Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier.

The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.

Triamcinolone acetonide nanoparticles incorporated in thermoreversible gels for age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the US affecting millions yearly. It is characterized by intraocular neovascularization, inflammation and retinal damage which can be ameliorated through intraocular injections of glucocorticoids. However, the complications that arise from repetitive injections as well as the difficulty posed by targeting the posterior segment of the eye make this interesting territory for the development of novel drug delivery systems (DDS). In the present study, we described the development of a DDS composed of triamcinolone acetonide-encapsulated PEGylated PLGA nanoparticles (NP) incorporated into PLGA-PEG-PLGA thermoreversible gel and its use against VEGF expression characteristic of AMD. We found that the NP with mean size of 208 ± 1.0 nm showed uniform size distribution and exhibited sustained release of the drug. We also demonstrated that the polymer can be injected as a solution and transition to a gel phase based on the biological temperature of the eye. Additionally, the proposed DDS was non-cytotoxic to ARPE-19 cells and significantly reduced VEGF expression by 43.5 ± 3.9% as compared to a 1.53 ± 11.1% reduction with triamcinolone. These results suggest the proposed DDS will contribute to the development of novel therapeutic strategies for AMD.

Web Conferencing Facilitation Within Problem-Based Learning Biomedical Engineering Courses.

Problem-based learning (PBL) has been effectively used within BME education, though there are several challenges in its implementation within courses with larger enrollments. Furthermore, the sudden transition to online learning from the COVID-19 pandemic introduced additional challenges in creating a similar PBL experience in an online environment. Online constrained PBL was implemented through asynchronous modules and synchronous web conferencing with rotating facilitators. Overall, facilitators perceived web conferencing facilitation to be similar to in-person, but noted that students were more easily "hidden" or distracted. Students did not comment on web conferencing facilitation specifically, but indicated the transition to online PBL was smooth. Course instructors identified that a fully synchronous delivery as well as modifications of Group Meeting Minutes assignments as potential modifications for future offerings. Future work will aim to address the perceptions and effectiveness of web conferencing facilitation for PBL courses within an undergraduate BME curriculum, as web conferencing could prove to be another significant breakthrough in addressing challenges of problem-based learning courses.

Protective effects of genistein on proinflammatory pathways in human brain microvascular endothelial cells.

Proinflammatory cerebromicrovascular environment has been implicated in the critical early pathologic events in a variety of neurodegenerative diseases. Recent studies also have demonstrated the potential beneficial effects of soy isoflavones. However, cellular and molecular mechanisms underlying these processes are not fully understood. The present study was designed to examine the hypothesis that soy isoflavone genistein may attenuate cytokine-induced proinflammatory pathways in human brain microvascular endothelial cells. The quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that pretreatment of HBMEC with increasing concentrations of genistein significantly and dose-dependently inhibited cytokine-induced up-regulation of mRNA and protein expression of proinflammatory mediators such as tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, interleukin-8, and intercellular adhesion molecule-1. In addition, genistein pretreatment significantly reduced cytokine-mediated up-regulation of transmigration of blood leukocytes in a dose-dependent manner. Our results suggest that genistein may attenuate proinflammatory pathways through inhibition of cytokine-induced overexpression of proinflammatory mediators and inflammatory reactions in human brain microvascular endothelial cells.

The comparison of three high-frequency chest compression devices.

High-frequency chest compression (HFCC) is shown to enhance clearance of pulmonary airway secretions. Several HFCC devices have been designed to provide this therapy. Standard equipment consists of an air pulse generator attached by lengths of tubing to an adjustable, inflatable vest/jacket (V/J) garment. In this study, the V/Js were fitted over a mannequin. The three device air pulse generators produced characteristic waveform patterns. The variations in the frequency and pressure setting of devices were consistent with specific device design features. These studies suggest that a better understanding of the effects of different waveform, frequency, and pressure combinations may improve HFCC therapeutic efficacy of three different HFCC machines. The V/J component of HFCC devices delivers the compressive pulses to the chest wall to produce both airflow through and oscillatory effects in the airways. The V/J pressures of three HFCC machines were measured and analyzed to characterize the frequency, pressure, and waveform patterns generated by each of three device models. The dimensions of all V/Js were adjusted to a circumference of approximately 110% of the chest circumference. The V/J pressures were measured, and maximum, minimum, and mean pressure, pulse pressure, and root mean square of three pulse generators were calculated. Jacket pressures ranged between 2 and 34 mmHg. The 103 and 104 models' pulse pressures increased with the increase in HFCC frequency at constant dial pressure. With the ICS the pulse pressure decreased when the frequency increased. The waveforms of models 103 and 104 were symmetric sine wave and asymmetric sine wave patterns, respectively. The ICS had a triangular waveform. At 20 Hz, both the 103 and 104 were symmetric sine waveform but the ICS remained triangular. Maximum crest factors emerged in low-frequency and high-pressure settings for the ICS and in the high-frequency and low-pressure settings for models 103 and 104. Recognizing the significant differences in frequency and pressure amplitude may help clinicians and patients optimize the efficacy of HFCC therapy. Evidence-based therapeutic guidelines are needed.

Development of an In Vitro 3D Brain Tissue Model Mimicking In Vivo-Like Pro-inflammatory and Pro-oxidative Responses.

To analyze complex inflammatory responses in an in vitro system, we constructed a new 3D in vitro brain tissue model that exhibits in vivo-like tissue responses (e.g. immune cell phenotypes, and molecular response) to inflammatory stimuli. Finite element modeling of oxygen diffusion and cellular oxygen consumption predicted the oxygen profile within 3D structures, consisting of Type I collagen hydrogel embedded with murine microglia. Viability and cytotoxicity analyses supported the mathematical analysis, determining optimal cell growth conditions for 3D construct development. Real-time RT-PCR and ELISA demonstrated significant up-regulation of pro-inflammatory mediators, such as TNF-α, MCP-1, IL-6 and IL-1ß, in lipopolysaccharide (LPS)-stimulated in vitro cell culture (2D and 3D) and in vivo mouse model systems. Interestingly, levels of inflammatory responses from the in vitro 3D model system were more similar to in vivo than in vitro 2D. Additionally, in situ dihydroethidium (DHE) assay and immunofluorescence staining revealed that levels of LPS-stimulated reactive oxygen species (ROS) generation and microglial activation from in vitro 3D model system were closer to in vivo than in vitro 2D. These results demonstrated that an in vitro 3D model provides more physiologically relevant pro-oxidative and pro-inflammatory environments in brain than an in vitro 2D model.

Investigation of non-uniform airflow signal oscillation during high frequency chest compression.

BACKGROUND: High frequency chest compression (HFCC) is a useful and popular therapy for clearing bronchial airways of excessive or thicker mucus. Our observation of respiratory airflow of a subject during use of HFCC showed the airflow oscillation by HFCC was strongly influenced by the nonlinearity of the respiratory system. We used a computational model-based approach to analyse the respiratory airflow during use of HFCC. METHODS: The computational model, which is based on previous physiological studies and represented by an electrical circuit analogue, was used for simulation of in vivo protocol that shows the nonlinearity of the respiratory system. Besides, airflow was measured during use of HFCC. We compared the simulation results to either the measured data or the previous research, to understand and explain the observations. RESULTS AND DISCUSSION: We could observe two important phenomena during respiration pertaining to the airflow signal oscillation generated by HFCC. The amplitudes of HFCC airflow signals varied depending on spontaneous airflow signals. We used the simulation results to investigate how the nonlinearity of airway resistance, lung capacitance, and inertance of air characterized the respiratory airflow. The simulation results indicated that lung capacitance or the inertance of air is also not a factor in the non-uniformity of HFCC airflow signals. Although not perfect, our circuit analogue model allows us to effectively simulate the nonlinear characteristics of the respiratory system. CONCLUSION: We found that the amplitudes of HFCC airflow signals behave as a function of spontaneous airflow signals. This is due to the nonlinearity of the respiratory system, particularly variations in airway resistance.

Folate Conjugated Cellulose Nanocrystals Potentiate Irreversible Electroporation-induced Cytotoxicity for the Selective Treatment of Cancer Cells.

Cellulose nanocrystals are rod-shaped, crystalline nanoparticles that have shown prom-ise in a number of industrial applications for their unique chemical and physical properties. However, investigations of their abilities in the biomedical field are limited. The goal of this study is to show the potential use of folic acid-conjugated cellulose nanocrystals in the potentiation of irreversible electroporation-induced cell death in folate receptor (FR)-positive cancers. We optimized key pulse parameters including pulse duration, intensity, and incubation time with nanoparticles prior to electroporation. FR-positive cancer cells, KB and MDA-MB-468, were preincubated with cellulose nanocrystals (CNCs) conjugated with the targeting molecule folic acid (FA), 10 and 20 min respectively, prior to application of the optimized pulse electric field (PEF), 600 and 500 V/cm respectively. We have shown cellulose nanocrystals' ability to potentiate a new technique for tumor ablation, irreversible electroporation. Pre-incubation with FA-conjugated CNCs (CNC-FA) has shown a significant increase in cytotoxicity induced by irreversible electroporation in FR-positive cancer cells, KB and MDA-MB-468. Non-targeted CNCs (CNC-COOH) did not potentiate IRE when preincubated at the same parameters as previously stated in these cell types. In addition, CNC-FA did not potentiate irreversible electroporation-induced cytotoxicity in a FR-negative cancer cell type, A549. Without changing irreversible electroporation parameters it is possible to increase the cytotoxic effect on FR-positive cancer cells by exploiting the specific binding of FA to the FR, while not causing further damage to FR-negative tissue.

Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth.

Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 µs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 µs, containing individual pulses 1, 2, or 5 µs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.

A population pharmacokinetic-pharmacodynamic model for simvastatin that predicts low-density lipoprotein-cholesterol reduction in patients with primary hyperlipidaemia.

Simvastatin (SV), a HMG-CoA reductase inhibitor, is widely used for the treatment of hyperlipidaemia. The objectives of the present study were to develop a population pharmacokinetic-pharmacodynamic (PK-PD) model for simvastatin and to evaluate its usefulness in predicting the dose-response relationship of simvastatin in patients with hyperlipidaemia. The data were obtained from a drug-drug interaction study to assess the effect of aspirin on the PK of simvastatin. Twenty-seven healthy volunteers were given simvastatin 40 mg daily for 14 days in whom aspirin 100 mg q.d. was co-administered after day 8. Full PK studies were performed on days 1, 7 and 14 in addition to trough sampling on days 5, 6, 12 and 13. Low-density lipoprotein-cholesterol (LDL-C) levels were also measured serially. Then, a population PK-PD model for simvastatin and its active metabolite, simvastatin acid (SVA), was developed using mixed effect methods (NONMEM Ver. 6.2). A simple linear PK model with parent and metabolite compartments provided the best fit for the 2647 concentrations of simvastatin and simvastatin acid, and a turnover model was used to describe the change in LDL-C levels. The dose-response curve simulated from the final model and those obtained from the literature overlapped very closely. No influence of aspirin was observed in PK or PD. A simple PK-PD model developed using only 2-week study data from fewer than 30 healthy volunteers successfully predicted the dose-response relationship of simvastatin in patients when compared with published data.

Effect of low-frequency pulsatile flow on expression of osteoblastic genes by bone marrow stromal cells.

Perfusion culture of osteoprogenitor cells is a promising means to form a bone-like extracellular matrix for tissue engineering applications, but the mechanism by which hydrodynamic shear stimulates expression of bone extracellular matrix (ECM) proteins is not understood. Osteoblasts are mechanosensitive and respond differently to steady and pulsatile flow. Therefore, to probe the effect of flow, bone marrow stromal cells (BMSCs)--cultured under osteogenic conditions--were exposed to steady or pulsatile flow at frequencies of 0.015, 0.044, or 0.074 Hz. Following 24 h of stimulus, cells were cultured statically for an additional 13 days and then analyzed for the expression of bone ECM proteins collagen 1alpha1 (Col1alpha1), osteopontin, osteocalcin (OC), and bone sialoprotein (BSP). All mRNA levels were elevated by flow, but OC and BSP were enhanced modestly with pulsatile flow. To determine if these effects were related to gene induction during flow, BMSCs were again exposed to steady or pulsatile flow for 24 h, but then analyzed immediately for expression of growth and differentiation factors bone morphogenetic proteins (BMP)-2, -4, and -7, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor-A. All growth and differentiation factors were significantly elevated by flow, except BMP-4 which was suppressed. In addition, expression of BMP-2 and -7 were enhanced and TGF-beta1 suppressed by pulsatile flow relative to steady flow. These results demonstrate that pulsatile flow modulates expression of BMP-2, -7, and TGF-beta1 and suggest that enhanced expression of bone ECM proteins by pulsatile flow may be mediated through the induction of BMP-2 and -7.

Free-knot spline model for analysis of pulmonary function.

The pulmonary function test (PFT) is used to evaluate and monitor respiratory function. The PFT is critical for the care of patients having cystic fibrosis (CF) and adjusting their clinical treatments. We analyzed the percent predicted value of forced expiratory volume in one second (FEV(1)%) from PFT of CF patients collected four times a year from 1966. Longitudinal FEV(1)% for each patient was fitted with linear free-knot spline (FKS) model. We explained as the time when the PFT trend changes. We classified the patients' pulmonary function trend using eight groups of FEV(1)% based on the angle of linear FKS model. The overlapped majority of knots in groups located at 1978, 1991, and 1993 for worsening and at 1983, 1988, and 2000 for not worsening.

High frequency chest compression effects heart rate variability.

High frequency chest compression (HFCC) supplies a sequence of air pulses through a jacket worn by a patient to remove excessive mucus for the treatment or prevention of lung disease patients. The air pulses produced from the pulse generator propagates over the thorax delivering the vibration and compression energy. A number of studies have demonstrated that the HFCC system increases the ability to clear mucus and improves lung function. Few studies have examined the change in instantaneous heart rate (iHR) and heart rate variability (HRV) during the HFCC therapy. The purpose of this study is to measure the change of HRV with four experimental protocols: (a) without HFCC, (b) during Inflated, (c)HFCC at 6Hz, and (d) HFCC at 21Hz. The nonlinearity and regularity of HRV was assessed by approximate entropy (ApEn), a method used to quantify the complexities and randomness. To compute the ApEn, we sectioned with a total of eight epochs and displayed the ApEn over the each epoch. Our results show significant differences in the both the iHR and HRV between the experimental protocols. The iHR was elevated at both the (c) 6Hz and (d) 21Hz condition from without HFCC (10%, 16%, respectively). We also found that the HFCC system tends to increase the HRV. Our study suggests that monitoring iHR and HRV are very important physiological indexes during HFCC therapy.