Isolation and Characterization of Synovial Mesenchymal Stem Cell Derived from Hip Joints: A Comparative Analysis with a Matched Control Knee Group

Abstract

Purpose. To determine the characteristics of MSCs from hip and compare them to MSCs from knee. Methods. Synovial tissues were obtained from both the knee and the hip joints in 8 patients who underwent both hip and knee arthroscopies on the same day. MSCs were isolated from the knee and hip synovial samples. The capacities of MSCs were compared between both groups. Results. The number of cells per unit weight at passage 0 of synovium from the knee was significantly higher than that from the hip (P < 0.05). While it was possible to observe the growth of colonies in all the knee synovial fluid samples, it was impossible to culture cells from any of the hip samples. In adipogenesis experiments, the frequency of Oil Red-O-positive colonies and the gene expression of adipsin were significantly higher in knee than in hip. In osteogenesis experiments, the expression of COL1A1 and ALPP was significantly less in the knee synovium than in the hip synovium. Conclusions. MSCs obtained from hip joint have self-renewal and multilineage differentiation potentials. However, in matched donors, adipogenesis and osteogenesis potentials of MSCs from the knees are superior to those from the hips. Knee synovium may be a better source of MSC for potential use in hip diseases.

Figure 1

Data on cell samples obtained from the synovium of each case on 8 donors.

Figure 2

(a) Arthroscopic observation and sample harvesting. (b), (c) Macroscopic features of from knee and hip synovium were examined on a 1 mm scare and in PBS. (d), (e) Histological analysis was performed on tissues stained with hematoxylin and eosin (HE). (f) Representative cell colonies derived from synovium of a donor stained with crystal violet showing large colonies when the nucleated cells were plated at 10⁴ cells/dish. There was no significant difference in the number of colonies between both MSCs from knee and hip joints. (g) Representative morphologic features of the cells magnified at 14 days (passage 0) showing fibroblastic spindle cell morphology. Bar = 100 μm. (h) Representative cell colonies stained with crystal violet derived from the synovial fluid of a donor showing large colonies from the knee, but no colonies from the hip.

Figure 3

Proliferating potential of both hip and knee cells on 3 donors. Both passage-1 cells were plated at 50 cells/cm² on 150 cm² dishes. Thereafter, the cells were replated at 50 cells/cm² every 14 days until their expansion potential was lost. The expandabilities were lost at passage 8 in both MSCs derived from knee and hip joints.

Figure 4

(a) LIVE/DEAD Viability/Cytotoxicity Kit assay. At passage 1, green fluorescence was observed in all cells derived from both joints, confirming that these cells were alive. Passage-4 cells derived from knee were alive, but the part of cells from hip was dead. At passage 7, the half of both cells was dead. (b) Cell Counting Kit-8 assay for passages 4 and 7 cells on 3 donors. The bar means standard deviation. At passage 4, viability of cells derived from knee was significantly higher than those from hip (P < 0.01, Mann–Whitney U test). At passage 7, there was no significant difference of cell viability between knee and hip. ^∗Significant difference. (c) The correlation between the fold increase and cell viability on each sample at passages 4 and 7.

Figure 5

Adipogenic potential about each synovial cell. (a) Colonies staining positively with Oil Red-O and crystal violet in each cell. (b) Proportion of Oil Red-O-positive colonies in relation to the total number of colonies in each MSC. Values are shown as the mean and standard deviation of 6 donors. ^∗The rate of Oil Red-O-positive colonies from the knee was significantly higher than that from hip (Mann–Whitney U test, P < 0.0001). (c) Quantitative real-time PCR data from 3 representative donors are shown. The bar means standard deviation.

Figure 6

Osteogenic potential of each type of MSCs. (a) Colonies stained positively with von Kossa and ALP as well as crystal violet. (b) Proportion of von Kossa and ALP-positive colonies in relation to the total number of colonies. Values are the mean and standard deviation of 6 donors. ^∗There was no significant difference in the rate of von Kossa and ALP-positive colonies between knee and hip (Mann–Whitney U test, P = 0.20). (c) Real-time RT-PCR. Gene expressions of COL1 and ALP in knee were significantly lower than those in hip (Mann–Whitney U test, P < 0.05); on the other hand gene expressions of RUNX2 and BGLAP in the knee were significantly higher than in the hip (Mann–Whitney U test, P < 0.05). The bar means standard deviation.

Figure 7

Chondrogenic potential of each type of synovial cell. (a) The pellets from each sample were shown. There was no significant difference in the diameter of pellets between knee and hip (P = 0.82, Mann–Whitney U test). Histological analysis was performed on tissues stained with Toluidine Blue staining and immunocytochemical analysis with type II collagen by tissue source and human chondrosarcoma for positive control. (b) Real-time RT-PCR. The bar means standard deviation. Expression of COL2 gene in knee was significantly higher than that in hip (P < 0.05, Mann–Whitney U test). ^∗Significant difference.

Figure 8

Flow cytometry analysis of the expression of cell surface markers related to stem cells, hematopoietic stem cells, and endothelial cells in each population of cells derived from each sample. Passage-2 cells from 3 donors were examined, and similar results were obtained. Positive expression rates are shown.