Stretchable and Micropatterned Membrane for Osteogenic Differentation of Stem Cells

Stem cells have emerged as potentially useful cells for regenerative medicine applications. To fully harness this potential, it is important to develop in vitro cell culture platforms with spatially regulated mechanical, chemical, and biological cues to induce the differentiation of stem cells. In this study, a cell culture platform was constructed that used polydopamine (PDA)-coated parafilm. The modified parafilm supports cell attachment and proliferation. In addition, because of the superb plasticity and ductility of the parafilm, it can be easily micropatterned to regulate the spatial arrangements of cells, and can exert different mechanical tensions. Specifically, we constructed a PDA-coated parafilm with grooved micropatterns to induce the osteogenic differentiation of stem cells. Adipose-derived mesenchymal stem cells that were cultured on the PDA-coated parafilm exhibited significantly higher osteogenic commitment in response to mechanical and spatial cues compared to the ones without stretch. Our findings may open new opportunities for inducing osteogenesis of stem cells in vitro using the platform that combines mechanical and spatial cues

Biomimetic Microfluidic Device for in Vitro Antihypertensive Drug Evaluation

Microfluidic devices have emerged as revolutionary, novel platforms for in vitro drug evaluation. In this work, we developed a facile method for evaluating antihypertensive drugs using a microfluidic chip. This microfluidic chip was generated using the elastic material poly­(dimethylsiloxane) (PDMS) and a microchannel structure that simulated a blood vessel as fabricated on the chip. We then cultured human umbilical vein endothelial cells (HUVECs) inside the channel. Different pressures and shear stresses could be applied on the cells. The generated vessel mimics can be used for evaluating the safety and effects of antihypertensive drugs. Here, we used hydralazine hydrochloride as a model drug. The results indicated that hydralazine hydrochloride effectively decreased the pressure-induced dysfunction of endothelial cells. This work demonstrates that our microfluidic system provides a convenient and cost-effective platform for studying cellular responses to drugs under mechanical pressure

Microfluidic Generation of Polydopamine Gradients on Hydrophobic Surfaces

Engineered surface-bound molecular gradients are of great importance for a range of biological applications. In this paper, we fabricated a polydopamine gradient on a hydrophobic surface. A microfluidic device was used to generate a covalently conjugated gradient of polydopamine (PDA), which changed the wettabilty and the surface energy of the substrate. The gradient was subsequently used to enable the spatial deposition of adhesive proteins on the surface. When seeded with human adipose mesenchymal stem cells, the PDA-graded surface induced a gradient of cell adhesion and spreading. The PDA gradient developed in this study is a promising tool for controlling cellular behavior and may be useful in various biological applications

Online Monitoring of Superoxide Anions Released from Skeletal Muscle Cells Using an Electrochemical Biosensor Based on Thick-Film Nanoporous Gold

Online detection and accurate quantification of superoxide anions released from skeletal muscle tissue is important in both physiological and pathological contexts. Above certain physiologically redundant levels, superoxides may exert toxic effects. Here we present design, fabrication, and successful testing of a highly sensitive electrochemical superoxide biosensor based on nanoporous gold (NPG) immobilized with cytochrome-<i>c</i> (cyt-<i>c</i>). A significant 14-fold enhancement in the biosensor sensitivity was achieved using NPG instead of nonporous gold, enabling the device to quantify minuscule levels of superoxides. Such improvement was attributed to the very large surface-to-volume ratio of the NPG network. The average values of superoxide sensitivity and analytical limit of detection (LOD) were 1.90 ± 0.492 nA nM^–1 cm^–2 and 3.7 nM, respectively. The sensor was employed to measure the rates of superoxide release from C2C12 myoblasts and differentiated myotubes upon stimulation with an endogenous superoxide-producing drug. To account for the issue of sensor-to-sensor sensitivity variations, each sensor was individually calibrated prior to measurements of biologically released superoxides. For the drug concentrations studied, C2C12 superoxide generation rates varied from 0.03 to 0.2 pM min^–1 cell^–1, within the range of superoxide release rates from normally contracting to fatiguing skeletal muscle tissue. Electrochemically obtained results were validated using a fluorescent superoxide probe. Compared to other destructive methods, the NPG-based electrochemical biosensor provides unique advantages in tissue engineering because of its higher sensitivity and the ability to measure the levels of biologically released superoxides in real-time

Development of Flexible Cell-Loaded Ultrathin Ribbons for Minimally Invasive Delivery of Skeletal Muscle Cells

Cell transplantation therapy provides a potential solution for treating skeletal muscle disorders, but cell survival after transplantation is poor. This limitation could be addressed by grafting donor cells onto biomaterials to protect them against harsh environments and processing, consequently improving cell viability in situ. Thus, we present here the fabrication of poly­(lactic-<i>co</i>-glycolic acid) (PLGA) ultrathin ribbons with “canal-like” structures using a microfabrication technique to generate ribbons of aligned murine skeletal myoblasts (C2C12). We found that the ribbons functionalized with a solution of 3,4-dihydroxy-l-phenylalanine (DOPA) and then coated with poly-l-lysine (PLL) and fibronectin (FN) improve cell attachment and support the growth of C2C12. The viability of cells on the ribbons is evaluated following the syringe-handling steps of injection with different needle sizes. C2C12 cells readily adhere to the ribbon surface, proliferate over time, align (over 74%), maintain high viability (over 80%), and differentiate to myotubes longer than 400 μm. DNA content quantification carried out before and after injection and myogenesis evaluation confirm that cell-loaded ribbons can safely retain cells with high functionality after injection and are suitable for minimally invasive cell transplantation

Engineered Nanomembranes for Directing Cellular Organization Toward Flexible Biodevices

Controlling the cellular microenvironment can be used to direct the cellular organization, thereby improving the function of synthetic tissues in biosensing, biorobotics, and regenerative medicine. In this study, we were inspired by the microstructure and biological properties of the extracellular matrix to develop freestanding ultrathin polymeric films (referred as “nanomembranes”) that were flexible, cell adhesive, and had a morphologically tailorable surface. The resulting nanomembranes were exploited as flexible substrates on which cell-adhesive micropatterns were generated to align C2C12 skeletal myoblasts and embedded fibril carbon nanotubes enhanced the cellular elongation and differentiation. Functional nanomembranes with tunable morphology and mechanical properties hold great promise in studying cell–substrate interactions and in fabricating biomimetic constructs toward flexible biodevices

Gelatin–Polyaniline Composite Nanofibers Enhanced Excitation–Contraction Coupling System Maturation in Myotubes

In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced myotube maturation. Thus, in myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation