Stukel Jessica M, Goss Monika, Zhou Haoyan, Zhou Wenda, Willits Rebecca Kuntz, Exner Agata A
Department of Biomedical Engineering, The University of Akron, Akron, OH, 44325-0302, USA.
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA.
Ann Biomed Eng. 2016 Mar;44(3):793-802. doi: 10.1007/s10439-015-1507-0. Epub 2015 Nov 17.
Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90 ± 2.58 × 10(-5) a.u. compared to non-fiber forming intensity at 2.74 ± 0.36 × 10(-5) a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5.00 ± 0.80 × 10(-5) a.u. in 1 day live cells compared to 2.26 ± 0.39 × 10(-5) a.u.in fixed cells at a concentration of 1 × 10(6) cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings.
基于水凝胶的组织工程构建体的发展速度很快，但向患者应用的转化却很缓慢。需要多年昂贵的临床前测试来预测这些装置的临床性能和安全性。这些测试具有侵入性，会破坏样本，而且在许多情况下并不能代表最终的体内情况。生物医学成像有潜力通过直接在原位进行纵向非侵入性装置表征来促进生物材料的开发。在各种可用的成像方式中，超声因其低成本、广泛可用性和良好的安全性而成为一个优秀的候选者。这项工作的总体目标是证明临床超声在纵向表征支持细胞生长的3D水凝胶基质中的效用。具体而言，我们开发了一种使用临床B型超声的定量技术，以区分聚乙二醇（PEG）水凝胶中的胶原蛋白含量和成纤维细胞密度，并在体外模型环境中对其进行了验证。通过控制水凝胶的凝胶化过程，发现含有胶原纤维的凝胶与不形成纤维的胶原蛋白的凝胶之间在超声信号强度上存在差异，这表明该技术对蛋白质的构型敏感。在胶原蛋白密度为2.5mg/mL时，形成纤维的胶原蛋白的信号强度显著增加，为14.90±2.58×10(-5) a.u.，而不形成纤维的胶原蛋白的强度为2.74±0.36×10(-5) a.u.。此外，还发现活的和成纤维细胞之间在强度上存在差异，活细胞中的信号强度增加（在含有胶原蛋白的凝胶中，1天的活细胞中为5.00±0.80×10(-5) a.u.，而固定细胞中为2.26±0.39×10(-5) a.u.，细胞浓度为1×10(6)个细胞/mL）。总体而言，随着细胞密度的增加，超声强度的线性相关性>0.90。结果证明了在组织模拟模型中使用临床超声表征基于PEG的水凝胶的可行性。该方法具有临床相关性，经过进一步验证后，可用于在临床前和临床环境中随时间无损监测植入的组织工程支架的体内性能。
