Chondroitinase ABC treatment results in greater tensile properties of self-assembled tissue-engineered articular cartilage

Abstract

Collagen content and tensile properties of engineered articular cartilage have remained inferior to glycosaminoglycan (GAG) content and compressive properties. Based on a cartilage explant study showing greater tensile properties after chondroitinase ABC (C-ABC) treatment, C-ABC as a strategy for cartilage tissue engineering was investigated. A scaffold-less approach was employed, wherein chondrocytes were seeded into non-adherent agarose molds. C-ABC was used to deplete GAG from constructs 2 weeks after initiating culture, followed by 2 weeks culture post-treatment. Staining for GAG and type I, II, and VI collagen and transmission electron microscopy were performed. Additionally, quantitative total collagen, type I and II collagen, and sulfated GAG content were measured, and compressive and tensile mechanical properties were evaluated. At 4 wks, C-ABC treated construct ultimate tensile strength and tensile modulus increased 121% and 80% compared to untreated controls, reaching 0.5 and 1.3 MPa, respectively. These increases were accompanied by increased type II collagen concentration, without type I collagen. As GAG returned, compressive stiffness of C-ABC treated constructs recovered to be greater than 2 wk controls. C-ABC represents a novel method for engineering functional articular cartilage by departing from conventional anabolic approaches. These results may be applicable to other GAG-producing tissues functioning in a tensile capacity, such as the musculoskeletal fibrocartilages.

FIG. 1.

Representative gross and histological pictures of self-assembled tissue constructs for all groups. Two-week control (A–E), 2-week chondroitinase C-ABC treated (F–J), 4-week control (K–O), 4-week C-ABC treated (P–T). Ruler markings = 1 mm in A, F, K, P, and the scale bar = 200 μm in T applies to all histological images. Histological images were taken at 100X, and safranin-O/fast green was used to stain for glycosaminoglycan (GAG). Note the loss of GAG after C-ABC treatment (G), the return of GAG staining at 4 weeks (Q), and the absence of type I collagen staining in all treatment groups (C, H, M, R). A picture of an immature native articular cartilage explant and staining of immature native articular cartilage tissue are provided for comparison. The inset in the lower left of W is bovine tendon stained for collagen type I. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Representative transmission electron microscopy images: 2-week control (A, E, I), 2-week C-ABC treatment (B, F, J), 4-week control (C, G, K), 4-week C-ABC treatment (D, H, L). (A–D) Magnification 5,000X, (E–H) magnification 30,000X, (I–L) magnification 75,000X. Cupromeronic blue stains the GAGs black.

FIG. 3.

(A) Construct sulfated GAG (sGAG) concentration and (B) compressive stiffness. Total sGAG normalized to wet weight of each group was significantly different from all others. The same was seen for the aggregate modulus. In both panels, groups not connected by the same letter are significantly different from one another (p < 0.05).

FIG. 4.

Type II collagen normalized to wet weight in the 4-week C-ABC-treated group was significantly greater than in 4-week controls. Groups not connected by the same letter are significantly different from one another (p < 0.05).

FIG. 5.

(A) Ultimate tensile strength and (B) tensile modulus. The ultimate tensile strength (UTS) and apparent Young's modulus (E_Y) were significantly greater at 4 weeks in the C-ABC-treated group (121% and 80%, respectively). (A) Groups not connected by the same letter are significantly different from one another. (B) *indicates significantly different from all other groups (p < 0.05).