Highly porous multiple-cell-laden collagen/hydroxyapatite scaffolds for bone tissue engineering.

Efficient vascularization within a scaffold is an essential criterion for evaluating the success of volumetric bone formation. Various strategies using angiogenic growth factors and cell-based approaches to induce effective osteogenic and angiogenic activities have been investigated. In this study, we propose a new highly porous multiple-cell-laden collagen/hydroxyapatite scaffold fabricated using a whipped bioink. After in vitro culturing of cells in the porous scaffolds for an extended culture period, osteogenic and angiogenic activities were significantly enhanced owing to the well-developed microporous cell-supporting matrix inducing efficient crosstalk between the adipose stem cells and endothelial cells compared to those of the normally bioprinted cell-constructs. Furthermore, the in vitro results were thoroughly evaluated by in vivo experiments using a posterolateral lumbar spinal fusion model of an ovariectomized mouse. Based on these results, the porous multiple-cell-laden scaffolds enhanced spine fusion in the event of osteoporosis.

Bioprinted hASC-laden collagen/HA constructs with meringue-like macro/micropores.

Extrusion-based bioprinting is one of the most effective methods for fabricating cell-laden mesh structures. However, insufficient cellular activities within the printed cylindrical cell-matrix blocks, inducing low cell-to-cell interactions due to the disturbance of the matrix hydrogel, remain to be addressed. Hence, various sacrificial materials or void-forming methods have been used; however, most of them cannot solve the problem completely or require complicated fabricating procedures. Herein, we suggest a bioprinted cell-laden collagen/hydroxyapatite (HA) construct comprising meringue-like porous cell-laden structures to enhance osteogenic activity. A porous bioink is generated using a culinary process, i.e., the whipping method, and the whipping conditions, such as the material concentration, time, and speed, are selected appropriately. The constructs fabricated using the meringue-like bioink with MG63 cells and human adipose stem cells exhibit outstanding metabolic and osteogenic activities owing to the synergistic effects of the efficient cell-to-cell interactions and HA stimulation released from the porous structure. The in vitro cellular responses indicate that the meringue-like collagen bioink for achieving an extremely porous cell-laden construct can be a highly promising cell-laden material for various tissue regeneration applications.

ASC/chondrocyte-laden alginate hydrogel/PCL hybrid scaffold fabricated using 3D printing for auricle regeneration.

Tissue engineering using adipose derived stem cells (ASCs) has become one of the most promising treatments for defective articular cartilage owing to the stability and dynamic differentiation of ASCs. In this study, we fabricated a 3D hybrid scaffold using poly(ε-caprolactone) (PCL) to support the mechanical properties of the regenerating auricle cartilage, and injected a cell-laden alginate hydrogel, containing a mixture of ASCs and chondrocytes, into the PCL scaffold. Using the cell-laden 3D auricle structure, the in vitro chondrogenesis of the ASCs with and without the presence of chondrocytes was examined. Additionally, the feasibility of utilizing the 3D cell-laden auricle structure for cartilage tissue engineering was evaluated in a rat model. In our in vitro and in vivo experiments, we observed that as the ASCs were co-cultured with the chondrocytes, chondrogenic differentiation was encouraged, and the regeneration of cartilage was significantly increased.

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.

QStatin, a Selective Inhibitor of Quorum Sensing in Vibrio Species.

Pathogenic Vibrio species cause diseases in diverse marine animals reared in aquaculture. Since their pathogenesis, persistence, and survival in marine environments are regulated by quorum sensing (QS), QS interference has attracted attention as a means to control these bacteria in aquatic settings. A few QS inhibitors of Vibrio species have been reported, but detailed molecular mechanisms are lacking. Here, we identified a novel, potent, and selective Vibrio QS inhibitor, named QStatin [1-(5-bromothiophene-2-sulfonyl)-1H-pyrazole], which affects Vibrio harveyi LuxR homologues, the well-conserved master transcriptional regulators for QS in Vibrio species. Crystallographic and biochemical analyses showed that QStatin binds tightly to a putative ligand-binding pocket in SmcR, the LuxR homologue in V. vulnificus, and changes the flexibility of the protein, thereby altering its transcription regulatory activity. Transcriptome analysis revealed that QStatin results in SmcR dysfunction, affecting the expression of SmcR regulon required for virulence, motility/chemotaxis, and biofilm dynamics. Notably, QStatin attenuated representative QS-regulated phenotypes in various Vibrio species, including virulence against the brine shrimp (Artemia franciscana). Together, these results provide molecular insights into the mechanism of action of an effective, sustainable QS inhibitor that is less susceptible to resistance than other antimicrobial agents and useful in controlling the virulence of Vibrio species in aquacultures.IMPORTANCE Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus, and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures.

Innovative Cryopreservation Process Using a Modified Core/Shell Cell-Printing with a Microfluidic System for Cell-Laden Scaffolds.

This work investigated the printability and applicability of a core/shell cell-printed scaffold for medium-term (for up to 20 days) cryopreservation and subsequent cultivation with acceptable cellular activities including cell viability. We developed an innovative cell-printing process supplemented with a microfluidic channel, a core/shell nozzle, and a low-temperature working stage to obtain a cell-laden 3D porous collagen scaffold for cryopreservation. The 3D porous biomedical scaffold consisted of core/shell struts with a cell-laden collagen-based bioink/dimethyl sulfoxide mixture in the core region and an alginate/poly(ethylene oxide) mixture in the shell region. Following 2 weeks of cryopreservation, the cells (osteoblast-like cells or human adipose stem cells) in the scaffold showed good viability (over 90%), steady growth, and mineralization similar to those of a control scaffold fabricated using a conventional cell-printing process without cryopreservation. We believe that these results are attributable to the optimized fabrication processes the cells underwent, including safe freezing/thawing processes. On the basis of these results, this fabrication process has great potential for obtaining core/shell cell-laden collagen scaffolds for cryopreservation, which have various tissue engineering applications.

Development of a tannic acid cross-linking process for obtaining 3D porous cell-laden collagen structure.

Cell-printing is an emerging technique that enables to build a customized structure using biomaterials and living cells for various biomedical applications. In many biomaterials, alginate has been widely used for rapid gelation, low cost, and relatively high processability. However, biocompatibilities enhancing cell adhesion and proliferation were limited, so that, to overcome this problem, an outstanding alternative, collagen, has been extensively investigated. Many factors remain to be proven for cell-printing applications, such as printability, physical sustainability after printing, and applicability of in vitro cell culture. This study proposes a cell-laden collagen scaffold fabricated via cell-printing and tannic acid (TA) crosslinking process. The effects of the crosslinking agent (0-3wt% TA) in the cell-laden collagen scaffolds on physical properties and cellular activities using preosteoblasts (MC3T3-E1) were presented. Compared to the cell-laden collagen scaffold without TA crosslinking, the scaffold with TA crosslinking was significantly enhanced in mechanical properties, while reasonable cellular activities were observed. Concisely, this study introduces the possibility of a cell-printing process using collagen and TA crosslinking and in vitro cell culture for tissue regeneration.

New strategy for enhancing in situ cell viability of cell-printing process via piezoelectric transducer-assisted three-dimensional printing.

Tissue engineering has become one of the great applications of three-dimensional cell printing because of the possibility of fabricating complex cell-laden scaffolds. Three typical methods (inkjet, micro-extrusion, and laser-assisted bio-printing) have been used to fabricate structures. Of these, micro-extrusion is a comparatively easy method, but has some drawbacks such as low in situ cell viability after fabricating cell-laden structures because of the high wall shear stress in micro-sized nozzles. To overcome this shortcoming, we suggest an innovative cell printing method, which is assisted by a piezoelectric transducer (PZT). The PZT assistance in the dispensing process enhances the printing efficiency and cell viability by decreasing the wall shear stress within a nozzle because the PZT effect can lower the shear viscosity of the bioink via micro-scale vibration. In this study, 5 wt% cell-laden alginate was used as a bioink, and various PZT conditions (frequencies up to ∼400 Hz and amplitudes up to ∼40.5 µm) were simultaneously applied to the cell-printing process to examine the effectiveness of the PZT. The PZT-assisted cell-printing method was found to be highly effective in direct cell printing and could achieve cell-laden structures with high in situ cell viability.