Modulation of Gene Expression in Infrapatellar Fat Pad-Derived Mesenchymal Stem Cells in Osteoarthritis

Abstract

Aim In the osteoarthritis (OA) disease, all structures of the joint are involved. The infrapatellar Hoffa fat pad is rich in macrophages and granulocytes, which also represents a source of adipose mesenchymal progenitor cells (ASC) cells. In our study, we analyze how OA affects the ability of ASC-derived from Hoffa's fat pad to differentiate into chondrocytes. Material and methodology We took knee Hoffa's pad samples and adipose tissue from the proximal thigh from 6 patients diagnosed with severe OA and from another 6 patients with an anterior cruciate ligament (ACL) rupture without OA. From all the patients, we took subcutaneous adipose tissue from the thigh, as the control group. Samples of synovial fluid (SF) were also extracted. The gene expression was analyzed by real-time quantitative polymerase chain reaction. Results PTH1R and MMP13 expression during chondrogenic differentiation were similar between OA and ACL groups, while the expression of OPG, FGF2, TGFβ, MMP3 were significantly lower in the OA group. Exposure of differentiated ASC to OA SF induced an increase in the expression of OPG, PTH1R, and MMP13 and a decrease in the expression of FGF2 in cell culture of the ACL group. However, expression of none of these factors was altered by the OA synovial fluid in ASC cells of the OA group. Conclusion OA of the knee also affects the mesenchymal stem cells of Hoffa fat, suggesting that Hoffa fat is a new actor in the OA degenerative process that can contribute to the origin, onset, and progression of the disease.

Keywords: cells; chondrocytes; cytokines and growth factors; diagnosis; mesenchymal stem cells; osteoarthritis; synovial fluid.

Figure 1.

Flow diagram.

Figure 2.

Contrast-phase microscopic image from fleshly isolated and primary cultured adipose stem cells (ASCs) on day 1 (A), day 7 (B), and day 14 (C). Response of primary cultured ASCs to specific cell lineage differentiation media. Control ASCs with basal medium (D); adipogenic differentiation by Oil Red staining of mesenchymal stem cells (MSCs) (E) Detection of osteogenic differentiation by Alizarin Red staining of MSCs (F).

Figure 3.

Flow cytometric analysis at day 7 on the surface expression of CD29-CD45 (a) and CD34-CD-90 (b) with the use of FITC and PE-conjugated antibodies. Representative data of primary cultured adipose stem cells (ASCs) obtained from one single patient are shown. The same results were verified in at least ASC samples from 5 different patients.

Figure 4.

Gene expression of OPG (A), PTHr1 (B), FGF2 (C), TGFβ (D), MMP3 (E), and MMP13 (F) at day 0, day 15, and day 28 of chondrogenic differentiation of mesenchymal stem cell (MSC) culture from Hoffa fat pad in osteoarthritic group (OA) (n = 6 patients) and anterior cruciate ligament rupture group (ACL) (n = 6 patients) (*P < 0.05).

Figure 5.

Gene expression of OPG (A), PTHr1 (B), FGF2 (C), TGFβ (D), MMP-3 (E), and MMP13 (F) at day 0, day 28, and day 29 after including osteoarthritic synovial fluid in the media of chondrogenic differentiation of mesenchymal stem cell (MSC) culture from Hoffa fat pad in the control group (extra-articular subcutaneous adipose tissue) (n = 6 patients), osteoarthritic group (OA) (n = 6 patients), and anterior cruciate ligament rupture group (ACL) (n = 6 patients) (*P < 0.05).