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Recent studies have begun to highlight the diverse and tumor-specific microbiomes across multiple cancer types. We believe this work raises the important question of whether the classical “Hallmarks of Cancer” should be expanded to include tumor microbiomes. To answer this question, the causal relationships and co-evolution of these microbiotic tumor ecosystems must be better understood. Because host-microbe interactions should be studied in a physiologically relevant context, animal models have been preferred. Yet these models are often poor mimics of human tumors and are difficult to interrogate at high spatiotemporal resolution. We believe that in vitro tissue engineered platforms could provide a powerful alternative approach that combines the high-resolution of in vitro studies with a high degree of physiological relevance. This review will focus on tissue engineered approaches to study host-microbe interactions and to establish their role as an emerging hallmark of cancer with potential as a therapeutic target.

Multi-agent biohybrid microrobotic systems, owing to their small size and distributed nature, offer powerful solutions to challenges in biomedicine, bioremediation, and biosensing. Synthetic biology enables programmed emergent behaviors in the biotic component of biohybrid machines, expounding vast potential benefits for building biohybrid swarms with sophisticated control schemes. The design of synthetic genetic circuits tailored toward specific performance characteristics is an iterative process that relies on experimental characterization of spatially homogeneous engineered cell suspensions. However, biohybrid systems often distribute heterogeneously in complex environments, which will alter circuit performance. Thus, there is a critically unmet need for simple predictive models that describe emergent behaviors of biohybrid systems to inform synthetic gene circuit design. Here, we report a data-driven statistical model for computationally efficient recapitulation of the motility dynamics of two types of Escherichia coli bacteria-based biohybrid swarms-NanoBEADS and BacteriaBots. The statistical model was coupled with a computational model of cooperative gene expression, known as quorum sensing (QS). We determined differences in timescales for programmed emergent behavior in BacteriaBots and NanoBEADS swarms, using bacteria as a comparative baseline. We show that agent localization and genetic circuit sensitivity strongly influence the timeframe and the robustness of the emergent behavior in both systems. Finally, we use our model to design a QS-based decentralized control scheme wherein agents make independent decisions based on their interaction with other agents and the local environment. We show that synergistic integration of synthetic biology and predictive modeling is requisite for the efficient development of biohybrid systems with robust emergent behaviors.

Multicellular tumor spheroid (MCTS) systems provide an in vitro cell culture model system which mimics many of the complexities of an in vivo solid tumor and tumor microenvironment, and are often used to study cancer cell growth and drug efficacy. Here, we present a coupled experimental-computational framework to estimate phenotypic growth and biophysical tumor microenvironment properties. This novel framework utilizes standard microscopy imaging of MCTS systems to drive a biophysical mathematical model of MCTS growth and mechanical interactions. By extending our previous in vivo mechanically-coupled reaction-diffusion modeling framework we developed a microscopy image processing framework capable of mechanistic characterization of MCTS systems. Using MDA-MB-231 breast cancer MCTS, we estimated biophysical parameters of cellular diffusion, rate of cellular proliferation, and cellular tractions forces. We found significant differences in these model-based biophysical parameters throughout the treatment time course between untreated and treated MCTS systems, whereas traditional size-based morphometric parameters were inconclusive. The proposed experimental-computational framework estimates mechanistic MCTS growth and invasion parameters with significant potential to assist in better and more precise assessment of in vitro drug efficacy through the development of computational analysis methodologies for three-dimensional cell culture systems to improve the development and evaluation of antineoplastic drugs.

Pulsed electric fields (PEFs) have become clinically important through the success of Irreversible Electroporation (IRE), Electrochemotherapy (ECT), and nanosecond PEFs (nsPEFs) for the treatment of tumors. PEFs increase the permeability of cell membranes, a phenomenon known as electroporation. In addition to well-known membrane effects, PEFs can cause profound cytoskeletal disruption. In this review, we summarize the current understanding of cytoskeletal disruption after PEFs. Compiling available studies, we describe PEF-induced cytoskeletal disruption and possible mechanisms of disruption. Additionally, we consider how cytoskeletal alterations contribute to cell–cell and cell–substrate disruption. We conclude with a discussion of cytoskeletal disruption-induced anti-vascular effects of PEFs and consider how a better understanding of cytoskeletal disruption after PEFs may lead to more effective therapies.

Treatment of intracranial disorders suffers from the inability to accumulate therapeutic drug concentrations due to protection from the blood–brain barrier (BBB). Electroporation-based therapies have demonstrated the capability of permeating the BBB, but knowledge of the longevity of BBB disruption (BBBD) is limited. In this study, we quantify the temporal, high-frequency electroporation (HFE)-mediated BBBD in an in vivo healthy rat brain model. 40 male Fisher rats underwent HFE treatment; two blunt tipped monopolar electrodes were advanced into the brain and 200 bursts of HFE were delivered at a voltage-to-distance ratio of 600 V/cm. BBBD was verified with contrast enhanced T1W MRI (gadopentetate dimeglumine) and pathologically (Evans blue dye) at time points of 1, 24, 48, 72, and 96 h after HFE. Contrast enhanced T1W scans demonstrated BBBD for 1 to 72 h after HFE but intact BBB at 96 h. Histologically, tissue damage was restricted to electrode insertion tracks. BBBD was induced with minimal muscle contractions and minimal cell death attributed to HFE. Numerical modeling indicated that brief BBBD was induced with low magnitude electric fields, and BBBD duration increased with field strength. These data suggest the spatiotemporal characteristics of HFE-mediated BBBD may be modulated with the locally applied electric field.

The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin-covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (similar to 2.4-fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine-liver cultures can be used as a platform for future investigations.

High-frequency irreversible electroporation (H-FIRE) is a technique that uses pulsed electric fields that have been shown to ablate malignant cells. In order to evaluate the clinical potential of H-FIRE to treat glioblastoma (GBM), a primary brain tumor, we have studied the effects of high-frequency waveforms on therapy-resistant glioma stem-like cell (GSC) populations. We demonstrate that patient-derived GSCs are more susceptible to H-FIRE damage than primary normal astrocytes. This selectivity presents an opportunity for a degree of malignant cell targeting as bulk tumor cells and tumor stem cells are seen to exhibit similar lethal electric field thresholds, significantly lower than that of healthy astrocytes. However, neural stem cell (NSC) populations also exhibit a similar sensitivity to these pulses. This observation may suggest that different considerations be taken when applying these therapies in younger versus older patients, where the importance of preserving NSC populations may impose different restrictions on use.We also demonstrate variability in threshold among the three patient-derived GSC lines studied, suggesting the need for personalized cell-specific characterization in the development of potential clinical procedures. Future work may provide further useful insights regarding this patient-dependent variability observed that could inform targeted and personalized treatment.

Immunotherapies have demonstrated limited efficacy in pancreatic ductal adenocarcinoma (PDAC) patients despite their success in treating other tumor types. This limitation is largely due to the relatively immunosuppressive environment surrounding the tumor. A focal ablative technique called irreversible electroporation (IRE) has been shown to modulate this environment, enhancing the efficacy of immunotherapy. One enhancing factor related to improved prognosis is a decrease in regulatory T cells (T-reg). This decrease has been previously unpredictable for clinicians using IRE, who currently have limited real-time metrics for determining the activation of the patient's immune response. Here, we report that larger overall changes in output current are correlated with larger decreases in T cell populations 24 hours post-treatment. This result suggests that clinicians can make real-time decisions regarding optimal follow-up therapy based on the range of output current delivered during treatment. This capability could maximize the immunomodulating effect of IRE in synergy with follow-up immunotherapy. Additionally, these results suggest that feedback from a preliminary IRE treatment of the local tumor may help inform clinicians regarding the timing and choice of subsequent therapies, such as resection, immunotherapy, chemotherapy, or follow-up thermal or non-thermal ablation.

The early treatment and rapid closure of acute or chronic wounds is essential for normal healing and prevention of hypertrophic scarring. The use of split thickness autografts is often limited by the availability of a suitable area of healthy donor skin to harvest. Cellular and non-cellular biological skin-equivalents are commonly used as an alternative treatment option for these patients, however these treatments usually involve multiple surgical procedures and associated with high costs of production and repeated wound treatment. Here we describe a novel design and a proof-of-concept validation of a mobile skin bioprinting system that provides rapid on-site management of extensive wounds. Integrated imaging technology facilitated the precise delivery of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into an injured area, replicating the layered skin structure. Excisional wounds bioprinted with layered autologous dermal fibroblasts and epidermal keratinocytes in a hydrogel carrier showed rapid wound closure, reduced contraction and accelerated re-epithelialization. These regenerated tissues had a dermal structure and composition similar to healthy skin, with extensive collagen deposition arranged in large, organized fibers, extensive mature vascular formation and proliferating keratinocytes.

The most ubiquitous gap junction protein within the body, connexin 43 (Cx43), is a target of interest for modulating the dermal wound healing response. Observational studies found associations between Cx43 at the wound edge and poor healing response, and subsequent studies utilizing local knockdown of Cx43 found improvements in wound closure rate and final scar appearance. Further preclinical work conducted using Cx43-based peptide therapeutics, including alpha connexin carboxyl terminus 1 (αCT1), a peptide mimetic of the Cx43 carboxyl terminus, reported similar improvements in wound healing and scar formation. Clinical trials and further study into the mode of action have since been conducted on αCT1, and Phase III testing for treatment of diabetic foot ulcers is currently underway. Therapeutics targeting connexin activity show promise in beneficially modulating the human body’s natural healing response for improved patient outcomes across a variety of injuries.

A bioengineered skeletal muscle tissue as an alternative for autologous tissue flaps, which mimics the structural and functional characteristics of the native tissue, is needed for reconstructive surgery. Rapid progress in the cell-based tissue engineering principle has enabled in vitro creation of cellularized muscle-like constructs; however, the current fabrication methods are still limited to build a three-dimensional (3D) muscle construct with a highly viable, organized cellular structure with the potential for a future human trial. Here, we applied 3D bioprinting strategy to fabricate an implantable, bioengineered skeletal muscle tissue composed of human primary muscle progenitor cells (hMPCs). The bioprinted skeletal muscle tissue showed a highly organized multi-layered muscle bundle made by viable, densely packed, and aligned myofiber-like structures. Our in vivo study presented that the bioprinted muscle constructs reached 82% of functional recovery in a rodent model of tibialis anterior (TA) muscle defect at 8 weeks of post-implantation. In addition, histological and immunohistological examinations indicated that the bioprinted muscle constructs were well integrated with host vascular and neural networks. We demonstrated the potential of the use of the 3D bioprinted skeletal muscle with a spatially organized structure that can reconstruct the extensive muscle defects.

High-frequency irreversible electroporation is a nonthermal method of tissue ablation that uses bursts of 0.5- to 2.0-microsecond bipolar electric pulses to permeabilize cell membranes and induce cell death. High-frequency irreversible electroporation has potential advantages for use in neurosurgery, including the ability to deliver pulses without inducing muscle contraction, inherent selectivity against malignant cells, and the capability of simultaneously opening the blood–brain barrier surrounding regions of ablation. Our objective was to determine whether high-frequency irreversible electroporation pulses capable of tumor ablation could be delivered to dogs with intracranial meningiomas. Three dogs with intracranial meningiomas were treated. Patient-specific treatment plans were generated using magnetic resonance imaging-based tissue segmentation, volumetric meshing, and finite element modeling. Following tumor biopsy, high-frequency irreversible electroporation pulses were stereotactically delivered in situ followed by tumor resection and morphologic and volumetric assessments of ablations. Clinical evaluations of treatment included pre- and posttreatment clinical, laboratory, and magnetic resonance imaging examinations and adverse event monitoring for 2 weeks posttreatment. High-frequency irreversible electroporation pulses were administered successfully in all patients. No adverse events directly attributable to high-frequency irreversible electroporation were observed. Individual ablations resulted in volumes of tumor necrosis ranging from 0.25 to 1.29 cm3. In one dog, nonuniform ablations were observed, with viable tumor cells remaining around foci of intratumoral mineralization. In conclusion, high-frequency irreversible electroporation pulses can be delivered to brain tumors, including areas adjacent to critical vasculature, and are capable of producing clinically relevant volumes of tumor ablation. Mineralization may complicate achievement of complete tu

Wearable sensors and systems have become increasingly popular in recent years. Two prominent wearable technologies for human activity monitoring are smart textile systems (STSs) and inertial measurement units (IMUs). Despite ongoing advances in both, the usability aspects of these devices require further investigation, especially to facilitate future use. In this study, 18 participants evaluate the preferred placement and usability of two STSs, along with a comparison to a commercial IMU system. These evaluations are completed after participants engaged in a range of activities (e.g., sitting, standing, walking, and running), during which they wear two representatives of smart textile systems: (1) a custom smart undershirt (SUS) and commercial smart socks; and (2) a commercial whole-body IMU system. We first analyze responses regarding the usability of the STS, and subsequently compared these results to those for the IMU system. Participants identify a short-sleeved shirt as their preferred activity monitor. In additional, the SUS in combination with the smart socks is rated superior to the IMU system in several aspects of usability. As reported herein, STSs show promise for future applications in human activity monitoring in terms of usability.

This study demonstrates the feasibility of using a seat sensor designed for occupant classification from a production passenger vehicle to measure an occupant’s respiration rate (RR) and heart rate (HR) in a laboratory setting. Relaying occupant vital signs after a crash could improve emergency response by adding a direct measure of the occupant state to an Advanced Automatic Collision Notification (AACN) system. Data was collected from eleven participants with body weights ranging from 42 to 91 kg using a Ford Mustang passenger seat and seat sensor. Using a ballistocardiography (BCG) approach, the data was processed by time domain filtering and frequency domain analysis using the fast Fourier transform to yield RR and HR in a 1-min sliding window. Resting rates over the 30-min data collection and continuous RR and HR signals were compared to laboratory physiological instruments using the Bland-Altman approach. Differences between the seat sensor and reference sensor were within 5 breaths per minute for resting RR and within 15 beats per minute for resting HR. The time series comparisons for RR and HR were promising with the frequency analysis technique outperforming the peak detection technique. However, future work is necessary for more accurate and reliable real-time monitoring of RR and HR outside the laboratory setting.

The present invention relates to the field of medical treatment of diseases and disorders, as well as the field of biomedical engineering. Embodiments of the invention relate to the delivery of Irreversible Electroporation (IRE) through the vasculature of organs to treat tumors embedded deep within the tissue or organ, or to decellularize organs to produce a scaffold from existing animal tissue with the existing vasculature intact. In particular, methods of administering non-thermal irreversible electroporation (IRE) in vivo are provided for the treatment of tumors located in vascularized tissues and organs. Embodiments of the invention further provide scaffolds and tissues from natural sources created using IRE ex vivo to remove cellular debris, maximize recellularization potential, and minimize foreign body immune response. The engineered tissues can be used in methods of treating subjects, such as those in need of tissue replacement or augmentation.

Devices and methods for performing dielectrophoresis are described. The devices contain sample channel which is separated by physical barriers from electrode channels which receive electrodes. The devices and methods may be used for the separation and analysis of particles in solution, including the separation and isolation of cells of a specific type. As the electrodes do not make contact with the sample, electrode fouling is avoided and sample integrity is better maintained.

A system and method that comprises an X-ray source configured to emit an X-ray beam; an object positioned to receive the X-ray beam; a detector configured to receive an attenuated beam of the X-rays through the object for measuring projection data from various orientations that can be used to generate spectral images in terms of energy-dependent linear attenuation coefficients, and a detector, as described above, comprising one or more energy-integrating detector elements and one or more photon counting detector elements in one or more detectors array or separate detector arrays. The photon counting detector elements may also use dynamic thresholds to define energy windows.

Ovarian cancer cells are exposed to physical stress in the peritoneal cavity during both tumor growth and dissemination. Ascites build-up in metastatic ovarian cancer further increases the exposure to fluid shear stress. Here, we used a murine, in vitro ovarian cancer progression model in parallel with immortalized human cells to investigate how ovarian cancer cells of increasing aggressiveness respond to < 1 dyne/cm2 of fluid-induced shear stress. This biophysical stimulus significantly reduced cell viability in all cells exposed, independent of disease stage. Fluid shear stress induced spheroid formation and altered cytoskeleton organization in more tumorigenic cell lines. While benign ovarian cells appeared to survive in higher numbers under the influence of fluid shear stress, they exhibited severe morphological changes and chromosomal instability. These results suggest that exposure of benign cells to low magnitude fluid shear stress can induce phenotypic changes that are associated with transformation and ovarian cancer progression. Moreover, exposure of tumorigenic cells to fluid shear stress enhanced anchorage-independent survival, suggesting a role in promoting invasion and metastasis.

Variability in patient response to anti-cancer drugs is currently addressed by relating genetic mutations to chemotherapy through precision medicine. However, practical benefits of precision medicine to therapy design are less clear. Even after identification of mutations, oncologists are often left with several drug options, and for some patients there is no definitive treatment solution. There is a need for model systems to help predict personalized responses to chemotherapeutics. We have microengineered 3D tumor organoids directly from fresh tumor biopsies to provide patient-specific models with which treatment optimization can be performed before initiation of therapy. We demonstrate the initial implementation of this platform using tumor biospecimens surgically removed from two mesothelioma patients. First, we show the ability to biofabricate and maintain viable 3D tumor constructs within a tumor-on-a-chip microfluidic device. Second, we demonstrate that results of on-chip chemotherapy screening mimic those observed in subjects themselves. Finally, we demonstrate mutation-specific drug testing by considering the results of precision medicine genetic screening and confirming the effectiveness of the non-standard compound 3-deazaneplanocin A for an identified mutation. This patient-derived tumor organoid strategy is adaptable to a wide variety of cancers and may provide a framework with which to improve efforts in precision medicine oncology.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art.

Purpose: Irreversible electroporation (IRE) has been developed as a promising minimally invasive treatment to ablate spontaneous brain tumors with pulsed electric fields in canine patients. The purpose of the study is to determine the Peleg-Fermi parameters needed to incorporate pulse number and pulse duration into the therapeutic planning of IRE. Methods: Seven canine patients were treated with IRE for spontaneous malignant glioma with MRIbased treatment planning. The treatment planning method consists of building patient-specific finite element models and using them to compute electric fields used in the IRE treatment. We evaluate the predictive power of tumor coverage with electric field alone vs. cell kill probability using radiographically confirmed clinical outcomes. Results: Results of post-treatment diagnostic imaging, tumor biopsies, and neurological examinations indicated successful tumor ablation without significant direct neurotoxicity in six of the seven dogs. Objective tumor responses were seen in four (80%) of five dogs with quantifiable target lesions according to RANO criteria. Two dogs experienced survivals in excess of 1 yr, including one dog that resulted in complete response to IRE treatment for 5+ years to date. Tumor fraction exposed to electric field over 600 V/cm was between 0.08 and 0.73, while tumor fraction exposed to electric field over 300 V/cm was between 0.17 and 0.95. Probability of cell kill of ≥ 90% was found in tumor volume fractions between 0.21 and 0.99. Conclusions: We conclude that IRE is a safe and effective minimally invasive treatment for malignant glioma and can be predicted with the Peleg-Fermi cell kill probability function. A tumor coverage of ≥ 0.9 at a cell kill probability ≥ 90% can be used to guide IRE treatments of spontaneous malignant glioma based on the radiographically confirmed clinical outcomes achieved.

It is well documented that the tumor microenvironment profoundly impacts the etiology and progression of breast cancer, yet the contribution of the resident microbiome within breast tissue remains poorly understood. Tumor microenvironmental conditions, such as hypoxia and dense tumor stroma, predispose progressive phenotypes and therapy resistance, however the role of bacteria in this interplay remains uncharacterized. We hypothesized that the effect of individual bacterial secreted molecules on breast cancer viability and proliferation would be modulated by these tumor-relevant stressors differentially for cells at varying stages of progression. To test this, we incubated human breast adenocarcinoma cells (MDA-MB-231, MCF-DCIS.com) and non-malignant breast epithelial cells (MCF-10A) with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule from Pseudomonas aeruginosa that regulates bacterial stress responses. This molecule was selected because Pseudomonas was recently characterized as a significant fraction of the breast tissue microbiome and OdDHL is documented to impact mammalian cell viability. After OdDHL treatment, we demonstrated the greatest decrease in viability with the more malignant MDA-MB-231 cells and an intermediate MCF-DCIS.com (ductal carcinoma in situ) response. The responses were also culture condition (i.e. microenvironment) dependent. These results contrast the MCF-10A response, which demonstrated no change in viability in any culture condition. We further determined that the observed trends in breast cancer viability were due to modulation of proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact with the host tissue environment to modulate breast cancer viability and proliferation, and that the effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the i

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Approximately 9180 primary GBM tumors are diagnosed in the United States each year, in which median survival is up to 16 months. GBM eludes and resists typical cancer treatments due to the presence of infiltrative cells beyond the solid tumor margin, heterogeneity within the tumor microenvironment, and protection from the blood-brain barrier. Conventional treatments for GBM, such as surgical resection, radiotherapy, and chemotherapy, have shown limited efficacy; therefore, alternate treatments are needed. Tumor chemoresistance and its proximity to critical structures make GBM a prime theoretical candidate for nonthermal ablation with irreversible electroporation (IRE) and high-frequency IRE (H-FIRE). IRE and H-FIRE are treatment modalities that utilize pulsed electric fields to permeabilize the cell membrane. Once the electric field magnitude exceeds a tissue-specific lethal threshold, cell death occurs. Benefits of IRE and H-FIRE therapy include, but are not limited to, the elimination of cytotoxic effects, sharp delineation from treated tissue and spared tissue, a nonthermal mechanism of ablation, and sparing of nerves and major blood vessels. Preclinical studies have confirmed the safety and efficacy of IRE and H-FIRE within their experimental scope. In this chapter, studies will be collected and information extrapolated to provide possible treatment regimens for use in high-grade gliomas, specifically in GBM.

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases -before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Cell emergence onto damaged or organized fibrous extracellular matrix (ECM) is a crucial precursor to collective cell migration in wound closure and cancer metastasis, respectively. However, there is a fundamental gap in our quantitative understanding of the role of local ECM size and arrangement in cell emergence-based migration and local gap closure. Here, using ECM-mimicking nanofibers bridging cell monolayers, we describe a method to recapitulate and quantitatively describe these in vivo behaviors over multispatial (single cell to cell sheets) and temporal (minutes to weeks) scales. On fiber arrays with large interfiber spacing, cells emerge (invade) either singularly by breaking cell-cell junctions analogous to release of a stretched rubber band (recoil), or in groups of few cells (chains), whereas on closely spaced fibers, multiple chains emerge collectively. Advancing cells on fibers form cell streams, which support suspended cell sheets (SCS) of various sizes and curvatures. SCS converge to form local gaps that close based on both the gap size and shape. We document that cell stream spacing of 375 mu m and larger hinders SCS advancement, thus providing abilities to engineer closing and nonclosing gaps. Altogether we highlight the importance of studying cell-fiber interactions and matrix structural remodeling in fundamental and translational cell biology.

A number of innovative methods exist to measure cell-matrix adhesive forces, but they have yet to accurately describe and quantify the intricate interplay of a cell and its fibrous extracellular matrix (ECM). In cardiovascular pathologies, such as aortic aneurysm, new knowledge on the involvement of cell-matrix forces could lead to elucidation of disease mechanisms. To better understand this dynamics, we measured primary human aortic single smooth muscle cell (SMC) forces using nanonet force microscopy in both inside-out (I-O intrinsic contractility) and outside-in (O-I external perturbation) modes. For SMC populations, we measured the I-O and O-I forces to be 12.9 +/- 1.0 and 57.9 +/- 2.5 nN, respectively. Exposure of cells to oxidative stress conditions caused a force decrease of 57 and 48% in I-O and O-I modes, respectively, and an increase in migration rate by 2.5-fold. Finally, in O-I mode, we cyclically perturbed cells at constant strain of varying duration to simulate in vivo conditions of the cardiac cycle and found that I-O forces decrease with increasing duration and O-I forces decreased by half at shorter cycle times. Thus our findings highlight the need to study forces exerted and felt by cells simultaneously to comprehensively understand force modulation in cardiovascular disease.

The Virginia Tech - Wake Forest University Biomedical Engineering Society (BMES) Student Chapter hosts an annual Graduate Student Research Symposium to provide students with the opportunity to interact and exchange research ideas with colleagues and industry leaders. This symposium promotes research collaboration between the two campuses and cutting edge facilities, such as the Wake Forest Institute for Regenerative Medicine, the Comprehensive Cancer Center of Wake Forest University Baptist Medical Center, the Virginia-Maryland Regional College of Veterinary Medicine, and the Virginia Tech Carilion Research Institute. 20 oral presentations from graduating Ph.D. and M.S. students. 70 poster presentations from non-graduating students. Topics include injury and musculoskeletal biomechanics, biomedical imaging, cell and tissue engineering, and nanotechnology for medical applications.

The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200–300 nm of each other in the xy-plane and within 500–700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy–based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and <50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins.

Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43) in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

Despite abundant research conducted on cancer biomarker discovery and validation, to date, less than two-dozen biomarkers have been approved by the FDA for clinical use. One main reason is attributed to inadvertent use of low quality biospecimens in biomarker research. Most proteinaceous biomarkers are extremely susceptible to pre-analytical factors such as collection, processing, and storage. For example, cryogenic storage imposes very harsh chemical, physical, and mechanical stresses on biospecimens, significantly compromising sample quality. In this communication, we report the development of an electrospun lyoprotectant matrix and isothermal vitrification methodology for non-cryogenic stabilization and storage of liquid biospecimens. The lyoprotectant matrix was mainly composed of trehalose and dextran (and various low concentration excipients targeting different mechanisms of damage), and it was engineered to minimize heterogeneity during vitrification. The technology was validated using five biomarkers; LDH, CRP, PSA, MMP-7, and C3a. Complete recovery of LDH, CRP, and PSA levels was achieved post-rehydration while more than 90% recovery was accomplished for MMP-7 and C3a, showing promise for isothermal vitrification as a safe, efficient, and low-cost alternative to cryogenic storage.

High throughput sorting of micro/nanoparticles of similar sizes is of significant interest in many biological and chemical applications. In this work, we report a simple and cost-effective sorting technique for separation of similarly-sized particles of dissimilar surface properties within a diffusion-based microfluidic platform using chemotaxis in Escherichia coli bacteria. Differences in surface chemistry of two groups of similarly-sized nanoparticles in a mixture were exploited to selectively assemble one particle group onto motile E. coli, through either specific or non-specific adhesion, and separate them from the remaining particle group via chemotaxis of the attached bacteria. To enable optimal operation of the sorting platform, the chemotaxis behavior of E. coli bacteria in response to casamino acids, the chemoeffector of choice was first characterized. The chemical concentration gradient range within which the bacteria exhibit a positive chemotactic response was found to be within 0.25 × 10−7–1.0 × 10−3 g ml−1 mm−1. We demonstrate that at the optimum concentration gradient of 5.0 × 10−4 g ml−1 mm−1, a sorting efficiency of up to 81% at a throughput of 2.4 × 105 particles per min can be achieved. Sensitivity of the sorting efficiency to the adhesion mechanism and particle size in the range of 320–1040 nm was investigated.

Slipping and tripping contribute to a large number of falls and fall-related injuries. While the vestibular system is known to contribute to balance and fall prevention, it is unclear whether it contributes to detecting slip or trip onset. Therefore, the purpose of this study was to investigate the effects of slipping and tripping on head acceleration during walking. This information would help determine whether individuals with vestibular dysfunction are likely to be at a greater risk of falls due to slipping or tripping, and would inform the potential development of assistive devices providing augmented sensory feedback for vestibular dysfunction. Twelve young men were exposed to an unexpected slip or trip. Head acceleration was measured and transformed to an approximate location of the vestibular system. Peak linear acceleration in anterior, posterior, rightward, leftward, superior, and inferior directions were compared between slipping, tripping, and walking. Compared to walking, peak accelerations were up to 4.68 m/s2 higher after slipping, and up to 10.64 m/s2 higher after tripping. Head acceleration first deviated from walking 100-150ms after slip onset and 0-50ms after trip onset. The temporal characteristics of head acceleration support a possible contribution of the vestibular system to detecting trip onset, but not slip onset. Head acceleration after slipping and tripping also appeared to be sufficiently large to contribute to the balance recovery response.

5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.

The SBES Graduate Student Research Symposium was developed to provide students and faculty the opportunity to interact and exchange research ideas with colleagues and industry personnel. This program book includes a schedule of events and abstracts from student oral and poster presentations.

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10⁻⁴ M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10−7 M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10⁻⁵ M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of bet

When a cell is exposed to a sufficiently intense electric field (for a sufficient duration), local defects in the cell membrane appear and become permeable to molecules that otherwise cannot pass through it. This phenomenon is commonly known as electroporation but additional terms electropermeabilization and PEF (i.e. pulsed electric field) treatment are commonly used as well (the latter mainly in food processing area). In reversible electroporation, cell membrane permeabilization to molecules otherwise deprived of transmembrane transport mechanisms is a transient phenomenon as after some time membrane reseals and reestablishes its homeostatic semipermeable properties, allowing ions and other small molecules to passively cross...

Both beta adrenergic and musoarinio receptor stimulation independently potentiate arrhy-thmogenesis. However, the effect of simultaneous stimulation on arrhy-thmogenesis is not well known. The purpose of this study was to determine the temporal response of arrhythmia risk to individual and combined autonomic agonists. Guinea pig hearts were excised and Langendorff-perfused. The beta adrenergic receptor and musoarinio receptor agonists were isoproterenol (ISO, 0.6 mu M) and acetylcholine (ACh, 10 mu M), respectively. All measurements with agonists occurred over 21 min. ISO induced ectopic activity for the first 8 min. ISO also transiently shortened and then prolonged R-R interval over a similar time course. ACh added after ISO transiently induced ectopic activity for 12 min, while R-R interval invariantly prolonged. ACh alone produced few ectopic beats, while invariantly prolonging R-R interval. In contrast to ISO alone, ISO following ACh significantly increased ectopic activity and shortened R-R interval for the duration of the experiment. Animals aged 17-19 months exhibited sustained arrhythmogenesis while those aged 11-14 did not. When ACh was removed in older hearts while ISO perfused, a transient increase in ectopic activity and decreased R-R interval was observed, similar to ISO alone. These data suggest that pre-treating with and maintaining ACh perfusion can sustain ISO sensitivity, in contrast to ISO perfusion alone.

Increasing sensitivity of modern evaluation tools allows for the study of weaker electric stimulation effects on neural populations. In the current study we examined the effects of sham continuous theta burst (cTBS) transcranial magnetic stimulation to the left dorsolateral prefrontal cortex (DLPFC) upon somatosensory evoked potentials (SEP) and frontal-parietal phase coupling of alpha and beta bands. Sham TMS results in an induced electric field amplitude roughly 5% that of real TMS with a similar spatial extent in cortex. Both real and sham cTBS reduced the amplitude of the frontal P14-N30 SEP and increased local phase coupling in the alpha-beta frequency bands of left frontal cortex. In addition, both sham and real cTBS increased frontal-parietal phase coupling in the alpha-beta bands concomitant with an increase in amplitude of parietal P50-N70 complex. These data suggest that weak electric fields from sham cTBS can affect both local and downstream neuronal circuits, though in a different manner than high strength TMS.

This report describes the methodology, diagnostic yield, and adverse events (AE) associated with frame-based stereotactic brain biopsies (FBSB) obtained from 26 dogs with solitary forebrain lesions. Medical records were reviewed from dogs that underwent FBSB using two stereotactic headframes designed for use in small animals and compatible with computed tomographic (CT) and magnetic resonance (MR) imaging. Stereotactic plans were generated from MR and CT images using commercial software, and FBSB performed both with (14/26) and without intraoperative image guidance. Records were reviewed for diagnostic yield, defined as the proportion of biopsies producing a specific neuropathological diagnosis, AE associated with FBSB, and risk factors for the development of AE. Postprocedural AE were evaluated in 19/26 dogs that did not proceed to a therapeutic intervention immediately following biopsy. Biopsy targets included intra-axial telencephalic masses (24/26), one intra-axial diencephalic mass, and one extra-axial parasellar mass. The median target volume was 1.99 cm(3). No differences in patient, lesion, or outcome variables were observed between the two headframe systems used or between FBSB performed with or without intraoperative CT guidance. The diagnostic yield of FBSB was 94.6%. Needle placement error was a significant risk factor associated with procurement of non-diagnostic biopsy specimens. Gliomas were diagnosed in 24/26 dogs, and meningioma and granulomatous meningoencephalitis in 1 dog each. AE directly related to FBSB were observed in a total of 7/26 (27%) of dogs. Biopsy-associated clinical morbidity, manifesting as seizures and transient neurological deterioration, occurred in 3/19 (16%) of dogs. The case fatality rate was 5.2% (1/19 dogs), with death attributable to intracranial hemorrhage. FBSB using the described apparatus was relatively safe and effective at providing neuropathological diagnoses in dogs with focal forebrain lesions.

This program book includes a schedule of events and abstracts from the 14th annual School of Biomedical Engineering & Sciences Graduate Student Research Symposium.

A high-performance public-domain Monte Carlo optical simulator is highly desirable to solve complex heterogeneous optical problems in biomedical engineering. Recently, we developed a Tetrahedron-based Inhomogeneous Monte- Carlo Optical Simulator (TIM-OS) pubware, which addresses this challenge efficiently and accurately based on the tetrahedral mesh.

Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 +/- 4.0%, n = 10) without changing QRS duration. Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34 degrees C) produced significantly earlier excited beats (53 +/- 14%, n = 5) with wider QRS durations (58 +/- 6.3%, n = 5) than suppressed beats. Digoxin + hypothermia (0.75 mu M) produced the most excited beats with significantly earlier RCL (44 +/- 3.2%, n = 6) and wider QRS (60 +/- 3.1%, n = 6) ratios relative to suppressed beats. Increasing pacing further shortened RCL (30 +/- 7.8%, n = 6). In a prospective study, TTX (100 nM) increased RCL ratio (15 +/- 6.0%, n = 10) without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.

Background For electroporation-based therapies, accurate modeling of the electric field distribution within the target tissue is important for predicting the treatment volume. In response to conventional, unipolar pulses, the electrical impedance of a tissue varies as a function of the local electric field, leading to a redistribution of the field. These dynamic impedance changes, which depend on the tissue type and the applied electric field, need to be quantified a priori, making mathematical modeling complicated. Here, it is shown that the impedance changes during high-frequency, bipolar electroporation therapy are reduced, and the electric field distribution can be approximated using the analytical solution to Laplace's equation that is valid for a homogeneous medium of constant conductivity. Methods Two methods were used to examine the agreement between the analytical solution to Laplace's equation and the electric fields generated by 100 µs unipolar pulses and bursts of 1 µs bipolar pulses. First, pulses were applied to potato tuber tissue while an infrared camera was used to monitor the temperature distribution in real-time as a corollary to the electric field distribution. The analytical solution was overlaid on the thermal images for a qualitative assessment of the electric fields. Second, potato ablations were performed and the lesion size was measured along the x- and y-axes. These values were compared to the analytical solution to quantify its ability to predict treatment outcomes. To analyze the dynamic impedance changes due to electroporation at different frequencies, electrical impedance measurements (1 Hz to 1 MHz) were made before and after the treatment of potato tissue. Results For high-frequency bipolar burst treatment, the thermal images closely mirrored the constant electric field contours. The potato tissue lesions differed from the analytical solution by 39.7 ± 1.3 % (x-axis) and 6.87 ± 6.26 % (y-axis) for conventional unipolar pulses, and 15

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.