Micropatterned Neurovascular Interface to Mimic the Blood-Brain Barrier's Neurophysiology and Micromechanical Function: A BBB-on-CHIP Model.

A hybrid blood-brain barrier (BBB)-on-chip cell culture device is proposed in this study by integrating microcontact printing and perfusion co-culture to facilitate the study of BBB function under high biological fidelity. This is achieved by crosslinking brain extracellular matrix (ECM) proteins to the transwell membrane at the luminal surface and adapting inlet-outlet perfusion on the porous transwell wall. While investigating the anatomical hallmarks of the BBB, tight junction proteins revealed tortuous zonula occludens (ZO-1), and claudin expressions with increased interdigitation in the presence of astrocytes were recorded. Enhanced adherent junctions were also observed. This junctional phenotype reflects in-vivo-like features related to the jamming of cell borders to prevent paracellular transport. Biochemical regulation of BBB function by astrocytes was noted by the transient intracellular calcium effluxes induced into endothelial cells. Geometry-force control of astrocyte-endothelial cell interactions was studied utilizing traction force microscopy (TFM) with fluorescent beads incorporated into a micropatterned polyacrylamide gel (PAG). We observed the directionality and enhanced magnitude in the traction forces in the presence of astrocytes. In the future, we envisage studying transendothelial electrical resistance (TEER) and the effect of chemomechanical stimulations on drug/ligand permeability and transport. The BBB-on-chip model presented in this proposal should serve as an in vitro surrogate to recapitulate the complexities of the native BBB cellular milieus.

Sperm Cell Driven Microrobots-Emerging Opportunities and Challenges for Biologically Inspired Robotic Design.

With the advent of small-scale robotics, several exciting new applications like Targeted Drug Delivery, single cell manipulation and so forth, are being discussed. However, some challenges remain to be overcome before any such technology becomes medically usable; among which propulsion and biocompatibility are the main challenges. Propulsion at micro-scale where the Reynolds number is very low is difficult. To overcome this, nature has developed flagella which have evolved over millions of years to work as a micromotor. Among the microscopic cells that exhibit this mode of propulsion, sperm cells are considered to be fast paced. Here, we give a brief review of the state-of-the-art of Spermbots - a new class of microrobots created by coupling sperm cells to mechanical loads. Spermbots utilize the flagellar movement of the sperm cells for propulsion and as such do not require any toxic fuel in their environment. They are also naturally biocompatible and show considerable speed of motion thereby giving us an option to overcome the two challenges of propulsion and biocompatibility. The coupling mechanisms of physical load to the sperm cells are discussed along with the advantages and challenges associated with the spermbot. A few most promising applications of spermbots are also discussed in detail. A brief discussion of the future outlook of this extremely promising category of microrobots is given at the end.

Stimulation of osteoblast differentiation with guided ultrasound waves.

BACKGROUND: Ultrasound induces mechanical vibration and heat, causing differentiation and proliferation in osteoblasts. All known in vitro evaluations of ultrasound are, however, performed with longitudinal ultrasound waves. We addressed a question: Do other forms of ultrasound waves, such as guided waves (longitudinal and guided flexural) transduced at a remote location, enhance differentiation of osteoblast cells? METHODS: In this study, we employed guided Lamb waves that were induced in a borosilicate glass slide (cortical bone mimic). An average energy of 10-30 mW/cm(2) for 20 min per day was applied to MC3T3 osteoblast-like cells, which were placed 30-75 mm distant from the transducer. RESULTS: The result revealed that guided waves significantly stimulated the differentiation and mineralization of MC3T3 cells. In particular, guided waves elevated mRNA expression levels of bone formation-related genes such as alkaline phosphatase, osteopontin, osteocalcin, osteoprotegerin, and bone sialoprotein on days 8 and 16. In addition, the amount of mineralization found via Alizarin red staining was increased by 157 % (p = 0.034). The amount of mineralization was found to be independent of distance from the transducer (p = 0.967). CONCLUSION: We demonstrate herein that ultrasound in a form of guided Lamb waves is capable of inducing osteoblast differentiation in vitro, and it may enable the stimulation of osteoblasts in vivo over a distance from the site of ultrasound application.

Multilayered Nanoparticles for Gene Delivery Used to Reprogram Human Foreskin Fibroblasts to Neurospheres.

Polycationic nanocomplexes are a robust means for achieving nucleic acid condensation and efficient intracellular gene deliveries. To enhance delivery, a multilayered nanoparticle consisting of a core of electrostatically bound elements was used. These included a histone-mimetic peptides, poly-l-arginine and poly-d-glutamic acid was coated with silicate before surface functionalization with poly-l-arginine. Transfection efficiencies and duration of expression were similar when using green fluorescent protein (GFP) plasmid DNA (pDNA) or GFP mRNA. These nanoparticles demonstrated significantly higher (>100%) and significantly longer (15 vs. 4 days) transfection efficiencies in comparison to a commercial transfection agent (Lipofectamine 2000). Reprogramming of human foreskin fibroblasts using mRNA to the Sox2 transcription factor resulted in three-fold higher neurosphere formation in comparison to the commercial reagent. These results demonstrate the potential of these nanoparticles as ideal vectors for gene delivery.

Combinatorial therapy using negative pressure and varying lithium dosage for accelerated wound healing.

In this work, we investigated the effects of negative pressure, applied using a pump designed for Negative Pressure Wound Therapy (NPWT), on the process of wound healing in vitro via initiation of the Wnt signaling pathway. Results indicate that negative pressure enhanced Wnt signaling and migration into a simulated wound in vitro in NIH-3T3 murine fibroblast cells. Increasing doses of lithium (upto 15 mM) increased basal Wnt signaling and enhanced cell migration into the simulated wound site. A combination of negative pressure and increased doses of lithium synergistically increased Wnt signaling and demonstrated further enhanced cell migration into simulated wound sites, with maximal filling of the simulated wound observed at lithium concentrations of at least 10mM.

Evaluation of multifunctional polysaccharide hydrogels with varying stiffness for bone tissue engineering.

The use of hydrogels for bone regeneration has been limited due to their inherent low modulus to support cell adhesion and proliferation as well as their susceptibility to bacterial infections at the wound site. To overcome these limitations, we evaluated multifunctional polysaccharide hydrogels of varying stiffness to obtain the optimum stiffness at which the gels (1) induce proliferation of human dermal fibroblasts, human umbilical vascular endothelial cells (HUVECs), and murine preosteoblasts (MC3T3-E1), (2) induce osteoblast differentiation and mineralization, and (3) exhibit an antibacterial activity. Rheological studies demonstrated that the stiffness of hydrogels made of a polysaccharide blend of methylcellulose, chitosan, and agarose was increased by crosslinking the chitosan component to different extents with increasing amounts of genipin. The gelation time decreased (from 210 to 60 min) with increasing genipin concentrations. Proliferation of HUVECs decreased by 10.7 times with increasing gel stiffness, in contrast to fibroblasts and osteoblasts, where it increased with gel stiffness by 6.37 and 7.8 times, respectively. At day 14 up to day 24, osteoblast expression of differentiation markers-osteocalcin, osteopontin-and early mineralization marker-alkaline phosphatase, were significantly enhanced in the 0.5% (w/v) crosslinked gel, which also demonstrated enhanced mineralization by day 25. The antibacterial efficacy of the hydrogels decreased with the increasing degree of crosslinking as demonstrated by biofilm formation experiments, but gels crosslinked with 0.5% (w/v) genipin still demonstrated significant bacterial inhibition. Based on these results, gels crosslinked with 0.5% (w/v) genipin, where 33% of available groups on chitosan were crosslinked, exhibited a stiffness of 502±64.5 Pa and demonstrated the optimal characteristics to support bone regeneration.

Large-scale and highly efficient synthesis of micro- and nano-fibers with controlled fiber morphology by centrifugal jet spinning for tissue regeneration.

PLLA fibrous tissue scaffolds with controlled fiber nanoscale surface roughness are fabricated with a novel centrifugal jet spinning process. The centrifugal jet spinning technique is a highly efficient synthesis method for micron- to nano-sized fibers with a production rate up to 0.5 g min(-1). During the centrifugal jet spinning process, a polymer solution jet is stretched by the centrifugal force of a rotating chamber. By engineering the rheological properties of the polymer solution, solvent evaporation rate and centrifugal force that are applied on the solution jet, polyvinylpyrrolidone (PVP) and poly(l-lactic acid) (PLLA) composite fibers with various diameters are fabricated. Viscosity measurements of polymer solutions allowed us to determine critical polymer chain entanglement limits that allow the generation of continuous fiber as opposed to beads or beaded fibers. Above a critical concentration at which polymer chains are partially or fully entangled, lower polymer concentrations and higher centrifugal forces resulted in thinner fibers. Etching of PVP from the PLLA-PVP composite fibers doped with increasing PVP concentrations yielded PLLA fibers with increasing nano-scale surface roughness and porosity, which increased the fiber hydrophilicity dramatically. Scanning electron micrographs of the etched composite fibers suggest that PVP and PLLA were co-contiguously phase separated within the composite fibers during spinning and nano-scale roughness features were created after the partial etching of PVP. To study the tissue regeneration efficacy of the engineered PLLA fiber matrix, human dermal fibroblasts are used to simulate partial skin graft. Fibers with increased PLLA surface roughness and porosity demonstrated a trend towards higher cell attachment and proliferation.

Design and analysis of a novel mechanical loading machine for dynamic in vivo axial loading.

This paper describes the construction of a loading machine for performing in vivo, dynamic mechanical loading of the rodent forearm. The loading machine utilizes a unique type of electromagnetic actuator with no mechanically resistive components (servotube), allowing highly accurate loads to be created. A regression analysis of the force created by the actuator with respect to the input voltage demonstrates high linear correlation (R(2) = 1). When the linear correlation is used to create dynamic loading waveforms in the frequency (0.5-10 Hz) and load (1-50 N) range used for in vivo loading, less than 1% normalized root mean square error (NRMSE) is computed. Larger NRMSE is found at increased frequencies, with 5%-8% occurring at 40 Hz, and reasons are discussed. Amplifiers (strain gauge, linear voltage displacement transducer (LVDT), and load cell) are constructed, calibrated, and integrated, to allow well-resolved dynamic measurements to be recorded at each program cycle. Each of the amplifiers uses an active filter with cutoff frequency at the maximum in vivo loading frequencies (50 Hz) so that electronic noise generated by the servo drive and actuator are reduced. The LVDT and load cell amplifiers allow evaluation of stress-strain relationships to determine if in vivo bone damage is occurring. The strain gauge amplifier allows dynamic force to strain calibrations to occur for animals of different sex, age, and strain. Unique features are integrated into the loading system, including a weightless mode, which allows the limbs of anesthetized animals to be quickly positioned and removed. Although the device is constructed for in vivo axial bone loading, it can be used within constraints, as a general measurement instrument in a laboratory setting.