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Mizuno, Shuichi

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Mizuno

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Shuichi

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Mizuno, Shuichi
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    Reproduction of Characteristics of Extracellular Matrices in Specific Longitudinal Depth Zone Cartilage within Spherical Organoids in Response to Changes in Osmotic Pressure
    (MDPI, 2018) Takada, Eiichiro; Mizuno, Shuichi
    Articular cartilage is compressed with joint-loading and weight-bearing stresses, followed by a bulging of the tissue during times of off-loading. This loading and off-loading causes changes in water content, and thus alterations in osmotic pressure. Another unique characteristic of articular cartilage is that it has longitudinal depth: surface, middle, and deep zones. Since each zone is composed of unique components of highly negative extracellular matrices, each zone has a different level of osmotic pressure. It was unclear how changes in osmotic pressure affected chondrocyte matrix turnover in specific longitudinal zones. Therefore, we hypothesized that a change in extrinsic osmotic pressure would alter the production of extracellular matrices by zone-specific chondrocytes. We incubated spheroidal cartilage organoids, formed by specific longitudinal depth zone-derived chondrocytes, under different levels of osmotic pressure. We compared the gene expression and the immunohistology of the matrix proteins produced by the zone-specific chondrocytes. We found that high osmotic pressure significantly upregulated the transient expression of aggrecan and collagen type-II by all zone-derived chondrocytes (p < 0.05). At a high osmotic pressure, surface-zone chondrocytes significantly upregulated the expression of collagen type-I (p < 0.05), and middle- and deep-zone chondrocytes significantly upregulated matrix metalloproteinase-13 (p < 0.05). The spheroids, once exposed to high osmotic pressure, accumulated extracellular matrices with empty spaces. Our findings show that chondrocytes have zone-specific turnover of extracellular matrices in response to changes in osmotic pressure.
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    The Effect of Hydrostatic Pressure on Three-Dimensional Chondroinduction of Human Adipose–Derived Stem Cells
    (Mary Ann Liebert Inc, 2009) Ogawa, Rei; Mizuno, Shuichi; Murphy, George; Orgill, Dennis
    Background: The optimal production of three-dimensional cartilage in vitro requires both inductive factors and specified culture conditions (e.g., hydrostatic pressure [HP], gas concentration, and nutrient supply) to promote cell viability and maintain phenotype. In this study, we optimized the conditions for human cartilage induction using human adipose–derived stem cells (ASCs), collagen scaffolds, and cyclic HP treatment. Methods: Human ASCs underwent primary culture and three passages before being seeded into collagen scaffolds. These constructs were incubated for 1 week in an automated bioreactor using cyclic HP at 0–0.5 MPa, 0.5 Hz, and compared to constructs exposed to atmospheric pressure. In both groups, chondrogenic differentiation medium including transforming growth factor-β1 was employed. One, 2, 3, and 4 weeks after incubation, the cell constructs were harvested for histological, immunohistochemical, and gene expression evaluation. Results: In histological and immunohistochemical analyzes, pericellular and extracellular metachromatic matrix was observed in both groups and increased over 4 weeks, but accumulated at a higher rate in the HP group. Cell number was maintained in the HP group over 4 weeks but decreased after 2 weeks in the atmospheric pressure group. Chondrogenic-specific gene expression of type II and X collagen, aggrecan, and SRY-box9 was increased in the HP group especially after 2 weeks. Conclusion: Our results demonstrate chondrogenic differentiation of ASCs in a three-dimensional collagen scaffolds with treatment of a cyclic HP. Cyclic HP was effective in enhancing accumulation of extracellular matrix and expression of genes indicative of chondrogenic differentiation.
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    Methods of high integrity RNA extraction from cell/agarose construct
    (BioMed Central, 2015) Ogura, Takahiro; Tsuchiya, Akihiro; Minas, Tom; Mizuno, Shuichi
    Background: Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis. Results: With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities. Conclusions: The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering.