| Objective: To observe the effect of human knee infrapatellar fat pad with osteoarthritis (OA) on normal and chondrocytes. Methods: Twenty-four cases of human knee infrapatellar fat pads and cartilages were provided by orthopedics operating room, including 12 cases of acute knee injury, and 12 cases of OA undergoing knee joint replacement. The fat pads were cut and stained with hematoxylin-eosin (HE) staining, and subjected to immunohistochemical staining with leptin. The OA fat pads were used to prepare fat conditioned medium (FCM). The normal chondrocytes were isolated from the integrate specimens of acute knee injury. The OA chondrocytes were isolated from the knee joint cartilage in OA. The cells were cultured and identified in vitro, and were randomly divided into four groups: normal chondrocytes group, OA chondrocytes group, normal chondrocytes + FCM group, OA chondrocytes + FCM group. The normal chondrocytes group and OA chondrocytes group were added with full Dulbecco's Modified Eagle's Medium (DMEM) medium, and the normal chondrocytes + FCM group and OA chondrocytes + FCM group were added with FCM. Each group was observed under a microscope. Cells were stained with type Ⅱ collagen (COL2) immunohistochemical staining and oil red O staining after 14 days. Western blotting was used to examine the expression of COL2, aggrecan, and MMP-13. Results: (1) COL2 immunohistochemical staining: The average absorbance of COL2 immunohistochemical staining in OA group was significantly lower than that in the normal group (P<0.05). As compared with OA group, the average absorbance of COL2 immunohistochemical staining showed no significant difference between OA group and normal + FCM group (P>0.05), and that in OA + FCM group was significantly lower than in OA group (P<0.05). As compared with normal + FCM group, the average absorbance of COL2 immunohistochemical staining in OA + FCM group was significantly decreased (P<0.05). (2) Oil red O staining: The average absorbance of oil red O staining showed no significant difference between OA group and normal group (P>0.05). As compared with OA group, the average absorbance of oil red O staining in normal + FCM group and OA + FCM group was significantly increased, with more significant increase in OA + FCM group (P<0.05). (3) Western blotting showed that the expression of COL2 and aggrecan in the OA chondrocytes group was significantly lower than that in the normal chondrocytes group (P<0.05), but the expression of MMP-13 was significantly higher in the OA chondrocytes group than in the normal chondrocytes group (P<0.05). There was no significant difference in levels of type Ⅱ collagen, aggrecan and MMP-13 between OA chondrocytes group and normal chondrocytes + FCM group (P<0.05). As compared with normal group and normal + FCM group, the expression of type Ⅱ collagen and aggrecan in OA chondrocytes + FCM group was significantly reduced (P<0.05), but the expression of MMP-13 was significantly increased (P<0.05). Conclusions: FCM of OA can induce fatty degeneration of the normal and OA chondrocytes, and it also increases the expression of MMP-13 and decreases the expression of COL2 and aggrecan in chondrocytes through metabolic pathway. Moreover, the damage in OA chondrocytes is more significant. |
