Influence of temporary chondroitinase ABC-induced glycosaminoglycan suppression on maturation of tissue-engineered cartilage

Abstract

Objective: A fundamental challenge of cartilage tissue engineering has been the inability to promote collagen synthesis up to native levels. In contrast, recent protocols have demonstrated that glycosaminoglycans (GAG) can be synthesized to native levels in 4-6 weeks of in vitro culture. We hypothesize that rapid GAG synthesis may be an impediment to collagen synthesis, possibly by altering transport pathways of nutrients or synthesis products. In this study, this hypothesis is tested by inducing enzymatic GAG loss in the early culture period of cartilage tissue constructs, and monitoring collagen content at various time points after cessation of enzymatic treatment.

Methods: In Study 1, to induce breakdown of proteoglycans, chondroitinase ABC (CABC, 0.002U/mL) was continuously added into the culture media for the initial 4 weeks of culture or for 2 weeks starting on day 14 of culture. In Study 2, multiple transient CABC treatments (0.15U/mL, for 2 days) were applied to the matured tissue-engineered constructs.

Results: Continuous and transient CABC treatments significantly increased the collagen concentration of the constructs, improving their tensile properties. The GAG content of the treated constructs recovered quickly to the pretreatment level after 2-3 weeks.

Conclusions: This study demonstrates that tissue-engineered cartilage constructs with improved tensile properties can be achieved by temporarily suppressing the GAG content enzymatically.

FIG. 1.

Temporal application of CABC or agarase in the treatment groups in Study 1 (A) and Study 2 (B). Study 2 was extended to 105 days to permit recovery from the second enzymatic treatment. The thick line indicates transient TGF β3 administration.

FIG. 2.

Study 1: (A) equilibrium modulus and (B) GAG content normalized to wet weight of the construct. (C) Total collagen content per construct. (D) Collagen content normalized to wet weight. *p < 0.005 versus Control, n = 4.

FIG. 3.

Study 1: Safranin-O staining of the histological sections on day 28 and day 56. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Study 2: (A) equilibrium modulus and (B) dynamic modulus (0.1 Hz) of the constructs. (C) GAG content and (D) collagen content of the constructs (normalized to the wet weight of the constructs). (E) DNA content of the constructs. (F) Volume of the constructs normalized by the day 0 level. *p < 0.005 versus pretreatment level (day 35 Cont, and day 58 CABC1T and Agarase1T). **p < 0.005 versus control. ^†p < 0.005 versus all other groups (n = 4).

FIG. 5.

Study 2: failure stress (A) and tensile modulus (B) of the constructs on day 105. ^♦p < 0.05 versus all other groups, n = 5.

FIG. 6.

Study 2: Safranin-O staining of the Control (A), CABCT2T (B, C), and Agarase2T (D, E). Picrosirius Red staining of the Control (F), CABCT2T (G, H), and Agarase2T (I, J). Color images available online at www.liebertonline.com/ten.