Nanocomposite scaffold for chondrocyte growth and cartilage tissue engineering: effects of carbon nanotube surface functionalization

Abstract

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair.

FIG. 1.

(A) Time-dependent viability (% live cells) of chondrocytes in 2D culture after 3, 7, or 14 days of coculture with functionalized SWNTs (-COOH or -PEG) at 0.01 or 0.1 mg/mL (n=5 per group; *p<0.05 versus SM and 0.1 mg/mL group of corresponding functionalization, within time point. ^p<0.05 versus D3 within group). (B-G) Representative live (green) images of chondrocytes in 2D culture at D7 (scale bar=40 μm). 2D, two-dimensional; SWNTs, single-wall carbon nanotubes. Color images available online at www.liebertpub.com/tea

FIG. 2.

Histological evaluation of chondrocytes grown in pellet culture with functionalized SWNTs (-COOH or -PEG) at 0.01 or 0.1 mg/mL at day 14. (A–E) Alcian blue staining, indicative of proteoglycan deposition; (F–J) Picrosirius red staining, indicative of collagen deposition (scale bar=50 μm); (K–M) SEM evaluation of chondrocytes in pellet culture in control or SWNT groups (at 0.1 mg/mL). Color images available online at www.liebertpub.com/tea

FIG. 3.

Representative images of construct morphology (A–C), live (D–F), dead (G–I), and composite live/dead (J–L) staining at day 7 of chondrocytes cultured in hydrogel-SWNT nanocomposite scaffolds (scale bar=50 μm). No significant change in construct morphology or cell viability was observed at D7 in culture in nanocomposite scaffolds at 0.01% w/v (0.1 mg/mL) SWNTs. Color images available online at www.liebertpub.com/tea

FIG. 4.

Quantitative PCR gene expression of chondrocytes cultured in Control (no SWNTs), 0.1 mg/mL SWNT-COOH, or 0.1 mg/mL SWNT-PEG nanocomposite scaffolds up to 35 days in culture. Expression of (a) Agc, (b) Col2a1, (c) Col10a1, (d) Col1a1, (e) Fn, (f) CD44, and (g) Tp53 was quantified (n=3 per group, *p<0.05 versus control, **p<0.01 versus control, ***p<0.001 versus control).

FIG. 5.

(A) Glycosaminoglycan (GAG) content (% of wet weight) and (B) compressive material properties (Young's modulus) of nanocomposite scaffolds composed of control (no SWNTs) or functionalized SWNT group (-COOH or -PEG) at 0.1 mg/mL up to 35 days in culture (n=4–6 per group). ^p<0.05 versus day 0 within group; *p<0.05 versus control at corresponding time point; ⁺p<0.05 versus PEG at corresponding time point.