Gudur Madhu Sudhan Reddy, Rao Rameshwar R, Peterson Alexis W, Caldwell David J, Stegemann Jan P, Deng Cheri X
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS One. 2014 Jan 22;9(1):e85749. doi: 10.1371/journal.pone.0085749. eCollection 2014.
Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5-15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10(4) at day 1 to 0.9±0.2×10(4) at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.
为了将这些产品从实验室转化到临床，需要对工程组织进行无损监测。在本研究中，使用非侵入性高分辨率光谱超声成像（SUSI）监测接种在胶原水凝胶中的MC3T3前成骨细胞的分化。SUSI用于测量在对照或成骨培养基中培养21天的此类构建体中散射体的直径、浓度和声衰减。对平行样本进行传统生化分析以确定DNA含量和钙沉积。使用超声准确成像构建体的体积和形态。使用SUSI估计细胞直径约为12.5 - 15.5 µm，这与荧光染色细胞的测量结果非常吻合。通过DNA含量定量评估的每个构建体的细胞总数从第1天的5.6±2.4×10⁴减少到第21天的0.9±0.2×10⁴。SUSI对等效声散射体数量的估计显示出类似的下降趋势，但在成骨样本的第21天除外，此时散射体数量和声阻抗均显著增加，提示分化的MC3T3细胞发生了矿物质沉积。SUSI估计的钙含量为41.7±11.4 µg/ml，与生化测量值38.7±16.7 µg/ml非常吻合。参数值的彩色编码图叠加在B模式图像上，以显示细胞直径和钙沉积的时空变化。本研究证明了使用无损超声成像来提供关于嵌入三维工程构建体中细胞数量和分化状态的定量信息，因此为工程组织发育的纵向监测提供了一个有价值的工具。
