Functional attachment of primary neurons and glia on radiopaque implantable biomaterials for nerve repair.

Repairing peripheral nerve injuries remains a challenge, even with use of auxiliary implantable biomaterial conduits. After implantation the location or function of polymeric devices cannot be assessed via clinical imaging modalities. Adding nanoparticle contrast agents into polymers can introduce radiopacity enabling imaging using computed tomography. Radiopacity must be balanced with changes in material properties impacting device function. In this study radiopaque composites were made from polycaprolactone and poly(lactide-co-glycolide) 50:50 and 85:15 with 0-40 wt% tantalum oxide (TaOx) nanoparticles. To achieve radiopacity, ≥5 wt% TaOx was required, with ≥20 wt% TaOx reducing mechanical properties and causing nanoscale surface roughness. Composite films facilitated nerve regeneration in an in vitro co-culture of adult glia and neurons, measured by markers for myelination. The ability of radiopaque films to support regeneration was driven by the properties of the polymer, with 5-20 wt% TaOx balancing imaging functionality with biological response and proving that in situ monitoring is feasible.

Comparisons of 3D printed materials for biomedical imaging applications.

In biomedical imaging, it is desirable that custom-made accessories for restraint, anesthesia, and monitoring can be easily cleaned and not interfere with the imaging quality or analyses. With the rise of 3D printing as a form of rapid prototyping or manufacturing for imaging tools and accessories, it is important to understand which printable materials are durable and not likely to interfere with imaging applications. Here, 15 3D printable materials were evaluated for radiodensity, optical properties, simulated wear, and capacity for repeated cleaning and disinfection. Materials that were durable, easily cleaned, and not expected to interfere with CT, PET, or optical imaging applications were identified.

A guide for selecting 3D printed materials for custom research tools through characterization of their merits and limitations in biomedical imaging.

Effect of mouse strain and diet on feasibility of MRI-based cell tracking in the liver.

PURPOSE: MRI-based cell tracking identifies the location of magnetically labeled cells with hypointense voxels. Here we demonstrate a strain-dependent effect of liver MRI background on the feasibility of MRI-based cell tracking of transplanted cells in the mouse liver. METHODS: FVB mice (GFP-LUC and NOG) and C57BL/6 mice (GFP+ and wild-type) were fed 3 different diets with varying iron content. In vivo T2∗ -weighted images and T2∗ maps of the liver were acquired at different ages. Magnetically labeled cancer cells were injected intrasplenically for hepatic migration; then, mice were imaged by in vivo MRI and bioluminescence imaging. Livers were also imaged ex vivo by magnetic particle imaging. RESULTS: R2∗ increased with age in FVBNOG and FVBGFP-LUC mice that were fed diets sufficient in iron. FVBNOG mice developed a mottled appearance in their livers with age that did not occur in FVBGFP-LUC mice. R2∗ was unchanging with age in C57BL/6GFP mice, and the liver remained bright and homogenous. Labeled cells were not detectable by MRI in some livers despite successful engraftment as shown by bioluminescence imaging and magnetic particle imaging. CONCLUSION: Strain, diet, and age are important considerations for MRI-based cell tracking in the liver. If a model with excessive liver iron must be used, alternative imaging methods such as magnetic particle imaging can be considered.

Ductal tree ablation by local delivery of ethanol prevents tumor formation in an aggressive mouse model of breast cancer.

BACKGROUND: Prophylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant. METHODS: After ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively. RESULTS: We report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value < 0.0001) and reduced tumor incidence (34% of glands with tumors [85 of 250] in the untreated control group vs. 7.3% of glands with tumor [7 of 95] in the ethanol-treated group, risk ratio = 4.76 [95% CI 1.89 to 11.97, p value < 0.0001]). CONCLUSIONS: This preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.

T1 , T1 contrast, and Ernst-angle images of four rat-lung pathologies.

PURPOSE: To initiate the archive of relaxation-weighted images that may help discriminate between pulmonary pathologies relevant to acute respiratory distress syndrome. MRI has the ability to distinguish pathologies by providing a variety of different contrast mechanisms. Lungs have historically been difficult to image with MRI but image quality is sufficient to begin cataloging the appearance of pathologies in T1 - and T2 -weighted images. This study documents T1 and the use of T1 contrast with four experimental rat lung pathologies. METHODS: Inversion-recovery and spoiled steady state images were made at 1.89 T to measure T1 and document contrast in rats with atelectasis, lipopolysaccharide-induced inflammation, ventilator-induced lung injury (VILI), and injury from saline lavage. Higher-resolution Ernst-angle images were made to see patterns of lung infiltrations. RESULTS: T1 -weighted images showed minimal contrast between pathologies, similar to T1 -weighted images of other soft tissues. Images taken shortly after magnetization inversion and displayed with inverted contrast highlight lung pathologies. Ernst-angle images distinguish the effects of T1 relaxation and spin density and display distinctive patterns. T1 for pathologies were: atelectasis, 1.25 ± 0.046 s; inflammation from instillation of lipopolysaccharide, 1.24 ± 0.015 s; VILI, 1.55 ± 0.064 s (p = 0.0022 vs. normal lung); and injury from saline lavage, 1.90±0.080 s (p = 0.0022 vs. normal lung; p = 0.0079 vs. VILI). T1 of normal lung and erector spinae muscle were 1.25 ± 0.028 s and 1.02 ± 0.027 s, respectively (p = 0.0022). CONCLUSIONS: Traditional T1 -weighting is subtle. However, images made with inverted magnetization and inverted contrast highlight the pathologies and Ernst-angle images aid in distinguishing pathologies.

Chimeric mouse model for MRI contrast agent evaluation.

PURPOSE: While rodents are the primary animal models for contrast agent evaluation, rodents can potentially misrepresent human organ clearance of newly developed contrast agents. For example, gadolinium (Gd)-BOPTA has ~50% hepatic clearance in rodents, but ~5% in humans. This study demonstrates the benefit of chimeric mice expressing human hepatic OATPs (organic anion-transporting polypeptides) to improve evaluation of novel contrast agents for clinical use. METHODS: FVB (wild-type) and OATP1B1/1B3 knock-in mice were injected with hepatospecific MRI contrast agents (Gd-EOB-DTPA, Gd-BOPTA) and nonspecific Gd-DTPA. T1 -weighted dynamic contrast-enhanced MRI was performed on mice injected intravenously. Hepatic MRI signal enhancement was calculated per time point. Mass of gadolinium cleared per time point and percentage elimination by means of feces and urine were also measured. RESULTS: Following intravenous injection of Gd-BOPTA in chimeric OATP1B1/1B3 knock-in mice, hepatic MRI signal enhancement and elimination by liver was more reflective of human hepatic clearance than that measured in wild-type mice. Gd-BOPTA hepatic MRI signal enhancement was reduced to 22% relative to wild-type mice. Gd-BOPTA elimination in wild-type mice was 83% fecal compared with 32% fecal in chimeric mice. Hepatic MRI signal enhancement and elimination for Gd-EOB-DTPA and Gd-DTPA were similar between wild-type and chimeric cohorts. CONCLUSION: Hepatic MRI signal enhancement and elimination of Gd-EOB-DTPA, Gd-BOPTA, and Gd-DTPA in chimeric OATP1B1/1B3 knock-in mice closely mimics that seen in humans. This study provides evidence that the chimeric knock-in mouse is a more useful screening tool for novel MRI contrast agents destined for clinical use as compared to the traditionally used wild-type models.

Magnetic resonance imaging provides sensitive in vivo assessment of experimental ventilator-induced lung injury.

Animal models play a critical role in the study of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). One limitation has been the lack of a suitable method for serial assessment of acute lung injury (ALI) in vivo. In this study, we demonstrate the sensitivity of magnetic resonance imaging (MRI) to assess ALI in real time in rat models of VILI. Sprague-Dawley rats were untreated or treated with intratracheal lipopolysaccharide or PBS. After 48 h, animals were mechanically ventilated for up to 15 h to induce VILI. Free induction decay (FID)-projection images were made hourly. Image data were collected continuously for 30 min and divided into 13 phases of the ventilatory cycle to make cinematic images. Interleaved measurements of respiratory mechanics were performed using a flexiVent ventilator. The degree of lung infiltration was quantified in serial images throughout the progression or resolution of VILI. MRI detected VILI significantly earlier (3.8 ± 1.6 h) than it was detected by altered lung mechanics (9.5 ± 3.9 h, P = 0.0156). Animals with VILI had a significant increase in the Index of Infiltration (P = 0.0027), and early regional lung infiltrates detected by MRI correlated with edema and inflammatory lung injury on histopathology. We were also able to visualize and quantify regression of VILI in real time upon institution of protective mechanical ventilation. Magnetic resonance lung imaging can be utilized to investigate mechanisms underlying the development and propagation of ALI, and to test the therapeutic effects of new treatments and ventilator strategies on the resolution of ALI.

Material matters: Degradation products affect regenerating Schwann cells.

Devices to treat peripheral nerve injury (PNI) must balance many considerations to effectively guide regenerating nerves across a gap and achieve functional recovery. To enhance efficacy, design features like luminal fillers have been explored extensively. Material choice for PNI devices is also critical, as the determining factor of device mechanics, and degradation rate and has increasingly been found to directly impact biological response. This study investigated the ways in which synthetic polymer materials impact the differentiation state and myelination potential of Schwann cells, peripheral nerve glia. Microporous substrates of polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15, or PLGA 50:50 were chosen, as materials already used in nerve repair devices, representing a wide range of mechanics and degradation profiles. Schwann cells co-cultured with dorsal root ganglion (DRG) neurons on the substrates expressed more mature myelination proteins (MPZ) on PLGA substrates compared to PCL. Changes to myelination and differentiation state of glia were reflected in adhesion proteins expressed by glia, including ß-dystroglycan and integrin α6, both laminin binding proteins. Importantly, degradation products of the polymers affected glial expression independently of direct attachment. Fast degrading PLGA 50:50 substrates released measurable amounts of degradation products (lactic acid) within the culture period, which may push Schwann cells towards glycolytic metabolism, decreasing expression of early transcription factors like sox10. This study shows the importance of understanding not only material effects on attachment, but also on cellular metabolism which drives myelination responses.

In vivo micro-computed tomography evaluation of radiopaque, polymeric device degradation in normal and inflammatory environments.

Polymeric biomedical implants are an important clinical tool, but degradation remains difficult to determine post-implantation. Computed tomography (CT) could be a powerful tool for device monitoring, but polymers require incorporation of radiopaque contrast agents to be distinguishable from tissue. In addition, immune response to radiopaque devices must be characterized as it modulates device function. Radiopaque devices and films were produced by incorporating 0-20 wt% TaOx nanoparticles into polymers: polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA). In vitro inflammatory responses of mouse bone marrow-derived macrophages to polymer matrix incorporating TaOx nanoparticles was determined by monitoring cytokine secretion. Nanoparticle addition stimulated a slight inflammatory reaction, increasing TNFα secretion, mediated by changes in polymer matrix properties. Subsequently, devices (PLGA 50:50 + 20 wt% TaOx) were implanted subcutaneously in a mouse model of chronic inflammation, that featured a sustained increase in inflammatory response local to the implant site over 12 weeks. No changes to device degradation rates or foreign body response were noted between a normal and chronically stimulated inflammatory environment. Serial CT device monitoring post-implantation provided a detailed timeline of device collapse, with no rapid, spontaneous release of nanoparticles that occluded matrix visualization. Importantly, repeat CT sessions did not ablate the immune system or alter degradation kinetics. Thus, polymer devices incorporating radiopaque nanoparticles can be used for in situ monitoring and be readily combined with other medical imaging techniques, for a dynamic view biomaterial and tissue interactions. STATEMENT OF SIGNIFICANCE: A growing number of implantable devices are in use in the clinic, exposing patients to inherent risks of implant movement, collapse, and infection. The ability to monitor implanted devices would enable faster diagnosis of failure and open the door for personalized rehabilitation therapies - both of which could vastly improve patient outcomes. Unfortunately, polymeric materials which make up most biomedical devices are not radiologically distinguishable from tissue post-implantation. The introduction of radiopaque nanoparticles into polymers allows for serial monitoring via computed tomography, without affecting device degradation. Here we demonstrate for the first time that nanoparticles do not undergo burst release from devices post-implantation and that inflammatory responses - a key determinant of device function in vivo - are also unaffected by nanoparticle addition.

Device Design and Diagnostic Imaging of Radiopaque 3D Printed Tissue Engineering Scaffolds.

3D printed biomaterial implants are revolutionizing personalized medicine for tissue repair, especially in orthopedics. In this study, a radiopaque Bi 2 O 3 doped polycaprolactone ( PCL ) composite is developed and implemented to enable the use of diagnostic X-ray technologies, especially photon counting X-ray computed tomography ( PCCT ), for comprehensive in vivo device monitoring. PCL filament with homogeneous Bi 2 O 3 nanoparticle ( NP ) dispersion (0.8 to 11.7 wt%) are first fabricated. Tissue engineered scaffolds ( TES ) are then 3D printed with the composite filament, optimizing printing parameters for small feature size and severely overhung geometries. These composite TES are characterized via micro-computed tomography ( µ CT ), tensile testing, and a cytocompatibility study, with Bi 2 O 3 mass fractions as low as 2 wt% providing excellent radiographic distinguishability, improved tensile properties, and equivalent cytocompatibility of neat PCL. The excellent radiographic distinguishability is validated in situ by imaging 4 and 7 wt% TES in a mouse model with µCT, showing excellent agreement with in vitro measurements. Subsequently, CT image-derived swine menisci are 3D printed with composite filament and re-implanted in their corresponding swine legs ex vivo . Re-imaging the swine legs via clinical CT allows facile identification of device location and alignment. Finally, the emergent technology of PCCT unambiguously distinguishes implanted menisci in situ.

Radiopaque Implantable Biomaterials for Nerve Repair.

Repairing peripheral nerve injuries remains a clinical challenge. To enhance nerve regeneration and functional recovery, the use of auxiliary implantable biomaterial conduits has become widespread. After implantation, there is currently no way to assess the location or function of polymeric biomedical devices, as they cannot be easily differentiated from surrounding tissue using clinical imaging modalities. Adding nanoparticle contrast agents into polymer matrices can introduce radiopacity and enable imaging using computed tomography (CT), but radiopacity must be balanced with changes in material properties that impact device function and biological response. In this study radiopacity was introduced to porous films of polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA) 50:50 and 85:15 with 0-40wt% biocompatible tantalum oxide (TaO x ) nanoparticles. To achieve radiopacity, at least 5wt% TaO x was required, with ≥ 20wt% TaO x leading to reduced mechanical properties and increased nano-scale surface roughness of films. As polymers used for peripheral nerve injury devices, films facilitated nerve regeneration in an in vitro co-culture model of glia (Schwann cells) and dorsal root ganglion neurons (DRG), measured by expression markers for myelination. The ability of radiopaque films to support nerve regeneration was determined by the properties of the polymer matrix, with a range of 5-20wt% TaO x balancing both imaging functionality with biological response and proving that in situ monitoring of nerve repair devices is feasible.

In vivo Biomedical Imaging of Immune Tolerant, Radiopaque Nanoparticle-Embedded Polymeric Device Degradation.

Biomedical implants remain an important clinical tool for restoring patient mobility and quality of life after trauma. While polymers are often used for devices, their degradation profile remains difficult to determine post-implantation. CT monitoring could be a powerful tool for in situ monitoring of devices, but polymers require the introduction of radiopaque contrast agents, like nanoparticles, to be distinguishable from native tissue. As device function is mediated by the immune system, use of radiopaque nanoparticles for serial monitoring therefore requires a minimal impact on inflammatory response. Radiopaque polymer composites were produced by incorporating 0-20wt% TaOx nanoparticles into synthetic polymers: polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA). In vitro inflammatory response to TaOx was determined by monitoring mouse bone marrow derived macrophages on composite films. Nanoparticle addition stimulated only a slight inflammatory reaction, namely increased TNFα secretion, mediated by changes to the polymer matrix properties. When devices (PLGA 50:50 + 20wt% TaOx) were implanted subcutaneously in a mouse model of chronic inflammation, no changes to device degradation were noted although macrophage number was increased over 12 weeks. Serial CT monitoring of devices post-implantation provided a detailed timeline of device structural collapse, with no burst release of the nanoparticles from the implant. Changes to the device were not significantly altered with monitoring, nor was the immune system ablated when checked via blood cell count and histology. Thus, polymer devices incorporating radiopaque TaOx NPs can be used for in situ CT monitoring, and can be readily combined with multiple medical imaging techniques, for a truly dynamic view biomaterials interaction with tissues throughout regeneration, paving the way for a more structured approach to biomedical device design.

Omniparticle Contrast Agent for Multimodal Imaging: Synthesis and Characterization in an Animal Model.

PURPOSE: Individual imaging modalities have certain advantages, but each suffers from drawbacks that other modalities may overcome. The goal of this study was to create a novel contrast agent suitable for various imaging modalities that after a single administration can bridge and strengthen the collaboration between the research fields as well as enrich the information obtained from any one modality. PROCEDURES: The contrast agent platform is based on dextran-coated iron oxide nanoparticles (for MRI and MPI) and synthesized using a modified co-precipitation method, followed by a series of conjugation steps with a fluorophore (for fluorescence and photoacoustic imaging), thyroxine (for CT imaging), and chelators for radioisotope labeling (for PET imaging). The fully conjugated agent was then tested in vitro in cell uptake, viability, and phantom studies and in vivo in a model of intraductal injection and in a tumor model. RESULTS: The agent was synthesized, characterized, and tested in vitro where it showed the ability to produce a signal on MRI/MPI/FL/PA/CT and PET images. Studies in cells showed the expected concentration-dependent uptake of the agent without noticeable toxicity. In vivo studies demonstrated localization of the agent to the ductal tree in mice after intraductal injection with different degrees of resolution, with CT being the best for this particular application. In a model of injected labeled tumor cells, the agent produced a signal with all modalities and showed persistence in tumor cells confirmed by histology. CONCLUSIONS: A fully functional omniparticle contrast agent was synthesized and tested in vitro and in vivo in two animal models. Results shown here point to the generation of a potent signal in all modalities tested without detrimental toxicity. Future use of this agent includes its exploration in various models of human disease including image-guided diagnostic and therapeutic applications.

Incorporating Tantalum Oxide Nanoparticles into Implantable Polymeric Biomedical Devices for Radiological Monitoring.

Longitudinal radiological monitoring of biomedical devices is increasingly important, driven by the risk of device failure following implantation. Polymeric devices are poorly visualized with clinical imaging, hampering efforts to use diagnostic imaging to predict failure and enable intervention. Introducing nanoparticle contrast agents into polymers is a potential method for creating radiopaque materials that can be monitored via computed tomography. However, the properties of composites may be altered with nanoparticle addition, jeopardizing device functionality. Thus, the material and biomechanical responses of model nanoparticle-doped biomedical devices (phantoms), created from 0-40 wt% tantalum oxide (TaOx ) nanoparticles in polycaprolactone and poly(lactide-co-glycolide) 85:15 and 50:50, representing non, slow, and fast degrading systems, respectively, are investigated. Phantoms degrade over 20 weeks in vitro in simulated physiological environments: healthy tissue (pH 7.4), inflammation (pH 6.5), and lysosomal conditions (pH 5.5), while radiopacity, structural stability, mechanical strength, and mass loss are monitored. The polymer matrix determines overall degradation kinetics, which increases with lower pH and higher TaOx content. Importantly, all radiopaque phantoms could be monitored for a full 20 weeks. Phantoms implanted in vivo and serially imaged demonstrate similar results. An optimal range of 5-20 wt% TaOx nanoparticles balances radiopacity requirements with implant properties, facilitating next-generation biomedical devices.

Incorporating Radiopacity into Implantable Polymeric Biomedical Devices for Clinical Radiological Monitoring.

Longitudinal radiological monitoring of biomedical devices is increasingly important, driven by risk of device failure following implantation. Polymeric devices are poorly visualized with clinical imaging, hampering efforts to use diagnostic imaging to predict failure and enable intervention. Introducing nanoparticle contrast agents into polymers is a potential method for creating radiopaque materials that can be monitored via computed tomography. However, properties of composites may be altered with nanoparticle addition, jeopardizing device functionality. This, we investigated material and biomechanical response of model nanoparticle-doped biomedical devices (phantoms), created from 0-40wt% TaO x nanoparticles in polycaprolactone, poly(lactide-co-glycolide) 85:15 and 50:50, representing non-, slow and fast degrading systems, respectively. Phantoms degraded over 20 weeks in vitro, in simulated physiological environments: healthy tissue (pH 7.4), inflammation (pH 6.5), and lysosomal conditions (pH 5.5), while radiopacity, structural stability, mechanical strength and mass loss were monitored. The polymer matrix determined overall degradation kinetics, which increased with lower pH and higher TaO x content. Importantly, all radiopaque phantoms could be monitored for a full 20-weeks. Phantoms implanted in vivo and serially imaged, demonstrated similar results. An optimal range of 5-20wt% TaO x nanoparticles balanced radiopacity requirements with implant properties, facilitating next-generation biomedical devices.

Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming.

Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, delayed cellular changes were observed that are not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric l-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. The results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models.

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most effective intervention is prophylactic mastectomy. This is a drastic surgical procedure in which the mammary epithelial cells that can give rise to breast cancer are completely removed along with the surrounding tissue. The goal of this protocol is to demonstrate the feasibility of a minimally invasive intraductal procedure that could become a new primary intervention for breast cancer prevention. This local procedure would preferentially ablate mammary epithelial cells before they can become malignant. Intraductal methods to deliver solutions directly to these epithelial cells in rodent models of breast cancer have been developed at Michigan State University and elsewhere. The rat mammary gland consists of a single ductal tree that has a simpler and more linear architecture compared to the human breast. However, chemically induced rat models of breast cancer offer valuable tools for proof-of-concept studies of new preventive interventions and scalability from mouse models to humans. Here, a procedure for intraductal delivery of an ethanol-based ablative solution containing tantalum oxide nanoparticles as X-ray contrast agent and ethyl cellulose as gelling agent into the rat mammary ductal tree is described. Delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) by intraductal injection has been described previously in mouse and rat models. This protocol description emphasizes methodological changes and steps that pertain uniquely to delivering an ablative solution, formulation consideration to minimize local and systemic side effects of the ablative solution, and X-ray imaging for in vivo assessment of ductal tree filling. Fluoroscopy and micro-CT techniques enable to determine the success of ablative solution delivery and the extent of ductal tree filling thanks to compatibility with the tantalum-containing contrast agent.

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models.

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

Design Considerations to Facilitate Clinical Radiological Evaluation of Implantable Biomedical Structures.

Clinical effectiveness of implantable medical devices would be improved with in situ monitoring to ensure device positioning, determine subsequent damage, measure biodegradation, and follow healing. While standard clinical imaging protocols are appropriate for diagnosing disease and injury, these protocols have not been vetted for imaging devices. This study investigated how radiologists use clinical imaging to detect the location and integrity of implanted devices and whether embedding nanoparticle contrast agents into devices can improve assessment. To mimic the variety of devices available, phantoms from hydrophobic polymer films and hydrophilic gels were constructed, with and without computed tomography (CT)-visible TaOx and magnetic resonance imaging (MRI)-visible Fe3O4 nanoparticles. Some phantoms were purposely damaged by nick or transection. Phantoms were implanted in vitro into tissue and imaged with clinical CT, MRI, and ultrasound. In a blinded study, radiologists independently evaluated whether phantoms were present, assessed the type, and diagnosed whether phantoms were damaged or intact. Radiologists identified the location of phantoms 80% of the time. However, without incorporated nanoparticles, radiologists correctly assessed damage in only 54% of cases. With an incorporated imaging agent, the percentage jumped to 86%. The imaging technique which was most useful to radiologists varied with the properties of phantoms. With benefits and drawbacks to all three imaging modalities, future implanted devices should be engineered for visibility in the modality which best fits the treated tissue, the implanted device's physical location, and the type of required information. Imaging protocols should also be tailored to best exploit the properties of the imaging agents.

Complex Relationship Between Iron Oxide Nanoparticle Degradation and Signal Intensity in Magnetic Particle Imaging.

Magnetic particle imaging (MPI), using superparamagnetic nanoparticles as an imaging tracer, is touted as a quantitative biomedical imaging technology, but MPI signal properties have never been characterized for magnetic nanoparticles undergoing biodegradation. We show that MPI signal properties can increase or decrease as iron oxide nanoparticles degrade, depending on the nanoparticle formulation and nanocrystal size, and degradation rate and mechanism. Further, we show that long-term in vitro MPI experiments only roughly approximate long-term in vivo MPI signal properties. Further, we demonstrate for the first time, an environmentally sensitive MPI contrast mechanism opening the door to smart contrast paradigms in MPI.