Recapitulation of the microstructural organization of cellular and extracellular components found in natural tissues is an important but challenging feat for tissue engineering, which demands innovation across both process and material fronts. In this work, a highly versatile ultrasound‐assisted biofabrication (UAB) approach is demonstrated that utilizes radiation forces generated by superimposing ultrasonic bulk acoustic waves to rapidly organize arrays of cells and other biomaterial additives within single and multilayered hydrogel constructs. UAB is used in conjunction with a novel hybrid bioink system, comprising of cartilage‐forming cells (human adipose‐derived stem cells or chondrocytes) and additives to promote cell adhesion (collagen microaggregates or polycaprolactone microfibers) encapsulated within gelatin methacryloyl (GelMA) hydrogels, to fabricate cartilaginous tissue constructs featuring bulk anisotropy. The hybrid matrices fabricated under the appropriate synergistic thermo‐reversible and photocrosslinking conditions demonstrate enhanced mechanical stiffness, stretchability, strength, construct shape fidelity and aligned encapsulated cell morphology and collagen II secretion in long‐term culture. Hybridization of UAB is also shown with extrusion and stereolithography printing to fabricate constructs featuring 3D perfusable channels for vasculature combined with a crisscross or circumferential organization of cells and adhesive bioadditives, which is relevant for further translation of UAB toward complex physiological‐scale biomimetic tissue fabrication.

Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks

Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Development of accurate temperature regulation culture system with metallic culture vessel demonstrates different thermal cytotoxicity in cancer and normal cells.

The ability to physically manipulate specific cells is critical for the fields of biomedicine, synthetic biology, and living materials. Ultrasound has the ability to manipulate cells with high spatiotemporal precision via acoustic radiation force (ARF). However, because most cells have similar acoustic properties, this capability is disconnected from cellular genetic programs. Here, we show that gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—can serve as genetically encodable actuators for selective acoustic manipulation. Because of their lower density and higher compressibility relative to water, GVs experience strong ARF with opposite polarity to most other materials. When expressed inside cells, GVs invert the cells’ acoustic contrast and amplify the magnitude of their ARF, allowing the cells to be selectively manipulated with sound waves based on their genotype. GVs provide a direct link between gene expression and acoustomechanical actuation, opening a paradigm for selective cellular control in a broad range of contexts.

Biomolecular actuators for genetically selective acoustic manipulation of cells

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.

https://www.mdpi.com/1422-0067/22/13/6780/pdf

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue.

The levitation methodology, which enables us to operate a contactless reaction without a container, is likely to be a revolutionary technology in the fields of chemistry and biology to reduce the plastic waste in life science laboratories. Here, the authors show that plasmid DNA can be effectively transfected into animal cells in a floating droplet of culture medium levitated using ultrasonic standing waves. The data indicate that there is no significant damage to the plasmid and cells during the levitating transfection time, and the transgene expression efficiency and cellular uptake in the droplet are significantly higher than those in the conventional tube, with and without shaking. These results suggest the consolidation of the endocytic uptake pathway into macropinocytosis, indicating that ultrasonic levitation induced a change in cell characteristics. This study suggests that transfection methodology using ultrasonic levitation has the potential to advance the current experimental procedures in the field of cell engineering, in addition to presenting a revolutionary containerless reactor for sustainable technology.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/advs.202203576

Effective Cell Transfection in An Ultrasonically Levitated Droplet for Sustainable Technology

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration.

Tribochemical reaction and wear mechanism of MoDTC based friction modifier

Abstract A one-step method was developed to create a highly biocompatible micropatterned surface on a diamond-like carbon (DLC) through irradiation with a nitrogen ion beam and thus enhance the biocompatibility of osseointegrated surfaces and biotribological performance of articular surfaces. The biocompatibility and biotribological mechanisms were analyzed in terms of the structure and morphology of DLC. It was demonstrated that a layer enriched in sp 3 C−N bonds was formed on the surface of the DLC after nitrogen ion beam irradiation. Moreover, with an increase in the radiation dose, the content of sp 3 C−N on the DLC surface increased significantly, and the biocompatibility was positively correlated with it. The adhesion of the MC3T3 osteoblasts increased significantly from 32% to 86% under an irradiation dose of 8 × 10 15 ions/cm 2 . In contrast, the micropattern had a significant negative effect on the adhesion of the osteoblasts as it physically hindered cell expansion and extension. The micropattern with a depth of 37 nm exhibited good friction properties, and the coefficient of friction was reduced by 21% at relatively high speeds. 

/pdf/one-step-method-to-enhance-biotribological-properties-and-1m8qqdlg.pdf

One-step method to enhance biotribological properties and biocompatibility of DLC coating by ion beam irradiation

The reliability and durability of devices that operate in vacuum and are subjected to wear are critical issues. The concerns rise at the macroscale wherein the contact area is large, yet the contact stress is in the GPa range. This paper reports on the design and fabrication of a coating comprising nitridized molybdenum disulfide (MoSN) supported by a novel nanolayered coating composed of carbon with subnanometer‐thick periodic albeit discrete Cr interlayers. The MoSN coatings are deposited onto mating surfaces using pulsed‐DC magnetron sputtering in an atmosphere with varying N2 to argon (Ar) ratios. Pulsed‐DC parameters, such as pulse duty and frequency, together with nitrogen content, are systematically optimized to achieve superior mechanical and tribological properties. The results reveal the importance of optimizing pulsing parameters during deposition as they significantly alter the properties of MoS2. The outcome is fabrication of a coating that despite having a low thickness, exhibits extremely low friction and record‐breaking macroscale wear rate in high vacuum compared to the values reported in the literature by date. Lastly, the analysis confirms the prediction of theoretical studies regarding the release of entrapped nitrogen in gaseous form during the wear process without disrupting the formation of a stable solid lubricant tribofilm.

Youn Hoo Hwang

Papers

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration.

Tribochemical reaction and wear mechanism of MoDTC based friction modifier

One-step method to enhance biotribological properties and biocompatibility of DLC coating by ion beam irradiation

Ultralow Macroscale Friction and Wear of MoS2 in High‐Vacuum Through DC Pulsing Optimization, Nitridization, and Sublayer Engineering