Thyroxine Increases Collagen Type II Expression and Accumulation in Scaffold-Free Tissue-Engineered Articular Cartilage

Abstract

Low collagen accumulation in the extracellular matrix is a pressing problem in cartilage tissue engineering, leading to a low collagen-to-glycosaminoglycan (GAG) ratio and poor mechanical properties in neocartilage. Soluble factors have been shown to increase collagen content, but may result in a more pronounced increase in GAG content. Thyroid hormones have been reported to stimulate collagen and GAG production, but reported outcomes, including which specific collagen types are affected, are variable throughout the literature. Here we investigated the ability of thyroxine (T4) to preferentially stimulate collagen production, as compared with GAG, in articular chondrocyte-derived scaffold-free engineered cartilage. Dose response curves for T4 in pellet cultures showed that 25 ng/mL T4 increased the total collagen content without increasing the GAG content, resulting in a statistically significant increase in the collagen-to-GAG ratio, a fold change of 2.3 ± 1.2, p < 0.05. In contrast, another growth factor, TGFβ1, increased the GAG content in excess of threefold more than the increase in collagen. In large scaffold-free neocartilage, T4 also increased the total collagen/DNA at 1 month and at 2 months (fold increases of 2.1 ± 0.8, p < 0.01 and 2.1 ± 0.4, p < 0.001, respectively). Increases in GAG content were not statistically significant. The effect on collagen was largely specific to collagen type II, which showed a 2.8 ± 1.6-fold increase of COL2A1 mRNA expression (p < 0.01). Western blots confirmed a statistically significant increase in type II collagen protein at 1 month (fold increase of 2.2 ± 1.8); at 2 months, the fold increase of 3.7 ± 3.3 approached significance (p = 0.059). Collagen type X protein was less than the 0.1 μg limit of detection. T4 did not affect COL10A1 and COL1A2 gene expression in a statistically significant manner. Biglycan mRNA expression increased 2.6 ± 1.6-fold, p < 0.05. Results of this study show that an optimized dosage of T4 is able to increase collagen type II content, and do so preferential to GAG. Moreover, the upregulation of COL2A1 gene expression and type II collagen protein accumulation, without a concomitant increase in collagens type I or type X, signifies a direct enhancement of chondrogenesis of hyaline articular cartilage without the induction of terminal differentiation.

Keywords: chondrogenesis; collagen type II; collagen-to-GAG ratio; engineered cartilage; extracellular matrix composition; thyroxine.

Conflict of interest statement

The authors have no financial interests to disclose. This study, less the histomorphometry results, was first published in dissertation form in Characterization of the Frictional Shear Damage Properties of Scaffold-Free Engineered Cartilage and Reduction of Damage Susceptibility by Upregulation of Collagen Content, by G. Adam Whitney, PhD, Case Western Reserve University, November 2014.

Figures

FIG. 1.

Growth factor dose response in micromass culture. T4 (A, C, D) and TGFβ1 (B) dose response for HYP and GAG. In both (A) and (B), change relative to control is fold change, treatment:control. T4 increased the HYP/DNA but not the GAG/DNA at 25 ng/mL (arrow). Asterisks indicated statistical significance compared with the control (0 ng/mL), *p < 0.05, ***p < 0.001, ****p < 0.0001. The mean response of each donor is shown in (C) and (D). GAG, glycosaminoglycan; HYP, hydroxyproline. Color images available online at www.liebertpub.com/tea

FIG. 2.

Tissue characterization and ECM composition. Lines between −T4 and +T4 samples indicate matched neocartilages (same donor) paired for statistical analysis. The mean and standard deviation of each group are overlaid in gray. Table 2 contains p-values from paired t-tests for all groups. ECM, extracellular matrix. Color images available online at www.liebertpub.com/tea

FIG. 3.

ECM gene expression at 1 month. ECM gene expression is relative to 18S gene expression. Lines between −T4 and +T4 samples indicate matched constructs (same donor) paired for statistical analysis. Differences between control and +T4 samples in genes without an indicated p-value were not significant.

FIG. 4.

Histological evaluation. Micrographs of safranin O (saf O) and collagen type I, type II, and type X, immunostained histological cross-sections of native rabbit and TEC. In immunostained images, blue and green coloring is a result of a fast green counterstain, and darker coloring (red, brown, purple) is the result of the peroxidase reaction, indicating detection of the specific collagen type. B, bone; C, cartilage; GP, growth plate; PC, perichondrium; TEC, tissue-engineered cartilage; TM, tide mark. Color images available online at www.liebertpub.com/tea

FIG. 5.

Magnified histological cross-sections showing flattened cells near the construct periphery. Both −T4 (A) and +T4 (B) constructs exhibited some flattened cells (arrows) near the periphery of the constructs. (A, B) are magnified views of the 1-month safranin O-stained images from Figure 4. Color images available online at www.liebertpub.com/tea

FIG. 6.

Histomorphometry of control and T4-treated constructs at 1 and 2 months. (A, C, E) 1-month metrics, (B, D, F) 2-month metrics. A 0–20% depth corresponds to the upper surface of the construct during the initial membrane attached culture, whereas 80–100% depth corresponds to the lower membrane contacting region. Cell area (A, B) increased toward the central regions of the constructs for both control (−T4) and T4-treated (+T4) constructs, whereas cell density was nearly uniform throughout (C, D). Cell area fraction (E, F) also increased in the central region for both −T4 and +T4 constructs, apparently as a result of increased cell area rather than increased cell density. Differences between −T4 and +T4 constructs were minimal, with differences only reaching statistical significance in the cell area fraction metric, and only at the membrane-contacting surface of the construct (80–100% region). There were no statistically significant cell size differences between control and treatment groups when comparing matched depth regions. Color images available online at www.liebertpub.com/tea

FIG. 7.

The effect of T4 on types of collagen in neocartilage. (A) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis showing the presence of type II and type XI collagen in pepsin extracts of TEC. Bovine type II collagen was electrophoresed as a control. (B) Densitometric semiquantitative analysis of type II and type XI collagen in TEC. AUC is the area under the pixel intensity plot curve. (C) Western blot probed with type X collagen antibody in 4 M GuHCl and pepsin extracts of TEC and native auricular cartilage as a positive control. In (A, C) the molecular weights of globular protein standards are shown. GuHCl, guanidine hydrochloride. Color images available online at www.liebertpub.com/tea