Shintaroh Iwanaga

Metre-long cell-laden microfibres exhibit tissue morphologies and functions

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Three-dimensional inkjet biofabrication based on designed images

Tissue engineering has been developed with the ultimate aim of manufacturing human organs, but success has been limited to only thin tissues and tissues with no significant structures. In order to construct more complicated tissues, we have developed a three-dimensional (3D) fabrication technology in which 3D structures are directly built up by layer-by-layer printing with living cells and several tissue components. We developed a custom-made inkjet printer specially designed for this purpose. Recently, this printer was improved, and the on-demand printing mode was developed and installed to fabricate further complicated structures. As a result of this version, 3D layer-by-layer printing based on complicated image data has become possible, and several 2D and 3D structures with more complexity than before were successfully fabricated. The effectiveness of the on-demand printing mode in the fabrication of complicated 3D tissue structures was confirmed. As complicated 3D structures are essential for biofunctional tissues, inkjet 3D biofabrication has great potential for engineering complicated bio-functional tissues.

Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

Image printing on the surface of anti-biofouling zwitterionic polymer brushes by ion beam irradiation.

A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly hydrophilic and showed a strong resistance against non-specific adsorption of proteins and cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a hollow space with a designed shape could be made to which HEK293 cells (from human embryonic kidney) and Hep G2 (from human hepatoma) cells non-specifically adhered, while no adhesion of these cells to the non-treated area on the brush was observed. The present results clearly indicate the usefulness of ion beam-printed patterns of anti-biofouling zwitterionic polymer brushes in the biomedical field.

Facile fabrication of uniform size-controlled microparticles and potentiality for tandem drug delivery system of micro/nanoparticles

This article describes a rapid and facile method for manufacturing various size-controlled gel particles with utilizing inkjet printing technology. Generally, the size of droplets could be controlled by changing nozzle heads of inkjet printer, from which ink solution is ejected. However, this method uses drying process before gelling microparticles, and with that, the size of microparticles was easily controlled by only altering the concentration of ejected solution. When sodium alginate solution with various concentrations was ejected from inkjet printer, we found that the concentration of alginate solution vs. the volume of dried alginate particle showed an almost linear relationship in the concentration range from 0.1 to 3.0%. After dried alginate particles were soaked into calcium chloride solution, the size of microgel beads were obtained almost without increasing their size. The microparticles including various sizes of nanoparticles were easily manufactured by ejecting nanoparticle-dispersed alginate solution. The release of 25-nm sized nanoparticles from alginate microgel beads was finished in a relatively-rapid manner, whereas 100-nm sized nanoparticles were partially released from those ones. Moreover, most of 250-nm sized nanoparticles were not released from alginate microgel beads even after 24-h soaking. This particle fabricating method would enable the tandem drug delivery system with a combination of the release from nano and microparticles, and be expected for the biological and tissue engineering application.

Neural stem/progenitor cell-laden microfibers promote transplant survival in a mouse transected spinal cord injury model

Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen‐based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC–microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC‐laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC‐laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation‐based approaches to regenerative medicine.

Bioprinting with pre-cultured cellular constructs towards tissue engineering of hierarchical tissues

The fabrication of physiologically active tissue constructs from various tissue elements are vital for establishing integrated bioprinting and transfer printing techniques as vital tools for biomedical research. Physiologically functional tissues are hierarchically constructed from a variety of tissue subunits with different feature sizes and topographies. For example, skeletal muscles are composed of many muscle bundles, muscle fibers, and muscle cells respectively. The fundamental constituents of all types of muscle tissues include various sized blood vessels, and vascular related cells. Nature has designed the direction of all the aforementioned components to have unidirectional alignment, so that muscle contractions can effectively generate the mechanical functions. In this study, we demonstrate a promising approach to fabricating such hierarchical tissues by applying bioprinting and a transfer patterning technique. Linear-patterned smooth muscle cells were obtained by culturing on the surface patterned discs, before being transferred onto the Matrigel substrate. The fiber-like tissues structures were successfully formed on the substrate after a few days of culturing; these are partially aligned smooth muscle cells. Additionally, stacked structures were also successfully fabricated using laminating printing technique. Our results indicate that bioprinting and transfer printing strategy of pre-cultured aligned muscular fiber-like tissues is very promising method to assemble tissue elements for biofabrication of hierarchical tissues.

Cells on arrays of microsprings: An approach to achieve triaxial control of substrate stiffness

Microenvironmental biophysical stimuli influence diverse cellular functions, such as directional motility and stem cell differentiation. Previously, researchers have tuned the linear stiffness of microposts to investigate cell mechanobiological processes and direct cellular behavior; however, microposts suffer from an inherent, yet critical drawback - regulation of micropost stiffness is fundamentally limited to “biaxial” control. To overcome this issue, here we utilize three-dimensional (3D) direct-write laser lithography processes to fabricate arrays of microscale springs (μSprings). By adjusting the geometric characteristics of individual μSprings, the x-, y-, and z-axis stiffness of the cellular substrate can be customized at the microscale. COMSOL simulations were performed to characterize the theoretical “triaxial” stiffness associated with a variety of μSpring designs. Endothelial cells seeded on μSpring arrays were found to successfully deform the μSprings via cell-generated forces. By enabling user-control over the triaxial stiffness of discrete, microscale substrate features, the presented μSpring methodology could offer a powerful platform for cellular studies and applications in fields including tissue engineering, biomaterials, and regenerative medicine.

Dermal cell damage induced by topical application of non-steroidal anti-inflammatory drugs is suppressed by trehalose co-lyophilization in ex vivo analysis.

ABSTRACT Topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) is generally considered safer than oral administration, although the former can occasionally induce cutaneous irritation. We hypothesized that the cutaneous irritation by topical NSAIDs might be suppressed by trehalose, which has protective effects on biological membranes. Using the three-dimensional cultured human skin model, Living Skin Equivalent-high, we found that cutaneous damage due to NSAIDs was reduced by concomitant use of trehalose and that this effect of trehalose was reinforced by co-lyophilization of NSAIDs with trehalose. The anti-inflammatory effect of co-lyophilized NSAIDs with trehalose was comparable to that seen with NSAIDs alone in a rat model. Our results suggest that co-lyophilization of NSAIDs with trehalose might be a novel procedure that can help prevent NSAIDs-induced skin irritation.

Prevention of intubation-induced mucosal damage using a tube coated with 2-methacryloyloxyethyl phosphorylcholine polymer.

Background and objective Tracheal intubation is associated with various complications that include epithelial injury. Abrasion of the fragile tracheal epithelium can occur at the points of contact between the tube and mucosa subject to respiratory movement. In this original experiment, we examined the mucosal protective effect of coating endotracheal tubes with poly[2-methacryloyloxyethl phosphorylcholine (MPC)-co-n-butyl methacrylate] (PMB). Methods We prepared four types of tubes: tube A (control, no coating), tube B (two coats, 0.5% PMB), tube C (10 coats, 0.5% PMB) or tube D (one coat, 5% PMB). Twenty-nine beagle dogs were divided into four groups and orally intubated with tube A, B, C or D for 4 ± 0.5 h. The cuffs of extubated tubes were stained with haematoxylin. Paraffin sections from tracheal walls in contact with the inflated cuff were stained with haematoxylin/eosin and periodic acid-Schiff. Results Cuffs of tubes A and B were strongly stained with haematoxylin because of attached epithelial cells. Stained areas in those of tubes C and D were significantly reduced. Histological analysis showed that a single coat of 5% PMB prevented epithelial abrasion and proliferation of goblet cells. Excess tracheal mucus was observed in the tube A group, but not in the tube D group. Conclusion Tracheal epithelial damage caused by intubation was greatly reduced or eliminated by PMB coating on the surface of the tracheal tube.