Biomaterial scaffolds are the cornerstone to supporting 3D tissue growth. Optimized scaffold design is critical to successful regeneration, and this optimization requires accurate knowledge of the scaffold's interaction with living tissue in the dynamic in vivo milieu. Unfortunately, non‐invasive methods that can probe scaffolds in the intact living subject are largely underexplored, with imaging‐based assessment relying on either imaging cells seeded on the scaffold or imaging scaffolds that have been chemically altered. In this work, the authors develop a broadly applicable magnetic resonance imaging (MRI) method to image scaffolds directly. A positive‐contrast “bright” manganese porphyrin (MnP) agent for labeling scaffolds is used to achieve high sensitivity and specificity, and polydopamine, a biologically derived universal adhesive, is employed for adhering the MnP. The technique was optimized in vitro on a prototypic collagen gel, and in vivo assessment was performed in rats. The results demonstrate superior in vivo scaffold visualization and the potential for quantitative tracking of degradation over time. Designed with ease of synthesis in mind and general applicability for the continuing expansion of available biomaterials, the proposed method will allow tissue engineers to assess and fine‐tune the in vivo behavior of their scaffolds for optimal regeneration.

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.

Magnetic resonance imaging (MRI) provides superior resolution of anatomical features and the best soft tissue contrast, and is one of the predominant imaging modalities. With this technique, contrast agents are often used to aid discrimination by enhancing specific features. Over the years, a rich diversity of such agents has evolved and with that, so has a need to systematically sort contrast agents based on their efficiency, which directly determines sensitivity. Herein, we present a scale to rank MRI contrast agents. The scale is based on analytically determining the minimum detectable concentration of a contrast agent, and employing a ratiometric approach to standardize contrast efficiency to a benchmark contrast agent. We demonstrate the approach using several model contrast agents and compare the relative sensitivity of these agents for the first time. As the first universal metric of contrast agent sensitivity, this scale will be vital to easily assessing contrast agent efficiency and thus important to promoting use of some of the elegant and diverse contrast agents in research and clinical practice.

MRI for non-invasive cell tracking is recognized for enabling pre-clinical research on stem cell therapy. Yet, adoption of cellular imaging in stem cell research has been restricted to sites with experience in MR contrast agent synthesis and to small animal models that do not require scaled-up synthesis. In this study, we demonstrate the use of a gadolinium-free T1 contrast agent for tracking human embryonic stem cells. The agent, MnPNH2, is an easily synthesized manganese porphyrin that can be scaled for large cell numbers. MRI was performed on a 3 T clinical scanner. Cell pellets labeled at different MnPNH2 concentrations for 24 hours demonstrated a decrease in T1 relaxation time of nearly two-fold (P < 0.05), and cellular contrast was maintained for 24 hours (P < 0.05). Cell viability (Trypan blue) and differentiation (embryoid body formation) were unaffected. Cell uptake of Mn on inductively coupled plasma atomic emission spectroscopy corroborated MRI findings, and fluorescence microscopy revealed the agent localized mainly in cell-cell boundaries and cell nuclei. Labeled cells transplanted in rats demonstrated the superior sensitivity of MnPNH2 for in-vivo cell tracking.

BackgroundA noninvasive method to track implanted biomaterials is desirable for real‐time monitoring of material interactions with host tissues and assessment of efficacy and safety.PurposeTo explore quantitative in vivo tracking of polyurethane implants using a manganese porphyrin (MnP) contrast agent containing a covalent binding site for pairing to polymers.Study TypeProspective, longitudinal.Animal ModelRodent model of dorsal subcutaneous implants (10 female Sprague Dawley rats).Field Strength/SequenceA 3‐T; two‐dimensional (2D) T1‐weighted spin‐echo (SE), T2‐weighted turbo SE, three‐dimensional (3D) spoiled gradient‐echo T1 mapping with variable flip angles.AssessmentA new MnP‐vinyl contrast agent to covalently label polyurethane hydrogels was synthesized and chemically characterized. Stability of binding was assessed in vitro. MRI was performed in vitro on unlabeled hydrogels and hydrogels labeled at different concentrations, and in vivo on rats with unlabeled and labeled hydrogels implanted dorsally. In vivo MRI was performed at 1, 3, 5, and 7 weeks postimplantation. Implants were easily identified on T1‐weighted SE, and fluid accumulation from inflammation was distinguished on T2‐weighted turbo SE. Implants were segmented on contiguous T1‐weighted SPGR slices using a threshold of 1.8 times the background muscle signal intensity; implant volume and mean T1 values were then calculated at each timepoint. Histopathology was performed on implants in the same plane as MRI and compared to imaging results.Statistical TestsUnpaired t‐tests and one‐way analysis of variance (ANOVA) were used for comparisons. A P value <0.05 was considered to be statistically significant.ResultsHydrogel labeling with MnP resulted in a significant T1 reduction in vitro (T1 = 517 ± 36 msec vs. 879 ± 147 msec unlabeled). Mean T1 values of labeled implants in rats increased significantly by 23% over time, from 1 to 7 weeks postimplantation (651 ± 49 msec to 801 ± 72 msec), indicating decreasing implant density.Data ConclusionPolymer‐binding MnP enables in vivo tracking of vinyl‐group coupling polymers.Evidence Level1.Technical EfficacyStage 1.

Background: A three-dimensional (3D) bioprinted tissue scaffold is a promising therapeutic that goes beyond providing physical support for tissue regeneration by enabling precise spatial control over scaffold geometry and integration of different materials/cells. Critically important is in vivo confirmation of correct scaffold placement and retention during the initial 24 hours postimplantation, to detect unwanted implant migration. Purpose: To incorporate a safe, efficient MR contrast agent into a bioprinting workflow, and to achieve bright-contrast scaffold monitoring in vivo postimplantation. Study Type: In vitro and animal in vivo longitudinal study. Animal Model: Two female Sprague Dawley rats (~200 g) for labeled and unlabeled scaffold implantation in the subcutaneous dorsal space flanking the vertebral column. Field Strength/Sequence: A 7.0 T/T 1 -weighted spin echo (SE) sequence and T 1 mapping using turbo SE with variable repetition times (TRs). Assessment: Cell viability and proliferation were assessed over 2 weeks after labeling bioprinted gelatin/alginate scaffolds with MnPNH 2 (0.5 mM, 24 hours). In vitro MRI was performed 0, 12, and 24 hours postlabeling in nine labeled and three unlabeled (control) scaffolds to monitor T 1 evolution. In vivo MRI was performed immediately and 24 hours postimplantation to assess T 1 . Acute inflammation near surgical site was monitored in one rat to 3 days. Statistical Tests: One-way analysis of variance with Tukey-Kramer post hoc analysis (P < 0.01). Results: Cell viability was unaffected by bioprinting/labeling: viability exceeded 90% in all scaffolds after 1 week. In vitro T 1 's were significantly lower in labeled scaffolds compared to control (207 msec vs. 2257 msec) immediately postlabeling and 24 hours later (1227 msec vs. 2257 msec). In vivo T 1 's were significantly different (243.6 msec vs. 2414.6 msec) immediately postimplantation, and no differences emerged compared to respective in vitro control/labeled counterparts. The 24-hours imaging and gross pathology confirmed migration of scaffolds beyond the imaging field. Data Conclusion: We report an MR-detectable, cell-compatible bioprinted scaffold, utilizing a T 1 -weighting contrast agent for high-resolution, postimplantation scaffold tracking.

Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4-deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing.

Summary A major unresolved challenge in cell-based regenerative medicine is the absence of non-invasive technologies for tracking cell fate in deep tissue and with high spatial resolution over an extended interval. MRI is highly suited for this task, but current methods fail to provide longitudinal monitoring or high sensitivity, or both. In this study, we fill this technological gap with the first discovery and demonstration of in vivo cellular production of endogenous bright contrast via an MRI genetic reporter system that forms manganese-ferritin nanoparticles. We demonstrate this technology in human embryonic kidney cells genetically modified to stably overexpress ferritin and show that, in the presence of manganese, these cells produce far greater contrast than conventional ferritin overexpression with iron or manganese-permeable cells. In living mice, diffusely implanted bright-ferritin cells produce the highest and most sustained contrast in skeletal muscle. The bright-ferritin platform has potential for on-demand, longitudinal, and sensitive cell tracking in vivo .