Adult bone marrow stromal cell-based tissue-engineered aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage

Abstract

Objective: To quantify the structural characteristics and nanomechanical properties of aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to aggrecan from adult articular cartilage.

Design: Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-β1 to induce chondrogenic differentiation. BMSC-aggrecan was extracted and compared with aggrecan from age-matched adult equine articular cartilage. Single molecules of aggrecan were visualized by atomic force microscopy-based imaging and aggrecan nanomechanical stiffness was quantified by high resolution force microscopy. Population-averaged measures of aggrecan hydrodynamic size, core protein structures and CS sulfation compositions were determined by size-exclusion chromatography, Western analysis, and fluorescence-assisted carbohydrate electrophoresis (FACE).

Results: BMSC-aggrecan was primarily full-length while cartilage-aggrecan had many fragments. Single molecule measurements showed that core protein and GAG chains of BMSC-aggrecan were markedly longer than those of cartilage-aggrecan. Comparing full-length aggrecan of both species, BMSC-aggrecan had longer GAG chains, while the core protein trace lengths were similar. FACE analysis detected a ∼ 1:1 ratio of chondroitin-4-sulfate to chondroitin-6-sulfate in BMSC-GAG, a phenotype consistent with aggrecan from skeletally-immature cartilage. The nanomechanical stiffness of BMSC-aggrecan was demonstrably greater than that of cartilage-aggrecan at the same total sGAG (fixed charge) density.

Conclusions: The higher proportion of full-length monomers, longer GAG chains and greater stiffness of the BMSC-aggrecan makes it biomechanically superior to adult cartilage-aggrecan. Aggrecan stiffness was not solely dependent on fixed charge density, but also on GAG molecular ultrastructure. These results support the use of adult BMSCs for cell-based cartilage repair.