Pathogenic Hydrogel? A Novel-Entrapment Neuropathy Model Induced by Ultrasound-Guided Perineural Injections.

Entrapment neuropathy (EN) is a prevalent and debilitative condition caused by a complex pathogenesis that involves a chronic compression-edema-ischemia cascade and perineural adhesion that results in excessive shear stress during motion. Despite decades of research, an easily accessible and surgery-free animal model mimicking the mixed etiology is currently lacking, thus limiting our understanding of the disease and the development of effective therapies. In this proof-of-concept study, we used ultrasound-guided perineural injection of a methoxy poly(ethylene glycol)-b-Poly(lactide-co-glycoilide) carboxylic acid (mPEG-PLGA-BOX) hydrogel near the rat's sciatic nerve to induce EN, as confirmed sonographically, electrophysiologically, and histologically. The nerve that was injected with hydrogel appeared unevenly contoured and swollen proximally with slowed nerve conduction velocities across the injected segments, thus showing the compressive features of EN. Histology showed perineural cellular infiltration, deposition of irregular collagen fibers, and a possible early demyelination process, thus indicating the existence of adhesions. The novel method provides a surgery-free and cost-effective way to establish a small-animal model of EN that has mixed compression and adhesion features, thus facilitating the additional elucidation of the pathophysiology of EN and the search for promising treatments.

Evaluation of cytotoxic T lymphocyte-mediated anticancer response against tumor interstitium-simulating physical barriers.

Tumor antigen-specific cytotoxic T lymphocyte (CTL) is a promising agent for cancer therapy. Most solid tumors are characterized by increased interstitial fluid pressure (IFP) and dense collagen capsule, which form physical barriers to impede cancer treatment. However, it remains unclear how CTL-mediated anticancer response is affected at the presence of these obstacles. Using a microfluidic-based platform mimicking these obstacles, we investigated the migration characteristics and performance of anticancer response of CTLs targeting hepatic cancer cells via antigen-specific and allogeneic recognition. The device consisted of slit channels mimicking the narrow interstitial paths constrained by the fibrous capsule and increased IFP was simulated by applying hydrostatic pressure to the tumor center. We found that antigen-specificity of CTLs against the targeted cancer cells determined the cytotoxic efficacy of the CTLs but did not significantly affect the success rate in CTLs that attempted to infiltrate into the tumor center. When increased IFP was present in the tumor center, CTL recruitment to tumor peripheries was promoted but success of infiltration was hindered. Our results highlight the importance of incorporating the physical characteristics of tumor interstitum into the development of CTL-based cancer immunotherapy.

Shear-wave elasticity measurements of three-dimensional cell cultures for mechanobiology.

Studying mechanobiology in three-dimensional (3D) cell cultures better recapitulates cell behaviors in response to various types of mechanical stimuli in vivo Stiffening of the extracellular matrix resulting from cell remodeling potentiates many pathological conditions, including advanced cancers. However, an effective tool for measuring the spatiotemporal changes in elastic properties of such 3D cell cultures without directly contacting the samples has not been reported previously. We describe an ultrasonic shear-wave-based platform for quantitatively evaluating the spatiotemporal dynamics of the elasticity of a matrix remodeled by cells cultured in 3D environments. We used this approach to measure the elasticity changes of 3D matrices grown with highly invasive lung cancer cells and cardiac myoblasts, and to delineate the principal mechanism underlying the stiffening of matrices remodeled by these cells. The described approach can be a useful tool in fields investigating and manipulating the mechanotransduction of cells in 3D contexts, and also has potential as a drug-screening platform.

Protective Effect of Vanillic Acid against Hyperinsulinemia, Hyperglycemia and Hyperlipidemia via Alleviating Hepatic Insulin Resistance and Inflammation in High-Fat Diet (HFD)-Fed Rats.

Excess free fatty acid accumulation from abnormal lipid metabolism results in the insulin resistance in peripheral cells, subsequently causing hyperinsulinemia, hyperglycemia and/or hyperlipidemia in diabetes mellitus (DM) patients. Herein, we investigated the effect of phenolic acids on glucose uptake in an insulin-resistant cell-culture model and on hepatic insulin resistance and inflammation in rats fed a high-fat diet (HFD). The results show that vanillic acid (VA) demonstrated the highest glucose uptake ability among all tested phenolic acids in insulin-resistant FL83B mouse hepatocytes. Furthermore, rats fed HFD for 16 weeks were orally administered with VA daily (30 mg/kg body weight) at weeks 13-16. The results show that levels of serum insulin, glucose, triglyceride, and free fatty acid were significantly decreased in VA-treated HFD rats (p < 0.05), indicating the protective effects of VA against hyperinsulinemia, hyperglycemia and hyperlipidemia in HFD rats. Moreover, VA significantly reduced values of area under the curve for glucose (AUCglucose) in oral glucose tolerance test and homeostasis model assessment-insulin resistance (HOMA-IR) index, suggesting the improving effect on glucose tolerance and insulin resistance in HFD rats. The Western blot analysis revealed that VA significantly up-regulated expression of hepatic insulin-signaling and lipid metabolism-related protein, including insulin receptor, phosphatidylinositol-3 kinase, glucose transporter 2, and phosphorylated acetyl CoA carboxylase in HFD rats. VA also significantly down-regulated hepatic inflammation-related proteins, including cyclooxygenase-2 and monocyte chemoattractant protein-1 expressions in HFD rats. These results indicate that VA might ameliorate insulin resistance via improving hepatic insulin signaling and alleviating inflammation pathways in HFD rats. These findings also suggest the potential of VA in preventing the progression of DM.

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device.

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 µM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.

Substrate stiffness regulates filopodial activities in lung cancer cells.

Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light on the understanding and treatment of cancer progression and metastasis.

Increased hydrostatic pressure enhances motility of lung cancer cells.

Interstitial fluid pressures within most solid tumors are significantly higher than that in the surrounding normal tissues. Therefore, cancer cells must proliferate and migrate under the influence of elevated hydrostatic pressure while a tumor grows. In this study, we developed a pressurized cell culture device and investigated the influence of hydrostatic pressure on the migration speeds of lung cancer cells (CL1-5 and A549). The migration speeds of lung cancer cells were increased by 50-60% under a 20 mmHg hydrostatic pressure. We also observed that the expressions of aquaporin in CL1-5 and A549 cells were increased under the hydrostatic pressure. Our preliminary results indicate that increased hydrostatic pressure plays an important role in tumor metastasis.

Microfluidic device with dual mechanical cues for cell migration investigation.

Cell migration plays an important role in numerous physiological and pathological conditions, such as angiogenesis, wound healing and cancer metastasis. Understanding the fundamental mechanisms of cell migration is crucial to develop strategies for disease treatment and regenerative medicine. Several biomechanical cues have been well studied about their effects on guiding cell migration. However, the effects of dual or multiple cues on cell migration are barely addressed. In this work, we developed a microfluidic-based device to study the combinatory effects of osmotic and stiffness gradient on cell migration. Computer simulation and experimental validation showed that the device was capable of providing stable osmotic and stiffness gradient to cultured cells at the same time. Preliminary results suggest that our device has a valuable potential in studying cell migration in complex conditions which better recapitulate the complex environmental conditions in vivo.

Imaging monitored loosening of dense fibrous tissues using high-intensity pulsed ultrasound.

Pulsed high-intensity focused ultrasound (HIFU) is proposed as a new alternative treatment for contracture of dense fibrous tissue. It is hypothesized that the pulsed-HIFU can release the contracted tissues by attenuating tensile stiffness along the fiber axis, and that the stiffness reduction can be quantitatively monitored by change of B-mode images. Fresh porcine tendons and ligaments were adapted to an ex vivo model and insonated with pulsed-HIFU for durations ranging from 5 to 30 min. The pulse length was 91 µs with a repetition frequency of 500 Hz, and the peak rarefactional pressure was 6.36 MPa. The corresponding average intensities were kept around 1606 W cm(-2) for ISPPA and 72.3 W cm(-2) for ISPTA. B-mode images of the tissues were acquired before and after pulsed-HIFU exposure, and the changes in speckle intensity and organization were analyzed. The tensile stiffness of the HIFU-exposed tissues along the longitudinal axis was examined using a stretching machine. Histology examinations were performed by optical and transmission electron microscopy. Pulsed-HIFU exposure significantly decreased the tensile stiffness of the ligaments and tendons. The intensity and organization of tissue speckles in the exposed region were also decreased. The speckle changes correlated well with the degree of stiffness alteration. Histology examinations revealed that pulsed-HIFU exposure probably damages tissues via a cavitation-mediated mechanism. Our results suggest that pulsed-HIFU with a low duty factor is a promising tool for developing new treatment strategies for orthopedic disorders.

The migration speed of cancer cells influenced by macrophages and myofibroblasts co-cultured in a microfluidic chip.

We employ a microfluidic chip with three culture chambers to investigate the interactions among lung cancer cells, macrophages and myofibroblasts. By mixing the conditioned media of macrophages and myofibroblasts in this chip, we confirm that these two stromal cells have synergistic effects in accelerating the migration of cancer cells. However, as the myofibroblasts are pretreated with the conditioned medium of macrophages, the myofibroblasts' ability to enhance the migration of cancer cells is lowered. The tumour necrosis factor-α produced by macrophages reduces the expression of α-smooth muscle actin and the secretion of transforming growth factor-ß1 in myofibroblasts. Once the tumour necrosis factor-α in the macrophage conditioned medium is neutralized, the macrophage medium-pretreated myofibroblasts can still accelerate the migration of cancer cells.

Elastic modulus measurements of human liver and correlation with pathology.

Viral hepatitis causes fibrosis in the liver and may change mechanical properties of the liver. To evaluate the impact of fibrosis on elastic properties of human liver and to investigate potential benefits of ultrasonic elasticity imaging, 19 fresh human liver samples and 1 hepatic tumor (focal nodular hyperplasia) sample obtained during operations were studied. Simple 1-D estimates based on the cyclic compression-relaxation method were performed. Elastic modulus values were derived from the predetermined strain (controlled by a step motor system) and the stress values (measured by an electronic balance). Each specimen subsequently received histologic examination and a grade of liver fibrosis was scored from 0 to 5. Results show that the elastic modulus values were on the order of several hundreds to thousands of Pascals. The elastic modulus generally increased with the fibrosis grade, although some discrepancies existed at the middle grades of fibrosis (scores 1 to 3). The correlation between the fibrosis score and the elastic modulus was significant (p < 0.01) based on the statistical analysis using the Pearson correlation method. In addition, the relation between the elastic modulus and the fibrosis grade generally exhibited a quadratic trend. It was concluded that severity of fibrosis had a good correlation with stiffness of the liver. Results also indicated that the elasticity imaging of the liver may provide significant clinical values if the elastic modulus can be accurately measured.