Suspended Micro/Nanofiber Hierarchical Biological Scaffolds Fabricated Using Non-Electrospinning STEP Technique

Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro- and nanoarchitectures, which provide extracellular signaling cues and influence cell behaviors. Mimicking this three-dimensional microenvironment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies (diameter from micrometers to less than 100 nm) with accurately controlled fiber orientation and spacing are demonstrated as biological scaffolds fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique. Micro/nanofiber arrays were manufactured with high parallelism (relative angles between fibers were maintained less than 6°) and well controlled interfiber spacing (<15%). Using these controls, we demonstrate a bottom up hierarchical assembly of suspended six layer structures of progressively reduced diameters and spacing from several polymer systems. We then demonstrate use of STEP scaffolds to study single and multicell arrangement at high magnifications. Specifically, using double layer divergent (0°–90°) suspended nanofibers assemblies, we show precise quantitative control of cell geometry (change in shape index from 0.15 to 0.57 at similar cell areas), and through design of scaffold porosity (80 × 80 μm² to 5 × 5 μm²) quadruple the cell attachment density. Furthermore, using unidirectional or crisscross patterns of sparse and dense fiber arrays, we are able to control the cell spread area from ∼400 to ∼700 μm², while the nucleus shape index increases from 0.75 to 0.99 with cells nearly doubling their focal adhesion cluster lengths (∼15 μm) on widely spaced nanofiber arrays. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology

Tunneling Nanotubes between Cells Migrating in ECM Mimicking Fibrous Environments

Tunneling nanotubes (TNTs) comprise a unique class of actin-rich nanoscale membranous protrusions. They enable long-distance intercellular communication and may play an integral role in tumor formation, progression, and drug resistance. TNTs are three-dimensional, but nearly all studies have investigated them using two-dimensional cell culture models. Here, we applied a unique 3D culture platform consisting of crosshatched and aligned fibers to fabricate synthetic suspended scaffolds that mimic the native fibrillar architecture of tumoral extracellular matrix (ECM) to characterize TNT formation and function in its native state. TNTs are upregulated in malignant mesothelioma; we used this model to analyze the biophysical properties of TNTs in this 3D setting, including cell migration in relation to TNT dynamics, rate of TNT-mediated intercellular transport of cargo, and conformation of TNT-forming cells. We found that highly migratory elongated cells on aligned fibers formed significantly longer but fewer TNTs than uniformly spread cells on crossing fibers. We developed new quantitative metrics for the classification of TNT morphologies based on shape and cytoskeletal content using confocal microscopy. In sum, our strategy for culturing cells in ECM-mimicking bioengineered scaffolds provides a new approach for accurate biophysical and biologic assessment of TNT formation and structure in native fibrous microenvironments

Aligned Nanofiber Topography Directs the Tenogenic Differentiation of Mesenchymal Stem Cells

Tendon is commonly injured, heals slowly and poorly, and often suffers re-injury after healing. This is due to failure of tenocytes to effectively remodel tendon after injury to recapitulate normal architecture, resulting in poor mechanical properties. One strategy for improving the outcome is to use nanofiber scaffolds and mesenchymal stem cells (MSCs) to regenerate tendon. Various scaffold parameters are known to influence tenogenesis. We designed suspended and aligned nanofiber scaffolds with the hypothesis that this would promote tenogenesis when seeded with MSCs. Our aligned nanofibers were manufactured using the previously reported non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. We compared parallel versus perpendicular nanofiber scaffolds with traditional flat monolayers and used cellular morphology, tendon marker gene expression, and collagen and glycosaminoglycan deposition as determinants for tendon differentiation. We report that compared with traditional control monolayers, MSCs grown on nanofibers were morphologically elongated with higher gene expression of tendon marker scleraxis and collagen type I, along with increased production of extracellular matrix components collagen (p = 0.0293) and glycosaminoglycan (p = 0.0038). Further study of MSCs in different topographical environments is needed to elucidate the complex molecular mechanisms involved in stem cell differentiation

IMECE2008-68032 DRAFT: DESIGN AND NUMERICAL MODELING OF AN ON-BOARD CHEMICAL RELEASE MODULE FOR MOTION CONTROL OF BACTERIA-PROPELLED SWIMMING MICRO-ROBOTS mine the number, size, and location of the required micro-valves

ABSTRACT On/off motion control of bacteria-propelled synthetic bodies was previously achieved using a chemical switching technique. A chemical agent (CuSO 4 ) directly binds to the rotor of the flagellar motor inhibiting it. When desired, a second chemical agent (EDTA) is introduced, which binds to the CuSO 4 molecules, freeing the motor and allowing the bacteria to resume its motio

IMECE2008-67964 FABRICATION OF SINGLE AND MULTI-LAYER FIBROUS BIOMATERIAL SCAFFOLDS FOR TISSUE ENGINEERING IMECE2008-67964

ABSTRACT For regenerative medicine applications, we need to expand our understanding of the mechanisms by which nature assembles and functionalizes specialized complex tissues to form a complete organism. The first step towards this goal involves understanding the underlying complex mechanisms of highly organized behavior spanning not only diverse scientific fields, but also nano, micro and macro length-scales. For example, an engineered fibrous biomaterial scaffold possessing the hierarchal spatial properties of a native extracellular matrix (ECM) can serve as a building block upon which living cells are seeded for repair or regeneration. The hierarchical nature of ECM along with the inherent topological constraints of fiber diameter, fiber spacing, multi-layer configurations provide different pathways for living cells to adapt and conform to the surrounding environment. Our previously developed Spinneret based Tunable Engineered Parameters (STEP) technique to deposit biomaterial scaffolds in aligned configurations has been used for the first time to deposit single and multi-layer biological scaffolds of fibrinogen. Fibrinogen is a very well established tissue engineering scaffold material, as it improves cellular interactions and allows scaffold remodeling compared to synthetic polymers. Current state-of-the-art fiber deposition techniques lack the ability to fabricate scaffolds of desired fiber dimensions and orientations and in this study we present fabrication and aligned deposition of fibrinogen fiber arrays with diameters ranging from sub-200 nm to sub-microns and several millimeters in length. The fabricated scaffolds are then cultured with pluripotent mouse C2C12 cells for seven days and cells on the scaffolds are observed to elongate resembling myotube morphology along the fiber axis, spread along intersecting layers and fuse into bundles at the macroscale. Additionally, we demonstrate the ability to deposit poly (lacticco-glycolic acid) (PLGA), Polystyrene (PS) biomaterial scaffolds of different diameters to investigate the effects of topological variations on cellular adhesion, proliferation and migration. Previous studies have indicated cells making right angle transitions upon encountering perpendicular double layer fibers and cellular motion is thwarted in the vicinity o