Enhancement of Chondrogenic Markers by Exosomes Derived from Cultured Human Synovial Fluid-Derived Cells: A Comparative Analysis of 2D and 3D Conditions.

OBJECTIVE

The goal of this pilot study was to investigate the effects of exosomes derived from synovial fluid-derived cells (SFDCs) cultured under normoxic conditions in a two-dimensional (2D) monolayer or encapsulated within a three-dimensional (3D) matrix for chondrogenic differentiation in vitro and cartilage defect repair in vivo.

DESIGN

Synovial fluid samples were obtained from three patients, and SFDCs were isolated and expanded either in a 2D monolayer culture or seeded within a transglutaminase cross-linked gelatin (Col-Tgel) to create a 3D gel culture. Exosomes derived from each environment were isolated and characterized. Then, their effects on cartilage-cell proliferation and chondrogenic differentiation were assessed using an in vitro organoid model, and their potential for enhancing cartilage repair was evaluated using a rat cartilage defect model.

RESULTS

SFDCs obtained from different donors reached a state of senescence after four passages in 2D culture. However, transferring these cells to a 3D culture environment mitigated the senescence and improved cell viability. The 3D-cultured exosomes exhibited enhanced potency in promoting chondrogenic differentiation, as evidenced by the increased expression of chondrogenic genes and greater deposition of cartilage-specific extracellular matrix. Furthermore, the 3D-cultured exosomes demonstrated superior effectiveness in enhancing cartilage repair and exhibited better healing properties compared to exosomes derived from a 2D culture.

CONCLUSIONS

The optimized 3D culture provided a more favorable environment for the proliferation of human synovial cells and the secretion of exosomes compared to the 2D culture. The 3D-cultured exosomes exhibited greater potential for promoting chondrogenic gene expression in vitro and demonstrated improved healing properties in repairing cartilage defects compared to exosomes derived from the 2D culture.