Chondrocytes and meniscal fibrochondrocytes differentially process aggrecan during de novo extracellular matrix assembly

Abstract

Aggrecan is an extracellular matrix molecule that contributes to the mechanical properties of articular cartilage and meniscal fibrocartilage, but the abundance and processing of aggrecan in these tissues are different. The objective of this study was to compare patterns of aggrecan processing by chondrocytes and meniscal fibrochondrocytes in tissue explants and cell-agarose constructs. The effects of transforming growth factor-beta 1 (TGF-beta1) stimulation on aggrecan deposition and processing were examined, and construct mechanical properties were measured. Fibrochondrocytes synthesized and retained less proteoglycans than did chondrocytes in tissue explants and agarose constructs. In chondrocyte constructs, TGF-beta1 induced the accumulation of a 120-kDa aggrecan species previously detected in mature bovine cartilage. Fibrochondrocyte-seeded constructs contained high-molecular-weight aggrecan but lacked aggrecanase-generated fragments found in native, immature meniscus. In addition, reflecting the lesser matrix accumulation, fibrochondrocyte constructs had significantly lower compression moduli than did chondrocyte constructs. These cell type-specific differences in aggrecan synthesis, retention, and processing may have implications for the development of functional engineered tissue grafts.

FIG. 1.

Articular cartilage (A,C) and meniscal fibrocartilage (B,D) explants exhibit distinct patterns of proteoglycan distribution (A,B) and aggrecan processing in vitro. *Indicates total construct sGAG (extract + digest) is different from untreated day 10 controls (p < 0.05). Note 10-fold difference in scale between A and B. Data are mean + SEM, with n = 6. In C & D, equal portions of explant extracts (pooled from 6 samples) were loaded in each lane. Articular cartilage extracts were diluted 10-fold over fibrocartilage extracts to account for the substantially higher sGAG content. Numbers to the left ofeach blot indicate the migration of molecular weight markers in kDa. sGAG, sulfated glycosaminoglycan; TGF-β1, transforming growth factor-beta 1.

FIG. 2.

Chondrocyte-agarose (A) and meniscal fibrochondrocyte-agarose (B) constructs exhibit distinct patterns of proteoglycan distribution. Total sGAG (retained in constructs + released to the media) was normalized by DNA content in C. *Indicates a statistically significant difference between TGF-β1-stimulated and untreated day 16 constructs (p < 0.05). Note 4-fold difference in scale between A and B. Data are mean + SEM, with n = 6.

FIG. 3.

Cell type differences in deposition of newly synthesized sGAG are illustrated by Safranin-O staining. Cell-agarose constructs treated with (middle panels) or without (left panels) TGF-β1 for 16 days were compared with sections of native articular cartilage or midstubstance meniscal fibrocartilage (right panels). Sections were counterstained with fast green and hematoxylin. Scale bar = 100 μm; inset scale bar = 25 μm.

FIG. 4.

Highly processed aggrecan accumulates in TGF-β1-stimulated chondrocyte-agarose tissue constructs. Blots are of extracts of freshly-prepared constructs (Day 0) and constructs cultured for 16 days with or without TGF-β1. Aggrecan fragments bearing the G1, G2, and G3 domains or the NITEGE or GVA neoepitopes were examined. Extracts were also probed for the small proteoglycan, decorin. Numbers to the left of each blot indicate the migration of molecular weight markers (in kDa). Equal volumes of extracts pooled from 6 constructs were loaded in each lane.

FIG. 5.

Fibrochondrocyte constructs stimulated with TGF-β1 accumulate full length aggrecan. Blots are of extracts of freshly-prepared constructs (Day 0) and constructs cultured for 16 days with or without TGF-β1. Aggrecan fragments bearing the G1, G2, and G3 domains or the NITEGE neoepitope were examined. Extracts were also probed for the small proteoglycan, decorin. Numbers to the left of each blot indicate the migration of molecular weight markers (in kDa). Equal volumes of extracts pooled from 6 constructs were loaded in each lane.

FIG. 6.

Chondrocytes (A) and fibrochondrocytes (B) assemble ECMs with different compressive properties. The dynamic compressive moduli (at 0.1 Hz) were determined by oscillatory unconfined compression tests. # indicates different from untreated day 16 controls (p < 0.05). Data are mean + SEM, with n = 6.