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  • 1
    Online-Ressource
    Online-Ressource
    Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells
    MDPI AG ; 2023
    In:  International Journal of Molecular Sciences Vol. 24, No. 15 ( 2023-07-31), p. 12289-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 24, No. 15 ( 2023-07-31), p. 12289-
    Kurzfassung: To develop an off-the-shelf therapeutic product for intervertebral disc (IVD) repair using nucleus pulposus cells (NPCs), it is beneficial to mitigate dimethyl sulfoxide (DMSO)-induced cytotoxicity caused by intracellular reactive oxygen species (ROS). Hyaluronic acid (HA) has been shown to protect chondrocytes against ROS. Therefore, we examined the potential of HA on mitigating DMSO-induced cytotoxicity for the enhancement of NPC therapy. Human NPC cryopreserved in DMSO solutions were thawed, mixed with equal amounts of EDTA-PBS (Group E) or HA (Group H), and incubated for 3–5 h. After incubation, DMSO was removed, and the cells were cultured for 5 days. Thereafter, we examined cell viability, cell proliferation rates, Tie2 positivity (a marker of NP progenitor cells), and the estimated numbers of Tie2 positive cells. Fluorescence intensity of DHE and MitoSOX staining, as indicators for oxidative stress, were evaluated by flow cytometry. Group H showed higher rates of cell proliferation and Tie2 expressing cells with a trend toward suppression of oxidative stress compared to Group E. Thus, HA treatment appears to suppress ROS induced by DMSO. These results highlight the ability of HA to maintain NPC functionalities, suggesting that mixing HA at the time of transplantation may be useful in the development of off-the-shelf NPC products.
    Materialart: Online-Ressource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms241512289
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2023
    ZDB Id: 2019364-6
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 2
    Online-Ressource
    Online-Ressource
    Can Alterations in Cerebrovascular CO2 Reactivity Be Identified Using Transfer Function Analysis without the Requirement for Carbon Dioxide Inhalation?
    MDPI AG ; 2023
    In:  Journal of Clinical Medicine Vol. 12, No. 6 ( 2023-03-22), p. 2441-
    Details
    In: Journal of Clinical Medicine, MDPI AG, Vol. 12, No. 6 ( 2023-03-22), p. 2441-
    Kurzfassung: The present study aimed to examine the validity of a novel method to assess cerebrovascular carbon dioxide (CO2) reactivity (CVR) that does not require a CO2 inhalation challenge, e.g., for use in patients with respiratory disease or the elderly, etc. In twenty-one healthy participants, CVR responses to orthostatic stress (50° head-up tilt, HUT) were assessed using two methods: (1) the traditional CO2 inhalation method, and (2) transfer function analysis (TFA) between middle cerebral artery blood velocity (MCA V) and predicted arterial partial pressure of CO2 (PaCO2) during spontaneous respiration. During HUT, MCA V steady-state (i.e., magnitude) and MCA V onset (i.e., time constant) responses to CO2 inhalation were decreased (p 〈 0.001) and increased (p = 0.001), respectively, indicative of attenuated CVR. In contrast, TFA gain in the very low-frequency range (VLF, 0.005–0.024 Hz) was unchanged, while the TFA phase in the VLF approached zero during HUT (−0.38 ± 0.59 vs. 0.31 ± 0.78 radians, supine vs. HUT; p = 0.003), indicative of a shorter time (i.e., improved) response of CVR. These findings indicate that CVR metrics determined by TFA without a CO2 inhalation do not track HUT-evoked reductions in CVR identified using CO2 inhalation, suggesting that enhanced cerebral blood flow response to a change in CO2 using CO2 inhalation is necessary to assess CVR adequately.
    Materialart: Online-Ressource
    ISSN: 2077-0383
    URL: Article
    DOI: 10.3390/jcm12062441
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2023
    ZDB Id: 2662592-1
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 3
    Online-Ressource
    Online-Ressource
    Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro
    MDPI AG ; 2023
    In:  International Journal of Molecular Sciences Vol. 24, No. 23 ( 2023-11-24), p. 16713-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 24, No. 23 ( 2023-11-24), p. 16713-
    Kurzfassung: The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.
    Materialart: Online-Ressource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms242316713
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2023
    ZDB Id: 2019364-6
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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    Online-Ressource
  • 4
    Online-Ressource
    Online-Ressource
    Weekly Variation of Rotavirus A Concentrations in Sewage and Oysters in Japan, 2014–2016
    MDPI AG ; 2019
    In:  Pathogens Vol. 8, No. 3 ( 2019-06-26), p. 89-
    Details
    In: Pathogens, MDPI AG, Vol. 8, No. 3 ( 2019-06-26), p. 89-
    Kurzfassung: Concentrations of rotavirus A, in sewage and oysters collected weekly from September 2014 to April 2016 in Japan, were investigated using RT-qPCR; results showed up to 6.5 log10 copies/mL and 4.3 log10 copies/g of digestive tissue (DT) in sewage and oysters, respectively. No correlation was found between rotavirus concentration in sewage and oysters and cases of rotavirus-associated gastroenteritis.
    Materialart: Online-Ressource
    ISSN: 2076-0817
    URL: Article
    DOI: 10.3390/pathogens8030089
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2019
    ZDB Id: 2695572-6
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 5
    Online-Ressource
    Online-Ressource
    Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution
    MDPI AG ; 2019
    In:  Journal of Clinical Medicine Vol. 8, No. 10 ( 2019-09-30), p. 1561-
    Details
    In: Journal of Clinical Medicine, MDPI AG, Vol. 8, No. 10 ( 2019-09-30), p. 1561-
    Kurzfassung: Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.
    Materialart: Online-Ressource
    ISSN: 2077-0383
    URL: Article
    DOI: 10.3390/jcm8101561
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2019
    ZDB Id: 2662592-1
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 6
    Online-Ressource
    Online-Ressource
    Serum Fatty Acid Composition Balance by Fuzzy C-Means Method in Individuals with or without Metabolic Dysfunction-Associated Fatty Liver Disease
    MDPI AG ; 2023
    In:  Nutrients Vol. 15, No. 4 ( 2023-02-04), p. 809-
    Details
    In: Nutrients, MDPI AG, Vol. 15, No. 4 ( 2023-02-04), p. 809-
    Kurzfassung: Circulating fatty acid composition is assumed to play an important role in metabolic dysfunction-associated fatty liver disease (MAFLD) pathogenesis. This study aimed to investigate the association between the overall balance of serum fatty acid composition and MAFLD prevalence. This cross-sectional study involved 400 Japanese individuals recruited from a health-screening program. We measured fatty acids in serum lipids using gas chromatography–mass spectrometry. The serum fatty acid composition balance was evaluated using fuzzy c-means clustering, which assigns individual data points to multiple clusters and calculates the percentage of data points belonging to multiple clusters, and serum fatty acid mass%. The participants were classified into four characteristic subclasses (i.e., Clusters 1, 2, 3, and 4), and the specific serum fatty acid composition balance (i.e., Cluster 4) was associated with a higher MAFLD prevalence. We suggest that the fuzzy c-means method can be used to determine the circulating fatty acid composition balance and highlight the importance of focusing on this balance when examining the relationship between MAFLD and serum fatty acids.
    Materialart: Online-Ressource
    ISSN: 2072-6643
    URL: Article
    DOI: 10.3390/nu15040809
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2023
    ZDB Id: 2518386-2
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 7
    Online-Ressource
    Online-Ressource
    Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics
    MDPI AG ; 2021
    In:  Applied Sciences Vol. 11, No. 8 ( 2021-04-07), p. 3309-
    Details
    In: Applied Sciences, MDPI AG, Vol. 11, No. 8 ( 2021-04-07), p. 3309-
    Kurzfassung: After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.
    Materialart: Online-Ressource
    ISSN: 2076-3417
    URL: Article
    DOI: 10.3390/app11083309
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2021
    ZDB Id: 2704225-X
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 8
    Online-Ressource
    Online-Ressource
    Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells
    MDPI AG ; 2021
    In:  International Journal of Molecular Sciences Vol. 22, No. 9 ( 2021-04-29), p. 4723-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 22, No. 9 ( 2021-04-29), p. 4723-
    Kurzfassung: Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.
    Materialart: Online-Ressource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms22094723
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2021
    ZDB Id: 2019364-6
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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    Online-Ressource
  • 9
    Online-Ressource
    Online-Ressource
    Concentration-Dependent Activity of Pazufloxacin against Pseudomonas aeruginosa: An In Vivo Pharmacokinetic/Pharmacodynamic Study
    MDPI AG ; 2022
    In:  Antibiotics Vol. 11, No. 7 ( 2022-07-21), p. 982-
    Details
    In: Antibiotics, MDPI AG, Vol. 11, No. 7 ( 2022-07-21), p. 982-
    Kurzfassung: The bacterium Pseudomonas aeruginosa is known to be associated with nosocomial infections around the world. Pazufloxacin, a potent DNA gyrase inhibitor, is known to be an effective drug candidate. However, it has not been clarified whether the pharmacokinetic (PK)/pharmacodynamic (PD) of pazufloxacin was effective against P. aeruginosa. Herein, we demonstrated that the PK/PD index of pazufloxacin against P. aeruginosa infection is used to optimize the dosing regiments. We constructed an in vivo infection model by infecting P. aeruginosa into the thigh of a mouse to determine the PD, and we measured the serum concentration of pazufloxacin to construct the PK model using high-performance liquid chromatography. The therapeutic efficacy of pazufloxacin was correlated with the ratio of the area under the free concentration time curve at 24 h to the minimum inhibitory concentration (fAUC24/MIC), and the maximum free concentration to the MIC (fCmax/MIC). Each contribution rate (R2) was 0.72 and 0.65, respectively, whereas the time at which the free drug concentration remained above the MIC (R2 = 0.28). The target value of pazufloxacin fAUC24/MIC for stasis was 46.1, for 1 log10 it was 63.8, and for 2 log10 it was 100.8. Moreover, fCmax/MIC for stasis was 5.5, for 1 log10 it was 7.1, and for 2 log10 it was 10.8. We demonstrated that the in vivo concentration-dependent activity of pazufloxacin was effective against the P. aeruginosa infection, and successfully made the PK/PD model sufficiently bactericidal. The PK/PD model will be beneficial in preventing the spread of nosocomial infections.
    Materialart: Online-Ressource
    ISSN: 2079-6382
    URL: Article
    DOI: 10.3390/antibiotics11070982
    Sprache: Englisch
    Verlag: MDPI AG
    Publikationsdatum: 2022
    ZDB Id: 2681345-2
    SSG: 15,3
    Standort Signatur Einschränkungen Verfügbarkeit
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