A multi-channel collagen conduit with aligned Schwann cells and endothelial cells for enhanced neuronal regeneration in spinal cord injury.

Traumatic spinal cord injury (SCI) leads to Wallerian degeneration and the accompanying disruption of vasculature leads to ischemia, which damages motor and sensory function. Therefore, understanding the biological environment during regeneration is essential to promote neuronal regeneration and overcome this phenomenon. The band of Büngner is a structure of an aligned Schwann cell (SC) band that guides axon elongation providing a natural recovery environment. During axon elongation, SCs promote axon elongation while migrating along neovessels (endothelial cells [ECs]). To model this, we used extrusion 3D bioprinting to develop a multi-channel conduit (MCC) using collagen for the matrix region and sacrificial alginate to make the channel. The MCC was fabricated with a structure in which SCs and ECs were longitudinally aligned to mimic the sophisticated recovering SCI conditions. Also, we produced an MCC with different numbers of channels. The aligned SCs and ECs in the 9-channel conduit (9MCC-SE) were more biocompatible and led to more proliferation than the 5-channel conduit (5MCC-SE) in vitro. Also, the 9MCC-SE resulted in a greater healing effect than the 5MCC-SE with respect to neuronal regeneration, remyelination, inflammation, and angiogenesis in vivo. The above tissue recovery results led to motor function repair. Our results show that our 9MCC-SE model represents a new therapeutic strategy for SCI.

FeS2-incorporated 3D PCL scaffold improves new bone formation and neovascularization in a rat calvarial defect model.

199Three-dimensional (3D) scaffolds composed of various biomaterials, including metals, ceramics, and synthetic polymers, have been widely used to regenerate bone defects. However, these materials possess clear downsides, which prevent bone regeneration. Therefore, composite scaffolds have been developed to compensate these disadvantages and achieve synergetic effects. In this study, a naturally occurring biomineral, FeS2, was incorporated in PCL scaffolds to enhance the mechanical properties, which would in turn influence the biological characteristics. The composite scaffolds consisting of different weight fractions of FeS2 were 3D printed and compared to pure PCL scaffold. The surface roughness (5.77-fold) and the compressive strength (3.38-fold) of the PCL scaffold was remarkably enhanced in a dose-dependent manner. The in vivo results showed that the group with PCL/ FeS2 scaffold implanted had increased neovascularization and bone formation (2.9-fold). These results demonstrated that the FeS2 incorporated PCL scaffold might be an effective bioimplant for bone tissue regeneration.

Advances in the development of tubular structures using extrusion-based 3D cell-printing technology for vascular tissue regenerative applications.

Until recent, there are no ideal small diameter vascular grafts available on the market. Most of the commercialized vascular grafts are used for medium to large-sized blood vessels. As a solution, vascular tissue engineering has been introduced and shown promising outcomes. Despite these optimistic results, there are limitations to commercialization. This review will cover the need for extrusion-based 3D cell-printing technique capable of mimicking the natural structure of the blood vessel. First, we will highlight the physiological structure of the blood vessel as well as the requirements for an ideal vascular graft. Then, the essential factors of 3D cell-printing including bioink, and cell-printing system will be discussed. Afterwards, we will mention their applications in the fabrication of tissue engineered vascular grafts. Finally, conclusions and future perspectives will be discussed.

Overcome the barriers of the skin: exosome therapy.

Exosomes are nano-sized cargos with a lipid bilayer structure carrying diverse biomolecules including lipids, proteins, and nucleic acids. These small vesicles are secreted by most types of cells to communicate with each other. Since exosomes circulate through bodily fluids, they can transfer information not only to local cells but also to remote cells. Therefore, exosomes are considered potential biomarkers for various treatments. Recently, studies have shown the efficacy of exosomes in skin defects such as aging, atopic dermatitis, and wounds. Also, exosomes are being studied to be used as ingredients in commercialized skin treatment products. In this review, we discussed the need for exosomes in skin therapy together with the current challenges. Moreover, the functional roles of exosomes in terms of skin treatment and regeneration are overviewed. Finally, we highlighted the major limitations and the future perspective in exosome engineering.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

A skeleton muscle model using GelMA-based cell-aligned bioink processed with an electric-field assisted 3D/4D bioprinting.

The most important requirements of biomedical substitutes used in muscle tissue regeneration are appropriate topographical cues and bioactive components for the induction of myogenic differentiation/maturation. Here, we developed an electric field-assisted 3D cell-printing process to fabricate cell-laden fibers with a cell-alignment cue. Methods: We used gelatin methacryloyl (GelMA) laden with C2C12 cells. The cells in the GelMA fiber were exposed to electrical stimulation, which induced cell alignment. Various cellular activities, such as cell viability, cell guidance, and proliferation/myogenic differentiation of the microfibrous cells in GelMA, were investigated in response to parameters (applied electric fields, viscosity of the bioink, and encapsulated cell density). In addition, a cell-laden fibrous bundle mimicking the structure of the perimysium was designed using gelatin hydrogel in conjunction with a 4D bioprinting technique. Results: Cell-laden microfibers were fabricated using optimized process parameters (electric field intensity = 0.8 kV cm-1, applying time = 12 s, and cell number = 15 × 106 cells mL-1). The cell alignment induced by the electric field promoted significantly greater myotube formation, formation of highly ordered myotubes, and enhanced maturation, compared to the normally printed cell-laden structure. The shape change mechanism that involved the swelling properties and folding abilities of gelatin was successfully evaluated, and we bundled the GelMA microfibers using a 4D-conceptualized gelatin film. Conclusion: The C2C12-laden GelMA structure demonstrated effective myotube formation/maturation in response to stimulation with an electric field. Based on these results, we propose that our cell-laden fibrous bundles can be employed as in vitro drug testing models for obtaining insights into the various myogenic responses.

Cell-Electrospinning and Its Application for Tissue Engineering.

Electrospinning has gained great interest in the field of regenerative medicine, due to its fabrication of a native extracellular matrix-mimicking environment. The micro/nanofibers generated through this process provide cell-friendly surroundings which promote cellular activities. Despite these benefits of electrospinning, a process was introduced to overcome the limitations of electrospinning. Cell-electrospinning is based on the basic process of electrospinning for producing viable cells encapsulated in the micro/nanofibers. In this review, the process of cell-electrospinning and the materials used in this process will be discussed. This review will also discuss the applications of cell-electrospun structures in tissue engineering. Finally, the advantages, limitations, and future perspectives will be discussed.

Three-dimensional gelatin/PVA scaffold with nanofibrillated collagen surface for applications in hard-tissue regeneration.

The surface topography of a tissue-engineered scaffold is widely known to play an essential role in bone tissue engineering applications. Therefore, the cell-to-material interaction should be considered when developing scaffolds for bone tissue regeneration. Bone is a dynamic tissue with a distinct hierarchical structure composed of mostly collagen and bioceramics. In this study, the surface of gelatin/PVA scaffold (CF-G5P5) coated with fibrillated collagen was fabricated to enhance cell proliferation and osteogenic differentiation for bone tissue regeneration. The physical and biological properties of the fabricated scaffolds were investigated. As a result, the CF-G5P5 scaffold increased surface roughness and increased protein absorption compared to a gelatin/PVA scaffold (G5P5) by 1.6 times from OD value 0.43 to 0.71 after 12 h, cell proliferation increased 1.7 times from OD value 0.57 to 0.96, and differentiation increased by 1.5 times from 100 to 151%. Based on the results, the CF-G5P5 scaffold developed can be considered as a highly potential bone tissue regenerative material.

4D Bioprinting: Technological Advances in Biofabrication.

The development of the three-dimensional (3D) printer has resulted in significant advances in a number of fields, including rapid prototyping and biomedical devices. For 3D structures, the inclusion of dynamic responses to stimuli is added to develop the concept of four-dimensional (4D) printing. Typically, 4D printing is useful for biofabrication by reproducing a stimulus-responsive dynamic environment corresponding to physiological activities. Such a dynamic environment can be precisely designed with an understanding of shape-morphing effects (SMEs), which enables mimicking the functionality or intricate geometry of tissues. Here, 4D bioprinting is investigated for clinical use, for example, in drug delivery systems, tissue engineering, and surgery in vivo. This review presents the concept of 4D bioprinting and smart materials defined by SMEs and stimulus-responsive mechanisms. Then, biomedical smart materials and applications are discussed along with future perspectives.

The fabrication of uniaxially aligned micro-textured polycaprolactone struts and application for skeletal muscle tissue regeneration.

One of the most important factors in skeletal muscle tissue regeneration is the alignment of muscle cells to mimic the native tissue. In this study, we developed a PCL-based scaffold with uniaxially aligned surface topography by stretching a 3D-printed scaffold. We examined the formation of aligned patterns by stretching the samples at different temperatures and stretching rates. This was possible through the effects of crystalline and amorphous regions on micro-textured deformation during the stretching process. We characterized the physical and biological properties of unstretched and stretched PCL struts. The stretched PCL showed greater surface roughness, protein absorption ability, and wettability. Moreover, myoblasts were cultured on the stretched and unstretched samples to analyze cellular activity. The cells cultured on the stretched samples were aligned along the pattern and showed a more elongated morphology. Furthermore, proliferation and differentiation were increased on the stretched samples resulting in a greater number of myotubes. We also discuss the possible alternative applications of this developed scaffold in other tissues.

Gelatin/PVA scaffolds fabricated using a 3D-printing process employed with a low-temperature plate for hard tissue regeneration: Fabrication and characterizations.

Tissue engineering aims to repair or replace damaged tissues or organs using biomedical scaffolds cultured with cells. The scaffolds composed of biomaterials should guide the cells to mature into functional tissues or organs. An ideal scaffold to regenerate hard tissues should have mechanical stability as well as biocompatibilities. It has been well known that gelatin can provide outstanding biological activities, but its low mechanical stability can be one of obstacles to be used in hard tissue regeneration. To overcome the issue, we used PVA, which can reinforce the low mechanical stability of the gelatin. The gelatin/PVA scaffolds have been fabricated using a low temperature 3D-printing process. By manipulating various weight fractions of PVA/gelatin, we can obtain the optimal mixture ratio in aspect of the physical and biological properties of the scaffolds. As a result, a weight fraction of 5:5 showed appropriate mechanical strength and enhanced cell activities, such as cell proliferation and differentiation. The gelatin/PVA scaffold showed potential for future application as biomedical scaffold in soft and hard tissue regeneration.

Fabrication of micro/nanoporous collagen/dECM/silk-fibroin biocomposite scaffolds using a low temperature 3D printing process for bone tissue regeneration.

Biomaterials must be biocompatible, biodegradable, and mechanically stable to be used for tissue engineering applications. Among various biomaterials, a natural-based biopolymer, collagen, has been widely applied in tissue engineering because of its outstanding biocompatibility. However, due to its low mechanical properties, collagen has been a challenge to build a desired/complex 3D porous structure with appropriate mechanical strength. To overcome this problem, in this study, we used a low temperature printing process to create a 3D porous scaffold consisting of collagen, decellularized extracellular matrix (dECM) to induce high cellular activities, and silk-fibroin (SF) to attain the proper mechanical strength. To show the feasibility of the scaffold, pre-osteoblast (MC3T3-E1) cells were grown on the fabricated scaffold. Various in vitro cellular activities (cell-viability, MTT assay, and osteogenic activity) for pure collagen, collagen/dECM, and collagen/SF/dECM scaffolds were compared.

3D bioprinting and its in vivo applications.

The purpose of 3D bioprinting technology is to design and create functional 3D tissues or organs in situ for in vivo applications. 3D cell-printing, or additive biomanufacturing, allows the selection of biomaterials and cells (bioink), and the fabrication of cell-laden structures in high resolution. 3D cell-printed structures have also been used for applications such as research models, drug delivery and discovery, and toxicology. Recently, numerous attempts have been made to fabricate tissues and organs by using various 3D printing techniques. However, challenges such as vascularization are yet to be solved. This article reviews the most commonly used 3D cell-printing techniques with their advantages and drawbacks. Furthermore, up-to-date achievements of 3D bioprinting in in vivo applications are introduced, and prospects for the future of 3D cell-printing technology are discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 444-459, 2018.

Alternately plasma-roughened nanosurface of a hybrid scaffold for aligning myoblasts.

For successful skeletal muscle tissue regeneration, inducing alignment and fusion of myoblasts into multinucleated myotubes is critical. Many studies are ongoing to induce myoblast alignment using various micro/nanopatternings on scaffold surfaces, mechanically stretching scaffolds, or aligned micro/nanofibers. In this study, we have developed a simple method to induce myoblast alignment using a modified plasma treatment on a hybrid PCL scaffold consisting of melt-printed perpendicular PCL struts and an electrospun PCL fibrous mat. For the hybrid scaffold, the surface of the electrospun mat was selectively roughened with a plasma process supplemented with a template. The cell alignment of myoblasts using this system was enhanced significantly when compared to results from the use of a hybrid scaffold with a non-roughened electrospun fiber surface or a hybrid scaffold where the whole surface of the electrospun fibers was roughened. This new type of plasma-treated hybrid scaffold has strong potential as a biomaterial for use in muscle tissue regeneration.

Additive-manufactured polycaprolactone scaffold consisting of innovatively designed microsized spiral struts for hard tissue regeneration.

Three-dimensional biomedical polycaprolactone scaffolds consisting of microsized spiral-like struts were fabricated using an additive manufacturing process. In this study, various processing parameters such as applied pressure, polymer viscosity, printing nozzle-to-stage distance, and nozzle moving speed were optimized to achieve a unique scaffold consisting of spiral-like struts. Various physical and biological analyses, including the morphological structure of spirals, mechanical properties, cell proliferation, and osteogenic activities, were performed to evaluate the effect of the spirals of the scaffold. Osteoblast-like cells (MG63) were used to identify the various in vitro cellular responses on the scaffolds. The spiral-like struts, having unique spiral angles, had a more significant effect on cell attachment, proliferation, and differentiation compared to normal struts. The results suggest that the scaffold consisting of spiral struts can be a potential biomedical device for various applications in tissue engineering.

Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue.

Cell-printing technology has provided a new paradigm for biofabrication, with potential to overcome several shortcomings of conventional scaffold-based tissue regeneration strategies via controlled delivery of various cell types in well-defined target regions. Here we describe a cell-printing method to obtain mechanically reinforced multi-layered cell-embedded scaffolds, formed of micron-scale poly(ε-caprolactone) (PCL)/alginate struts coated with alginate-based bioink. To compare the physical and cellular activities, we used a scaffold composed of pure alginate (without cells) coated PCL/alginate struts as a control. We systematically varied the ratio of alginate cross-linking agent, and determined the optimal cell-coating conditions to form the PCL/alginate struts. Following fabrication of the cell (MG63)-laden PCL/alginate scaffold, the bioactivity was evaluated in vitro. The laden cells exhibited a substantially more developed cytoskeleton compared with those on a control scaffold consisting of the same material composition. Based on these results, the printed cells exhibited a significantly more homogenous distribution within the scaffold compared with the control. Cell proliferation was determined via MTT assays at 1, 3, 7, and 14 days of culture, and the proliferation of the cell-printed scaffold was substantially in excess (∼2.4-fold) of that on the control. Furthermore, the osteogenic activity such as ALP was measured, and the cell-laden scaffold exhibited significantly greater activity (∼3.2-fold) compared with the control scaffold.

A hybrid PCL/collagen scaffold consisting of solid freeform-fabricated struts and EHD-direct-jet-processed fibrous threads for tissue regeneration.

Hybrid biomedical structures have been used widely in various tissue-regenerating materials because they effectively induce exceptional physical and cellular responses. In this study, a new hybrid process was used to design a three-dimensional (3D) biomedical hybrid scaffold with a controlled pore-structure and high mechanical strength. A melt-dispensing method was used to obtain mechanical properties and electrohydrodynamic direct-jet (EHD-DJ) printing was used to provide microsized fibrous structures for the scaffold. Furthermore, the poly(ε-caprolactone) (PCL) hybrid scaffolds were coated biomimetically with type-I collagen to increase bioactive interactions between cells and scaffolds. The fabricated scaffolds showed similar mechanical properties to the two control scaffolds; however, the results of culturing osteoblast-like (MG63) cells showed significant increases in in vitro cellular activities (cell viability>twofold and calcium deposition>sevenfold). Based on these results, we propose a newly designed hybrid scaffold that can support significant in vitro cellular activities at the interface between cells and the 3D micro-pore structure for soft and hard tissue regeneration.

Tissue engineering bioreactor systems for applying physical and electrical stimulations to cells.

Bioreactor systems in tissue engineering applications provide various types of stimulation to mimic the tissues in vitro and in vivo. Various bioreactors have been designed to induce high cellular activities, including initial cell attachment, cell growth, and differentiation. Although cell-stimulation processes exert mostly positive effects on cellular responses, in some cases such stimulation can also have a negative effect on cultured cells. In this review, we discuss various types of bioreactor and the positive and negative effects of stimulation (physical, chemical, and electrical) on various cultured cell types.