Spatiotemporal Characterization of Extracellular Matrix Microstructures in Engineered Tissue: A Whole-Field Spectroscopic Imaging Approach.

Microstructures and integrity of the ECM of engineered tissue are critical to their quality and functionality [1–3]. Moreover, decellularized ECM scaffolds have also received considerable attention as a promising approach for engineering functional organ replacement [4,5]. Thus, quantitative assessments of the ECM and scaffold microstructures are of great significance for tissue engineering and regenerative medicine. However, currently available assessment methods, such as electron microscopy, optical microscopy, and equilibrium swelling, are often labor-intensive and destructive. Any assays using exogenous probes also reduce usable volumes of tissue and further require toxicity tests for in vivo implantation. In addition, a better understanding of the dynamic nature of ECM microstructures originating from cell-ECM interactions and matrix remodeling is critical to generate biomimetic tissue constructs [6]. Using conventional methods, it is, however, challenging to characterize spatiotemporal changes in ECM microstructures. Besides research and scientific purposes, rapid and high-throughput assessments of the ECM or scaffold microstructures are highly relevant to quality control of engineered tissue in industrial and clinical settings. For example, cryopreservation of engineered tissue has been actively pursued to provide reliable long-term storage and off-the-shelf availability of cell/tissue engineering products for clinical use. Since many of the functional properties of tissue are associated with ECM microstructures, post-thaw ECM microstructures can be a crucial marker for the functionality of the post-thaw tissue. Recent studies reported that ECM microstructures could be affected by freezing-induced cell-fluid-matrix interactions [3,7]. Thus, rapid and nondestructive assessments of post-thaw ECM structures can be valuable tools to confirm quality of cryopreserved tissue prior to their use. Moreover, engineered tissue in clinically relevant size is large, but conventional bioassays and imaging methods can only evaluate a small fraction of the total area. Conventional methods require a large number of images to be stitched together to cover a large tissue area, which can be computationally intensive and time-consuming. To address these challenges, we propose a new imaging method on the basis of an elastic light scattering approach. The underlying rationale is that elastic light scattering spectra (i.e., scattering without change in the wavelength) are sensitive to scattering structures in size comparable to the wavelength [8–10]. The principle of this method is that the spectral pattern of light elastically reflected from tissue is determined by the structures of light scatterers. In particular, spectral behavior of elastic light scattering depends on the size distribution of scatterers. Typically, the light scattering spectrum from biological tissue is a declining function of the wavelength and its steepness depends on the overall size distribution of scattering microstructures. Larger structures generally yield slower declining spectra, while smaller structures tend to increase the steepness of the decline. Spectral patterns of elastic light scattering have been intensively used to detect cancer [10–12]. It was also demonstrated that a light scattering approach could capture nanoscale prefailure deformation of bone at extremely low strains [13]. In this study, we employ this approach to spatiotemporally characterize ECM microstructures in engineered tissue. Specifically, it is tested whether spectroscopic measurements of light elastically backscattered from engineered tissue can be correlated to ECM structural changes. First, we combine this approach with a whole-field spectroscopic imaging platform to demonstrate the feasibility for a nondestructive, whole-field, and sensitive spatiotemporal characterization method. Acellular collagen scaffolds and dermal equivalents (i.e., fibroblast-embedded collagen gels) are used as model ECM structures. Second, backscattering spectra from collagen scaffolds are obtained and analyzed to characterize microstructural alterations in the collagen matrix due to collagen concentration and freezing-induced structural changes. Third, spatiotemporal changes in ECM structures during cell-driven compaction are assessed using dermal equivalents. Finally, we discuss the potential utilization of the proposed imaging approach for ECM microstructure characterization in engineered tissue.