Hepatic organic anion transporting polypeptides (OATPs) transport off-the-shelf, FDA-approved, hepatospecific Gd-based MRI contrast agents into cells that express the transporters enhancing signal on clinical T1-weighted MRI. Studies have used MRI to identify OATP-overexpressing tumors and metastases in mice following the delivery of Gd-EOB-DTPA at 27-67-fold higher than clinical doses. With safety and regulatory concern over Gd-based contrast agents, translation of this imaging paradigm to humans for regenerative medicine cell therapy will require substantially lower doses of agent. Here we complemented static T1-weighted MRI and/or T1-mapping with dynamic contrast enhanced (DCE)-MRI and show that even low intracellular accumulation of contrast agent results in a sustained signal enhancement in OATP-overexpressing tumors while control, non-expressing tumors have rapid wash-in and wash-out dynamics which could be distinguished by performing area-under-the-curve (AUC) analyses.

3D printed biomaterial implants are revolutionizing personalized medicine for tissue repair, especially in orthopedics. In this study, a radiopaque bismuth oxide (Bi2O3) doped polycaprolactone (PCL) composite is developed and implemented to enable the use of diagnostic X-ray technologies, especially spectral photon counting X-ray computed tomography (SPCCT), for comprehensive tissue engineering scaffold (TES) monitoring. PCL filament with homogeneous Bi2O3 nanoparticle (NP) dispersion (0.8 to 11.7 wt%) are first fabricated. TES are then 3D printed with the composite filament, optimizing printing parameters for small features and severely overhung geometries. These composite TES are characterized via micro-computed tomography (μCT), tensile testing, and a cytocompatibility study, with 2 wt% Bi2O3 NPs providing improved tensile properties, equivalent cytocompatibility to neat PCL, and excellent radiographic distinguishability. Radiographic performance is validated in situ by imaging 4 and 7 wt% Bi2O3 doped PCL 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 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 SPCCT unambiguously distinguishes the implanted meniscus in situ via means of color K-edge imaging.

Background: Protease-activated receptor-1 (PAR1) has emerged as an important link between coagulation and the complications of obesity including metabolic dysfunction- associated steatotic liver disease (MASLD). PAR1 is expressed by various cells and cleaved by different proteases to generate unique tethered agonists that activate distinct signaling pathways. Mice expressing PAR1 with an R41Q mutation have disabled canonical thrombin-mediated signaling, whereas R46Q mice express PAR1 resistant to noncanonical signaling by activated protein C. Methods: Mice with whole body and hepatocyte-selective PAR1 deficiency as well as PAR1 R41Q and R46Q mice were fed a high-fat diet (HFD) to induce MASLD. Results: HFD-fed R41Q mice displayed reduced hepatic steatosis and liver/body weight ratio. In contrast, HFD-fed R46Q mice displayed increased relative liver weight and hepatic steatosis alongside increased serum alanine aminotransferase activity. Surprisingly, despite the distinct impact of PAR1 mutations on steatosis, selective deletion of PAR1 in hepatocytes had no impact. To evaluate a viable PAR1-targeted approach, mice with HFD-induced obesity were treated with the allosteric PAR1 modulator NRD21, which inhibits canonical PAR1 inflammatory signaling but promotes PAR1 protective, noncanonical anti-inflammatory signaling. NRD-21 treatment reduced plasma tumor necrosis factor-alpha, serum alanine aminotransferase activity, hepatic steatosis, and insulin resistance (Homeostatic Model Assessment for Insulin Resistance) but increased plasma active glucagon-like peptide-1. Conclusion: The results suggest that nonhepatocellular canonical PAR1 cleavage drives MASLD in obese mice and provide translational proof-of-concept that selective pharmacologic modulation of PAR1 yields multiple metabolic benefits in experimental obesity.

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 β-dystroglycan and integrin α 6 , 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.

Abstract There are limited options for primary prevention of breast carcinoma (BC). Prophylactic mastectomy and systemic hormonal therapy are only recommended for high-risk individuals. Prophylactic mastectomy is a drastic surgery that removes the entire breast, including the epithelial cell-lined ductal trees where BC arises but also uninvolved stroma. Even with a 90% risk reduction, many women are deterred from prophylactic mastectomy due to its associated risk of infection, lymphedema, painful recovery time, and mental health toll. Hormonal therapies, tamoxifen and raloxifene, reduce BC risk by upwards of 50% but also increase the risk of uterine cancer and stroke. Thus, new interventions to reduce the risk of BC and minimize deterrents are necessary. Intraductal (ID) injection is a minimally invasive procedure that avoids systemic toxicity by cannulating a needle directly into the ductal tree opening to directly administer a solution to the epithelial cells. We previously reported that ID infusion of 70% ethanol (EtOH) as a cell-killing solution prevents tumor formation in an aggressive genetically engineered mouse model of BC with minimum collateral tissue damage. Here, we investigated the therapeutic efficacy of EtOH-based ablative solutions in a Methyl-N-Nitrosourea (MNU) chemically induced rat model of BC to assess scalability of this procedure towards clinical trials. Two weeks after tumor induction, rats received ID infusions of a solution containing 70% EtOH to ablate epithelial cells, ethyl cellulose (EC) to minimize collateral tissue damage, and/or Tantalum Oxide (TaOx) to visualize infused ductal trees via micro-CT imaging. EtOH-infused glands had the longest tumor latency (273 days vs 97 days in control group, p < 0.0001). No statistically significant differences were noted in epithelial ablation or collateral tissue damage between treatment groups receiving ID injections of 70% EtOH with varying concentrations of EC, but there was a trend to lower collateral damage with EC-containing solutions. Local clearance of TaOx in infused mammary glands enabled post-procedure MR imaging surveillance after 15 d of ID procedure. This preclinical study demonstrates that ID injection of a refined solution containing 70% EtOH and TaOx is an efficacious and scalable procedure for primary prevention of BC. Citation Format: Erin Zaluzec, Mohamed Ashry, Elizabeth G. Phelps, Katarzyna Kempinska, Elizabeth Kenyon, Legend Kenny, Max Volk, Katherine Powell, Jeremy ML Hix, Christiane Mallett, Matti Kiupel, Erik M. Shapiro, Lorenzo F. Sempere. Intraductal ablative procedure for local preventive intervention in rat models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3389.

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% TaO x 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 TaO x nanoparticles was determined by monitoring cytokine secretion. Nanoparticle addition stimulated a slight inflammatory reaction, increasing TNF alpha secretion, mediated by changes in polymer matrix properties. Subsequently, devices (PLGA 50:50 + 20 wt% TaO x ) 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. (c) 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

There are limited options for primary prevention of breast cancer (BC). Experimental procedures to locally prevent BC have shown therapeutic efficacy in animal models. To determine the suitability of FDA-approved iodine-containing and various metal-containing (bismuth, gold, iodine, or tantalum) preclinical nanoparticle-based contrast agents for image-guided intraductal (ID) ablative treatment of BC in rodent models, we performed a prospective longitudinal study to determine the imaging performance, local retention and systemic clearance, safety profile, and compatibility with ablative solution of each contrast agent. At least six abdominal mammary glands (>3 female FVB/JN mice and/or Sprague-Dawley rats, 10–11 weeks of age) were intraductally injected with commercially available contrast agents (Omnipaque® 300, Fenestra® VC, MVivoTM Au, MVivoTM BIS) or in-house synthesized tantalum oxide (TaOx) nanoparticles. Contrast agents were administered at stock concentration or diluted in 70% ethanol (EtOH) and up to 1% ethyl cellulose (EC) as gelling agent to assess their compatibility with our image-guided ablative procedure. Mammary glands were serially imaged by microCT for up to 60 days after ID delivery. Imaging data were analyzed by radiologists and deep learning to measure in vivo signal disappearance of contrast agents. Mammary glands and major organs were ultimately collected for histopathological examination. TaOx-containing solutions provided best imaging performance for nitid visualization of ductal tree immediately after infusion, low outward diffusion (<1 day) and high homogeneity. Of all nanoparticles, TaOx had the highest local clearance rate (46% signal decay as stock and 36% as ablative solution 3 days after ID injection) and exhibited low toxicity. TaOx-containing ablative solution with 1% EC caused same percentage of epithelial cell death (88.62% ± 7.70% vs. 76.38% ± 9.99%, p value = 0.089) with similar minimal collateral damage (21.56 ± 5.28% vs. 21.50% ± 7.14%, p value = 0.98) in mouse and rat mammary glands, respectively. In conclusion, TaOx-nanoparticles are a suitable and versatile contrast agent for intraductal imaging and image-guided ablative procedures in rodent models of BC with translational potential to humans.

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 ( µCT ), 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.

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 β-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.

Description of antibodies used for protein detection

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 (TaOx) nanoparticles. To achieve radiopacity, at least 5wt% TaOxwas required, with ≥ 20wt% TaOxleading 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% TaOxbalancing both imaging functionality with biological response and proving that in situ monitoring of nerve repair devices is feasible.

Immunohistochemical characterization of HS cell lines CD204 and CD18 are standard cell surface markers expressed by HS in dogs. Newly established HS cell lines were positive with both CD204 and CD18 by IHC of sections of cell pellets. CD3 and CD79a were used to rule out a lymphoid phenotype, for which all cell lines were negative. Calibration bar: 100 μm.

Images of metastatic sites of the HS xenograft mice

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.

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.

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.

Breast cancer (BC) is the leading cancer diagnosis in women. While many BC treatment options exist, only two methods are approved for prevention. Prophylactic mastectomy is an invasive surgery that removes the breast, thereby removing the targeted epithelial ductal tree cells, where most BC arises. Despite a 90% risk reduction, recovery time, risk of infection, pain management, and mental health toll on women deter them from this surgery. Hormonal preventatives, tamoxifen and raloxifene, reduce BC risk by up to 50%. However, increased risk of uterine cancer and intolerable sides effect deter women from this option. Therefore, new preventative methods are needed to maintain BC risk reduction while minimizing current BC prevention deterrents. Intraductal (ID) injection is a technique that can circumvent systemic toxicity by inserting a needle directly into the ductal tree opening. We study BC prevention using 70% ethanol (EtOH) as a cell killing, ablative solution for ID injections and have shown its effectiveness of ablation in an aggressive genetically engineered mouse model with minimum collateral tissue damage. EtOH is a safe, inexpensive reagent clinically used as a sclerosing and ablative agent. We introduced ethyl cellulose (EC), a gelling agent which further minimized damage outside the ductal tree. To confirm successful ductal tree filling without animal sacrifice, we introduced Tantalum Oxide (TaOx), a nanoparticle-based contrast agent for real time and long-term visualization of ductal tree filling. TaOx showed remarkable local retention, without impeding 70% EtOH ablative effects or EC compared to other contrast agents. Despite our refined ablative solution, ID injection is a difficult technique to perform with limited ability to confirm success of a fully filled ductal tree. To elucidate any impact of partial ductal tree filling, we utilized a Methyl-N-Nitrosourea (MNU) rat model. Rat models improve scalability toward clinical trials and increase ductal tree filling volume and control to study the impact of partial ductal tree filling. Our MNU rat model provides a time frame between carcinogen exposure and tumor formation optimal for prevention studies. Two weeks after intraperitoneal MNU injections, rats received full or half volume (partial) injections, fat pad (partial), or no injection into individual mammary glands. Rats were monitored for tumor formation from 2 weeks post injection until reaching euthanasia criteria. Fully injected glands had the highest tumor latency increase by 1 month (130 days, p < 0.0001) compared to non-injected controls (95 days). Partial and fat pad tumor latency also improved compared to control (122 days, p <0.01 and 110 days, p <0.05 respectively). No iatrogenic effects of EtOH were observed. Here, we show that ID injection of 70% EtOH is a safe, effective, and scalable technique for BC prevention, which can be enhanced with TaOx and EC for visualization and retention. Citation Format: Erin K. Zaluzec, Mohamed Ashry, Elizabeth Kenyon, Elizabeth G. Phelps, Legend Kenney, Katherine Powell, Maximilian Volk, Shatadru Chakravarty, Jeremy M.L. Hix, Matti Kiupel, Erik Shapiro, Lorenzo F. Sempere. Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4247.

Immunohistochemical characterization of histiocytic diseases in humans and dogs

General characteristics and genetic profile of cell lines