Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

Abstract

Purpose: Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties, and effectiveness of aggregated MSCs for cartilage regeneration. Methods: Synovial MSCs. Human synovium was harvested from donors during ACL reconstruction surgery. Rabbit synovium was harvested from the knee joint. After collagenase digestion, nucleated cells were expanded, and colony forming cells were harvested. Preparation of aggregates of MSCs. 2.5×105 synovial MSCs suspended in 35μl of culture medium were aggregated using the hanging drop technique. Microarray analysis. Total RNA was extracted from human synovial MSCs in a monolayer culture, aggregates of human synovial MSCs cultured for 3 days. In vitro chondrogenic differentiation assay. 2.5 × 105 human synovial MSCs cultured as monolayer were pelleted by trypsinization and centrifugation. The pellets or aggregate of human synovial MSCs cultured for 3 days in hanging drop were cultured in 400μl chondrogenic medium. The medium was changed every 3-4 days for 21 days. Transplantation of aggregates of synovial MSCs in rabbits. Osteochondral defects (5×5 mm wide, 1.5 mm deep) were created in the femoral groove, and 5, 10, 20, 40, 80 aggregates of autologous synovial MSCs were transplanted to the defect. MSCs labeled with DiI and MSCs derived from GFP rabbit were also used. The cartilage was evaluated at 1 day, 2, 4, 12 weeks after the operation (n = 5). Statistical analysis. To assess differences, the Kruskal-Wallis test and the Steel test were used. A P value of < 0.05 was considered statistically significant. Results: Aggregates of MSCs cultured for three days became visible, approximately 1mm in diameter, and solid and durable by manipulation, and most of the cells were viable. Microarray analysis revealed upregulation of chondrogenesis-related genes (SOX5, -6, -9, and BMP2) and anti-inflammatory (TSG-6, and STC-1) genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to apoptosis and nutrient deprivation of aggregates of MSCs. Conclusions: Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential, and efficient attachment.