Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration.

BACKGROUND: Three-dimensional cultured clumps of a mesenchymal stem cell (MSC)/extracellular matrix (ECM) complex (C-MSC) consists of cells and self-produced ECM. C-MSC can regulate the cellular function in vitro and induce successful bone regeneration using ECM as a cell scaffold. Potentiating the immunomodulatory capacity of C-MSCs, which can ameliorate the allo-specific immune response, may be helpful in developing beneficial "off-the-shelf" cell therapy for tissue regeneration. It is well reported that interferon (IFN)-γ stimulates the immunosuppressive properties of MSC via upregulation of the immunomodulatory enzyme IDO. Therefore, the aim of this study was to investigate the effect of IFN-γ on the immunomodulatory capacity of C-MSC in vitro and to test the bone regenerative activity of C-MSC or IFN-γ-pretreated C-MSC (C-MSCγ) xenografts in a mice calvarial defect model. METHODS: Human bone marrow-derived MSCs were seeded at a density of 2.0 × 105 cells/well into 24-well plates and cultured with growth medium supplemented with 50 µg/mL L-ascorbic acid for 4 days. To obtain C-MSC, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The cellular sheet was rolled to make a round clump of cells. C-MSC was stimulated with IFN-γ and IDO expression, immunosuppressive capacity, and immunophenotype were evaluated in vitro. Moreover, C-MSC or C-MSCγ was xenotransplanted into immunocompetent or immunodeficient mice calvarial defect models without artificial scaffold, respectively. RESULTS: IFN-γ stimulated IDO expression in C-MSC. C-MSCγ, but not C-MSC, attenuated CD3/CD28-induced T cell proliferation and its suppressive effect was reversed by an IDO inhibitor. C-MSCγ showed upregulation of HLA-DR expression, but its co-stimulatory molecule, CD86, was not detected. Xenotransplantation of C-MSCγ into immunocompetent mice calvarial defect induced bone regeneration, whereas C-MSC xenograft failed and induced T cell infiltration in the grafted area. On the other hand, both C-MSC and C-MSCγ xenotransplantation into immunodeficient mice caused bone regeneration. CONCLUSIONS: Xenotransplantation of C-MSCγ, which exerts immunomodulatory properties via the upregulation of IDO activity in vitro, may attenuate xenoreactive host immune response, and thereby induce bone regeneration in mice. Accordingly, C-MSCγ may constitute a promising novel allograft cell therapy for bone regeneration.

Clumps of a mesenchymal stromal cell/extracellular matrix complex can be a novel tissue engineering therapy for bone regeneration.

BACKGROUND AIMS: The transplantation of mesenchymal stromal cells (MSCs) to damaged tissue has attracted attention in scientific and medical fields as an effective regenerative therapy. Nevertheless, additional studies are required to develop an MSC transplant method for bone regeneration because the use of an artificial scaffold restricts the number of transplanted cells and their function. Furthermore, regulating the degree of cell differentiation in vitro is desirable for a more effective regenerative therapy. To address these unresolved issues, with the use of a self-produced extracellular matrix (ECM), we developed clumps of an MSC/ECM complex (C-MSCs). METHODS: MSCs isolated from rat femur were cultured in growth medium supplemented with 50 µg/mL of ascorbic acid for 7 days. To obtain C-MSCs, confluent cells were scratched with the use of a micropipette tip to roll up the cellular sheet, which consisted of ECM produced by the MSCs. The biological properties of C-MSCs were assessed in vitro and their bone regenerative activity was tested by use of a rat calvarial defect model. RESULTS: Immunofluorescent confocal microscopic analysis revealed that type I collagen formed C-MSCs. Osteopontin messenger RNA expression and amount of calcium content were higher in C-MSCs cultured in osteo-inductive medium than those of untreated C-MSCs. The transplantation of osteogenic-differentiated C-MSCs led to rapid bone regeneration in the rat calvarial defect model. CONCLUSIONS: These results suggest that the use of C-MSCs refined by self-produced ECM, which contain no artificial scaffold and can be processed in vitro, may represent a novel tissue engineering therapy.

[Effect on duration of antibiotic therapy and drug cost by introducing critical pathway for patients with CAP in Japan].

The aim of this study was to assess whether Marrie's critical pathway is an effective approach to reduce the duration of antibiotic intravenous therapy and drug cost in patients with community-acquired pneumonia (CAP) in Japan. We conducted a retrospective cohort study in patients with CAP who were admitted to a community hospital or a university hospital. We collected clinical and economic data from medical records and medical fee receipts and estimated drug cost for switching the dosage form using Marrie's critical pathway. Outcomes of this study were change in duration of intravenous therapy and drug cost. Fifty patients with CAP were selected from two hospitals. Actual days of antibiotic intravenous therapy were 9.5+/-4.2 days; in contrast, estimated days were 1.2+/-3.0 days (p<0.001). Actual drug cost was 37148+/-28791 yen; in contrast, estimated drug cost was 8364+/-18356 yen (p<0.001). Average reduction of days of therapy and drug cost were 8.3 days and 28704 yen, respectively. This study suggests that the implementation of Marrie's critical pathway may be an effective approach to reduce medical resources used for CAP treatment in Japan.

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.