Comparison of micro-CT image enhancement after use of different vascular casting agents.

Background: Microvascular visualization is crucial in understanding the mechanisms of several pathologies. For instance, visualization of the tumor microenvironment is important in understanding angiogenesis and role in cancer progression. Visualization would provide insights to cancer diagnosis, predicting metastatic growth, and evaluating therapeutic protocols. Similarly, understanding the microvascular network could be beneficial for study of degenerative diseases and tissue repair. The use of microscale computed tomography (micro-CT) and vascular casting agents provides high-resolution images of tissue vasculature in volumetric space. The purpose of this research was to compare a selection of commercially available contrast agents to determine the optimal solution for vascular visualization. Methods: A population of 16 female nude athymic mice (Charles Rivers Laboratories) were implanted with MDA-MB-231 breast cancer cells (ATCC) orthotopically in the lower left mammary fat pad to investigate the tumor microenvironment. Once tumors reach sufficient size, animals were equally divided into four groups based on the micro-CT agent to be administered, namely, control (no contrast agent), barium sulfate (BaSO4), Vascupaint, or Microfil. Animals were anesthetized prior to transcarotid micro-cannulation to infuse 2 mL of the specific contrast agent for intravascular distribution throughout the animal. The jugular vein on the other side of the carotid artery was opened to drain blood flow. Following successful perfusion, animals and extracted organs underwent high-resolution micro-CT scanning (OI/CT, MILabs). Images were reconstructed and analyzed using analysis software to extract mean intensity signals. Results: Preliminary post-mortem micro-CT results reveal Vascupaint and BaSO4 are useful for microvascular visualization. Both Vascupaint and BaSO4 produced significant contrast-enhanced micro-CT image enhancement in the brain (3.39±0.93 and 6.27±3.78, respectively) and kidney (12.85±1.98 and 32.87±10.03, respectively) as compared to Microfil (0.22±0.07 and 0.91±0.63, respectively; P<0.05). For the various contrast agents, there were no differences in image enhancement from the liver, spleen, or tumor tissue (P>0.21). Moreover, use of Vascupaint and BaSO4 allowed for visualization of smaller microvascular structures with average diameters of 20.54±4.15 and 25.82±3.75 µm, which were smaller compared to the 91.66±24.91 µm measurements from Microfil-enhanced micro-CT images (P<0.004). Conclusions: Our study suggests that the use of Vascupaint and BaSO4 is more than sufficient for ex vivo visualization of microvascular structures with contrast-enhanced micro-CT imaging as these contrast agents more effectively perfused smaller blood vessels.

Multiparametric ultrasound imaging for early-stage steatosis: Comparison with magnetic resonance imaging-based proton density fat fraction.

BACKGROUND: The prevalence of liver diseases, especially steatosis, requires a more convenient and noninvasive tool for liver diagnosis, which can be a surrogate for the gold standard biopsy. Magnetic resonance (MR) measurement offers potential, however ultrasound (US) has better accessibility than MR. PURPOSE: This study aims to suggest a multiparametric US approach which demonstrates better quantification and imaging performance than MR imaging-based proton density fat fraction (MRI-PDFF) for hepatic steatosis assessment. METHODS: We investigated early-stage steatosis to evaluate our approach. An in vivo (within the living) animal study was performed. Fat inclusions were accumulated in the animal livers by feeding a methionine and choline deficient (MCD) diet for 2 weeks. The animals (n = 19) underwent US and MR imaging, and then their livers were excised for histological staining. From the US, MR, and histology images, fat accumulation levels were measured and compared: multiple US parameters; MRI-PDFF; histology fat percentages. Seven individual US parameters were extracted using B-mode measurement, Burr distribution estimation, attenuation estimation, H-scan analysis, and shear wave elastography. Feature selection was performed, and the selected US features were combined, providing quantification of fat accumulation. The combined parameter was used for visualizing the localized probability of fat accumulation level in the liver; This procedure is known as disease-specific imaging (DSI). RESULTS: The combined US parameter can sensitively assess fat accumulation levels, which is highly correlated with histology fat percentage (R = 0.93, p-value < 0.05) and outperforms the correlation between MRI-PDFF and histology (R = 0.89, p-value < 0.05). Although the seven individual US parameters showed lower correlation with histology compared to MRI-PDFF, the multiparametric analysis enabled US to outperform MR. Furthermore, this approach allowed DSI to detect and display gradual increases in fat accumulation. From the imaging output, we measured the color-highlighted area representing fatty tissues, and the fat fraction obtained from DSI and histology showed strong agreement (R = 0.93, p-value < 0.05). CONCLUSIONS: We demonstrated that fat quantification utilizing a combination of multiple US parameters achieved higher performance than MRI-PDFF; therefore, our multiparametric analysis successfully combined selected features for hepatic steatosis characterization. We anticipate clinical use of our proposed multiparametric US analysis, which could be beneficial in assessing steatosis in humans.

A Model of Immediate Implant Placement to Evaluate Early Osseointegration in 129/Sv Diabetic Mice.

PURPOSE: To analyze the process of early oral osseointegration of titanium (Ti) implants in diabetic 129/Sv mice through microCT and histologic and immunohistochemical analysis. MATERIALS AND METHODS: A group of 30 male 129/Sv mice was equally subdivided into two groups: (1) nondiabetic (ND), in which mice did not undergo systemic alterations and received a standard diet, and (2) diabetic (D), in which mice were provided a high-fat diet from the age of 6 weeks until the conclusion of the study and received two intraperitoneal (IP) injections of streptozotocin (STZ) at a concentration of 100 mg/Kg each. Each mouse underwent extraction of a maxillary first molar, and customized Ti screws (0.50 mm diameter, 1.5 mm length) were placed in the residual alveolar sockets of the palatal roots. At 7 and 21 days after implant placement, the animals were euthanized for maxilla and pancreas collection. Maxillae containing Ti implants were analyzed with microCT, histology, and immunohistochemistry for cells that were positive for F4/80, CD146, runt-related transcription factor 2 (Runx2), and proliferating cell nuclear antigen (PCNA). Pancreata were histologically analyzed. Quantitative data were statistically analyzed with a significance level at 5% (P < .05). RESULTS: ND mice presented successful healing and osseointegration, with a significantly higher fraction of bone volume compared to D mice, both at the alveolar sockets (53.39 ± 5.93 and 46.08 ± 3.18, respectively) and at the implant sites (68.88 ± 7.07 and 44.40 ± 6.98, respectively) 21 days after implant placement. Histologic evaluation revealed that the ND mice showed a significant decrease in inflammatory infiltrate and a significant increase in newly formed bone matrix at 21 days, whereas peri-implant sites in the D mice were predominantly encapsulated by fibrous tissue and chronic inflammatory infiltrate. Immunohistochemical characterization revealed higher Runx2 osteoblast differentiation and higher cell proliferation activity in the ND mice at 7 days, while higher amounts of macrophages were present in D mice at 7 and 21 days. Interestingly, no differences were found in CD146-positive cells when comparing ND and D mice. CONCLUSIONS: This study evaluated the effects of immediate dental implant placement in 129/Sv diabetic mice by using specific healing markers to identify changes in cellular events involved in early oral osseointegration. This approach may serve as tool to evaluate new materials and surface coatings to improve osseointegration in diabetic patients.

Optical blood-brain-tumor barrier modulation expands therapeutic options for glioblastoma treatment.

The treatment of glioblastoma has limited clinical progress over the past decade, partly due to the lack of effective drug delivery strategies across the blood-brain-tumor barrier. Moreover, discrepancies between preclinical and clinical outcomes demand a reliable translational platform that can precisely recapitulate the characteristics of human glioblastoma. Here we analyze the intratumoral blood-brain-tumor barrier heterogeneity in human glioblastoma and characterize two genetically engineered models in female mice that recapitulate two important glioma phenotypes, including the diffusely infiltrative tumor margin and angiogenic core. We show that pulsed laser excitation of vascular-targeted gold nanoparticles non-invasively and reversibly modulates the blood-brain-tumor barrier permeability (optoBBTB) and enhances the delivery of paclitaxel in these two models. The treatment reduces the tumor volume by 6 and 2.4-fold and prolongs the survival by 50% and 33%, respectively. Since paclitaxel does not penetrate the blood-brain-tumor barrier and is abandoned for glioblastoma treatment following its failure in early-phase clinical trials, our results raise the possibility of reevaluating a number of potent anticancer drugs by combining them with strategies to increase blood-brain-tumor barrier permeability. Our study reveals that optoBBTB significantly improves therapeutic delivery and has the potential to facilitate future drug evaluation for cancers in the central nervous system.

Image-guided focused ultrasound-mediated molecular delivery to breast cancer in an animal model.

Tumors become inoperable due to their size or location, making neoadjuvant chemotherapy the primary treatment. However, target tissue accumulation of anticancer agents is limited by the physical barriers of the tumor microenvironment. Low-intensity focused ultrasound (FUS) in combination with microbubble (MB) contrast agents can increase microvascular permeability and improve drug delivery to the target tissue after systemic administration. The goal of this research was to investigate image-guided FUS-mediated molecular delivery in volume space. Three-dimensional (3-D) FUS therapy functionality was implemented on a programmable ultrasound scanner (Vantage 256, Verasonics Inc.) equipped with a linear array for image guidance and a 128-element therapy transducer (HIFUPlex-06, Sonic Concepts). FUS treatment was performed on breast cancer-bearing female mice (N= 25). Animals were randomly divided into three groups, namely, 3-D FUS therapy, two-dimensional (2-D) FUS therapy, or sham (control) therapy. Immediately prior to the application of FUS therapy, animals received a slow bolus injection of MBs (Definity, Lantheus Medical Imaging Inc.) and near-infrared dye (IR-780, surrogate drug) for optical reporting and quantification of molecular delivery. Dye accumulation was monitored viain vivooptical imaging at 0, 1, 24, and 48 h (Pearl Trilogy, LI-COR). Following the 48 h time point, animals were humanely euthanized and tumors excised forex vivoanalyzes. Optical imaging results revealed that 3-D FUS therapy improved delivery of the IR-780 dye by 66.4% and 168.1% at 48 h compared to 2-D FUS (p= 0.18) and sham (p= 0.047) therapeutic strategies, respectively.Ex vivoanalysis revealed similar trends. Overall, 3-D FUS therapy can improve accumulation of a surrogate drug throughout the entire target tumor burden after systemic administration.

Liver three-dimensional cellular models for high-throughput chemical testing.

Drug-induced hepatotoxicity is a leading cause of drug withdrawal from the market. High-throughput screening utilizing in vitro liver models is critical for early-stage liver toxicity testing. Traditionally, monolayer human hepatocytes or immortalized liver cell lines (e.g., HepG2, HepaRG) have been used to test compound liver toxicity. However, monolayer-cultured liver cells sometimes lack the metabolic competence to mimic the in vivo condition and are therefore largely appropriate for short-term toxicological testing. They may not, however, be adequate for identifying chronic and recurring liver damage caused by drugs. Recently, several three-dimensional (3D) liver models have been developed. These 3D liver models better recapitulate normal liver function and metabolic capacity. This review describes the current development of 3D liver models that can be used to test drugs/chemicals for their pharmacologic and toxicologic effects, as well as the advantages and limitations of using these 3D liver models for high-throughput screening.

Assessment of Transarterial Chemoembolization Using Super-resolution Ultrasound Imaging and a Rat Model of Hepatocellular Carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a highly prevalent form of liver cancer diagnosed annually in 600,000 people worldwide. A common treatment is transarterial chemoembolization (TACE), which interrupts the blood supply of oxygen and nutrients to the tumor mass. The need for repeat TACE treatments may be assessed in the weeks after therapy with contrast-enhanced ultrasound (CEUS) imaging. Although the spatial resolution of traditional CEUS has been restricted by the diffraction limit of ultrasound (US), this physical barrier has been overcome by a recent innovation known as super-resolution US (SRUS) imaging. In short, SRUS enhances the visible details of smaller microvascular structures on the 10 to 100 µm scale, which unlocks a host of new clinical opportunities for US. METHODS: In this study, a rat model of orthotopic HCC is introduced and TACE treatment response (to a doxorubicin-lipiodol emulsion) is assessed using longitudinal SRUS and magnetic resonance imaging (MRI) performed at 0, 7 and 14 d. Animals were euthanized at 14 d for histological analysis of excised tumor tissue and determination of TACE response, that is, control, partial response or complete response. CEUS imaging was performed using a pre-clinical US system (Vevo 3100, FUJIFILM VisualSonics Inc.) equipped with an MX201 linear array transducer. After administration of a microbubble contrast agent (Definity, Lantheus Medical Imaging), a series of CEUS images were collected at each tissue cross-section as the transducer was mechanically stepped at 100 µm increments. SRUS images were formed at each spatial position, and a microvascular density metric was calculated. Microscale computed tomography (microCT, OI/CT, MILabs) was used to confirm TACE procedure success, and tumor size was monitored using a small animal MRI system (BioSpec 3T, Bruker Corp.). RESULTS: Although there were no differences at baseline (p > 0.15), both microvascular density levels and tumor size measures from the complete responder cases at 14 d were considerably lower and smaller, respectively, than those in the partial responder or control group animals. Histological analysis revealed tumor-to-necrosis levels of 8.4%, 51.1% and 100%, for the control, partial responder and complete responder groups, respectively (p < 0.005). CONCLUSION: SRUS imaging is a promising modality for assessing early changes in microvascular networks in response to tissue perfusion-altering interventions such as TACE treatment of HCC.

H-Scan Ultrasound Monitoring of Breast Cancer Response to Chemotherapy and Validation With Diffusion-Weighted Magnetic Resonance Imaging.

OBJECTIVES: H-scan ultrasound (US) imaging is a novel tissue characterization technique to detect apoptosis-induced changes in cancer cells after the initiation of effective drug treatment. The objective of the proposed research was to assess the sensitivity of 3-dimensional (3D) H-scan US technique for monitoring the response of breast cancer-bearing animals to neoadjuvant chemotherapy and correlate results to diffusion-weighted magnetic resonance imaging (DW-MRI) measurements of programmed cancer cell death. METHODS: Experimental studies used female mice (N = 18) implanted with human breast cancer cells. Animals underwent H-scan US and DW-MRI imaging on days 0, 1, 3, 7, and 10. After imaging at day 0, breast tumor-bearing nude mice were treated biweekly with an apoptosis-inducing drug. Texture analysis of H-scan US images explored spatial relationships between local US scattering. At day 10, H-scan measurements were compared with DW-MRI-derived apparent diffusion coefficient (ADC) values and histological findings. RESULTS: H-scan US imaging of low and high dose cisplatin-treated cancer-bearing animals revealed changes in image intensity suggesting a progressive decrease in aggregate US scatterer size that was not observed in control animals. Longitudinal trends discovered in H-scan US result matched with texture analysis and DW-MRI (P < .01). Further, analysis of the H-scan US image intensity and corresponding DW-MRI-derived ADC values revealed a strong linear correlation (R2  = .93, P < .001). These changes were due to cancer cell apoptotic activity and consider as early detectable biomarker during treatment. CONCLUSIONS: The 3D H-scan technique holds promise for assisting clinicians in monitoring the early response of breast cancer tumor to neoadjuvant chemotherapy and adding value to traditional diagnostic ultrasound examinations.

An open-source bioink database for microextrusion 3D printing.

3D printing has rapidly become a critical enabling technology in tissue engineering and regenerative medicine for the fabrication of complex engineered tissues. 3D bioprinting, in particular, has advanced greatly to facilitate the incorporation of a broad spectrum of biomaterials along with cells and biomolecules of interest forin vitrotissue generation. The increasing complexity of novel bioink formulations and application-dependent printing conditions poses a significant challenge for replicating or innovating new bioprinting strategies. As the field continues to grow, it is imperative to establish a cohesive, open-source database that enables users to search through existing 3D printing formulations rapidly and efficiently. Through the efforts of the NIH/NIBIB Center for Engineering Complex Tissues, we have developed, to our knowledge, the first bioink database for extrusion-based 3D printing. The database is publicly available and allows users to search through and easily access information on biomaterials and cells specifically used in 3D printing. In order to enable a community-driven database growth, we have established an open-source portal for researchers to enter their publication information for addition into the database. Although the database has a broad range of capabilities, we demonstrate its utility by performing a comprehensive analysis of the printability domains of two well-established biomaterials in the printing world, namely poly(ϵ-caprolactone) and gelatin methacrylate. The database allowed us to rapidly identify combinations of extrusion pressure, temperature, and speed that have been used to print these biomaterials and more importantly, identify domains within which printing was not possible. The data also enabled correlation analysis between all the printing parameters, including needle size and type, that exhibited compatibility for cell-based 3D printing. Overall, this database is an extremely useful tool for the 3D printing and bioprinting community to advance their research and is an important step towards standardization in the field.

Multiscale computational modeling of cancer growth using features derived from microCT images.

Advances in medical imaging technologies now allow noninvasive image acquisition from individual patients at high spatiotemporal resolutions. A relatively new effort of predictive oncology is to develop a paradigm for forecasting the future status of an individual tumor given initial conditions and an appropriate mathematical model. The objective of this study was to introduce a comprehensive multiscale computational method to predict cancer and microvascular network growth patterns. A rectangular lattice-based model was designed so different evolutionary scenarios could be simulated and for predicting the impact of diffusible factors on tumor morphology and size. Further, the model allows prediction-based simulation of cell and microvascular behavior. Like a single cell, each agent is fully realized within the model and interactions are governed in part by machine learning methods. This multiscale computational model was developed and incorporated input information from in vivo microscale computed tomography (microCT) images acquired from breast cancer-bearing mice. It was found that as the difference between expansion of the cancer cell population and microvascular network increases, cells undergo proliferation and migration with a greater probability compared to other phenotypes. Overall, multiscale computational model agreed with both theoretical expectations and experimental findings (microCT images) not used during model training.

Membrane composition is a functional determinant of NIR-activable liposomes in orthotopic head and neck cancer.

Near-infrared (NIR)-activable liposomes containing photosensitizer (PS)-lipid conjugates are emerging as tunable, high-payload, and tumor-selective platforms for photodynamic therapy (PDT)-based theranostics. To date, the impact that the membrane composition of a NIR-activable liposome (the chemical nature and subsequent conformation of PS-lipid conjugates) has on their in vitro and in vivo functionality has not been fully investigated. While their chemical nature is critical, the resultant physical conformation dictates their interactions with the immediate biological environments. Here, we evaluate NIR-activable liposomes containing lipid conjugates of the clinically-used PSs benzoporphyrin derivative (BPD; hydrophobic, membrane-inserting conformation) or IRDye 700DX (hydrophilic, membrane-protruding conformation) and demonstrate that membrane composition is critical for their function as tumor-selective PDT-based platforms. The PS-lipid conformations were primarily dictated by the varying solubilities of the two PSs and assisted by their lipid conjugation sites. Conformation was further validated by photophysical analysis and computational predictions of PS membrane partitioning (topological polar surface area [tPSA], calculated octanol/water partition [cLogP], and apparent biomembrane permeability coefficient [Papp]). Results show that the membrane-protruding lipo-IRDye700DX exhibits 5-fold more efficient photodynamic generation of reactive molecular species (RMS), 12-fold expedited phototriggered burst release of entrap-ped agents, and 15-fold brighter fluorescence intensity as compared to the membrane-inserting lipo-BPD-PC (phosphatidylcholine conjugate). Although the membrane-inserting lipo-BPD-PC exhibits less efficient photo-dynamic generation of RMS, it allows for more sustained phototriggered release, 10-fold greater FaDu cancer cell phototoxicity, and 7.16-fold higher tumor-selective delivery in orthotopic mouse FaDu head and neck tumors. These critical insights pave the path for the rational design of emerging NIR-activable liposomes, whereby functional consequences of membrane composition can be tailored toward a specific therapeutic purpose.

Evaluating Microelectrode Arrays in Peripheral Nerve Using Micro Computed Tomography.

Many advances have been made with imaging of implanted neural devices; however, the ability to image whole nerve samples remains limited. Further, few imaging modalities are well suited for visualizing both whole devices in vivo and individual microelectrodes within a nerve. In this study, we used micro-computed tomography (micro-CT) to evaluate Wireless Floating Microelectrode Arrays (WMFAs) implanted in rat sciatic nerve at the level of whole devices and individual electrodes. WFMAs were also used to track selective recruitment of plantar flexion and dorsiflexion of the rear paw, which was achieved by each implanted device (n=6) during chronic implantation. Evoked limb motion was correlated to end-of-study assessments using micro-CT to visualize electrode locations within the fascicular structure of the sciatic nerve. Results of this study show that micro-CT imaging can provide valuable assessments of microelectrode arrays implanted in peripheral nerves for both whole devices visualized in vivo and individual electrodes visualized in whole nerve tissue samples.Clinical relevance- This work informs the use of micro-computed tomography as a tool for correlating neural device performance with physical attributes of the implant location.

Monitoring Progression of Ductal Carcinoma In Situ Using Photoacoustics and Contrast-Enhanced Ultrasound.

Breast cancer is the leading form of cancer in women, accounting for approximately 41,400 deaths in 2018. While a variety of risk factors have been identified, physical exercise has been linked to reducing both the risk and aggressiveness of breast cancer. Within breast cancer, ductal carcinoma in situ (DCIS) is a common finding. However, less than 25% of DCIS tumors actually progress into invasive breast cancer, resulting in overtreatment. This overtreatment is due to a lack of predictive precursors to assess aggressiveness and development of DCIS. We hypothesize that tissue oxygenation and perfusion measured by photoacoustic and contrast-enhanced ultrasound imaging, respectively, can predict DCIS aggressiveness. To test this, 20 FVB/NJ and 20 SV40Tag mice that genetically develop DCIS-like breast cancers were divided evenly into exercise and control groups and imaged over the course of 6 weeks. Tissue oxygenation was a predictive precursor to invasive breast cancer for FVB/NJ mice (P = 0.015) in the early stages of tumor development. Meanwhile, perfusion results were inconclusive (P > 0.2) as a marker for disease progression. Moreover, voluntary physical exercise resulted in lower weekly tumor growth and significantly improved median survival (P = 0.014).