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

Lee, H.-Y.; Kopesky, P.W.; Plaas, A.; Sandy, J.; Kisiday, J.; Frisbie, D.; Grodzinsky, A.J.; Ortiz, C.

doi:10.1016/j.joca.2010.07.015

« Previous Next »Osteoarthritis and Cartilage
Volume 18, Issue 11 , Pages 1477-1486, November 2010

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

H.-Y. Lee
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, United States
P.W. Kopesky
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States
A. Plaas
- Department of Biochemistry, Rush University Medical Center, Chicago, IL, United States
J. Sandy
- Department of Biochemistry, Rush University Medical Center, Chicago, IL, United States
J. Kisiday
- Equine Orthopaedic Research Center, Colorado State University, Fort Collins, CO, United States
D. Frisbie
- Equine Orthopaedic Research Center, Colorado State University, Fort Collins, CO, United States
A.J. Grodzinsky
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, United States
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States
- Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States
C. Ortiz
- Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States
- Address correspondence and reprint requests to: C. Ortiz, Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States. Tel: 1-617-452-3084.

Received 21 January 2010; accepted 30 July 2010. published online 09 August 2010.

Summary

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 microcopy-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.

Keywords: Aggrecan, Bone marrow stromal cell, Cartilage repair, Tissue engineering, Self-assembling peptide, Molecular nanomechanical properties