Shogo Miyata

Evaluation of negative fixed-charge density in tissue-engineered cartilage by quantitative MRI and relationship with biomechanical properties

Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties. To reconstruct cartilage tissue, articular chondrocytes from bovine humeral heads were embedded in agarose gel and cultured in vitro for up to 4 weeks. The nFCD of the cartilage was determined using the MRI gadolinium exclusion method. The equilibrium modulus was determined using a compressive stress relaxation test, and the dynamic modulus was determined by a dynamic compression test. The equilibrium compressive modulus and dynamic modulus of the tissue-engineered cartilage increased with an increase in culture time. The nFCD value--as determined with the [Gd-DTPA(2-)] measurement using the MRI technique--increased with culture time. In the regression analysis, nFCD showed significant correlations with equilibrium compressive modulus and dynamic modulus. From these results, gadolinium-enhanced MRI measurements can serve as a useful predictor of the biomechanical properties of tissue-engineered cartilage.

Influence of cartilaginous matrix accumulation on viscoelastic response of chondrocyte/agarose constructs under dynamic compressive and shear loading

A method has been developed to restore cartilage defects by culturing autologous chondrocytes to create a three dimensional tissue and then implanting the cultured tissue. In this kind of approach, it is important to characterize the dynamic mechanical behavior of the regenerated cartilaginous tissue, because these tissues need to bear various dynamic loadings in daily life. The objectives of this study were to evaluate in detail the dynamic viscoelastic responses of chondrocyte-seeded agarose gel cultures in compression and torsion (shear) and to determine the relationships between these mechanical responses and biochemical composition. The results showed that both the dynamic compressive and shear stiffness of the cultured constructs increased during culture. The relative energy dissipation in dynamic compression decreased, whereas that in dynamic shear increased during culture. Furthermore, correlation analyses showed that the sulfated glycosaminoglycan (sGAG) content of the cultured construct showed significant correlations with the dynamic modulus in both compression and shear situations. On the other hand, the loss tangent in dynamic compression, which represents the relative energy dissipation capability of the constructs, showed a low correlation with the sGAG content, whereas this capability in shear exhibited moderate correlation. In conclusion, we explored the dynamic viscoelasticity of the tissue-engineered cartilage in dynamic compression and shear, and determined correlations between viscoelasticity and biochemical composition.

Effective cell collection method using collagenase and ultrasonic vibration

This study proposes a novel cell collection method based on collagenase treatment and ultrasonic vibration. The method collects calf chondrocytes from a reusable metal cell culture substrate. To develop our concept, we calculated the natural vibration modes of the cell culture substrate by a finite element method, and conducted eigenvalue and piezoelectric-structural analyses. Selecting the first out-of-plane vibration mode of the substrate, which has a single nodal circle, we designed and fabricated the cell collection device. The excited vibration mode properly realized our intentions. We then evaluated the cell collection ratio and the growth response, and observed the morphology of the collected cells. The collagenase and ultrasonic vibration treatment collected comparable numbers of cells to conventional trypsin and pipetting treatment, but improved the proliferating cell statistics. Morphological observations revealed that the membranes of cells collected by the proposed method remain intact; consequently, the cells are larger and rougher than cells collected by the conventional method. Therefore, we present a promising cell collection method for adhesive cell culturing process.

Improvement of adhesion and proliferation of mouse embryonic stem cells cultured on ozone/UV surface-modified substrates

Culturing pluripotent stem cells effectively requires feeder cell layers or cell adhesion matrix coating. However, the feeder cell layers or animal-derived factors have to be removed to apply the pluripotent stem cells as resources for regenerative medicine. To enable xeno-free culture conditions, we focused on the UV/ozone surface treatment technique for polystyrene cell culture substrates to improve the adhesion and proliferation of pluripotent stem cells. In this study, as a fundamental research for the feeder- and matrix coating-free culture system for embryonic stem cells (ESCs), mouse ESCs were cultured on UV/ozone-modified polystyrene substrates without feeder layers. We observed that UV/ozone surface-modified polystyrene substrates made it possible to culture mESCs under feeder-free conditions without any chemical treatment for the substrates.

Efficient Subculture Process for Adherent Cells by Selective Collection Using Cultivation Substrate Vibration

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 µm, 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.

Microscale organization of chondrocyte array in hydrogel by dielectrophoresis

Recently, microfabrication tools have been utilized to quantify the role of the cellular microenvironment on cell activity and function. Improving tissue regeneration by cell culture on scaffold material will also require tools to control cellular organization in 3-dimentional (3-D) condition. Our objective was to improve cartilage tissue engineering using 3-D cell organization technology. In this study, we developed an anisotropic cartilaginous tissue by cell patterning within hydrogel slabs using dielectrophoretic (DEP) forces. Our data indicate that the embedded chondrocytes remained viable and reconstructed cartilaginous tissue along the patterned cell array. DEP cell patterning may become a useful approach for reconstructing anisotropic structure in cartilage regeneration.

Extrinsic Camera Calibration Without Visible Corresponding Points Using Omnidirectional Cameras

This paper proposes a novel algorithm that calibrates multiple cameras scattered across a broad area. The key idea of the proposed method is “using the position of an omnidirectional camera as a reference point.” The common approach to calibrating multiple cameras assumes that the cameras capture at least some common points. This means calibration becomes quite difficult if there are no shared points in each camera’s field of view (FOV). The proposed method uses the position of an omnidirectional camera to determine point correspondence. The position of an omnidirectional camera relative to the calibrated camera is estimated by the theory of epipolar geometry, even if the omnidirectional camera is placed outside the camera’s FOV. This property makes our method applicable to multiple cameras scattered across a broad area. Qualitative and quantitative evaluations using synthesized and real data, e.g., a sports field, demonstrate the advantages of the proposed method.

Cell manipulation by nodal circle resonance vibration of a cell cultivation substrate

In this paper, we propose a novel cell culture method to generate an organ without scaffold. The concept of our study is to apply the principle of Chladnis figures in cell manipulation. To confirm this concept, we developed cell cultivation device that can excite resonance vibration of the cell cultivation substrate. After the fabrication of the device, we estimated the resonance frequency and vibration amplitude distribution of our device. Since the fabricated device successfully produced the designed vibration mode, we conducted cell manipulation experiment to confirm our concept. In our experiment, we varied the initial number of cells that were seeded into our device. Cells were manipulated by resonance vibration for 120 min. After the manipulation, we checked cell distribution on the substrate. As a result, cells were successfully manipulated by the resonance vibration when the initial number of cells was appropriate.

Non-Invasive Evaluation Method for Cartilage Tissue Regeneration Using Quantitative-MRI

Articular cartilage is an avascular tissue covering articulating surfaces of bones and it functions to bear loads and reduce friction in diarthrodial joints. The cartilage can be regarded as a porous gel, mainly composed of large proteoglycan (PG) aggregates having a negative fixed-charge density (nFCD), a water-swollen network of collagen fibrils, and interstitial water, all of which play important roles in load-bearing properties (Lee et al., 1981; Mow et al., 1980).

Feeder-free culture for mouse induced pluripotent stem cells by using UV/ozone surface-modified substrates

Pluripotent stem cells (PSCs), especially induced PSCs (iPSCs), have great potential for regenerative medicine. Conventionally, PSCs are cultured and expanded efficiently on feeder cell layers or on cell-adhesive matrices. Large-scale iPSC expansion in an undifferentiated state without laborious culturing procedures and high manufacturing costs for the adhesive matrix is urgently required to integrate iPSCs into therapeutic applications. For this, feeder layers or cell-adhesive matrix coating have to be removed from the iPSC culture system. To enable feeder- and matrix coating-free culture conditions, we focused on a UV/ozone surface treatment technique for polystyrene cell culture substrates to improve PSC adhesion and proliferation. In this study, changes in the molecular structure of UV/ozone-modified polystyrene were characterized to optimize the surface chemistry for iPSC. Mouse iPSCs (miPSCs) were cultured on the UV/ozone-modified polystyrene substrates without feeder layers. As a result, large polymeric chains of polystyrene were dissociated into small polymeric chains and oxidized to form ester and carboxylic acid functional groups by the UV/ozone treatment. Moreover, it was suggested that optimal valance of these modified molecules enabled the feeder- and matrix coating-free culture of miPSC with maintaining pluripotency.