Matrigel-encapsulated articular cartilage derived fibronectin adhesion assay derived chondroprogenitors for enhanced chondrogenic differentiation: An in vitro evaluation

Abstract

Purpose: In cartilage research, three-dimensional (3D) culture models are pivotal for assessing chondrogenic differentiation potential. Standard pellet cultures, despite their utility, pose challenges like uneven differentiation and handling difficulties. This study explores the use of Matrigel, an extracellular matrix-based hydrogel, to encapsulate fibronectin adhesion assay-derived chondroprogenitors (FAA-CPs) and evaluate their chondrogenic differentiation potential.

Methods: FAA-CPs, isolated from human articular cartilage and expanded to passage 2, were either polymerized in Matrigel or cultured as standard pellets. Both groups underwent chondrogenic differentiation for 28 days and osteogenic differentiation for 21 days. Comprehensive analyses included histological staining, gene expression (SOX-9, ACAN, COL2A1 for chondrogenesis; COL1A1, RUNX2, COL10A1 for osteogenesis), and biochemical assays for glycosaminoglycans (GAG) and Collagen type II.

Results: The results demonstrated that Matrigel-encapsulated FAA-CPs achieved greater GAG accumulation, as evidenced by enhanced Alcian Blue and Safranin O staining, compared to standard pellets. However, the Collagen type II deposition, both histologically and quantitatively, was reduced in Matrigel constructs. Gene expression analysis showed no significant differences in key chondrogenic and osteogenic markers between the two groups. Despite improved handling and GAG deposition, Matrigel did not enhance uniform chondrogenic differentiation nor offer significant benefits for osteogenic differentiation, showing comparable hypertrophic markers to the standard method.

Conclusion: While Matrigel encapsulation offers advantages in handling and enhances GAG accumulation quantitatively, these benefits were not reflected in staining results. Furthermore, Matrigel did not significantly outperform standard pellet cultures in chondrogenic or osteogenic differentiation. These findings suggest a need for further refinement and in vivo validation.

Keywords: Chondrogenesis; Chondrogenic differentiation; Chondroprogenitors; Glycosaminoglycan; Matrigel; Pellet culture.