Although many DMOAD have already been studied, there have been no clinically successful cases, suggesting that none tested so far have had convincingly disease-modifying efficacy [3, 25, 26]. The most effective DMOAD approach is either to increase ECM production by activating chondrocyte metabolism or to inhibit ECM degradation by controlling inflammation [27, 28]. In this study, we successfully developed a novel DMOAD, non-immunogenic ShFCPC, containing many ECM molecules and biomolecules that modify the progression of osteoarthritis through clear management of the inflammatory atmosphere towards a pro-chondrogenic microenvironment protecting chondrocytes from OA-induced hypertrophy and apoptosis, which might increase ECM production to enhance cartilage repair (Fig. 8).
hFCPC is known to have clear advantages in immune-modulation, chondrogenic repair potential, and inhibitory effects on inflammation mediated by macrophages compared to MSC [18–23]. However, current knowledge regarding the underlying mechanisms by which hFCPC relays signals to the inflammatory environment in OA is limited. In this study, we describe the possibility of utilizing lyophilized ShFCPC as a practical option for the enrichment of its secretome, which simultaneously optimizes its storage mode [29]. We found that hFCPC secretes a significantly higher diversity of proteins than hBMSC, and its secretome forms a sponge sheet after lyophilization and becomes a viscous solution when suspended in PBS, whereas SN does not (Fig. 1). These findings suggest that lyophilized ShFCPC may contain a large amount of ECM molecules. Subsequently, we performed mass spectrometry-based proteomic analysis of ShFCPC to uncover an extensive map of proteins (Fig. 2, 3). Interestingly, ShFCPC was significantly enriched with proteins associated with ECM molecules compared to ShBMSC. Unexpectedly, collagen type II was not detected in ShFCPC or ShBMSC. Our findings are consistent with previous studies showing that 3D culture of stem cells under chondrogenic conditions can recapitulate the early stage of prechondrogenic condensation, which is enriched in matrix proteins such as fibronectin, collagen type I, and the proteoglycan versican, which strongly supports the chondrogenesis of stem cells in vitro and in vivo. In contrast, mature cartilage is enriched in collagen type II [30–32]. We further found that protein-protein interaction analysis of the top 50 proteins suggests that the therapeutic effects of ShFCPC may result from the modulation of the TGF-β receptor signaling in EMT, regulation of insulin-like growth factor transport, and uptake by insulin-like growth factor binding proteins that are considered to be anabolic for cartilage, leading to enhanced repair of extensive cartilage defects and protection of the synovial membrane from chronic inflammation [33–35]. In addition, the pathways of focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, immune system, and interleukin-4 and interleukin-13 signaling were possibly modulated strongly by ECM proteins such as FN1, THBS1, collagen type VI alpha 1 chain (COL6A1), COL6A3, collagen type I alpha 1 chain (COL1A1), COL1A2, and laminin subunit gamma-1 (LAMC1) in ShFCPC (Fig. 2G and Table S7G). These pathways may underlie the many effects of cellular processes essential to homeostasis, including the cell cycle, cell survival, inflammation, metabolism, and apoptosis in OA progression [34–39], where interleukin-4 and interleukin-13 signaling pathways have been shown to underlie cartilage repair during OA progression through anti-inflammatory and chondroprotective mechanism [40–42]. The content of ShFCPC, in addition to a possible delivery mechanism through ECM molecules, provides us with an information database for the development of targeted therapeutics for specific arthritis diseases such as OA. Therefore, in vitro and in vivo OA models were established to confirm the ShFCPC mechanisms underlying our findings.
The ECM is mainly synthesized and produced by chondrocytes [35], which means that a sufficient number of healthy chondrocytes with suitable physiological functions are required for repairing injured cartilage tissue. However, during the development of OA, quiescent chondrocytes become activated by mechanical and inflammatory stresses and undergo proliferation and hypertrophic differentiation, characterized by enlarged cell size, enhanced expression of MMP1, ADAMTS-5, COLX, and the catabolic factors of primary inflammation: IL-1β, TNF-α and IL-6, which can cause hypertrophic chondrocytes to eventually undergo apoptosis and are replaced by calcification, leading to the degeneration of articular cartilage and are responsible for the progression of OA [1, 24, 43]. Thus, effective inhibition of chondrocyte hypertrophy and apoptosis is critical for the structural restoration of degenerative cartilage tissues. In this study, we observed that ShFCPC significantly enhanced the viability, proliferation, and migration of chondrocytes compared to ShBMSCs (Fig. S1, S2). Moreover, in in vitro model of inflammation by coculturing inflammatory chondrocytes and SW982-induced LPS (Fig. 4), ShFCPC exerted an obvious inhibitory effect on chondrocyte apoptosis and markedly downregulated the expression of IL-6, TNF-α, IL-1β, MMP-1, and ADAMTS-5, while markedly increasing the expression of SOX-9, ACAN, and COL2A1 and the secretion of IGF-1 and TGF-β3 in chondrocytes, which could then activate TGF-β receptor signaling, and insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins to regulate the expression of these chondrogenic genes [34, 44, 45]. In addition, in a rat OA model, we further demonstrated that intra-articular injection of ShFCPC attenuated the progression of cartilage matrix degradation caused by a combination of collagenase with forcing a specific amount of running. ShFCPC-treated OA rats demonstrated greatly improved structural integrity of articular cartilage by increased proteoglycan and COLII content, which are widely used as markers for cartilage repair [46]. Moreover, the expression of hypertrophy markers such as COLX was reduced, leading to inhibition of chondrocyte apoptosis and decrease in OA scores by ShFCPC treatment, which were observed to be better than those of the ShBMSC and HA treatment groups (Fig. 5). These data demonstrate that ShFCPC could modify the OA process by improving autophagy to decrease the hypotrophy and apoptosis of chondrocytes, which further enhances the integrity of the cartilage matrix and protects articular cartilage from mechanical stresses every day.
In addition to its beneficial effects on cartilage, ShFCPC exhibits a potent and broad anti-inflammatory profile in vitro and in vivo. Cartilage degeneration is well documented, and inflammation is a key factor in OA pathogenesis because it exacerbates joint destruction [47]. In the present study, ShFCPC markedly downregulated the expression of pro-inflammatory factors including IL-6, TNF-α and IL-1β in chondrocytes and SW982 in the model of inflammation in vitro (Fig. 4). In OA, a localized synovial inflammatory response, indicated by inflammatory cell accumulation and their effector cytokines in the locally inflammatory microenvironment, causes the initiation and deterioration of cartilage lesions [48, 49]. However, the evaluation of inflammatory cell changes, such as macrophages, lymphocytes, and leukocytes in arthritis inflammation has been widely studied in rheumatoid arthritis [50], whereas such data are scarce for OA. In the present investigation, we found that OA induction by using a combination of collagenase with forcing a specific amount of running following PBS treatment, which resulted in a high accumulation of leukocytes, and macrophages with a predominant number of M1 macrophages, a low number of M2 macrophages, and a high M1/M2 ratio, followed by CD3 + T cells but not CD19 + B cells, led to an increase in TNF-α, IL-1β, IL-6, COMP, and CTX-II in serum (Fig. 6, 7). Growing evidence indicates that synovitis in patients at risk for OA involves the recruitment of monocytes, lymphocytes, and other leukocytes. Typically, a high number of leukocytes is associated with infectious or ongoing acute synovitis, whereas lymphocytes have been suggested to increase the number of T cells, followed by an increase in the number of synovial macrophages [51, 52]. In particular, macrophages are important mediators of synovial inflammatory activity and pathologic cartilage responses with an imbalance of M1/M2 macrophages in the synovium, which is caused by an increase in M1 macrophages. A decrease in M2 macrophages is strongly involved in high levels of IL-1β, TNF-α, and IL-6, which result in the degradation of ECM components, including COMP and CTX-II. Similarly, ECM degradation components stimulate inflammatory cell activation and increase synovial inflammation, resulting in a repeated cycle of inflammation and cartilage degradation [52–54]. Therefore, one possible treatment strategy for OA is the modification of inflammatory cell infiltration. Our present study demonstrated that compared to the ShBMSC and HA treatment groups, decreased inflammatory cell infiltration and a higher number of M2 macrophages led to a decrease in synovitis scores in ShFCPC-treated OA rats (Fig. 6). Consistent with these results, the levels of inflammatory cytokines and ECM degradation products in the serum and urine were decreased in OA rats treated with ShFCPC (Fig. 7). Taken together, these results indicate that ShFCPC modified the OA process by reducing the recruitment of immune cells and promoting the polarization of synovial macrophages into the M2 phenotype, which might directly contribute to an increased immunomodulatory environment for the repair of injured cartilage.