Efficient Myogenic Activities Achieved through Blade-Casting-Assisted Bioprinting of Aligned Myoblasts Laden in Collagen Bioink.

This study investigated mechanical stimulation combined with three-dimensional (3D) bioprinting as a new approach for introducing biophysical and biological cues for tissue regeneration. A blade-casting method in conjunction with bioprinting was employed to fabricate bioengineered skeletal muscle constructs using a bioink composed of C2C12 myoblasts and collagen type-I. Various printing process parameters were selected and optimized to achieve a highly organized cell alignment within the constructs. The resulting cell-aligned constructs demonstrated remarkable improvement in actin filament alignment and cell proliferation compared with conventionally printed cell-laden constructs. This improvement can be attributed to the synergistic effects of mechanotransduction, facilitating the cellular response to mechanical cues and the alignment of fibrillated collagen, which plays a significant role in modulating cellular functions and promoting muscle tissue regeneration. Furthermore, we assessed the impact of blade casting combined with 3D bioprinting on gene expression. The expression levels of myogenesis-related genes were substantially upregulated, with an approximately 1.6-fold increase compared to the constructs fabricated without the blade-casting technique. The results demonstrated the effectiveness of combining mechanical stimulation through blade casting with 3D bioprinting in promoting aligned cell structures, enhancing cellular functions, and driving muscle tissue regeneration.

Three-Dimensional Artificial Skin Construct Bioprinted with a Marine-Based Biocomposite.

A variety of artificial skin scaffolds, including 3D-bioprinted constructs, have been widely studied for regenerating injured skin tissue. Here, we devised a new composite biomaterial ink using fish-skin-based decellularized extracellular matrices (dECM) from tilapia and cod fish. The composition of the biocomposite mixture was carefully selected to obtain a mechanically stable and highly bioactive artificial cell construct. In addition, the decellularized extracellular matrices were methacrylated, followed by exposure to UV light to initiate photo-cross-linking. Porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa) biomaterials were used as controls. Assessment of various biophysical parameters and in vitro cellular activities, including cytotoxicity, wound healing ability, and angiogenesis, showed that the biocomposite exhibited much higher cellular activities compared to the controls owing to the synergistic effect of the favorable biophysical properties of tdECMMa and bioactive components (collagen, glycosaminoglycans (GAGs), elastin, and free fatty acids) from the decellularized cod skin. Furthermore, the skin constructs bioprinted using the bioinks exhibited more than 90% cell viability, performed with 3 days of submerged culture and then 28 days of air-liquid culture. For all cell constructs, the expression of cytokeratin 10 (CK10) was observed on the top surface of the epidermal layer, and cytokeratin 14 (CK14) was detected in the lower section of the keratinocyte layer. However, more developed CK10 and CK14 antibodies were observed in the cell-laden biocomposite construct [tilapia-skin-based dECMMa with cod-skin-based dECM] than in the controls [porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa)]. Based on these results, we believe that the fish-skin-based biocomposite construct is a potential biomaterial ink for skin regeneration.

A Microfluidic Device to Fabricate One-Step Cell Bead-Laden Hydrogel Struts for Tissue Engineering.

Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cell-laden bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases the degree of cell-to-cell interaction to a level comparable to cell spheroids. The hybrid structure is obtained using a one-step process without the exhausting procedure. It consists of cell bead fabrication and an extrusion process for the cell-bead laden structure. Different flow rates are appropriately selected to develop cell-laden struts with homogeneously distributed cell beads for each hydrogel in the process. The hybrid struts exhibit significantly higher cellular activities than those of conventional alginate/GelMa struts, which are bioprinted using similar cell densities and bioink formulations. Furthermore, hybrid struts with adipose stem cells are implanted into mice, resulting in significantly higher myogenesis in comparison to normally bioprinted struts.

Engineered Myoblast-Laden Collagen Filaments Fabricated Using a Submerged Bioprinting Process to Obtain Efficient Myogenic Activities.

The skeletal muscle tissue comprises a hierarchical fibrous structure with fully aligned myofibers. To obtain a unique aligned engineering construct for regenerating muscle tissue, we adopted a submerged bioprinting process. Here, 3 wt % collagen and 6 wt % alginate solutions were used as a matrix cell-encapsulating bioink and supporting solution in the printing bath, respectively. By manipulating the processing parameters (various alginate weight fractions in the bath, nozzle moving speed, and hydrostatic pressure), cell-laden filaments (∼50 µm in diameter) were successfully fabricated. They presented a high degree of alignment of the fibrillated collagen and meaningful initial viability (∼90%) of the C2C12 myoblasts. In vitro cellular responses indicated that fully aligned F-actin filaments of myoblasts were developed, resulting in a high degree of alignment/formation of myotubes, compared to that in the controls (>100 µm diameter of cell-laden filaments). Furthermore, the expression levels of various myogenic genes (Myod1, Myh2, and Myog) were measured using a reverse transcription polymerase chain reaction on day 21 of the cell culture, and the results showed that the cell-laden filaments with a small diameter had considerably greater gene expression levels (2.2-8-fold) than those with a relatively large diameter. Thus, the printing process described herein can provide a new potential biofabricating platform to obtain cell-laden engineering constructs for various tissues.

Fabrication of Mechanically Reinforced Gelatin/Hydroxyapatite Bio-Composite Scaffolds by Core/Shell Nozzle Printing for Bone Tissue Engineering.

In tissue engineering, biocompatible scaffolds are used as 3D cell niches to provide a similar environment to that of native tissue for seeded cells to regenerate the target tissue. When engineering bone tissue, high mechanical strength and calcium phosphate composition are essential factors to consider. In this study, we fabricated biocompatible composite scaffolds composed of synthetic polymers (polycaprolactone (PCL) and poly (vinyl alcohol) (PVA)), natural polymers (gelatin and collagen) and bioceramic (hydroxyapatite; HA) for bone tissue engineering. The synthetic polymers were used to enhance the mechanical properties of the composite scaffolds while the natural protein-based polymers were used to enhance various cellular activities, such as cell adhesion and proliferation. Meanwhile, the bioceramic was introduced to promote osteogenic differentiation. Composite scaffolds were evaluated for their physical characteristics, such as mechanical, swelling and protein absorbing properties as well as biological properties (cell proliferation, alkaline phosphatase (ALP) activities and calcium deposition) with human osteoblast-like cells (MG63). Consequently, incorporation of hydroxyapatite into the gelatin/PVA (C-GPH) scaffold showed 5-fold and 1.5-fold increase in calcium deposition and ALP activities, respectively compared to gelatin/PVA scaffold (C-GP). Moreover, compressive modulus also increased 1.8-fold. Integration of PCL core into gelatin/PVA/hydroxyapatite scaffold (C-PGPH) further amplified the compressive modulus 1.5-fold. In conclusion, the scaffold that is reinforced with HA particles and integrated with PCL core of the struts showed significant potential in field of bone tissue engineering.

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.

Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: fabrication and cellular activities.

In this study, we propose a new scaffold fabrication method, "direct electro-hydrodynamic jet process," using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multilayered microsized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes. The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with preosteoblasts to observe the initial cellular activities in comparison with a solid-freeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding microcellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).

Engineered 3D liver-tissue model with minispheroids formed by a bioprinting process supported with in situ electrical stimulation.

Three-dimensional (3D) bioprinting, an effective technique for building cell-laden structures providing native extracellular matrix environments, presents challenges, including inadequate cellular interactions. To address these issues, cell spheroids offer a promising solution for improving their biological functions. Particularly, minispheroids with 50-100 µm diameters exhibit enhanced cellular maturation. We propose a one-step minispheroid-forming bioprinting process incorporating electrical stimulation (E-MS-printing). By stimulating the cells, minispheroids with controlled diameters were generated by manipulating the bioink viscosity and stimulation intensity. To validate its feasibility, E-MS-printing process was applied to fabricate an engineered liver model designed to mimic the hepatic lobule unit. E-MS-printing was employed to print the hepatocyte region, followed by bioprinting the central vein using a core-shell nozzle. The resulting constructs displayed native liver-mimetic structures containing minispheroids, which facilitated improved hepatic cell maturation, functional attributes, and vessel formation. Our results demonstrate a new potential 3D liver model that can replicate native liver tissues.

Macroscale pseudo-spheroids fabricated using methacrylated collagen-coated cells.

Rationale: Cell spheroids have shown great promise as tools for creating effective three-dimensional (3D) tissue models, facilitating tissue reconstruction and organoid development, due to their high cell density and efficient cellular interactions. However, a significant challenge persists in creating large-scale tissue structures with a 3D geometrical architecture using spheroids, due to the continual condensation and reorganization of cells and their environments. Methods: The spherical cell aggregates (pseudo-cell spheroids) or macroscale cell aggregates were obtained by coating each adipose-derived stem cell (hASC) with methacrylated collagen (Col-Ma). Subsequently, the coated cells were printed into an alginate supporting bath and photocrosslinked through exposure to UV light. To assess the effectiveness of this procedure on regenerative potential, the generated cell aggregates were compared with conventional cell spheroids and bioprinted cell constructs using immunofluorescent staining and quantification of myogenic-related gene expressions. Moreover, the bioconstructs were implanted into a mouse model with volumetric muscle loss to further elucidate their regenerative and functional recovery properties. Results: The use of Col-Ma as a cell-coating material enables the rapid and physical aggregation of cells within several hours, regardless of the cell type. Furthermore, Col-Ma-coated cell aggregates can provide relatively lower hypoxic conditions than cell spheroids fabricated using the hanging drop method owing to the thin porous Col-Ma layer coated on the cells. In addition, the resulting structures maintain their geometrical architecture following cell fusion and possess the potential for efficient scale-up and 3D complex shape formation, making them more suitable for clinical applications than conventional cell spheroids. Finally, the feasibility of the Col-Ma-coated cylindrical human adipose-derived stem cells aggregates was assessed through implantation in a mouse volumetric muscle loss model, showing a significantly higher regenerative ability of muscle tissue than the normally bioprinted cell construct. Conclusion: Our newly proposed method has meaningful potential for various tissue engineering applications, supported by the improved cellular activities and efficient muscle regeneration observed in both in vitro and in vivo studies, and organ-chip models.

Fabrication of fully aligned self-assembled cell-laden collagen filaments for tissue engineering via a hybrid bioprinting process.

Cell-laden structures play a pivotal role in various tissue engineering applications, particularly in tissue restoration. Interactions between cells within bioprinted structures are crucial for successful tissue development and regulation of stem cell fate through intricate cell-to-cell signaling pathways. In this study, we developed a new technique that combines polyethylene glycol (PEG)-infused submerged bioprinting with a stretching procedure. This approach facilitated the generation of fully aligned collagen structures consisting of myoblasts and a low concentration (2 wt%) of collagen to efficiently encourage muscle tissue regeneration. By adjusting several processing parameters, we obtained biologically safe and mechanically stable cell-laden collagen filaments with uniaxial alignment. Notably, the cell filaments exhibited markedly elevated cellular activities compared to those exhibited by conventional bioprinted filaments, even at similar cell densities. Moreover, when we implanted structures containing adipose stem cells into mice, we observed a significantly increased level of myogenesis compared to that in normally bioprinted struts. Thus, this promising approach has the potential to revolutionize tissue engineering by fostering enhanced cellular interactions and promoting improved outcomes in regenerative medicine.

Cell(MC3T3-E1)-printed poly(ϵ-caprolactone)/alginate hybrid scaffolds for tissue regeneration.

A new cell-printed scaffold consisting of poly(ϵ-caprolactone) (PCL) and cell-embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three-dimensional (3D) shape. The hybrid scaffold exhibits a two-phase structure consisting of cell (MC3T3-E1)-laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell-laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%.

Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

Bioprinted hASC-laden cell constructs with mechanically stable and cell alignment cue for tenogenic differentiation.

3D bioprinting is a technology that enables the precise and controlled deposition of cells and an artificial extracellular matrix (ECM) to create functional tissue constructs. However, current 3D bioprinting methods still struggle to obtain mechanically stable and unique cell-morphological structures, such as fully aligned cells. In this study, we propose a new 3D bioprinting approach that utilizes a high concentration of bioink without cells to support mechanical properties and drag flow to fully align cells in a thin bath filled with cell-laden bioink, resulting in a hybrid cell-laden construct with a mechanical stable and fully aligned cell structure. To demonstrate the feasibility of this approach, we used it to fabricate a cell-laden construct using human adipose stem cells (hASCs) for tendon tissue engineering. To achieve appropriate processing conditions, various factors such as the bioink concentration, nozzle moving speed, and volume flow rate were considered. To enhance the biocompatibility of the cell-laden construct, we used porcine decellularized tendon ECM.In vitrocellular responses, including tenogenic differentiation of the fabricated hybrid cell structures with aligned or randomly distributed cells, were evaluated using hASCs. In addition, the mechanical properties of the hybrid cell-laden construct could be adjusted by controlling the concentration of the mechanically reinforcing strut using methacrylated tendon-decellularized extracellular matrix. Based on these results, the hybrid cell-laden structure has the potential to be a highly effective platform for the alignment of musculoskeletal tissues.

Hybrid cell constructs consisting of bioprinted cell-spheroids.

Bioprinted cell constructs have been investigated for regeneration of various tissues. However, poor cell-cell interactions have limited their utility. Although cell-spheroids offer an alternative for efficient cell-cell interactions, they complicate bioprinting. Here, we introduce a new cell-printing process, fabricating cell-spheroids and cell-loaded constructs together without preparation of cell-spheroids in advance. Cells in mineral oil droplets self-assembled to form cell-spheroids due to the oil-aqueous interaction, exhibiting similar biological functions to the conventionally prepared cell-spheroids. By controlling printing parameters, spheroid diameter and location could be manipulated. To demonstrate the feasibility of this process, we fabricated hybrid cell constructs, consisting of endothelial cell-spheroids and stem cells loaded decellularized extracellular matrix/ß-tricalcium phosphate struts for regenerating vascularized bone. The hybrid cell constructs exhibited strong angiogenic/osteogenic activities as a result of increased secretion of signaling molecules and synergistic crosstalk between the cells.

Fabrication of self-assembled core-sheath microfibers via formulation of alginate-based bioinks.

Core-sheath microfibrous structures are widely used in various tissue engineering applications and drug delivery systems. However, the fabrication of the various core-sheath structures using a 3D printing process supplemented with a coaxial nozzle has been challenging due to the center positioning of the core nozzle enclosed in the bigger shell nozzle. In this work, we developed a new 3D printing process using an alginate-based bioink (a mixture of photo-crosslinkable hydrogel and alginate) and its in situ crosslinking process within a single glass nozzle of the 3D printer. By manipulating the alginate weight fraction, UV intensity, flow rate, and nozzle moving speed, we could fabricate various self-assembled core-sheath structures (straight, wavy, and crimped microfibers in the core region of the structure) in which the photocrosslinked hydrogel resided in the core, and alginate was positioned in the sheath region, like a virtual coaxial nozzle.

The one-step fabrication of porous hASC-laden GelMa constructs using a handheld printing system.

The fabrication of highly porous cell-loaded structures in tissue engineering applications has been a challenging issue because non-porous cell-laden struts can cause severe cell necrosis in the middle region owing to poor transport of nutrients and oxygen. In this study, we propose a versatile handheld 3D printer for the effective fabrication of porous cell-laden methacrylated gelatin (GelMa) with high porosity (≈97%) by air injection and a bubble-making system using mesh filters through which a mixture of air/GelMa bioink is passed. In particular, the pore size and foamability of the cell constructs could be manipulated using various processing parameters (rheological properties of GelMa, filter size and number, and air-bioink volume ratio). To demonstrate the feasibility of the cell construct as a tissue engineering substitute for muscle regeneration, in vitro cellular activities and in vivo regeneration ability of human adipose stem cells were assessed. The in vitro results demonstrated that the human adipose stem cells (hASCs) fabricated using the handheld 3D printer were alive and well-proliferated. Furthermore, the in vivo results showed that the hASCs-constructs directly printed from the handheld 3D printer showed significant restoration of functionality and efficient muscle regeneration in the volumetric muscle loss model of mice. Based on these results, the fabrication method of the porous cell-laden construct could be a promising tool for regenerating muscle tissues.

Effects of a cell-imprinted poly(dimethylsiloxane) surface on the cellular activities of MG63 osteoblast-like cells: preparation of a patterned surface, surface characterization, and bone mineralization.

To understand the relationship between surface patterns and cellular activities, various types of pattern models have been investigated. In this study, we suggest a new surface pattern model, which replicates proliferated cells. We used osteoblast-like cells (MG63) as a target cell pattern and constructed various cell-imprinted surfaces using an electric field assisted casting method for different culturing times (4 h and 7 and 14 days). On the basis of scanning electron microscopy images and three-dimensional topographical optical images, we acquired the cells' unique patterns and used them for replicating patterned substrates. We then cultured MG63 cells in the patterned surfaces for 7 and 14 days to observe various cellular activities, cell viability, alkaline phosphatase (ALP) activity, and mineralization. Higher cellular activities were observed on the roughened surface as compared with the smooth surface. In particular, we obtained the most appropriate roughness value (R(a) = 702 ± 87 nm) from proliferated cells cultured over 14 days. On the basis of these findings, we demonstrate a new biomimical surface model that enhances cellular activities at the cell-substrate interface.

Cells (MC3T3-E1)-laden alginate scaffolds fabricated by a modified solid-freeform fabrication process supplemented with an aerosol spraying.

In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl(2)), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 µm) and an aerosol spray flow rate (0.93 ± 0.12 mL min(-1)), 10 mm s(-1) nozzle moving speed, a 10 wt % concentration of CaCl(2) in the aerosol solution, and 2 wt % concentration of CaCl(2) in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, pore-structured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm(3) and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well.

Mechanically and biologically enhanced 3D-printed HA/PLLA/dECM biocomposites for bone tissue engineering.

Poly (L-lactic acid) (PLLA)-based biocomposites have been used in tissue engineering applications because of their reasonable biocompatibility and mechanical properties. However, the imperfect bioactive and mechanical properties of the composite make it difficult to be used in the region of bone defects that require high load-bearing. Therefore, this study introduced two fabricating strategies to induce mechanically and biologically enhanced hydroxyapatite (HA)/PLLA biocomposites. By introducing an in situ plasma treatment, which was simultaneously applied during the 3D-printing process, followed by the thermal annealing process, the flexural modulus of the composite was increased by 2.1-fold compared to the normal HA/PLLA composite. Furthermore, using the combinational process, efficient coating of bioactive material [decellularized extracellular matrix (dECM) derived from porcine bones] was possible. The fabricated biocomposite scaffold was assessed for various in vitro cellular activities such as cell proliferation and osteogenic activity. Based on the mechanical and biological studies, the HA/PLLA/dECM biocomposite scaffold is one of the promising scaffolds that can be applied in bone tissue regeneration.

Bone tissue engineering supported by bioprinted cell constructs with endothelial cell spheroids.

In bone tissue engineering, efficient formation of vascularized bone tissue is a challenging issue. Here, we introduce a new strategy for effectively using multiple cells laden in a hybrid structure, such as endothelial cell (EC) spheroids and homogeneously distributed human adipose stem cells (hASCs) for bone regeneration. Methods: To fabricate the EC spheroids, cell-mixed mineral oil was used, and microscale droplets of the cell mixture were interlayered between the bioprinted hASC-laden struts. In vitro cellular responses of spheroid-laden multiple-cell constructs have been evaluated by comparing with the cell constructs bioprinted with the mixture of hASCs and ECs. In addition, mastoid obliterated rat model has been used to observe in vivo bone formation of those cell constructs. Results: The spheroid-laden multiple-cell constructs induced outstanding angiogenesis and osteogenic activities compared to a conventionally bioprinted multiple-cell construct. The enhanced biological results were clearly due to the EC spheroids, which triggered highly cooperative crosstalk between ECs and stem cells. The co-culture of the hASC constructs with the EC spheroids exhibited enhanced osteogenic- and angiogenic-related gene expression in vitro. In addition, in a rat obliterated mastoid model, considerably greater new bone formation and more competent development of new blood vessels were observed compared to those achieved with the normally bioprinted multiple cell-loaded structure. Conclusion:In vitro and in vivo results demonstrated that the bioprinted spheroid-laden multiple-cell construct is a potential candidate for use in bone tissue engineering.