Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes

Abstract

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.

Figure 1

Cell Viability. Live (green) and dead (red) staining of self-assembling peptide hydrogels cultured with TGF-β1 at day 1.

Figure 2

Hydrogel Dynamic Stiffness. Chondrocyte (Chd) and BMSC seeded peptide hydrogels after 21 days of culture in control (Cntl) or TGF-β1 supplemented (TGF) medium. Stats: mean ± sem, n=2 foals × 3 samples each or n=3 adults × 3 samples each; ‡ vs. foal chondrocyte TGF, † vs. foal chondrocyte Cntl, # vs. No Cell; p<0.05.

Figure 3

Hydrogel DNA Content. DNA content for chondrocyte and BMSC hydrogels at day 0, or after 21 days. Stats: mean ± sem, n=2 foals × 4 samples each or n=3 adults × 4 samples each; for a given cell type and age significance is indicated by: # vs. Day 0; * vs. Cntl D21; p<0.001.

Figure 4

Hydrogel ECM Content and Biosythesis Rates at Day 21. sGAG content (A) per hydrogel (B) per wet weight. (C) Proteoglycan biosynthesis. (D) %sGAG retention. Hydroxyproline content (E) per hydrogel (F) per wet weight. (G) Protein biosynthesis (H) %Solid matrix. Stats: mean±sem, n=2 foals × 4 samples each or n=3 adults × 4 samples each; * vs. Cntl; ‡ vs. foal chondrocyte; † vs. adult chondrocyte; p<0.05.

Figure 5

Superose 6, Size-Exclusion, Proteoglycan Chromatography. Proteoglycans extracted from either chondrocyte and BMSC seeded peptide hydrogels after 21 days of culture with TGF-β1 or from native cartilage tissue harvested from newborn bovine calves.

Figure 6

AFM Single-Molecule Height Images of Aggrecan Ultrastructure. Proteoglycans extracted from cell-seeded peptide hydrogels after 21 days of culture with TGF-β1. (A-C) Foal chondrocytes, (D-F) Foal BMSCs, (G-I) Adult chondrocytes, (J-L) Adult BMSCs. Blue arrows in A,D,G,J denote ends of full-length aggrecan monomers. Example individual CS-GAG chains highlighted in blue in C,F,I,L.

Figure 7

Aggrecan Core Protein Quantification. Aggrecan extracted from chondrocyte (Chd) and BMSC seeded peptide hydrogels after 21 days of culture with TGF-β1. (A) Core-protein average length. Stats: mean ± sem; n=110-231 aggrecan molecules; ‡ vs. foal chondrocyte; † vs. adult chondrocyte; p<0.05. (B) Histograms of core-protein length.

Figure 8

Aggrecan CS-GAG Chain Quantificaiton. Aggrecan extracted from chondrocyte (Chd) and BMSC seeded peptide hydrogels after 21 days of culture with TGF-β1. (A) Average CS-GAG length per molecule. Stats: mean ± sem; n=28-35 aggrecan molecules; ‡ vs. foal chondrocyte; † vs. adult chondrocyte; p<0.05. (B) Histograms of CS-GAG distribution on the single pictured molecule. Scale bar = 100 nm.