The tissue engineering community has been vocal regarding the need for noninvasive instruments to assess the development of tissue-engineered constructs. Medical imaging has helped fulfill this role. However, specimens allocated to a test tube for imaging cannot be tested for a prolonged period or returned to the incubator. Therefore, samples are essentially wasted due to potential contamination and transfer in a less than optimal growth environment. In turn, we present a standalone, miniature, magnetic resonance imaging-compatible incubator, termed the e-incubator. This incubator uses a microcontroller unit to automatically sense and regulate physiological conditions for tissue culture, thus allowing for concurrent tissue culture and evaluation. The e-incubator also offers an innovative scheme to study underlying mechanisms related to the structural and functional evolution of tissues. Importantly, it offers a key step toward enabling real-time testing of engineered tissues before human transplantation. For validation purposes, we cultured tissue-engineered bone constructs for 4 weeks to test the e-incubator. Importantly, this technology allows for visualizing the evolution of temporal and spatial morphogenesis. In turn, the e-incubator can filter deficient constructs, thereby increasing the success rate of implantation of tissue-engineered constructs, especially as construct design grows in levels of complexity to match the geometry and function of patients' unique needs.
Myocarditis is an inflammation of the myocardium, but only -10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-? 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-? 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.
Traumatic brain injury (TBI) presents a variety of causes and symptoms, thus making the development of reliable diagnostic methods and therapeutic treatments challenging. Magnetic resonance elastography (MRE) is a technique that allows for a noninvasive assessment of the mechanical properties of soft biological tissue, such as tissue stiffness, storage modulus, and loss modulus. Importantly, by quantifying the changes in the stiffness of tissue that is often associated with disease, MRE is able to detect tissue pathologies at early stages. Recent improvements in instrumentation have allowed for the investigation of small samples with microscopic resolution (?MRE). We hypothesize that ?MRE can sensitively detect variations in micromechanical properties in the brain caused by the compressive and shearing forces sustained during TBI. To test this hypothesis, we randomized 13 C57BL mice to receive a controlled cortical impact at a 0.5?mm or 0.75?mm depth, with both sham and naïve mice as controls. Our objective was to propagate mechanical shear waves throughout the brain for in vivo TBI ?MRE imaging. The mechanical properties of the injured brain tissue were determined at days 0, 1, 7, and 28 post-injury. For both groups, we observed a significant drop in the stiffness of the impacted region immediately following the injury; the 0.75?mm animals experienced increased tissue softness that lasted longer than that for the 0.5?mm group. Although the shear stiffness, storage modulus, and loss modulus parameters all followed the same trend, the tissue stiffness yielded the most statistically significant results. Overall, this article introduces a transformative technique for mechanically mapping the brain and detecting brain diseases and injury.
Millions of cases of bone injury or loss due to trauma, osteoporosis, and cancer occur in the United States each year. Because bone is limited in its ability to regenerate, alternative therapy approaches are needed. Bone tissue engineering has the potential to correct musculoskeletal disorders through the development of cell-based substitutes for osteogenic tissue replacement. Multiple medical imaging techniques such as magnetic resonance microscopy (MRM) were investigated recently; these techniques are able to provide useful information on the anatomical and structural changes of developing bone. However, there is a need for noninvasive approaches to evaluate biochemical constituents and consequent compositional development associated with growing osteogenic constructs. In this study, near-infrared (NIR) optical imaging with a bone-specific NIR-targeted probe, IRDye(®) 800CW BoneTag™ (800CW BT), was applied in this study to longitudinally visualize regions of mineralization of tissue-engineered bone constructs in vivo. A fluorescent cell-based assay was performed to confirm the preferential binding of 800CW BT to the mineralized matrix of differentiated osteogenically driven human mesenchymal stem cells (hMSCs) in vitro. The hMSCs were seeded onto a biocompatible gelatin scaffold, allowed to develop, and implanted into a mouse model. Engineered constructs were examined in vivo using NIR imaging for bone mineralization, paired with MRM for verification of developing tissue. Results showed that NIR imaging with 800CW BT labeling can effectively assess the calcification of the developing osteogenic constructs, which is consistent with the analysis of excised tissue using NIR microscopy and histology. In conclusion, this study evaluated bone-like function of regenerating bone through tracking calcium deposition via NIR optical imaging with a fluorophore-labeled probe in a noninvasive manner.
Primary brain tumors present significant challenges for surgical resection because of their location and the frequent occurrence of malignant projections extending beyond the primary tumor. Visualization of the tumor margins during surgery is critical for a favorable outcome. We report the use of IRDye 800CW chlorotoxin (CLTX) as a targeted imaging agent for brain tumors in a spontaneous mouse model of medulloblastoma, ND2:SmoA1. Specificity and functionality of the targeted agent were confirmed in cell-based assays. Tumors were detected by magnetic resonance imaging and IRDye 800CW CLTX administered to individual animals for optical imaging at 1-month increments. The integrity of the blood-brain barrier (BBB) was measured by Evans Blue perfusion prior to sacrifice. Results show that IRDye 800CW CLTX specifically targeted tumor tissue. The extravasation of Evans Blue was observed in all tumors, suggesting that the presence of the tumors can introduce alterations in the permeability of the BBB. Because increased vascular permeability was observed early in the disease model, larger dye-labeled imaging agents that exceed current BBB size restrictions may warrant renewed consideration as candidates for tumor detection and surgical resection. Our study provides data characterizing in vitro and in vivo use of IRDye 800CW CLTX as a broadly applicable tumor imaging agent.
Traumatic brain injury (TBI) is a major cause of death and disability for which there is no cure. One of the issues inhibiting clinical trial success is the lack of targeting specific patient populations due to inconsistencies between clinical diagnostic tools and underlying pathophysiology. The development of reliable, noninvasive markers of TBI severity and injury mechanisms may better identify these populations, thereby improving clinical trial design. Magnetic resonance elastography (MRE), by assessing tissue mechanical properties, can potentially provide such marker. MRE synchronizes mechanical excitations with a phase contrast imaging pulse sequence to noninvasively register shear wave propagation, from which local values of tissue viscoelastic properties can be deduced. The working hypothesis of this study is that TBI involves a compression of brain tissue large enough to bring the material out of its elastic range, sufficiently altering mechanical properties to generate contrast on MRE measurements. To test this hypothesis, we combined microscopic MRE with brain tissue collected from adult male rats subjected to a controlled cortical impact injury. Measurements were made in different regions of interest (somatosensory cortex, hippocampus, and thalamus), and at different time points following the injury (immediate, 24 h, 7 days, 28 days). Values of stiffness in the somatosensory cortex were found to be 23-32% lower in the injured hemisphere than in the healthy one, when no significant difference was observed in the case of sham brains. A preliminary in vivo experiment is also presented, as well as alternatives to improve the faithfulness of stiffness recovery.
The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high-field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue-engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue-engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE.
We have developed a multi-layer approach for the synthesis of water-dispersible superparamagnetic iron oxide nanoparticles for hyperthermia, magnetic resonance imaging (MRI) and drug delivery applications. In this approach, iron oxide core nanoparticles were obtained by precipitation of iron salts in the presence of ammonia and provided ?-cyclodextrin and pluronic polymer (F127) coatings. This formulation (F127250) was highly water dispersible which allowed encapsulation of the anti-cancer drug(s) in ?-cyclodextrin and pluronic polymer for sustained drug release. The F127250 formulation has exhibited superior hyperthermia effects over time under alternating magnetic field compared to pure magnetic nanoparticles (MNP) and ?-cyclodextrin coated nanoparticles (CD200). Additionally, the improved MRI characteristics were also observed for the F127250 formulation in agar gel and in cisplatin resistant ovarian cancer cells (A12780CP) compared to MNP and CD200 formulations. Furthermore, the drug-loaded formulation of F127250 exhibited many folds of imaging contrast properties. Due to the internalization capacity of the F127250 formulation, its curcumin-loaded formulation (F127250-CUR) exhibited almost equivalent inhibition effects on A2780CP (ovarian), MDA-MB-231 (breast), and PC-3 (prostate) cancer cells even though curcumin release was only 40%. The improved therapeutic effects were verified by examining molecular effects using Western blotting and transmission electron microscopic (TEM) studies. F127250-CUR also exhibited haemocompatibility, suggesting a nanochemo-therapeutic agent for cancer therapy.
The next generation magnetic nanoparticles (MNPs) with theranostic applications have attracted significant attention and will greatly improve nanomedicine in cancer therapeutics. Such novel MNP formulations must have ultra-low particle size, high inherent magnetic properties, effective imaging, drug targeting, and drug delivery properties. To achieve these characteristic properties, a curcumin-loaded MNP (MNP-CUR) formulation was developed.
Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development. The method proposed in the following protocol uses microscopic magnetic resonance elastography (?MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner. Recently, ?MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map. The resulting measurements at each voxel have been shown to strongly correlate (R(2)>0.9914) with data collected using dynamic mechanical analysis. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.