Magnetization transfer imaging provides a quantitative measure of chondrogenic differentiation and tissue development

Abstract

The goal of the present investigation was to test whether quantitative magnetization transfer imaging can be used as a noninvasive evaluation method for engineered cartilage. In this work, we used magnetic resonance imaging (MRI) to monitor the chondrogenesis of stem-cell-based engineered tissue over a 3-week period by measuring on a pixel-by-pixel basis the relaxation times (T₁ and T₂), the apparent diffusion coefficient, and the magnetization transfer parameters: bound proton fraction and cross-relaxation rate (k). Tissue-engineered constructs for generating cartilage were created by seeding mesenchymal stem cells in a gelatin sponge. Every 7 days, tissue samples were analyzed using MRI, histological, and biochemical methods. The MRI measurements were verified by histological analysis, and the imaging data were correlated with biochemical analysis of the developing cartilage matrix for glycosaminoglycan content. The MRI analysis for bound proton fraction and k showed a statistically significant increase that was correlated with the increase of glycosaminoglycan (R = 0.96 and 0.87, respectively, p < 0.05), whereas T₁, T₂, and apparent diffusion coefficient results did not show any significant changes over the 3-week measurement period.

FIG. 1.

Magnetization transfer-weighted images of the constructs cultured over a 3-week period, with an in-plane resolution of 62.5 × 62.5 μm and a slice thickness of 125 μm. The offset frequency of the magnetization transfer pulse is 2 kHz. (A) Magnetic resonance image of a single construct after seeding with mesenchymal stem cells but before culture (week 0). (B–D) Magnetic resonance images of pairs of constructs after 1, 2, and 3 weeks in culture, respectively. The growth-stimulated constructs (arrows) have lower overall image intensities compared with the control.

FIG. 2.

Safranin-O staining for GAG and hematoxylin staining for cell nuclei. (A, C, E) Sections of control samples for weeks 1, 2, and 3, respectively. (B, D, F) Sections of the corresponding stimulated samples. The tissue blocks were sectioned with a slice thickness of 5 μm, and photographed under a light microscope (magnification, 40 ×). GAG, glycosaminoglycan. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Collagen type II immunohistochemistry at weeks 1 and 3. (A, C) Sections of control samples at weeks 1 and 3, respectively. (B, D) Sections of the corresponding stimulated construct samples. The tissue blocks were sectioned with a slice thickness of 5 μm and photographed under a light microscope (magnification, 20 ×). Color images available online at www.liebertonline.com/ten.

FIG. 4.

Changes in GAG, BPF, k, T₁, T₂, and ADC during the 3-week growth period (n = 6 for each group). (A) GAG content measured by biochemical analysis. (B) BPF measured by magnetization transfer imaging. (C) Cross-relaxation rate (k) measured by magnetization transfer imaging. (D) Longitudinal relaxation time measured using MRI (T₁). (E) Transverse relaxation time measured using MRI (T₂). (F) ADC measured using MRI. The error bars indicate ± 1 standard deviation. BPF, bound proton fraction; ADC, apparent diffusion coefficient; MRI, magnetic resonance imaging.

FIG. 5.

Graphs showing the relationship between qMTI parameters and GAG content of constructs cultured in the chondrogenic differentiation medium (stimulated group) during the 3-week growth period. (A) BPF plotted versus GAG content. (B) Cross-relaxation rate (k) plotted versus GAG content. The correlation coefficient and its p-value are shown in each graph. The error bars indicate ± 1 standard deviation.