Transplantation of patient-specific bile duct bioengineered with chemically reprogrammed and microtopographically differentiated cells.

Cholangiopathy is a diverse spectrum of chronic progressive bile duct disorders with limited treatment options and dismal outcomes. Scaffold- and stem cell-based tissue engineering technologies hold great promise for reconstructive surgery and tissue repair. Here, we report a combined application of 3D scaffold fabrication and reprogramming of patient-specific human hepatocytes to produce implantable artificial tissues that imitate the mechanical and biological properties of native bile ducts. The human chemically derived hepatic progenitor cells (hCdHs) were generated using two small molecules A83-01 and CHIR99021 and seeded inside the tubular scaffold engineered as a synergistic combination of two layers. The inner electrospun fibrous layer was made of nanoscale-macroscale polycaprolactone fibers acting to promote the hCdHs attachment and differentiation, while the outer microporous foam layer served to increase mechanical stability. The two layers of fiber and foam were fused robustly together thus creating coordinated mechanical flexibility to exclude any possible breaking during surgery. The gene expression profiling and histochemical assessment confirmed that hCdHs acquired the biliary epithelial phenotype and filled the entire surface of the fibrous matrix after 2 weeks of growth in the cholangiocyte differentiation medium in vitro. The fabricated construct replaced the macroscopic part of the common bile duct (CBD) and re-stored the bile flow in a rabbit model of acute CBD injury. Animals that received the acellular scaffolds did not survive after the replacement surgery. Thus, the artificial bile duct constructs populated with patient-specific hepatic progenitor cells could provide a scalable and compatible platform for treating bile duct diseases.

Additive manufacturing of the core template for the fabrication of an artificial blood vessel: the relationship between the extruded deposition diameter and the filament/nozzle transition ratio.

An artificial blood vessel with a tubular structure was additively manufactured via fused deposition modeling (FDM) starting from a single strand of polyvinyl alcohol (PVA) filament coated with a specific thickness of biocompatible polydimethylsiloxane (PDMS), followed by removal of the inner core via hydrogen peroxide leaching under sonication. In particular, we examined the relationship between the extruded deposition diameter and the filament migration speed/nozzle control speed (referred to as the filament/nozzle transition ratio), which is almost independent of the extruded deposition flow rate due to the weak die-swelling and memory effects of the extruded PVA arising from its intrinsically low viscoelasticity. The chemical stability of the PDMS during sonication in the hydrogen peroxide solution was then determined by spectroscopic techniques. The PDMS displayed no mechanical degradation in the hydrogen peroxide solution, resulting in similar fracture elongation and yield strength to those of the pristine specimen without the leaching treatment. As a further advantage, the inside surface of the PDMS was smooth regardless of the hydrogen peroxide leaching under sonication. The potential application of the as-developed scaffold in soft tissue engineering (particularly that involving vascular tissue regeneration) was demonstrated by the successful transplantation of the artificial blood vessel in a right-hand surgical replica used in a clinical simulation.

3D bioprinted complex constructs reinforced by hybrid multilayers of electrospun nanofiber sheets.

Despite the usefulness of hydrogels for cell-based bioprinting, the fragility of their resulting constructs has hindered their practical applications in tissue engineering research. Here, we suggest a hybrid integration method based on cell-hydrogel bioprinting that includes alternate layering of flexible nanofiber (NF) sheets. Because the bioprinting was implemented on a nanofibrous surface, the hydrogel-based materials could be printed with enhanced shape resolution compared to printing on a bare hydrogel. Furthermore, the insertion of NF sheets was effective for alleviating the shrinkage distortion of the hydrogel construct, which is inherently generated during the crosslinking process, thereby enhancing shape fidelity throughout the three-dimensional (3D) architecture. In addition to the structural precision, the NF-embedded constructs improved the mechanical properties in terms of compressive strength, modulus, and resilience limit (up to four-fold enhancement). With structural and mechanical supports, we could 3D fabricate complex constructs, including fully opened internal channels, which provided a favorable perfusion condition for cell growth. We confirmed the enhanced bioactivity of the NF-embedded bioprinted construct via cell culture experiments with 80% enhanced proliferation compared to the monolithic one. The synergistic combination of the two flexible materials, NFs and hydrogels, is expected to have extensive applicability in soft tissue engineering.

Detection of Stress Levels from Biosignals Measured in Virtual Reality Environments Using a Kernel-Based Extreme Learning Machine.

Virtual reality (VR) is a computer technique that creates an artificial environment composed of realistic images, sounds, and other sensations. Many researchers have used VR devices to generate various stimuli, and have utilized them to perform experiments or to provide treatment. In this study, the participants performed mental tasks using a VR device while physiological signals were measured: a photoplethysmogram (PPG), electrodermal activity (EDA), and skin temperature (SKT). In general, stress is an important factor that can influence the autonomic nervous system (ANS). Heart-rate variability (HRV) is known to be related to ANS activity, so we used an HRV derived from the PPG peak interval. In addition, the peak characteristics of the skin conductance (SC) from EDA and SKT variation can also reflect ANS activity; we utilized them as well. Then, we applied a kernel-based extreme-learning machine (K-ELM) to correctly classify the stress levels induced by the VR task to reflect five different levels of stress situations: baseline, mild stress, moderate stress, severe stress, and recovery. Twelve healthy subjects voluntarily participated in the study. Three physiological signals were measured in stress environment generated by VR device. As a result, the average classification accuracy was over 95% using K-ELM and the integrated feature (IT = HRV + SC + SKT). In addition, the proposed algorithm can embed a microcontroller chip since K-ELM algorithm have very short computation time. Therefore, a compact wearable device classifying stress levels using physiological signals can be developed.

Design and Fabrication of a Thin-Walled Free-Form Scaffold on the Basis of Medical Image Data and a 3D Printed Template: Its Potential Use in Bile Duct Regeneration.

Three-dimensional (3D) printing, combined with medical imaging technologies, such as computed tomography and magnetic resonance imaging (MRI), has shown a great potential in patient-specific tissue regeneration. Here, we successfully fabricated an ultrathin tubular free-form structure with a wall thickness of several tens of micrometers that is capable of providing sufficient mechanical flexibility. Such a thin geometry cannot easily be achieved by 3D printing alone; therefore, it was realized through a serial combination of processes, including the 3D printing of a sacrificial template, the dip coating of the biomaterial, and the removal of the inner template. We demonstrated the feasibility of this novel tissue engineering construct by conducting bile duct surgery on rabbits. Moving from a rational design based on MRI data to a successful surgical procedure for reconstruction, we confirmed that the presented method of fabricating scaffolds has the potential for use in customized bile duct regeneration. In addition to the specific application presented here, the developed process and scaffold are expected to have universal applicability in other soft-tissue engineering fields, particularly those involving vascular, airway, and abdominal tubular tissues.

Flexible and Stretchable Piezoelectric Sensor with Thickness-Tunable Configuration of Electrospun Nanofiber Mat and Elastomeric Substrates.

Here, we developed highly sensitive piezoelectric sensors in which flexible membrane components were harmoniously integrated. An electrospun nanofiber mat of poly(vinylidenefluoride-co-trifluoroethylene) was sandwiched between two elastomer sheets with sputtered electrodes as an active layer for piezoelectricity. The developed sensory system was ultrasensitive in response to various microscale mechanical stimuli and able to perceive the corresponding deformation at a resolution of 1 µm. Owing to the highly flexible and resilient properties of the components, the durability of the device was sufficiently stable so that the measuring performance could still be effective under harsh conditions of repetitive stretching and folding. When employing spin-coated thin elastomer films, the thickness of the entire sandwich architecture could be less than 100 µm, thereby achieving sufficient compliance of mechanical deformation to accommodate artery-skin motion of the heart pulse. These skin-attachable film- or sheet-type mechanical sensors with high flexibility are expected to enable various applications in the field of wearable devices, medical monitoring systems, and electronic skin.

Creation of a Hybrid Scaffold with Dual Configuration of Aligned and Random Electrospun Fibers.

A novel hybrid construct was developed by combining aligned fibers (AFs) and random fibers (RFs) to form a scaffolding system. Homogeneous fiber-based structures were fabricated by electrospinning, which produced both random and aligned fiber mats depending on the collection method. The upper part of the scaffold contained an AF layer, which possessed a well-organized configuration that provided uniaxial topographic guidance. For mechanical stability and support, the lower part of the scaffold was composed of an RF layer. Despite the presence of randomly distributed RFs, desirable alignment and differentiation could be achieved in cultured C2C12 myoblasts by controlling the density of AF layer. The fibrous structure of the hybrid scaffold also exhibited high porosity and therefore reasonable permeability. Owing to the structural stability provided by the underlying RFs, the cell-laden fibrous scaffolds were amenable to physical manipulation, such as multilayering. Collectively, the morphological features and manipulable architecture of the developed scaffolds suggest that they would perform well in practical applications.