Recombinant Human Parathyroid Hormone Biocomposite Promotes Bone-to-Tendon Interface Healing by Enhancing Tenogenesis, Chondrogenesis, and Osteogenesis in a Rabbit Model of Chronic Rotator Cuff Tears.

PURPOSE: To investigate the effect of recombinant human parathyroid hormone (rhPTH) biocomposite on bone-to-tendon interface (BTI) healing for surgical repair of a chronic rotator cuff tear (RCT) model of rabbit, focusing on genetic, histologic, biomechanical and micro-computed tomography (CT) evaluations. METHODS: Sixty-four rabbits were equally assigned to the 4 groups: saline injection (group A), nanofiber sheet alone (group B), rhPTH-soaked nanofiber sheet (nanofiber sheet was soaked with rhPTH, group C), and rhPTH biocomposite (rhPTH permeated the nanofiber sheet by coaxial electrospinning, group D). The release kinetics of rhPTH (groups C and D) was examined for 6 weeks in vitro. Nanofiber scaffolds were implanted on the surface of the repair site 6 weeks after the induction of chronic RCT. Genetic and histologic analyses were conducted 4 weeks after surgery. Furthermore, genetic, histologic, biomechanical, micro-CT, and serologic analyses were performed 12 weeks after surgery. RESULTS: In vivo, group D showed the highest collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), and bone morphogenetic protein 2 (BMP-2) messenger RNA (mRNA) expression levels (all P < .001) 4 weeks after surgery; however, there were no differences between groups at 12 weeks postsurgery. After 12 weeks postsurgery, group D showed better collagen fiber continuity and orientation, denser collagen fibers, more mature bone-to-tendon junction, and greater fibrocartilage layer formation compared with the other groups (all P < .05). Furthermore, group D showed the highest load-to-failure rate (28.9 ± 2.0 N/kg for group A, 30.1 ± 3.3 N/kg for group B, 39.7 ± 2.7 N/kg for group C, and 48.2 ± 4.5 N/kg for group D, P < .001) and micro-CT outcomes, including bone and tissue mineral density, and bone volume/total volume rate (all P < .001) at 12 weeks postsurgery. CONCLUSIONS: In comparison to rhPTH-soaked nanofiber sheet and the other control groups, rhPTH biocomposite effectively accelerated BTI healing by enhancing the mRNA expression levels of COL1A1, COL3A1, and BMP-2 at an early stage and achieving tenogenesis, chondrogenesis, and osteogenesis at 12 weeks after surgical repair of a chronic RCT model of rabbit. CLINICAL RELEVANCE: The present study might be a transitional study to demonstrate the efficacy of rhPTH biocomposites on BTI healing for surgical repair of chronic RCTs as an adaptable polymer biomaterial in humans.

Comprehensive feasibility evaluation of small-diameter 3D templated vascular graft via physical characterizations andin-vivoexperiments.

3D printing (3DP) technology for tissue engineering applications has been extensively studied for materials and processes. However, clinical application to the vascular system was limited owing to mechanical inconsistency and toxicity. Here, we characterized 3D templated artificial vascular grafts (3D grafts), which were fabricated by an integrative method involving 3DP, dip coating, and salt leaching method. The as-fabricated grafts were featured with micrometer-scale porosity enabling tissue-mimetic mechanical softness comparable with native blood vessels. In terms of mechanical properties and water permeability, the fabricated 3D grafts exhibited comparable or superior performances compared to the commercialized grafts. Furthermore, thein-vivostability of the 3D graft was validated through a toxicity test, and the small-diameter 3D graft was transplanted into a rat to confirm the implant's performance. Overall, the experimental results demonstrated the clinical feasibility of the 3D graft with retaining the mechanical biocompatibility and also revealed the possibility of patient-specific customization.

Surface tension-induced biomimetic assembly of cell-laden fibrous bundle construct for muscle tissue engineering.

The field of tissue engineering has been long seeking to develop functional muscle tissue that closely resembles natural muscle. This study used a bio-inspired assembly based on the surface tension mechanism to develop a novel method for engineering muscle tissue. This approach enabled uniaxially ordered electrospun fibers to naturally collide into an aligned bundle without the need for manual handling, thereby reducing cell damage during the cell culture procedure. During the assembly procedure, C2C12 myoblasts were cultured in a viscous collagen hydrogel that caused wetting while providing adequate structural stability for the cell-fiber construct. In addition, gene expression analysis of the resulting muscle-like fibril bundle revealed improved myogenic differentiation. These findings highlight the potential of using a collagen hydrogel and the surface tension mechanism to construct biologically relevant muscle tissue, offering a promising strategy that may outperform existing approaches. Overall, this study contributes to the development of advanced tissue engineering methods and brings us a step closer to creating functional muscle tissue for therapeutic and regenerative medicine applications.

In-situ ionic crosslinking of 3D bioprinted cell-hydrogel constructs for mechanical reinforcement and improved cell growth.

Hydrogels are commonly used in 3D bioprinting technology owing to their ability to encapsulate living cells. However, their inherent delicate properties limit their applicability in the fabrication of mechanically reliable tissue engineering constructs. Herein, we propose a novel reinvented layering integration method for the functional enhancement of 3D cell-hydrogel bioprinting. This was implemented by inserting electrospun microfiber sheets with a crosslinker between the 3D bioprinted layers. When surface-modified microfiber sheets were combined with Ca2+ ionic crosslinkers, the as-printed cell-hydrogel strand was immediately crosslinked when it contacted the sheet surface. The in-situ crosslinking in the bioprinting process not only improved the overall structural stability, but also reinforced the compressive strength and elastic modulus. The enhanced structural stability guaranteed the shape fidelity of the 3D architecture, which included the internal channel network, resulting in improved perfusion conditions for cell growth. The growth of NIH3T3 fibroblasts in 3D bioconstructs with in-situ crosslinking increased by up to five times compared to that of normally bioprinted constructs. The strengthened structural integrity was distinctly sustainable during the cell culture period owing to the sustained release of Ca2+ ions from the embedded microfiber sheets. The synergistic effect of the reinforced mechanical properties with enhanced cell growth is expected to extend the applicability of the proposed hydrogel-based bioprinting technique for soft tissue engineering.

Soft Biomimetic 3D Free-Form Artificial Vascular Graft Using a Highly Uniform Microspherical Porous Structure.

This study presents a biomimetic 3D customizable artificial vascular graft with a highly porous and uniform microscale structure. The structural features were obtained by dip coating of a highly close-packed microsphere suspension on a 3D printed sacrificial template. Considering the structured arrangement of microspherical porogens in the coating layer, the microsphere-leached constructs showed higher uniformity and porosity than the conventionally particulate-leached structures, leading to ultrasoft mechanical compliance. Considering biomechanical compatibility, the resulting elastic moduli were at the sub-MPa level, comparable with those of native vascular tissues. In addition, the developed porous graft was reinforced selectively at the edge regions using a nonporous coating to secure its practical sutureability for clinical use. The sufficiently low cytotoxicity was clinically confirmed to alleviate the stiffness mismatch issues at the anastomotic interface between the native tissue and the artificial graft, thus overcoming the relevant clinical complications. Furthermore, the overall superior properties could be implemented on the 3D printed template for patient-specific medicare, thus implying the manufacturability of patient-specific vascular grafts.

Three-dimensionally printed recombinant human parathyroid hormone-soaked nanofiber sheet accelerates tendon-to-bone healing in a rabbit model of chronic rotator cuff tear.

BACKGROUND: Recombinant human parathyroid hormone (rhPTH) promotes tendon-to-bone healing in humans and animals with rotator cuff tear (RCT). However, problems regarding repeated systemic rhPTH injections in humans exist. This study was conducted to evaluate the effect of topical rhPTH administration using 3-dimensionally (3D) printed nanofiber sheets on tendon-to-bone healing in a rabbit RCT model compared to that of direct topical rhPTH administration. METHODS: Eighty rabbits were randomly assigned to 5 groups (n = 16 each). To create the chronic RCT model, we induced complete supraspinatus tendon tears in both shoulders and left them untreated for 6 weeks. All transected tendons were repaired in a transosseous manner with saline injection in group A, hyaluronic acid (HA) injection in group B, 3D-printed nanofiber sheet fixation in group C, rhPTH and HA injection in group D, and 3D-printed rhPTH- and HA-soaked nanofiber sheet fixation in group E. Genetic (messenger RNA expression evaluation) and histologic evaluations (hematoxylin and eosin and Masson trichrome staining) were performed in half of the rabbits at 4 weeks postrepair. Genetic, histologic, and biomechanical evaluations (mode of tear and load to failure) were performed in the remaining rabbits at 12 weeks. RESULTS: For genetic evaluation, group E showed a higher collagen type I alpha 1 expression level than did the other groups (P = .008) at 4 weeks. However, its expression level was downregulated, and there was no difference at 12 weeks. For histologic evaluation, group E showed greater collagen fiber continuity, denser collagen fibers, and more mature tendon-to-bone junction than did the other groups (P = .001, P = .001, and P = .003, respectively) at 12 weeks. For biomechanical evaluation, group E showed a higher load-to-failure rate than did the other groups (P < .001) at 12 weeks. CONCLUSION: Three-dimensionally printed rhPTH-soaked nanofiber sheet fixation can promote tendon-to-bone healing of chronic RCT.

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.

Hepatic patch by stacking patient-specific liver progenitor cell sheets formed on multiscale electrospun fibers promotes regenerative therapy for liver injury.

Recently, use of cell sheets with bio-applicable fabrication materials for transplantation has been an attractive approach for the treatment of patients with liver failure. However, renewable and scalable cell sources for engineered tissue patches remain limited. We previously reported a new type of proliferating bipotent human chemically derived hepatic progenitor cells (hCdHs) developed by small molecule-mediated reprogramming. Here, we developed a patient-specific hepatic cell sheet constructed from liver biopsy-derived hCdHs on a multiscale fibrous scaffold by combining electrospinning and three-dimensional printing. Analysis of biomaterial composition revealed that the high-density electrospun sheet was superior in increasing the functional properties of hCdHs. Furthermore, the hepatic patch assembled by multilayer stacking with alternate cell sheets of hCdHs and human umbilical vein endothelial cells (HUVECs) recapitulated a liver tissue-like structure, with histological and morphological shape and size similar to those of primary human hepatocytes, and exhibited a significant increase in hepatic functions such as albumin secretion and activity of cytochrome P450 during in vitro hepatic differentiation compared with that in hCdH cells cultured in a two-dimensional monolayer. Interestingly, in the hepatic patch, the induction of functional hepatocytes was associated with both the electrospun fibrous-facilitated oncostatin M signaling and selective activation of AKT signaling by HUVECs. Notably, upon transplantation into a mouse model of therapeutic liver repopulation, the hepatic patch effectively repopulated the damaged parenchyma and induced the restoration of liver function with healthy morphology in the lobe and an improved survival rate (>70%) in mice. Overall, these results suggested that liver biopsy-derived hCdHs can be an efficient alternative source for developing hepatic cell sheets and patches with potential clinical applications in tissue engineering to advance liver regeneration.

Evaluation of 3D Templated Synthetic Vascular Graft Compared with Standard Graft in a Rat Model: Potential Use as an Artificial Vascular Graft in Cardiovascular Disease.

Although the number of vascular surgeries using vascular grafts is increasing, they are limited by vascular graft-related complications and size discrepancy. Current efforts to develop the ideal synthetic vascular graft for clinical application using tissue engineering or 3D printing are far from satisfactory. Therefore, we aimed to re-design the vascular graft with modified materials and 3D printing techniques and also demonstrated the improved applications of our new vascular graft clinically. We designed the 3D printed polyvinyl alcohol (PVA) templates according to the vessel size and shape, and these were dip-coated with salt-suspended thermoplastic polyurethane (TPU). Next, the core template was removed to obtain a customized porous TPU graft. The mechanical testing and cytotoxicity studies of the new synthetic 3D templated vascular grafts (3DT) were more appropriate compared with commercially available polytetrafluoroethylene (PTFE) grafts (ePTFE; standard graft, SG) for clinical use. Finally, we performed implantation of the 3DTs and SGs into the rat abdominal aorta as a patch technique. Four groups of the animal model (SG_7 days, SG_30 days, 3DT_7 days, and 3DT_30 days) were enrolled in this study. The abdominal aorta was surgically opened and sutured with SG or 3DT with 8/0 Prolene. The degree of endothelial cell activation, neovascularization, thrombus formation, calcification, inflammatory infiltrates, and fibrosis were analyzed histopathologically. There was significantly decreased thrombogenesis in the group treated with the 3DT for 30 days compared with the group treated with the SG for 7 and 30 days, and the 3DT for 7 days. In addition, the group treated with the 3DT for 30 days may also have shown increased postoperative endothelialization in the early stages. In conclusion, this study suggests the possibility of using the 3DT as an SG substitute in vascular surgery.

Optimization of the Projection Microstereolithography Process for a Photocurable Biomass-Based Resin.

Biomass materials, an important source of chemical feedstocks, could replace fossil fuels as a resource in the future. The chemical feedstocks from biomass materials are used in many medical and pharmaceutical products and in fuels, chemicals, and functional materials. Biomass materials are expected to be used in biomedical engineering fields, especially due to their low biotoxicity. By the way, most of the demand for bio-application fields is an application targeted for customized production, so a high formability is required for production. Research on three-dimensional (3D) printing technology capable of satisfying these requirements has been ongoing. Manufacturing additives need to be investigated to use biomass materials as a resin or bioink safely for 3D printing, which is a technique widely used in biomedical engineering fields. In this study, a projection microstereolithography (PµSL) system, a 3D printing technique, was made that uses a biomass-based resin. Biomass materials are designed to be photocurable for use in the PµSL process. Various PµSL process parameters were investigated using the biomass-based resin to determine the optimum fabrication conditions for 3D structures. This study demonstrated that a biomass-based resin can be used in the PµSL process. We provide a method for its application in various biomedical engineering fields.

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.

Enhanced bioactivity of titanium-coated polyetheretherketone implants created by a high-temperature 3D printing process.

Polyetheretherketone (PEEK), one of the potential alternatives to metallic materials for implants, necessarily involves high temperature process conditions to be three-dimensionally (3D) printed. We developed a 3D printing setup equipped with thermally stabilized modules of the printing nozzle and building chamber, by which the PEEK implants could be successfully manufactured. Under optimized printing conditions, the maximal mechanical strength of the 3D printed sample attained over 80% of the original bulk property of PEEK. To enhance the interfacial biocompatibility, the as-printed implants were postprocessed with titanium (Ti) sputtering. The Ti-coated surfaces were evaluated through characterization studies of x-ray diffraction spectra, microscopic topographies, and wetting properties. For the in vitro tests, preosteoblasts were cultured on the developed PEEK-Ti structures and evaluated in terms of cell adhesion, proliferation, and osteogenic differentiation. In addition, the bone regeneration capability of the PEEK-Ti implants was confirmed by animal experiments using a rabbit tibia defect model for a period of 12 weeks. In the overall in vitro and in vivo tests, we confirmed the superior bioactivities of the Ti-modified and 3D printed interface by comparisons between the samples of machined and printed samples with or without Ti coating. Taken together, the comprehensive manufacturing approaches that involve 3D printing and biocompatible postprocessing are expected to have universal applicability in a wide range of bone tissue engineering.

Fine Microstructured In-Sn-Bi Solder for Adhesion on a Flexible PET Substrate: Its Effect on Superplasticity and Toughness.

A novel In-Sn-Bi solder with a low electrical resistivity of 14.3 × 10-6 Ω cm and a melting temperature of 99.3 °C was produced for use in adhesive joining on a flexible poly(ethylene terephthalate) substrate. We determined that the fine microstructure of the In-based solder (which had an average phase size of 62.2 nm) strongly influenced its superplasticity and toughness at diffusive temperatures of 55-85 °C because the late-forming BiIn intermetallic compound (IMC) suppressed the growth of two other IMCs, In3Sn and In0.2Sn0.8, which formed earlier in the soldering process. Thus, an elongation of 858.3% and toughness of 36.0 MPa were obtained at a temperature of 85 °C and a strain rate of 0.0020 s-1. However, due to phase boundary fracturing, the phase-refined solder exhibited a slightly more brittle nature (with an elongation of 74.3%) at room temperature compared with a standard In-Sn solder consisting only of the In3Sn and In0.2Sn0.8 IMCs, which had a slightly larger phase size of 84.9 nm and higher ductility (with an elongation of 80.7%). In terms of superplastic deformation, the conventional fracture system based on the Hall-Petch effect transformed into phase boundary sliding at the solder operating temperature, significantly enhancing ductility.

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.

Fabrication of 3D freeform porous tubular constructs with mechanical flexibility mimicking that of soft vascular tissue.

Three-dimensional (3D) printing, with its capability for producing arbitrary shapes, has been extensively studied for tissue engineering applications. However, clinical applications, especially for soft tissues, have been limited due to mechanical mismatch between the 3D-printed artificial tissues and the native tissues. Here, we suggest an integrative method of 3D printing, dip coating, and salt leaching for the fabrication of soft 3D freeform porous tubes, which are expected to be applied to the engineering of vascular tissues. Owing to their porous morphology and controlled wall thickness, the processed tubular constructs had flexible properties comparable to those of native soft tissues with a modulus range of several MPa. When thermoplastic polyurethane (TPU) was used as the dip-coating material, the porous tube exhibited a low tensile modulus from 1.47 to 2.47 MPa and a high elongation limit of over 400%. These flexible properties, which were clearly differentiated from the stiffness of 3D-printed samples with moduli of tens or hundreds of MPa, were confirmed to mimic the mechanical properties of native tissues. Furthermore, by varying the material composition in the dip-coating process, the flexibility of the tube could be modulated when stiffer polycaprolactone (PCL) layers were combined. In addition, such a combination using biocompatible materials could be expected to provide safer interaction at surgical interfaces. Synergistically with the mechanical flexibility, since the proposed method was based on a 3D-printed template, the resulting construct would have extensive applicability in patient-specific tissue engineering.

A Modular Solder System with Hierarchical Morphology and Backward Compatibility.

A modular solder system with hierarchical morphology and micro/nanofeatures in which solder nanoparticles are distributed on the surface of template micropowders is reported. A core-shell structure of subsidiary nanostructures, which improved the intended properties of the modular solder is also presented. In addition, polymer additives can be used not only as an adhesive (like epoxy resin) but also to impart other functions. By combining all of these, it is determined that the modular solder system is able to increase reflowability on a heat-sensitive plastic substrate, oxidation resistance, and electrical conductivity. In this respect, the system could be readily modified by changing the structure and composition of each constituent and adopting backward compatibility with which the knowledge and information attained from a previously designed solder can offer feedback toward further improving the properties of a newly designed one. In practice, In-Sn-Bi nanoparticles engineered on the surface of Sn-Zn micropowders result in pronounced reflowing on a flexible Au-coated polyethylene terephthalate (PET) substrate even at the low temperature of 110 °C. Depending on their respective concentrations, the incorporation of CuO@CeO2 nanostructures and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) polymers increases oxidation resistance and electrical conductivity of the modular solder.

Enhanced Piezoelectricity in a Robust and Harmonious Multilayer Assembly of Electrospun Nanofiber Mats and Microbead-Based Electrodes.

Here, we present a simple yet highly efficient method to enhance the output performance of a piezoelectric device containing electrospun nanofiber mats. Multiple nanofiber mats were assembled together to harness larger piezoelectric sources in the as-spun fibers, thereby providing enhanced voltage and current outputs compared to those of a single-mat device. In addition to the multilayer assembly, microbead-based electrodes were integrated with the nanofiber mats to deliver a complexed compression and tension force excitation to the piezoelectric layers. A vacuum-packing process was performed to attain a tight and well-organized assembly of the device components even though the total thickness was several millimeters. The integrated piezoelectric device exhibited a maximum voltage and current of 10.4 V and 2.3 µA, respectively. Furthermore, the robust integrity of the device components could provide high-precision sensitivity to perceive small pressures down to approximately 100 Pa while retaining a linear input-output relationship.

3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh.

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4 µm. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3 × 3 cm2 or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ∼50 µm thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications.

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.