Transplanted artificial amnion membrane enhanced wound healing in third-degree burn injury diabetic mouse model.

Introduction: Wound healing is severely compromised in patients with diabetes owing to factors such poor blood circulation, delayed immune response, elevated blood sugar levels, and neuropathy. Although the development of new wound healing products and prevention of serious complications such as infections in wounds have received substantial interest, wound healing remains a challenge in regenerative medicine. Burn wounds, especially third-degree burns, are difficult to treat because they are associated with immune and inflammatory reactions and distributive shock. Wound care and treatment that protects the burn site from infection and allows wound healing can be achieved with bioengineered wound dressings. However, few studies have reported effective dressings for third-degree burn wounds, making it important to develop new dressing materials. Methods: In this study, we developed an artificial amniotic membrane (AM) using epithelial and mesenchymal cells derived from human amnion as a novel dressing material. The artificial AM was applied to the wound of a diabetic third-degree burn model and its wound healing ability was evaluated. Results: This artificial amnion produced multiple growth factors associated with angiogenesis, fibroblast proliferation, and anti-inflammation. In addition, angiogenesis and granulation tissue formation were promoted in the artificial AM-treated mouse group compared with the control group. Furthermore, the inflammatory phase was prolonged in the control group. Conclusions: Our preliminary results indicate that the artificial AM might be useful as a new dressing for refractory ulcers and third-degree burns. This artificial AM-based material represents great potential for downstream clinical research and treatment of diabetes patients with third-degree burns.

Whole Liver Derived Acellular Extracellular Matrix for Bioengineering of Liver Constructs: An Updated Review.

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

Mammalian-specific decellularized matrices derived bioink for bioengineering of liver tissue analogues: A review.

The absolute shortage of compatible liver donors and the growing number of potential recipients have led scientists to explore alternative approaches to providing tissue/ organ substitutes from bioengineered sources. Bioartificial regeneration of a fully functional tissue/organ replacement is highly dependent on the right combination of engineering tools, biological principles, and materiobiology horizons. Over the past two decades, remarkable achievements have been made in hepatic tissue engineering by converging various advanced interdisciplinary research approaches. Three-dimensional (3D) bioprinting has arisen as a promising state-of-the-art tool with strong potential to fabricate volumetric liver tissue/organ equivalents using viscosity- and degradation-controlled printable bioinks composed of hydrous microenvironments, and formulations containing living cells and associated supplements. Source of origin, biophysiochemical, or thermomechanical properties and crosslinking reaction kinetics are prerequisites for ideal bioink formulation and realizing the bioprinting process. In this review, we delve into the forecast of the potential future utility of bioprinting technology and the promise of tissue/organ- specific decellularized biomaterials as bioink substrates. Afterward, we outline various methods of decellularization, and the most relevant studies applying decellularized bioinks toward the bioengineering of in vitro liver models. Finally, the challenges and future prospects of decellularized material-based bioprinting in the direction of clinical regenerative medicine are presented to motivate further developments.

Design and Fabrication of Mature Engineered Pre-Cardiac Tissue Utilizing 3D Bioprinting Technology and Enzymatically Crosslinking Hydrogel.

The fabrication of mature engineered cardiac tissue is one of the major challenges in cardiac tissue engineering. For this purpose, we attempted to apply the 3D bioprinting approach. Aiming to construct an oriented tissue, a fine fiber-shaped scaffold with a support structure was first designed using CAD software. Then, a 3D bioprinter and cell-adhesive bio-inks were utilized to fabricate this structure. The cell-adhesive bio-inks were synthesized by combining sodium alginate and gelatin with tyramine, respectively, to form pre-gel materials that allow enzymatic crosslinking by horseradish peroxidase. By absorbance measurements, we confirmed that the tyramine modification rate of each polymer was 0.535 mmol/g-alginate and 0.219 mmol/g-gelatin. The width of the fiber-shaped scaffold was 216.8 ± 24.3 µm for the fabricated scaffold, while the design value was 200 µm. After 3D printing and adhesion-adding treatment of the scaffold with these bio-ink materials, cardiomyocytes were seeded and cultured. As a result, the cells spread onto the scaffold, and the entire pre-tissue contracted synchronously by day 6 of culture, showing a greater pulsatility than in the early days. Video analysis showed that the beating rate of pre-myocardial tissue on day 6 was 31 beats/min. In addition, we confirmed that the cardiomyocytes partially elongated along the long axis of the fiber-shaped scaffold in the pre-tissue cultured for 15 days by staining actin, suggesting the possibility of cell orientation. Furthermore, treatment with adrenaline resulted in a 7.7-fold increase in peak beating rate compared to that before treatment (from 6 beats/min to 46 beats/min), confirming the responsiveness of the pre-tissues to the drug. These results indicate that 3D bioprinting effectively produces mature cultured myocardial tissue that is oriented, contracts synchronously, and is responsive to drugs.

Biofabrication offers future hope for tackling various obstacles and challenges in tissue engineering and regenerative medicine: A Perspective.

Biofabrication is an emerging multidisciplinary field that makes a revolutionary impact on the researches on life science, biomedical engineering, and both basic and clinical medicine, has progressed tremendously over the past few years. Recently, there has been a big boom in three-dimensional (3D) printing or additive manufacturing (AM) research worldwide, and there is a significant increase not only in the number of researchers turning their attention to AM but also publications demonstrating the potential applications of 3D printing techniques in multiple fields. Biofabrication and bioprinting hold great promise for the innovation of engineering-based organ replacing medicine. In this mini review, various challenges in the field of tissue engineering are focused from the point of view of the biofabrication - strategies to bridge the gap between organ shortage and mission of medical innovation research seek to achieve organ-specific treatments or regenerative therapies. Four major challenges are discussed including (i) challenge of producing organs by AM, (ii) digitalization of tissue engineering and regenerative medicine, (iii) rapid production of organs beyond the biological natural course, and (iv) extracorporeal organ engineering.

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.

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.

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

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.

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.

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.

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.

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.

Anti-biofouling properties of an amphoteric polymer brush constructed on a glass substrate.

An amphoteric copolymer brush of methacrylic acid (MA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization using both a free chain transfer agent (n-butylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid) and a radical initiator (4,4'-azobis(4-cyanopentanoic acid)) covalently fixed to a glass substrate. An aqueous solution of the copolymer, Poly(MA-r-DMAEMA), which was simultaneously obtained in liquid phase, had a sufficiently small polydispersity in its molecular weight. The copolymer brush showed effective suppression of non-specific adsorption of bovine serum albumin and egg white lysozyme to the brush. In contrast, both negatively charged PolyMA and positively charged PolyDMAEMA brushes significantly adsorbed the proteins irrespective of their net charges. Upon ion beam irradiation, furthermore, a hollow space with a designed shape could be made on the glass substrate, and both HEK293 and HepG2 cells non-specifically adhered to the space, forming aggregates, while no adhesion to the non-treated area on the brush was observed. These results suggest that the amphoteric polymer brushes will be useful materials for biomedical applications.

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.

Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation.

Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10 degrees (costheta = 0.99) with PAAm grafted amounts of 1.6 microg/cm(2) as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 microg/mL fibronectin at 37 degrees C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.