The mechanical cue of fiber alignment plays a key role in the development of various tissues in the body. The ability to study the effect of these stimuli in vitro has been limited previously. Here, we present a microfluidic device capable of intrinsically generating aligned fibers using the microchannel geometry. The device also features tunable interstitial fluid flow and the ability to form a morphogen gradient. These aspects allow for the modeling of complex tissues and to differentiate cell response to different stimuli. To demonstrate the abilities of our device, we incorporated luminal epithelial cysts into our device and induced growth factor stimulation. We found the mechanical cue of fiber alignment to play a dominant role in cell elongation and the ability to form protrusions was dependent on cadherin-3. Together, this work serves as a springboard for future potential with these devices to answer questions in developmental biology and complex diseases such as cancers.

Semiflexible fiber gels such as collagen and fibrin have unique nonlinear mechanical properties that play an important role in tissue morphogenesis, wound healing, and cancer metastasis. Optical tweezers microrheology has greatly contributed to the understanding of the mechanics of fibrous gels at the microscale, including its heterogeneity and anisotropy. However, the explicit relationship between micromechanical properties and gel deformation has been largely overlooked. We introduce a unique gel-stretching apparatus and employ it to study the relationship between microscale strain and stiffening in fibrin and collagen gels, focusing on the development of anisotropy in the gel. We find that gels stretched by as much as 15 % stiffen significantly both in parallel and perpendicular to the stretching axis, and that the parallel axis is 2-3 times stiffer than the transverse axis. We also measure the stiffening and anisotropy along bands of aligned fibers created by aggregates of cancer cells, and find similar effects as in gels stretched with the tensile apparatus. Our results illustrate that the extracellular microenvironment is highly sensitive to deformation, with implications for tissue homeostasis and pathology. STATEMENT OF SIGNIFICANCE: The inherent fibrous architecture of the extracellular matrix (ECM) gives rise to unique strain-stiffening mechanics. The micromechanics of fibrous networks has been studied extensively, but the deformations involved in its stiffening at the microscale were not quantified. Here we introduce an apparatus that enables measuring the deformations in the gel as it is being stretched while simultaneously using optical tweezers to measure its microscale anisotropic stiffness. We reveal that fibrin and collagen both stiffen dramatically already at ∼10 % deformation, accompanied by the emergence of significant, yet moderate anisotropy. We measure similar stiffening and anisotropy in the matrix remodeled by the tensile apparatus to those found between cancer cell aggregates. Our results emphasize that small strains are enough to introduce substantial stiffening and anisotropy. These have been shown to result in directional cell migration and enhanced force propagation, and possibly control processes like morphogenesis and cancer metastasis.

Parts made through additive manufacturing (AM) often exhibit mechanical anisotropy due to the time-based deposition of material and processing parameters. In polymer material extrusion (MEX), printed parts have weak points at layer interfaces, perpendicular to the direction of deposition. Poly(lactic acid) with chopped carbon fiber was printed on a large-format pellet printer at various extrusion rates with the same tool pathing to measure the fiber alignment with deposition via two methods and relate it to the ultimate tensile strength (UTS). Within a singular printed bead, an X-ray microscopy (XRM) scan was conducted to produce a reconstruction of the internal microstructure and 3D object data on the length and orientation of fibers. From the scan, discrete images were used in an image analysis technique to determine the fiber alignment to deposition without 3D object data on each fiber\'s size. Both the object method and the discrete image method showed a negative relationship between the extrusion rate and fiber alignment, with -34.64% and -53.43% alignment per extrusion multiplier, respectively, as the slopes of the linear regression. Tensile testing was conducted to determine the correlation between the fiber alignment and UTS. For all extrusion rates tested, as the extrusion multiplier increased, the percent difference in the UTS decreased, to a minimum of 8.12 ± 14.40%. The use of image analysis for the determination of the fiber alignment provides a possible method for relating the microstructure to the meso-property of AM parts, and the relationship between the microstructure and the properties establishes process-structure-property relationships for large-format AM.

Ligaments and tendons undergo nonuniform deformation during movement. While deformations can be imaged, it remains challenging to use such information to infer regional tissue loading. Shear wave tensiometry is a promising noninvasive technique to gauge axial stress and is premised on a tensioned beam model. However, it is unknown whether tensiometry can predict regional stress in a nonuniformly loaded structure. The objectives of this study were to (1) determine whether regional shear wave speed tracks regional axial stress in nonuniformly loaded fibrous soft tissues, and (2) determine the sensitivity of regional axial stress and shear wave speed to nonuniform load distribution and fiber alignment. We created a representative set of 12,000 dynamic finite element models of a fibrous soft tissue with probabilistic variations in fiber alignment, stiffness, and aspect ratio. In each model, we applied a randomly selected nonuniform load distribution, and then excited a shear wave and tracked its regional propagation. We found that regional shear wave speed was an excellent predictor of the regional axial stress (RMSE = 0.57 MPa) and that the nature of the regional shear wave speed-stress relationship was consistent with a tensioned beam model (R2 = 0.99). Variations in nonuniform load distribution and fiber alignment did not substantially alter the wave speed-stress relationship, particularly at higher loads. Thus, these findings suggests that shear wave tensiometry could provide a quantitative estimate of regional tissue stress in ligaments and tendons.

Electrospinning has been widely employed to fabricate complex extracellular matrix-like microenvironments for tissue engineering due to its ability to replicate structurally biomimetic micro- and nanotopographic cues. Nevertheless, these nanofibrous structures are typically either confined to bidimensional systems or confined to three-dimensional ones that are unable to provide controlled multiscale patterns. Thus, an electrospinning modality was used in this work to fabricate chondrocyte-laden nanofibrous scaffolds with highly customizable three-dimensional (3D) architectures in an automated manner, with the ultimate goal of recreating a suitable 3D scaffold for articular cartilage tissue engineering. Three distinct architectures were designed and fabricated by combining multiple nanofibrous and chondrocyte-laden hydrogel layers and tested in vitro in a compression bioreactor system. Results demonstrated that it was possible to precisely control the placement and alignment of electrospun polycaprolactone and gelatin nanofibers, generating three unique architectures with distinctive macroscale porosity, water absorption capacity, and mechanical properties. The architecture organized in a lattice-like fashion was highly porous with substantial pore interconnectivity, resulting in a high-water absorption capacity but a poor compression modulus and relatively weaker energy dissipation capacity. The donut-like 3D geometry was the densest, with lower swelling, but the highest compression modulus and improved energy dissipation ability. The third architecture combined a lattice and donut-like fibrous arrangement, exhibiting intermediary behavior in terms of porosity, water absorption, compression modulus, and energy dissipation capacity. The properties of the donut-like 3D architecture demonstrated great potential for articular cartilage tissue engineering, as it mimicked key topographic, chemical, and mechanical characteristics of chondrocytes\' surrounding environment. In fact, the combination of these architectural features with a dynamically compressive mechanical stimulus triggered the best in vitro results in terms of viability and biosynthetic production.

Electrospinning has contributed substantially to the construction of nanofibrous scaffolds for potential tissue engineering and regenerative medicine applications. However, conventional electrospinning only has the ability to generate and collect nanofiber scaffolds with a randomly oriented fibrous pattern, which lack the necessary cell alignment guidance function. In this study, a novel electrospinning fiber-collecting device was designed and developed by setting a series of small pin-ring-structured collectors on a large plain plate. Specifically, we demonstrated that the pin-ring-structured collectors, which were constructed by inserting a metal pin into the center of a metal ring, could collect the as-electrospun nanofibers with radially oriented structures in an innovative manner. We first investigated the suitable polymeric concentration for electrospinning poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the optimum electrospinning concentration of PHBV was found to be 12% (w/v) PHBV dissolved in hexafluoroisopropyl alcohol (HFIP). Then, 12% (w/v) PHBV solution was electrospun into radially oriented nanofiber scaffolds using our novel electrospinning strategy, and their various performances were further compared with conventionally randomly oriented nanofiber scaffolds that were also produced from 12% (w/v) PHBV solution. The results showed that the radially oriented PHBV nanofiber scaffolds exhibited obviously enhanced mechanical properties and decreased hydrophobicity compared with the randomly oriented PHBV nanofiber scaffold controls. Importantly, the biological properties of radially oriented PHBV nanofiber scaffolds were also demonstrated to be enhanced, compared with randomly oriented PHBV nanofiber scaffolds, by effectively inducing cell alignment and significantly promoting cell proliferation. In sum, the present study indicates that our as-prepared nanofiber scaffolds with a radially oriented pattern are of great interest for advanced applications, such as wound dressings and tissue-engineered scaffolds.

One-dimensional (1D) fibers have been widely used in composites reinforcement for microwave attenuation due to their outstanding mechanical and electromagnetic properties, especially in the axial direction. However, the precise control of fiber alignment in a polymer matrix remains a challenge. In this work, we successfully demonstrated the well-controlled alignment of silicon carbide nanowires (SiCNW) in a silicone matrix by using direct ink writing (DIW)-based 3D printing. It is proven that the printed multilayer material with fiber alignment could show a dramatic improvement in both reflection loss (RL) and effective attenuation bandwidth (EAB, RL < -10 dB). In particular, a uniaxial in-plane orientation is found to be the optimal alignment among other planar and also out-of-plane orientations. Benefiting from the optimized alignment, the 3D-printed SiC composite could show an EAB (∼6.4 GHz)1.6 times broader than that of the randomly mixed composite at the same thickness without alignment, associated with a minimum RL of -48 dB at 14.3 GHz. In addition, it is demonstrated that DIW could print different materials, such as SiCNW and multiwall carbon nanotube (MWCNT), in alternating layers for multiple-frequency-band attenuation benefiting from the distinct property of each material. Considering the one-step control of fiber alignment and material selectivity, DIW could play an important role in materials design for high-efficiency microwave attenuation.

Fabrication of anisotropic materials is highly desirable in designing biomaterials and tissue engineered constructs. Electrospinning has been broadly adopted due to its versatility in producing non-woven fibrous meshes with tunable fiber diameters (from 10 nanometers to 10 microns), microarchitectures, and construct geometries. A myriad of approaches have been utilized to control fiber alignment of electrospun materials to achieve complex microarchitectures, improve mechanical properties, and provide topographical cellular cues. This review provides a comparative analysis of the techniques developed to generate fiber alignment in electrospun materials. A description of the underlying mechanisms that drive fiber alignment, setup variations for each technique, and the resulting impact on the aligned microarchitecture is provided. A critical analysis of the advantages and limitations of each approach is provided to guide researchers in method selection. Finally, future perspectives of advanced electrospinning methodologies are discussed in terms of developing a scalable method with precise control of microarchitecture.

Tissues are commonly defined as groups of cells that have similar structure and uniformly perform a specialized function. A lesser-known fact is that the placement of these cells within these tissues plays an important role in executing its functions, especially for neuronal cells. Hence, the design of a functional neural scaffold has to mirror these cell organizations, which are brought about by the configuration of natural extracellular matrix (ECM) structural proteins. In this review, we will briefly discuss the various characteristics considered when making neural scaffolds. We will then focus on the cellular orientation and axonal alignment of neural cells within their ECM and elaborate on the mechanisms involved in this process. A better understanding of these mechanisms could shed more light onto the rationale of fabricating the scaffolds for this specific functionality. Finally, we will discuss the scaffolds used in neural tissue engineering (NTE) and the methods used to fabricate these well-defined constructs.

3D printing of fiber-reinforced composites is expected to be the forefront technology for the next-generation high-strength, high-toughness, and lightweight structural materials. The intrinsic architecture of 3D-printed composites closely represents biomimetic micro/macrofibril-like hierarchical structure composed of intermediate filament assembly among the micron-sized reinforcing fibers, and thus contributes to a novel mechanism to simultaneously improve mechanical properties and structural features. Notably, it is found that an interfacial heterogeneity between numerous inner interfaces in the hierarchical structure enables an exceptional increase in the toughness of composites. The strong interfacial adhesion between the fibers and matrix, with accompanying the inherently weak interfacial adhesion between intermediate filaments and the resultant interfacial voids, provide a close representation of the toughness behavior of natural architectures relying on the localized heterogeneity. Given the critical embedment length of fiber reinforcement, extraordinary improvement has been attained not only in the strength but also in toughness taking advantage of the synergy effect from the aforementioned nature-inspired features. Indeed, the addition of a small amount of short fiber to the brittle bio-filaments results in a noticeable increase of more than 200% in the tensile strength and modulus with further elongation increment. This article highlights the inherent structural hierarchy of 3D-printed composites and the relevant sophisticated mechanism for anomalous mechanical reinforcement.