Bioinspired Three-Dimensional Human Neuromuscular Junction Development in Suspended Hydrogel Arrays.

The physical connection between motoneurons and skeletal muscle targets is responsible for the creation of neuromuscular junctions (NMJs), which allow electrical signals to be translated to mechanical work. NMJ pathology contributes to the spectrum of neuromuscular, motoneuron, and dystrophic disease. Improving in vitro tools that allow for recapitulation of the physiology of the neuromuscular connection will enable researchers to better understand the development and maturation of NMJs, and will help to decipher mechanisms leading to NMJ degeneration. In this work, we first describe robust differentiation of bungarotoxin-positive human myotubes, as well as a reproducible method for encapsulating and aligning human myoblasts in three-dimensional (3D) suspended culture using bioprinted silk fibroin cantilevers as cell culture supports. Further analysis with coculture of motoneuron-like cells demonstrates feasibility of fully human coculture using two-dimensional and 2.5-dimensional culture methods, with appropriate differentiation of both cell types. Using these coculture differentiation conditions with motoneuron-like cells added to monocultures of 3D suspended human myotubes, we then demonstrate synaptic colocalization in coculture as well as acetylcholine and glutamic acid stimulation of human myocytes. This method represents a unique platform to coculture suspended human myoblast-seeded 3D hydrogels with integrated motoneuron-like cells derived from human induced neural stem cells. The platform described is fully customizable using 3D freeform printing into standard laboratory tissue culture materials, and allows for human myoblast alignment in 3D with precise motoneuron integration into preformed myotubes. The coculture method will ideally be useful in observation and analysis of neurite outgrowth and myogenic differentiation in 3D with quantification of several parameters of muscle innervation and function.

Evolution of Bioinks and Additive Manufacturing Technologies for 3D Bioprinting.

3D printing is an additive manufacturing (AM) technique that has quickly disrupted traditional design and manufacturing strategies. New structures can be manufactured that could not be fabricated using other methods. These new capabilities are considered by many to hallmark a historic shift representative of a new industrial revolution. Exciting utilities of this evolving technology are the fields of biomedical engineering and translational medicine, particularly in applying three-dimensional (3D) printing toward enabling on-demand fabrication of customized tissue scaffolds and medical device geometries. AM techniques are promising a future where on-demand production of patient-specific living tissues is a reality. In this review, we cover the rapid evolution and widespread concepts of a bio-"ink" and bioprinted devices and tissues from the past two decades as well as review the various additive manufacturing methods that have been used toward 3D bioprinting of cells and scaffolds with a special look at the benefits and practical considerations for each method. Despite being a young technology, the evolution and impact of AM in the fields of tissue engineering and regenerative medicine has progressed rapidly. We finish the review by looking toward the future of bioprinting and identify some of the current bottlenecks facing the blossoming industry.

Three-dimensional Printing in Developing Countries.

The advent of 3-dimensional (3D) printing technology has facilitated the creation of customized objects. The lack of regulation in developing countries renders conventional means of addressing various healthcare issues challenging. 3D printing may provide a venue for addressing many of these concerns in an inexpensive and easily accessible fashion. These may potentially include the production of basic medical supplies, vaccination beads, laboratory equipment, and prosthetic limbs. As this technology continues to improve and prices are reduced, 3D printing has the potential ability to promote initiatives across the entire developing world, resulting in improved surgical care and providing a higher quality of healthcare to its residents.

Polyol-Silk Bioink Formulations as Two-Part Room-Temperature Curable Materials for 3D Printing.

Silk-based bioinks were developed for 2D and 3D printing. By incorporating nontoxic polyols into silk solutions, two-part formulations with self-curing features at room temperature were generated. By varying the formulations the crystallinity of the silk polymer matrix could be controlled to support printing in 2D and 3D formats interfaced with CAD geometry and with good feature resolution. The self-curing phenomenon was tuned and exploited in order to demonstrate the formation of both structural and support materials. Biocompatible aqueous protein inks for printing that avoid the need for chemical or photo initiators and that form aqueous-stable structures with good resolution at ambient temperatures provide useful options for biofunctionalization and a broad range of applications.

Rapid prototyped sutureless anastomosis device from self-curing silk bio-ink.

Sutureless anastomosis devices are designed to reduce surgical time and difficulty, which may lead to quicker and less invasive cardiovascular anastomosis. The implant uses a barb-and-seat compression fitting composed of one male and two female components. The implant body is resorbable and capable of eluting heparin. Custom robotic deposition equipment was designed to fabricate the implants from a self-curing silk solution. Curing did not require deleterious processing steps but devices demonstrated high crush resistance, retention strength, and leak resistance. Radial crush resistance is in the range of metal vascular implants. Insertion force and retention strength of the anastomosis was dependent on fit sizing of the male and female components and subsequent vessel wall compression. Anastomotic burst strength was dependent on the amount of vessel wall compression, and capable of maintaining higher than physiological pressures. In initial screening using a porcine implant, the devices remained intact for 28 days (the length of study). Histological sections revealed cellular infiltration within the laminar structure of the male component, as well as at the interface between the male and female components. Initial degradation and absorption of the implant wall were observed. The speed per anastomosis using this new device was much faster than current systems, providing significant clinical improvement.

Electroresponsive aqueous silk protein as "smart" mechanical damping fluid.

Here we demonstrate the effectiveness of an electroresponsive aqueous silk protein polymer as a smart mechanical damping fluid. The aqueous polymer solution is liquid under ambient conditions, but is reversibly converted into a gel once subjected to an electric current, thereby increasing or decreasing in viscosity. This nontoxic, biodegradable, reversible, edible fluid also bonds to device surfaces and is demonstrated to reduce friction and provide striking wear protection. The friction and mechanical damping coefficients are shown to modulate with electric field exposure time and/or intensity. Damping coefficient can be modulated electrically, and then preserved without continued power for longer time scales than conventional "smart" fluid dampers.

Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications.

Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.

Seamless, axially aligned, fiber tubes, meshes, microbundles and gradient biomaterial constructs.

A new electrospinning apparatus was developed to generate nanofibrous materials with improved organizational control. The system functions by oscillating the deposition signal (ODS) of multiple collectors, allowing significantly improved nanofiber control by manipulating the electric field which drives the electrospinning process. Other electrospinning techniques designed to impart deposited fiber organizational control, such as rotating mandrels or parallel collector systems, do not generate seamless constructs with high quality alignment in sizes large enough for medical devices. In contrast, the ODS collection system produces deposited fiber networks with highly pure alignment in a variety of forms and sizes, including flat (8 × 8 cm(2)), tubular (1.3 cm diameter), or rope-like microbundle (45 µm diameter) samples. Additionally, the mechanism of our technique allows for scale-up beyond these dimensions. The ODS collection system produced 81.6 % of fibers aligned within 5° of the axial direction, nearly a four-fold improvement over the rotating mandrel technique. The meshes produced from the 9 % (w/v) fibroin/PEO blend demonstrated significant mechanical anisotropy due to the fiber alignment. In 37 °C PBS, aligned samples produced an ultimate tensile strength of 16.47 ± 1.18 MPa, a Young's modulus of 37.33 MPa, and a yield strength of 7.79 ± 1.13 MPa. The material was 300 % stiffer when extended in the direction of fiber alignment and required 20 times the amount of force to be deformed, compared to aligned meshes extended perpendicular to the fiber direction. The ODS technique could be applied to any electrospinnable polymer to overcome the more limited uniformity and induced mechanical strain of rotating mandrel techniques, and greatly surpasses the limited length of standard parallel collector techniques.