Papers by Keyword: Apatite-Fiber Scaffold (AFS)

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Authors: Hide Ishii, Yuya Mukai, Mamoru Aizawa, Nobuyuki Kanzawa
Abstract: Heart disease is the second most common cause of mortality in Japan. Most cases of late stage heart failure can only be effectively treated by a heart transplant. Cardiac tissue engineering is emerging both as a new approach for improving the treatment of heart failure and for developing new cardiac drugs. Apatite-fiber scaffold (AFS) was originally designed as a substitute material for bone. AFS contains two sizes of pores and is appropriate for the three dimensional proliferation and differentiation of osteoblasts. To establish engineered heart tissue, a pluripotent embryonal carcinoma cell line, P19.CL6, was cultured in AFS. P19.CL6 cells seeded into AFS proliferated well. Generally, cardiac differentiation of P19.CL6 cells is induced by treating suspension-cultured cells with dimethyl sulfoxide (DMSO), after which the cells form spheroids. However, our results showed that P19.CL6 cells cultured in AFS differentiated into myocytes without forming spheroidal aggregates, and could be cultured for at least one month. Thus, we conclude that AFS is a good candidate as a scaffold for cardiac tissue engineering.
Authors: Michiyo Honda, Shigeki Izumi, Nobuyuki Kanzawa, Takahide Tsuchiya, Mamoru Aizawa
Abstract: Appropriate culture conditions cause bone marrow stem cells to differentiate into multilineage cells such as adipocytes, chondrocytes, and osteoblasts. One key factor that regulates intercellular signaling and cell differentiation is the extracellular matrix microenvironment. The composition of the extracellular matrix influences cellular functions. In the present study, we investigated the effects of a microenvironment comprising a three-dimensional apatite-fiber scaffold (AFS) that has two kinds of pores (micro- and macro pores) on proliferation and subsequent differentiation of bone marrow stem cells. Morphologic observation revealed that osteoblastic cells in the AFS were distributed primarily in the same location on the fibrous scaffold and formed bridges within micro- and macro pores. We used molecular approaches to evaluate cell proliferation and differentiation in detail. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that culturing bone marrow cells on AFS increases expression of osteocalcin (OC) mRNA compared with culture in a dish. Furthermore, cells cultured in AFS expressed type X collagen (Col X), which is a marker of hypertrophic cartilage. These data suggest that the three-dimensional microenvironment of AFS facilitates cell proliferation and differentiation, and promotes endochondral ossification of bone marrow cells.
Authors: Kei Yasuda, Hide Ishii, Masahiro Takahara, Mamoru Aizawa, Nobuyuki Kanzawa
Abstract: In the present study, we used an apatite-fiber scaffold (AFS) to culture P19.CL6 cells three-dimensionally. AFS was originally developed to use for the bone substitute and has high porosity and complex pore structure. The highly porous structure of AFS suggests it may be compatible for the in vitro reorganization of soft tissue. We previously showed the formation of small cell aggregates in AFS, and that culture in AFS increased the expression of cardiac-specific gene markers without the need for any inducing agent such as DMSO. However, it is difficult to evaluate the physiological function of cells in three-dimensional culture. In this study, we transformed P19.CL6 cells with the pTnnt2::GCaMP5G vector and observed the cells by fluorescence microscopy. pTnnt2::GCaMP5G consists of green fluorescent protein (GFP) fused with the Ca2+-sensitive domain of calmodulin; its expression is driven by a cardiomyocyte-specific promoter. We observed that the blinking of green fluorescence was synchronized to the beating of cardiomyocytes when the P19.CL6 cells were cultured in a dish, but blinking was not observed when the cells were cultured in AFS, even after 16 days. The expression of connexin 43 (Cx43) and enhanced Green Fluorescence Protein (EGFP) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cx43 is a gap junction protein expressed in cardiomyocytes that mediates cell-to-cell coupling. Although the expression of Cx43 and EGFP in transformed cells cultured in AFS was evident, fluorescence blinking of the cells was not observed. The results demonstrate that P19.CL6 cells cultured in AFS rapidly differentiated into early stage cardiomyocytes; however, additional modifications or developments are needed for further differentiation.
Authors: Mamoru Aizawa, A. Hiramoto, H. Maehashi, Tomokazu Matsuura
Abstract: We have previously developed apatite-fiber scaffolds (AFSs) for bone tissue engineering using single-crystal apatite fibers and carbon beads. In the present investigation, we examined the possibility of reconstruction of a liver organoid via three-dimensional (3D) culture of hepatocytes using the AFSs and the radial-flow bioreactor (RFB), aiming to apply the scaffold as a matrix for regeneration of a real organ. FLC-4 cells were used as a model of hepatocyte. The cells were well-viable in the RFB settled with AFSs over a period for 28 d, compared with the cases of cellulose beads and apatite beads with high porosity of 85%. We conclude that the present AFS may be a promising scaffold for tissue engineering of liver.
Authors: Maiko Miura, Jun Fukasawa, Yumiko Yasutomi, Haruka Maehashi, Tomokazu Matsuura, Mamoru Aizawa
Abstract: We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the tissue-engineered bone with 3D structure through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB), and examined effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone. Aim in the present work is to establish of the optimal conditions of flow rate in this construction method of 3D tissue-engineered bone. The flow rates were set to 0.4, 1.3, 6.3, 11.5 and 16.5 cm3min-1; tissue-engineered bones cultured by the individual flow rates are defined as bones#1~#5. The level of differentiation of osteoblasts in all the bones#1~#5 was examined by determining the content of two kinds of differentiation maker into osteoblast, alkaline phosphatase (ALP) for initial/middle stage and osteocalcin (OC) for late stage. The ALP activity normalized for DNA content of bone#3 showed the highest value among all of them. Moreover, the OC amount normalized for DNA content of bone#3 also indicated the highest among all the examined samples. These results demonstarate that the flow rate of 6.3 cm3min-1 may promote the differentiation into osteoblast. In conclusion, we determined that this flow rate was the optimal conditions for the bone regeneratrion in RFB.
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