processes which occur during OA in synovial membrane and menisci. This study, using a rabbit model of OA, was designed to determine the role of ASCs in the OA setting and their behavior on inflammatory environment within the affected articular joint. The ACLT model proposed is widely validated in investigating OA disease, because it determines biomechanical and pathological changes similar to those seen in humans [29
]. It has been reported from our group that eight weeks after ACLT, cartilage damages in rabbits occur mainly in the medial femoral condyle, showing a wide spectrum of OA changes, including fibrillation and delamination processes, altered cellular arrangement and proteoglycan depletion [30
]. The investigations performed on synovial membrane and menisci revealed that the ACLT procedure at eight weeks leads to a thickening of the lining layer in the synovial membrane associated with the presence of some inflammatory elements and to an increase in cell cluster formation in menisci. Direct intra-articular injection of cells is technically the simplest approach to the use of cells for OA therapy [13
]. In the current study, an intra-articular injection of ASCs was delivered in the hind limbs of the rabbits after OA induction. No animals showed swelling at the injection sites, signs of distress, or hyperalgesia after ASC administration. There were significant overall effects of ASC treatment in the cartilaginous, synovial and meniscal tissues at different levels. Our investigations gave evidence of the beneficial effect for the 2 × 106
ASC group, particularly at 16 weeks, showing a well-organized tissue with a low Laverty's score, an increased cartilage thickness compared to the OA group. Both cell doses provided good results showing a high expression of type II collagen in the cartilage matrix at 16 and 24 weeks. A positive contribution of the intra-articular delivery of both ASC concentrations was also noticed in the meniscal compartment at both experimental times, leading to a decrease of the number of cell clusters the fibro-cartilaginous area. Cell treatment inhibits the progression of OA, providing a reduction of the fibrillation index, Laverty's score and type I collagen in cartilage and favors the anabolic processes addressed to the formation of a new tissue. Moreover, ASC administration inhibits the development of thickening of the lining layer in the synovial membrane with major evidence at short-term follow-up. Since the release of cartilage matrix proteins in the articular environment contribute to cartilage damage through the production of inflammatory cytokines, chemokines and MMPs [3
], we decided to test the effects exerted by ASCs. To this regard, clear benefits of ASC treatment were observed with the reduction of the inflammation in cartilage tissue in terms of a decreased pattern of expression for TNF-α at both experimental times [42
]. In close correlation with the reduction of TNF-α, a decreased level of MMP-1 in cartilage tissue, responsible of proteoglycan degradation, was noticed in ASC-treated groups at 16 and 24 weeks [42
]. A time-dependent effect of ASC treatment was noticed for MMPs and TNF-α expression, particularly in the 2 × 106
ASC group providing the best results at short-term follow-up. Cell treatment inhibits the progression of OA, leading to a reduction of TNF-α and MMPs in menisci and synovial membrane at both experimental times. In general, the 4% RSA group displayed OA progression in the different compartments as indicated by an increased expression of catabolic and inflammatory markers. Both ASC doses gave evidence of the healing potential in cartilage, synovial membrane and menisci, even if the lowest cell concentration is more effective on cartilage repair at 16 weeks. The underlying mechanisms responsible of the best effects of 2 × 106
ASCs in cartilage are not fully understood. It could be related to the release of cytokines and other growth factors by ASCs that at low and high concentrations can determine contrasting biological effects on the immune system and/or on catabolic and inflammatory events. A time-dependent effect by ASCs was observed in the analysis performed particularly on cartilage tissue, detecting the best findings at short-term follow-up. These findings could be justified by the lack of repair of the anterior cruciate ligament, which could slow and/or inhibit some signaling pathways involved in the repair processes. The investigations performed on the local bio-distribution of ASCs by using a fluorescent tracking dye (CM-Dil) open some prospectives in the understanding of the mechanism of action by ASCs. The key advantage of CM-Dil and its derivatives is to represent a nontoxic fluorescent tracking system ready in a few hours, able to label ASCs without altering their multi-potential nature as observed by in vitro
differentiation protocols and avoiding genetic manipulation of the cells [33
]. Nevertheless, this system shows some disadvantages such as the loss of fluorescence signal over time during cell replication rendering difficult the cell tracking at long term follow-up, the transfer of the fluorescent dye to other cells.
After having injected labeled cells, no engraftment was noticed in the anterior cruciate ligament and in the cartilaginous tissue from the tibial plateau and femoral condyles. The homing of these cells was unexpectedly detected in the synovial and medial meniscus, probably due to the expression of specific receptors or ligands able to facilitate trafficking, adhesion and infiltration of ASCs to these sites and/or to the presence of a vascular fraction which could promote ASC migration. The distribution of ASCs in the lining layer of synovial tissue could also be due to the release of chemokines by macrophages located within the lining layer [44
]. Different hypotheses could be provided to explain the chondro-protective and healing effects exerted by ASCs. Cell administration could contribute to enhance anabolic signaling pathways and inhibit catabolic ones. These processes could be reasonably induced by growth factors and cytokines released by ASCs rather than their differentiation potential since biodistribution data showed the localization of ASCs in the synovial and meniscal tissues.
Another mechanism exerted by ASCs could be due to the inhibition of the release of catabolic and inflammatory molecules by macrophages in synovium or chondrocytes in cartilage. Experimental evidence supports this hypothesis, reporting a decrease of the catabolic and inflammatory molecules predominantly in cartilage. Other possibilities could be projected on the regulation of the immune system by ASCs as already observed for MSC [37
]. Several authors have shown how the inflammatory environment is an important parameter to consider, since it would seem to influence the behavior of ASCs by enhancing their immunosuppressive potential [46
]. This last consideration provides more attention on the pattern of molecules secreted by ASCs, which could have important implications in the resolution of inflammatory diseases. Further analysis of the molecules secreted by ASCs could be useful to identify key elements involved in repair processes. In conclusion, the findings of this study demonstrate that an intra-articular injection of ASCs exerts a chondro-protective role promoting a series of anabolic processes in order to allow the maintenance of a good collagen and proteoglycan network, and at the same time inhibiting catabolic events responsible for degenerative events in cartilage, synovial and meniscal tissues. ASC therapy could, therefore, represent a novel therapeutic tool for the treatment of osteoarthritis.