MRI method for labeling and imaging decellularized extracellular matrix scaffolds for tissue engineering

Abstract

Purpose To develop a facile method for labeling and imaging decellularized extracellular matrix (dECM) scaffolds intended for regenerating 3D tissues. Methods A small molecule manganese porphyrin, MnPNH2, was synthesized and used to label dECM scaffolds made from porcine bladder and trachea and murine whole lungs. The labeling protocol was optimized on bladder dECM, and imaging on a 3T clinical scanner was performed to assess reductions in T1 and T2 relaxation times. In vivo MRI was performed on dECM injected in the rat dorsum to verify sensitivity of detection. Toxicity assays for cell viability, metabolism, and proliferation were performed on human umbilical vein endothelial cells. The incorporation of MnPNH2 and its long‐term retention in dECM were assessed on transmission electron microscopy and ultraviolet absorbance of eluted MnPNH2 over time. Results All tissues, including thick whole 3D organs, were uniformly labeled and demonstrated high signal‐to‐noise on MRI. A nearly 10‐fold reduction in T1 was consistently obtained at a labeling dose of 0.4 mM, and even 0.2 mM provided sufficient contrast in vivo and ex vivo. No toxicity was observed up to 0.4 mM, the maximum tested. Binding studies suggested nonspecific association, and retention studies in the labeled whole decellularized lungs revealed less than 20% MnPNH2 loss over 30 days, the majority occurring in the first 3 days after labeling. Conclusion The proposed labeling method is the first report for visualizing dECM on MRI and has the potential for long‐term monitoring and optimization of dECM‐based organ tissue engineering.

Three‐Dimensional Bioprinted MR‐Trackable Regenerative Scaffold for Postimplantation Monitoring on T1‐Weighted MRI

“…The synthesis and preparation of MnPNH 2 , our MR contrast agent for scaffold labeling, were optimized and characterized in our previous studies. 15,16 Extending our previous protocol, we were able to label bioprinted scaffolds in a single step. Unlike other methods that utilize complex processing techniques and bio-incompatible solvents, 20,21 our protocol involves simply dissolving the contrast agent in PBS +/+ or cell media and submerging the bioprinted scaffold in the labeling solution.…”

“…The synthesis and characterization of the contrast agent used in this study, MnPNH 2 , are described in full in previously published studies. 15,16 In brief, the precursor 5-(4-aminophenyl)-10,15, 20-(triphenyl)porphyrin was dissolved in concentrated sulfuric acid and heated at 75 C to form the intermediate product 5-(4-aminophenyl)-10,15,20-(tri-4-sulfonatophenyl)porphyrin (Apo-PNH 2 ). Apo-PNH 2 was purified by dialysis (regenerated cellulose, MWCO: 1 kDa) and then reacted with MnCl 2 and DIPEA in dimethylformamide solvent at 135 C for 3 hours to form MnPNH 2 .…”

“…Longer lasting biomaterial (eg collagen) would not face the same challenge, and these materials have been described previously by our group. 15,16 Other limitations include small sample sizes in our in vivo study and the absence of a systemic toxicity assessment.…”

“…12 To avert toxicity issues associated with traditional gadolinium agents, 13 manganese-based contrast agents are a good alternative, being both safe and efficacious. 14 Several reports have described the application of manganese-based agents for labeling and tracking natural scaffolds, 15,16 but the extension to a 3D bioprinting workflow remains uncharted.…”

Capturing effects of blood flow on the transplanted decellularized nephron with intravital microscopy

“…Decellularization is a promising organ bioengineering strategy that supports the development of the bioartificial kidney[2]. Angiographic[3] and histologic[4] techniques have examined whole kidney scaffold structure and function in various settings. Such investigations have provided evidence that the decellularization process successfully removes native cellular components and preserves the macrovascular and extracellular matrix (ECM) architectures.…”