It is evident that the IFP might interact with cartilage to participate in the development of OA, however, the underlying mechanism remains unclear. In this study, by coculturing FCM derived from human OA IFP and chondrocytes from OA patients, we found that FCM might inhibit the proliferation of OA chondrocytes. Furthermore, a total of 131 metabolites obtained in the culture media of OA chondrocytes were significantly changed. Pathway analysis revealed that those metabolic pathways significantly affected in OA chondrocytes included the TCA cycle, amino acid metabolism, and glutamine metabolism. These results suggest that IFP has a deleterious effect on cartilage, and the involvement of the IFP in the development of OA might be associated with the effect of induced metabolic disturbances of chondrocytes.
Effect of FCM on TCA cycle pathways of human OA chondrocytes
The TCA cycle is a key biochemical pathway that provides cellular energy in the form of ATP by oxidative conversion of carbohydrates . The increased levels of citric acid and succinic acid and decreased levels of isocitrate in the culture medium OA chondrocytes after treatment of FCM indicate enhanced activity of the TCA cycle, possibly due to perturbed metabolism in the cartilage cells. Akhbari et al.’s  study on synovial fluid (SF) showed that when compared with the metabolites of the SF in rheumatoid arthritis patients, OA patients had higher levels of metabolites involved in both TCA and glycolysis. Another study examined the metabolites of SFs between early- and late-stage OA and found that TCA intermediate metabolites including malate, citrate, succinate, and fumarate levels were significantly higher in the late-stage OA group than in the early-stage OA group, suggesting that the upregulation of the TCA pathway is greater in the late stage of OA . In addition, a study on urine also indicated that the increased levels of TCA intermediate metabolites in the urine of inflammatory OA participants compared with non-OA controls suggested an enhanced activity of the TCA cycle . Briefly, these data suggest that the TCA pathway is upregulated in OA, and our results agree with these studies, demonstrating that the upregulated TCA pathway of OA is most likely to increase the production of ATP needed to repair damaged cartilage. On the other hand, most of the enzymes involved in the TCA cycle are located inside the mitochondrial matrix of the cartilage cells, and the enhanced TCA cycle activity provides metabolic evidence of mitochondrial dysfunction of the cartilage cells in OA . Back to our findings, these results showed that FCM treatment accelerated the injury of OA chondrocytes by enhancing the TCA cycle activity.
Effect of FCM on amino acid metabolism of human OA chondrocytes
Amino acids, small molecule metabolites, are considered the most important biological compounds, being involved in the synthesis of proteins and hormones and playing prominent metabolic and physiological roles (such as immune function, cytokine secretion) within organisms . The current literature data suggest that the metabolic pathogenesis of OA might be significantly related to perturbations of amino acid metabolism . In our study, after coculturing with FCM for 48 h, we observed increased levels of two main amino acids (threonine and glutamate) and declined levels of four main amino acids (cysteine, proline, arginine, and tyrosine) in the OA chondrocyte culture medium. Previous studies have found increased levels of glutamate and aspartate in dialysates from the injured joint of rats with anterior cruciate ligament transection (ACLT) . Furthermore, the contents of glutamate and aspartate in the SF of patients with synovitis are 54 times and 28 times higher than those of healthy controls, respectively . In addition, sheep with ACLT exhibited decreases in serum concentrations of tyrosine, valine, and isoleucine at week 4 after surgery, compared with the sham control . Moreover, a study evaluated the short-term metabolic response of human chondrocytes to mechanical loading and suggested that rates of serine and threonine metabolism were increased. In rabbits with an ACLT injury, there were negative correlations between histological assessments and changes in plasma concentrations of arginine . These findings indicate that the decrease of arginine plasma concentration was due to the process and progress of OA. In summary, the increase of glutamate and threonine levels and the decrease of tyrosine and arginine levels could induce the occurrence and development of OA. Furthermore, prolines are thought to inhibit cell oxidative damage and have anti-inflammatory effects . In our study, after coculturing with FCM, the levels of arginine, proline, and tyrosine decreased, and the levels of glutamate and threonine increased in the culture medium of OA chondrocytes, indicating that FCM promotes the inflammatory response of OA chondrocytes and IFP has a deleterious effect on cartilage.
Effect of FCM on glutamine metabolism of human OA chondrocytes
Glutamine is a key source of carbon secondary only to glucose for energy production and anabolic processes . Recently, a study found that glutamine suppressed OA progression . Glutamine is a precursor of glucosamine and can upregulate glucosamine synthesis in chondrocytes, and glucosamine could inhibit cartilage destruction and promote biosynthetic activity of chondrocytes . A study observed that the concentration of glutamine was lower in the SF from OA patients than in normal SF . In addition, 2-keto-glutaramic acid (a deaminated metabolite of glutamine under abnormal conditions) has been found to be increased in the urine of inflammatory OA participants, indicating that glutamine metabolism might be disturbed in the chondrocytes . Furthermore, Wang et al.  indicated that growth plate (GP) and articular cartilage chondrocytes exhibited metabolic plasticity that allowed these cells to switch to glutamine oxidation to support energy production when glucose metabolism was compromised. Glutamine oxidation enables GP chondrocytes to survive in glucose-limited conditions, however, normal proliferation and matrix production by these cells is impaired. In a word, in OA progression, glutamine metabolism could be disturbed in the chondrocytes, and lower glutamine might indicate the cartilage destruction of the joint. Our observations suggested that after coculturing with FCM, the level of glutamine declined. This result supports that IFP has a deleterious effect on cartilage. However, the mechanisms of glutamine involvement in OA pathogenesis are unclear. During stress loading, exogenous glutamine supplementation has been shown to increase the concentration of glutathione (an essential intracellular antioxidant) that protects cells from injury. Moreover, glutamine has been shown to be vital for fueling mitochondrial metabolism , and the IFT might disrupt glutamine metabolism, resulting in mitochondrial dysfunction, and finally lead to the development of OA. Further research is needed to determine the mechanisms of the crosstalk between the IFP and cartilage.
There are a couple of potential limitations of the current study that must be acknowledged. First, we only evaluated the changes to OA chondrocyte culture medium metabolites after FCM treatment. The underlying mechanism of the interaction between cartilage and IFP could be better understood together with the intracellular metabolites, which should be evaluated in the future. Furthermore, our results indicated that IFP could disturb the TCA cycle, amino acid metabolism, and glutamine metabolism of OA chondrocytes, however, further investigations are warranted to verify the results of metabolomics.