A microfluidic impedance platform for real-time, in vitro characterization of endothelial cells undergoing fluid shear stress.

We introduce a microfluidic impedance platform to electrically monitor in real-time, endothelium monolayers undergoing fluid shear stress. Our platform incorporates sensing electrodes (SEs) that measure cell behavior and cell-free control electrodes that measure cell culture media resistance simultaneously but independently from SEs. We evaluated three different cellular subpopulations sizes through 50, 100, and 200 µm diameter SEs. We tested their utility in measuring the response of human umbilical vein endothelial cells (HUVECs) at static, constant (17.6 dyne per cm2), and stepped (23.7-35-58.1 dyne per cm2) shear stress conditions. For 14 hours, we collected the impedance spectra (100 Hz-1 MHz) of sheared cells. Using equivalent circuit models, we extracted monolayer permeability (RTER), cell membrane capacitance, and cell culture media resistance. Platform evaluation concluded that: (1) 50 µm SEs (∼2 cells) suffered interfacial capacitance and reduced cell measurement sensitivity, (2) 100 µm SEs (∼6 cells) was limited to measuring cell behavior only and cannot measure cell culture media resistance, and (3) 200 µm SEs (∼20 cells) detected cell behavior with accurate prediction of cell culture media resistance. Platform-based shear stress studies indicated a shear magnitude dependent increase in RTER at the onset of acute flow. Consecutive stepped shear conditions did not alter RTER in the same magnitude after shear has been applied. Finally, endpoint staining of VE-cadherin on the actual SEs and endpoint RTER measurements were greater for 23.7-35-58.1 dyne per cm2 than 17.6 dyne per cm2 shear conditions.

Radiation-induced curcumin release from curcumin-chitosan polymer films.

The probability of human exposure to damaging radiation is increased in activities associated with long-term space flight, medical radiation therapies, and responses to nuclear accidents. However, the development of responsive countermeasures to combat radiation damage to biological tissue is lagging behind rates of human exposure. Herein, we report a radiation-responsive drug delivery system that releases doses of curcumin from a chitosan polymer/film in response to low level gamma radiation exposure. As a fibrous chitosan-curcumin polymer, 1 Gy gamma irradiation (137Cs) released 5 ± 1% of conjugated curcumin, while 6 Gy exposure releases 98 ± 1% of conjugated curcumin. The same polymer was formed into a film through solvent casting. The films showed similar, albeit attenuated behavior in water (100% released) and isopropyl alcohol (32% released) with statistically significant drug release following 2 Gy irradiation. ATR FT-IR studies confirmed glycosidic bond cleavage in the chitosan-curcumin polymer in response to gamma radiation exposure. Similar behavior was noted upon exposure of the polymer to 20 cGy (1 GeV amu-1, at 20 cGy min-1) high linear energy transfer (LET) 56Fe radiation based on FTIR studies. Density Functional Theory calculations indicate homolytic bond scission as the primary mechanism for polymer disintegration upon radiation exposure. Films did not change in thickness during the course of radiation exposure. The successful demonstration of radiation-triggered drug release may lead to new classes of radio-protective platforms for developing countermeasures to biological damage from ionizing radiation.

Release-Modulated Antioxidant Activity of a Composite Curcumin-Chitosan Polymer.

Curcumin is known to have immense therapeutic potential but is hindered by poor solubility and rapid degradation in solution. To overcome these shortcomings, curcumin has been conjugated to chitosan through a pendant glutaric anhydride linker using amide bond coupling chemistry. The hybrid polymer has been characterized by UV-visible, fluorescence, and infrared spectroscopies as well as zeta potential measurements and SEM imaging. The conjugation reactivity was confirmed through gel permeation chromatography and quantification of unconjugated curcumin. An analogous reaction of curcumin with glucosamine, a small molecule analogue for chitosan, was performed and the purified product characterized by mass spectrometry, UV-visible, fluorescence, and infrared spectroscopies. Conjugation of curcumin to chitosan has greatly improved curcumin aqueous solubility and stability, with no significant curcumin degradation detected after one month in solution. The absorbance and fluorescence properties of curcumin are minimally perturbed (λmax shifts of 2 and 5 nm, respectively) by the conjugation reaction. This conjugation strategy required use of one out of two curcumin phenols (one of the main antioxidant functional groups) for covalent linkage to chitosan, thus temporarily attenuating its antioxidant capacity. Hydrolysis-based release of curcumin from the polymer, however, is accompanied by full restoration of curcumin's antioxidant potential. Antioxidant assays show that curcumin radical scavenging potential is reduced by 40% after conjugation, but that full antioxidant potential is restored upon hydrolytic release from chitosan. Release studies show that curcumin is released over 19 days from the polymer and maintains a concentration of 0.23 ± 0.12 µM curcumin/mg polymer/mL solution based on 1% curcumin loading on the polymer. Release studies in the presence of carbonic anhydrase, an enzyme with known phenolic esterase activity, show no significant difference from nonenzymatic release studies, implying that simple ester hydrolysis is the dominant release mechanism. Conjugation of curcumin to chitosan through a phenol ester modification provides improved stability and solubility to curcumin, with ester hydrolysis restoring the full antioxidant potential of curcumin.

Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties.

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

Oriented matrix promotes directional tubulogenesis.

Detailed control over the structural organization of scaffolds and engineered tissue constructs is a critical need in the quest to engineer functional tissues using biomaterials. This work presents a new approach to spatially direct endothelial tubulogenesis. Micropatterned fibronectin substrates were used to control lung fibroblast adhesion and growth and the subsequent deposition of fibroblast-derived matrix during culture. The fibroblast-derived matrix produced on the micropatterned substrates was tightly oriented by these patterns, with an average variation of only 8.5°. Further, regions of this oriented extracellular matrix provided directional control of developing endothelial tubes to within 10° of the original micropatterned substrate design. Endothelial cells seeded directly onto the micropatterned substrate did not form tubes. A metric for matrix anisotropy showed a relationship between the fibroblast-derived matrix and the endothelial tubes that were subsequently developed on the same micropatterns with a resulting aspect ratio over 1.5 for endothelial tubulogenesis. Micropatterns in "L" and "Y" shapes were used to direct endothelial tubes to turn and branch with the same level of precision. These data demonstrate that anisotropic fibroblast-derived matrices instruct the alignment and shape of endothelial tube networks, thereby introducing an approach that could be adapted for future design of microvascular implants featuring organ-specific natural matrix that patterns microvascular growth.

Curcumin encapsulation in submicrometer spray-dried chitosan/Tween 20 particles.

Optimal curcumin delivery for medicinal applications requires a drug delivery system that both solubilizes curcumin and prevents degradation. To achieve this, curcumin has been encapsulated in submicrometer chitosan/Tween 20 particles via a benchtop spray-drying process. Spray-drying parameters have been optimized using a Taguchi statistical approach to minimize particle size and to favor spheroid particles with smooth surfaces, as evaluated with scanning electron microscopy (SEM) imaging. Nearly spherical particles with 285 ± 30 nm diameter and 1.21 axial ratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within the chitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 h period.

Microelastic properties of lung cell-derived extracellular matrix.

The mechanical properties of the extracellular microenvironment regulate cell behavior, including migration, proliferation and morphogenesis. Although the elastic moduli of synthetic materials have been studied, little is known about the properties of naturally produced extracellular matrix. Here we have utilized atomic force microscopy to characterize the microelastic properties of decellularized cell-derived matrix from human pulmonary fibroblasts. This heterogeneous three-dimensional matrix had an average thickness of 5 ± 0.4 µm and a Young's modulus of 105 ± 14 Pa. Ascorbate treatment of the lung fibroblasts prior to extraction produced a twofold increase in collagen I content, but did not affect the stiffness of the matrices compared with matrices produced in standard medium. However, fibroblast-derived matrices that were crosslinked with glutaraldehyde demonstrated a 67% increase in stiffness. This work provides a microscale characterization of fibroblast-derived matrix mechanical properties. An accurate understanding of native three-dimensional extracellular microenvironments will be essential for controlling cell responses in tissue engineering applications.

Single exposure to radiation produces early anti-angiogenic effects in mouse aorta.

Radiation exposure can increase the risk for many non-malignant physiological complications, including cardiovascular disease. We have previously demonstrated that ionizing radiation can induce endothelial dysfunction, which contributes to increased vascular stiffness. In this study, we demonstrate that gamma radiation exposure reduced endothelial cell viability or proliferative capacity using an in vitro aortic angiogenesis assay. Segments of mouse aorta were embedded in a Matrigel-media matrix 1 day after mice received whole-body gamma irradiation between 0 and 20 Gy. Using three-dimensional phase contrast microscopy, we quantified cellular outgrowth from the aorta. Through fluorescent imaging of embedded aortas from Tie2GFP transgenic mice, we determined that the cellular outgrowth is primarily of endothelial cell origin. Significantly less endothelial cell outgrowth was observed in aortas of mice receiving radiation of 5, 10, and 20 Gy radiation, suggesting radiation-induced endothelial injury. Following 0.5 and 1 Gy doses of whole-body irradiation, reduced outgrowth was still detected. Furthermore, outgrowth was not affected by the location of the aortic segments excised along the descending aorta. In conclusion, a single exposure to gamma radiation significantly reduces endothelial cell outgrowth in a dose-dependent manner. Consequently, radiation exposure may inhibit re-endothelialization or angiogenesis after a vascular injury, which would impede vascular recovery.

Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix.

Extracellular matrix plays a critical role in cellular development by providing signaling cues that direct morphogenesis. In order to study both the cues that natural matrix provides and endothelial cell responses to that information, human fetal lung fibroblasts were used to produce a fibrous three-dimensional matrix. Following the removal of the fibroblasts by detergent extraction, protein and proteoglycan constituents of the remaining matrix were identified by immunofluorescence and immunoblotting. Matrix components included fibronectin, tenascin-C, collagen I, collagen IV, collagen VI, versican, and decorin. Colocalization analysis suggested that fibronectin was a uniquely distributed matrix protein. Morphology, three-dimensional matrix adhesions, and integrin-mediated signaling during vasculogenesis were then studied in human endothelial cells seeded onto the fibroblast-derived matrix. Elongated morphology and decreased cell area were noted, as compared with cells on fibronectin-coated coverslips. Cell-matrix adhesions contained vinculin, pY397-FAK, and pY410-p130Cas, and all of these colocalized more with fibronectin than tenascin-C, collagen I, or collagen VI. Additionally, the endothelial cells remodeled the fibroblast-derived matrix and formed networks of tubes with demonstrable lumens. Matrix adhesions in these tubes also predominantly colocalized with fibronectin. The pattern of membrane type 1 matrix metalloprotease expression in the endothelial cells suggested its involvement in the matrix remodeling that occurred during tubulogenesis. These results indicated that information in fibroblast-derived matrix promoted vasculogenic behavior.