論文

Tissues formed by cells seeded in hydrogels are used in biotechnology, cell-based assays, and tissue engineering. We previously presented a cell micropatterning technique that localizes live cells within hydrogels using dielectrophoretic (DEP) forces. We also demonstrated the ability to modulate tissue function through the control of microscale cell architecture. In this study, we developed a novel cell patterning technology for accumulating cells around collagen microbeads using DEP forces. As a case study, we produced collagen-alginate microbeads as a cell-adhesive scaffold and accumulated bovine chondrocytes to cover the microbeads by combining the DEP forces with a flow of buffer solution. This approach allows studies to better examine the influence of three-dimensional cellular architecture on microscale levels.

Recently tissue engineering therapy culturing autologous chondrocytes in vitro to create three dimensional tissue for the replacement of damaged tissue has been developed. Although the tissue engineering is a useful approach to reconstruct cartilage in vitro, the mechanical property of the engineered cartilage is not sufficient to replace natural cartilage. Within the natural cartilage, cells and collagen fibers, which are main components of the cartilage, align in response to the direction of cyclic deformations induced by daily walking or other activities. Due to this collagen fiber alignment, the cartilage has mechanical anisotropy. In this study, we focus on the beneficial effect of dynamic compressive loading on tissue regeneration for articular cartilage. The purpose of this study is to create mechanically anisotropic cartilage by compressive stimuli during the culture. © 2013 The Japan Society of Mechanical Engineers.

Ultrasound sonification stimulates the release of pharmaceutical compounds from hydrogels. At the surface of hydrogels, cavitation, cavities formed in liquid, activates to stimulate that release under low-frequency ultrasound. Under high-frequency ultrasound, although cavitation activities are highly suppressed, the compounds are still released. Although it remains elusive how high-frequency ultrasound stimulates this release, one hypothesis is that the internal diffusivity is enhanced. In this study, internal diffusivities in agarose gels were estimated with fluorescent recovery after photobleaching (FRAP) analysis. Under 1-MHz ultrasound sonification, internal diffusivity in agarose gels was enhanced. The enhancement of internal diffusivity was larger than that with temperature elevation alone, although temperature elevation was also observed along with the ultrasound sonification. Thus, we found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation. © 2013 Elsevier Ltd. All rights reserved.

In this study, we investigated the growth response of chondrocytes on a metallic cell culture device with different microtopographies. Two different topographies were generated on 316L stainless steel by fine particle peening (FPP) treatment using either glass or alumina shot particles. Glass particles provided surface of micro asperities at low frequency spacing, whereas alumina provided surface micro asperities at high frequency spacing. Calf chondrocytes were seeded and cultured on both treated and smooth stainless steel surfaces. The cells were then counted. Based on the results, the FPP-treated surfaces showed better cell proliferation and denser filopodia compared to the polished surface, indicating that the micro asperities on the surface of the plate encouraged cell proliferation and adhesion. The number of cells observed on the FPP-treated surface depended on the shape of the asperities formed by FPP treatment, and alumina-treated surface had the highest cell counts. Cell distribution assay indicated that cells growing on the alumina-treated surface migrated easily. In addition, when the alumina-treated surface was used as a cell culture dish to incubate chondrocytes, cell proliferation was similar to that of obtained using polymeric culture dishes, demonstrating that FPP-treated surfaces are appropriate for use in cell culture dishes.

Articular cartilage has a poor ability to regenerate and repair itself. To restore cartilage defects, a method has been developed by culturing autologous chondrocytes to create a three dimensional tissue and then implanting the cultured tissue. However, articular chondrocytes easily leads to de-differentiation state and loses their ability to synthesize the functional cartilaginous matrixes during in vitro culture. Therefore, it is important to maintain their differentiated phenotype during the expansion culture of chondrocytes. The objective of this study is to develop a novel culturing methodology combined surface modification and mechanical stimuli on articular cartilage. To develop a surface-modified substrate, a SUS316L stainless plate was treated by Fine Particle Peening (FPP) technique using alumina particles. The chondrocytes were cultured on the modified stainless plate and stimulated by fluid-induced shear stress using a custom-made flow culture system. As a result, proliferation rate was increased and phenotypic-change of chondrocytes was also observed by culturing the chondrocytes on physically modified surface combined with fluid-induced shear stress. In addition, the synergetic effect on proliferation rate was observed by applying the surface modification and flow shear stress simultaneously.

Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture-without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 mu m, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72 h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.

© 2019 IEEE. This paper describes an analytical platform to investigate the cell response against mechanical stimuli in in vitro 3D tissues. The system is capable of live-imaging cells in 3D tissues at a single cell level under mechanical stimuli, which makes this system similar to the in vivo environment and suitable for analysis of maturation mechanism. We succeeded in imaging various shapes of cells in 3D tissue under mechanical stimuli and observed that the stretch-induced deformation was distributed non-uniformly inside cells. We believe that our system could contribute to a further understanding of the tissue maturation mechanism induced by mechanical stimuli, which is highly useful for the development of tissue reconstruction.