Magnetic resonance studies of macromolecular content in engineered cartilage treated with pulsed low-intensity ultrasound

Abstract

Noninvasive monitoring of matrix development in tissue-engineered cartilage constructs would permit ongoing assessment with the ability to modify culture conditions during development to optimize tissue characteristics. In this study, chondrocytes seeded in a collagen hydrogel were exposed for 20 min/day to pulsed low-intensity ultrasound (PLIUS) at 30 mWcm(-2) and cultured for up to 5 weeks. Biochemical assays, histology, immunohistochemistry, Fourier transform infrared spectroscopy, and magnetic resonance imaging (MRI) were performed at weeks 3 and 5 after initiation of growth. The noninvasive MRI measurements were correlated with those from the invasive studies. In particular, MRI transverse relaxation time (T2) and magnetization transfer rate (k(m)) correlated with macromolecular content, which was increased by application of PLIUS. This indicates the sensitivity of MR techniques to PLIUS-induced changes in matrix development, and highlights the potential for noninvasive assessment of the efficacy of anabolic interventions for engineered tissue.

FIG. 1.

Effect of pulsed low-intensity ultrasound (PLIUS) treatment on sulfated glycosaminoglycan (sGAG) content of chondrocyte-seeded collagen I scaffolds cultured for 3 and 5 weeks (n = 6 per group). There was an increase in sGAG content with time in culture and PLIUS treatment. *p < 0.05 versus 3-week control.

FIG. 2.

Effect of PLIUS treatment on total collagen (% wet weight) content of control and PLIUS-treated chondrocyte-seeded collagen I scaffolds cultured for 3 and 5 weeks (n = 6 per group). Collagen concentration increased with time in culture; however, the effect of PLIUS on collagen concentration varied with the age of the construct. *p < 0.05.

FIG. 3.

Water content (% wet weight) of control and PLIUS-treated chondrocyte-seeded collagen scaffolds cultured for 3 and 5 weeks (n = 10 per group). Water content decreased in the week 3 but increased in week 5 with PLIUS treatment. Overall, water content decreased significantly with time in culture. *p < 0.05.

FIG. 4.

Light micrographs of (A) Safranin O-stained and (B) Alcian blue-stained control and PLIUS-treated sections of chondrocyte-seeded collagen scaffolds cultured for 3 and 5 weeks (100 × ). Top row, 3 weeks; lower row, 5 weeks. Treated samples showed more intense staining than control samples. Also, the 5-week constructs showed more intense staining than the 3-week constructs. Both of these provide evidence of time-associated and PLIUS-induced increase in proteoglycan (PG) content. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Light micrographs of immunohistochemical stains for (A) collagen type I, (B) collagen type II, (C) collagen type X, and (D) matrix metalloproteinase 13 (MMP-13) in sections obtained from control and PLIUS-treated chondrocyte-seeded collagen scaffolds cultured for 3 and 5 weeks (100 × ). Top row, 3 weeks; lower row, 5 weeks. Overall staining for collagen types I and X and MMP-13 was weak. Collagen type X and MMP-13 stain intensities were further decreased with PLIUS treatment. In contrast, collagen II immunostain was strong for all groups and increased with time in culture. More intense pericellular collagen II staining was observed in the 5-week PLIUS-treated group. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Effect of PLIUS treatment on (A) PG content as measured by the area under the PG absorbance at 858 cm⁻¹ and (B) collagen content measured by area under the collagen absorbance at 1338 cm⁻¹ (n = 2 for week 3 and n = 3 for week 5). The integrated area under the PG absorbance increased in the PLIUS-treated samples. In contrast, the area under the collagen absorbance increased in week 3 but slightly decreased in week 5 after PLIUS treatment. ^#p < 0.05 versus 5-week control.

FIG. 7.

Effect of PLIUS treatment on (A) T1, (B) k_m, and (C) T2 measurements of 3- and 5-week collagen constructs (n = 10 per group). Although there were no significant changes in T1 with PLIUS, there was a trend toward decreased T1 with culture time. k_m increased while T2 decreased with time in culture, but the effects of PLIUS on k_m and T2 were dependent on the age of the construct. *p < 0.05 versus 3-week control, ^#p < 0.05 versus 5-week control.