Amrinder S. Nain

Bioprinting of growth factors onto aligned sub-micron fibrous scaffolds for simultaneous control of cell differentiation and alignment

The capability to spatially control stem cell orientation and differentiation simultaneously using a combination of geometric cues that mimic structural aspects of native extracellular matrix (ECM) and biochemical cues such as ECM-bound growth factors (GFs) is important for understanding the organization and function of musculoskeletal tissues. Herein, oriented sub-micron fibers, which are morphologically similar to musculoskeletal ECM, were spatially patterned with GFs using an inkjet-based bioprinter to create geometric and biochemical cues that direct musculoskeletal cell alignment and differentiation in vitro in registration with fiber orientation and printed patterns, respectively. Sub-micron polystyrene fibers (diameter ~ 655 nm) were fabricated using a Spinneret-based Tunable Engineered Parameters (STEP) technique and coated with serum or fibrin. The fibers were subsequently patterned with tendon-promoting fibroblast growth factor-2 (FGF-2) or bone-promoting bone morphogenetic protein-2 (BMP-2) prior to seeding with mouse C2C12 myoblasts or C3H10T1/2 mesenchymal fibroblasts. Unprinted regions of STEP fibers showed myocyte differentiation while printed FGF-2 and BMP-2 patterns promoted tenocyte and osteoblast fates, respectively, and inhibited myocyte differentiation. Additionally, cells aligned along the fiber length. Functionalizing oriented sub-micron fibers with printed GFs provides instructive cues to spatially control cell fate and alignment to mimic native tissue organization and may have applications in regenerative medicine.

Drawing suspended polymer micro-/nanofibers using glass micropipettes

This letter proposes a method for fabricating suspended micro-/nanoscale polymer fibers continuously, in which polymeric micro-/nanofibers are formed by drawing and solidification of a viscous liquid polymer solution which is pumped through a glass micropipette. By controlling the drawing parameters, this method is demonstrated to form networks of suspended fibers having amorphous internal structure and uniform diameters from micrometers down to sub-50-nm for different molecular weights of polystyrene dissolved in xylene.

Dry Spinning Based Spinneret Based Tunable Engineered Parameters (STEP) Technique for Controlled and Aligned Deposition of Polymeric Nanofibers

Polymeric nanofibers are finding increasing number of applications and hold the potential to revolutionize diverse fields such as tissue engineering, smart textiles, sensors, and actuators. Aligning and producing high aspect ratio fiber arrays (length/diameter > 2 000) in the sub-micron and nanoscale diameters has been challenging due to fragility of polymeric materials, thus making it difficult to deposit them as one dimensional structures functionally interfaced with other systems. Here, we present a pseudo dry spinning technique which allows precise control on fiber diameters and further allows deposition of fiber arrays in aligned configurations. Control on fiber diameters ranging from 50-500 nm and having lengths of several millimeters is achieved by altering the polymeric solution concentration. In the dilute and semi-dilute unentangled concentration domain droplets or beaded fibers are observed to form. Smooth uniform diameter fibers are observed to form at the onset of semi-dilute entangled concentration regime. For a given molecular weight, the increase in fiber diameter with increasing solution concentration is attributed to both the increase in the entanglement density and the decrease in the radius of gyration of solvated polymer molecules. Using this technique polymeric fiber arrays in single and multiple layers are demonstrated which can be used towards developing strong textiles, biological scaffolds, and sensor networks.

Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds

In the body, cells dynamically respond to chemical and mechanical cues from the extracellular matrix (ECM), yet precise mechanisms by which biophysical parameters (stiffness, topography and alignment) affect cell behavior remain unclear. Here, highly aligned and suspended multilayer polystyrene (PS) nanofiber scaffolds are used to study biophysical influences on focal adhesion complex (FAC) arrangement and associated migration behavior of mouse C2C12 cells arranged in specific shapes: spindle, parallel and polygonal. Furthermore, the role of cytoskeletal-altering drugs including blebbistatin, nocodazole and cytochalasin-D on FAC formation and migratory behavior is investigated. For the first time, this work reports that cells on suspended fiber networks, including cells with administered drugs, elongated along the fiber axes and developed longer (∼ 4×) and more concentrated FAC clusters compared to cells on flat PS control substrates. Additionally, substrate designs which topographically restrict sites of cell attachment and align adhesions were found to promote higher migration speeds (spindle: 52μmh(-1), parallel: 39μmh(-1), polygonal: 25μmh(-1), flat: 32μmh(-1)). This work demonstrates that suspended fiber topography-induced concentration of FACs along fiber axes generates increased migration potential as opposed to flat surfaces, which diffuse and randomly orient adhesions.

Proximal Probes Based Nanorobotic Drawing of Polymer Micro/Nanofibers

This paper proposes a nanorobotic fiber fabrication method which uses proximal probes to draw polymer fibers down to few hundred nanometers in diameter and several hundred micrometers in length. Using proximal probes such as Atomic Force Microscope (AFM) and Scanning Tunneling Microscope (STM) or glass micropipettes, liquid polymers dissolved in a solvent are drawn. During drawing, the solvent evaporates in real-time which solidifies the fiber. Controlling the drawn fibers trajectory and solidification in three-dimensions (3-D), suspended fibers, fiber cantilevers, custom 3-D fibers, and fiber networks, are proposed to be fabricated. Poly(methyl methacrylate) (PMMA) polymer dissolved in chlorobenzene is used to form a variety of suspended polymer fibers with diameters from few microns to 200nm. Fabrication of crossed and linear networks of fibers is also demonstrated. Viscoelastic modeling of polymer fiber drawing is realized using a finite element method to test the significance of the drawing speed and velocity profile on the extensional behavior of the drawn fiber. Since the mechanical properties of the drawn micro/nanofibers could vary from the bulk polymer material significantly, mechanical characterization of suspended fibers using an AFM and a Nanoindenter setup is proposed. Extending this technique to a variety of nonconductive and electroactive polymer fibers, many novel applications in micro/nanoscale sensors, actuators, fibrillar structures, and optical and electronic devices would become possible

Controlling bacterial adhesion to surfaces using topographical cues: a study of the interaction of Pseudomonas aeruginosa with nanofiber-textured surfaces

The state of adhesion of bacteria to nanofiber-textured model surfaces is analyzed at a single-cell level. The results reveal similarities between the effect of topography on bacteria–surface interactions and vesicle–surface interactions. The results are discussed in the context of controlling bacterial adhesion to surfaces using nanofibrous topographical features.

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 μm(2) to 5 × 5 μm(2)) 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 μm(2), 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.

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m(2) and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cells immediate mechanistic environment arising from alignment and geometry of fibers.

3-D nano-fiber manufacturing by controlled pulling of liquid polymers using nano-probes

In-addition to the applications of imaging and characterization, proximal probes are proposed to be used three-dimensional (3-D) nano-scale manufacturing tools in this paper. Commercially available Atomic Force Microscope (AFM) systems are mainly limited to 1-D or 2-D manipulation tasks, and advanced 3-D nano-manufacturing applications are not possible. Therefore, this paper proposes 3-D nano-scale manipulation of liquid polymer nano-fibers by using precise positioning and temperature control. AFM nano-probe is used to pull or extrude thermoset or thermoplastic polymers precisely to fabricate 3-D polymer nano-fiber structures. A liquid SU-8 polymer structure bridge between the probe tip and a substrate is held when pulling the probe from the surface with controlled speed and position. For thermoset polymer, by heating the substrate and moving the AFM probe tip in a precise 3-D trajectory, the liquid fiber is cured in real-time while a predetermined 3-D shape is constructed. To model the curing of thermoset resins a three dimensional transient heat transfer scheme using Alternate Direction Implicit (ADI) finite volume method has been developed which numerically simulates the kinetics of cure, specifically the exothermic heat given out during the cure reaction of thermosetting resin.

Microrobotically Fabricated Biological Scaffolds for Tissue Engineering

A microrobotic method for fabricating multilayered poly(lactic acid) (PLA) biological scaffolds using micropipettes for tissue engineering applications is presented. Biological scaffolds are fabricated over several different substrates by drawing and solidification of a viscous liquid polymer solution pumped continuously through a glass micropipette. The proposed method produces highly aligned, multilayered, crisscrossed fiber scaffolds with user specified pore sizes and diameters in the range from 1 to 10 micrometer. Attachment, proliferation and differentiation of C2C12 mouse pluripotential cells seeded on individual, parallel, and intersecting fibers is successfully demonstrated. The proposed robotic methodology consistently provides parameterized biological scaffolds to aid studies in tissue engineering and to develop novel MEMS, filtration and controlled drug delivery devices