Extracellular matrix modulates the biological effects of melatonin in mesenchymal stem cells

  1. Yihong Gong1⇑
  1. 1School of Engineering, Sun Yat-sen University, No. 132 East Waihuan Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, China
    2Orthopaedic Institute, Soochow University, No. 708 Renmin Road, Suzhou, Jiangsu 215007, China
    3Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006, China
    4Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
    5Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, West Virginia 26506, USA
  1. Correspondence should be addressed to F He or Y Gong; Emails: fanhe{at}suda.edu.cn or gongyih{at}mail.sysu.edu.cn
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    Figure 1

    Characterization of decellularized extracellular matrix (ECM). (A) Immunofluorescence staining of matrix proteins (green) in ECM, and of cell nuclei (blue) of original ECM-depositing cells. The cell-deposited ECM was examined before (upper panels) and after (lower panels) decellularization. Scale bars=50 μm. COL I, type I collagen; COL III, type III collagen; FL, fibronectin; LN, laminin; DCN, decorin. (B and C) Representative TEM images of decellularized ECM at low and high magnification. The collagen fibers (black arrowheads) and basement laminin (black arrows) were observed in decellularized ECM. Scale bar=1 μm in (B). Scale bar=100 nm in (C). (D and E) Representative scanning electron microscopy (SEM) images of decellularized ECM at low and high magnification. Bundles of fibrillar collagen fibers (white arrowheads) and beaded fibers (white arrows) were observed. Scale bar=5 μm in (D). Scale bar=3 μm in (E). A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 2

    The adsorption of melatonin to decellularized extracellular matrix (ECM) was assessed by immunofluorescence staining and HPLC. (A) Decellularized ECM was treated with 100 μM melatonin for 72 h, and the binding of melatonin to ECM was confirmed by immunofluorescence staining. Scale bar=50 μm. (B) The concentration of melatonin remaining in solution was assessed by HPLC assay at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h after addition of melatonin to tissue culture polystyrene (TCPS) plates or to plates layered with decellularized ECM. The percentage of melatonin was normalized to the initial concentration (concentration at 0 h). Data are expressed as the mean±s.e.m. of three independent experiments. A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 3

    Decellularized extracellular matrix (ECM) enhanced melatonin-mediated proliferative effects on bone-marrow-derived mesenchymal stem cells (BM-MSCs). (A) BM-MSCs were cultured in 12-well plates in a growth medium on tissue culture polystyrene (TCPS) or on decellularized ECM in the presence or absence of 100 μM melatonin. The cells were labeled with fluorescein diacetate to examine the cellular morphology and density. Scale bars=100 μm. (B) The BM-MSCs showed a dose response to melatonin, with respect to cell proliferation, when cultured on decellularized ECM, but not when cultured on TCPS. The BM-MSCs were cultured on TCPS or on decellularized ECM in the absence of melatonin or with 10 nM, 1 μM, or 100 μM melatonin. After continuous culture for 5 days, DNA quantification analysis was conducted to measure the cell proliferation of BM-MSCs. Melatonin did not affect proliferation of BM-MSCs grown on TCPS, but the cells grown on decellularized ECM exhibited a dose-dependent increase in cell proliferation with melatonin. Data are expressed as the mean±s.e.m. of five independent experiments (**P<0.01). A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 4

    Decellularized extracellular matrix (ECM)-modulated melatonin-mediated effects on intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) activity, and expression of SOD1/CuZn-SOD and SOD2/Mn-SOD. The dosage of melatonin was 100 μM. (A and B) Intracellular ROS production, as assessed by flow cytometry, was lower in bone-marrow-derived mesenchymal stem cells (BM-MSCs) grown on ECM and supplemented with melatonin. (C) Decellularized ECM increased SOD activity in BM-MSCs. (D) The levels of SOD1 mRNA (which encodes CuZn-SOD) in BM-MSCs were determined by real-time RT-PCR. (E) The levels of SOD2 mRNA (which encodes Mn-SOD) in BM-MSCs were determined by real-time RT-PCR. (F) The levels of SOD1/CuZn-SOD and SOD2/Mn-SOD protein were analyzed by western blotting analysis; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Melatonin treatment significantly increased the expression of both SOD proteins. (G) The intensity of bands was measured using the ImageJ Software and the levels of expression of SOD1/CuZn-SOD and SOD2/Mn-SOD were normalized to the level of expression of GAPDH. Data are expressed as the mean±s.e.m. of four independent experiments, except for data from western blotting analysis, which are expressed as the mean±s.e.m. of three independent experiments (*P<0.05 and **P<0.01). A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 5

    Decellularized extracellular matrix (ECM), when combined with melatonin, promoted calcium deposition in bone-marrow-derived mesenchymal stem cells (BM-MSCs) during osteogenic differentiation. (A) BM-MSCs were incubated in osteogenic differentiation medium for 14 days on tissue culture polystyrene (TCPS) or on decellularized ECM, with or without 100 μM melatonin. Alizarin Red S staining was used to detect calcium secretion. Scale bars=100 μm. (B) Quantification of the stained mineral layer, dissolved in 1% hydrochloric acid, determined using a spectrophotometer. Decellularized ECM significantly enhanced the effect of melatonin on calcium deposition. Data are expressed as the mean±s.e.m. of four independent experiments (**P<0.01). A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 6

    Decellularized extracellular matrix (ECM) modulates the effect of melatonin on the expression of mRNA encoding osteogenesis-specific markers and matrix degrading enzymes. The concentration of melatonin used was 100 μM. The mRNA levels of ALP (A), COL1A1 (B), BGLAP (C), SPP1 (D), SP7 (E), RUNX2 (F), MMP1 (G), and MMP2 (H) were measured by real-time RT-PCR. Decellularized ECM significantly enhanced melatonin-mediated effects on the transcription of osteogenic genes, while suppressing the levels of mRNAs encoding matrix metalloproteinases (MMPs). Data are expressed as the mean±s.e.m. of four independent experiments (**P<0.01).

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    Figure 7

    Treatment with superoxide dismutase (SOD) inhibitors affected oxidative stress in bone-marrow-derived mesenchymal stem cells (BM-MSCs) in the presence of extracellular matrix (ECM) and melatonin. (A) Production of intracellular reactive oxygen species (ROS) in BM-MSCs in the presence of diethyldithiocarbamate (DDC), an inhibitor of SOD1/CuZn-SOD. (B) Production of intracellular ROS by BM-MSCs in the presence of 2-methoxyestradiol (2-ME), an inhibitor of SOD2/Mn-SOD. Data are expressed as the mean±s.e.m. of four independent experiments (**P<0.01). A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0430.

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    Figure 8

    The effect of superoxide dismutase (SOD) inhibitors on gene expression of osteogenesis-specific markers, in differentiated bone-marrow-derived mesenchymal stem cells (BM-MSCs), in the presence of extracellular matrix (ECM) and melatonin. The mRNA levels of ALP (A and B), COL1A1 (C and D), SPP1 (E and F), and RUNX2 (G and H) were measured by real-time RT-PCR. Treatment with SOD inhibitors significantly suppressed the up-regulation of osteogenesis-specific marker genes by decellularized ECM and melatonin. Data are expressed as the mean±s.e.m. of four independent experiments (*P<0.05 and **P<0.01).

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    Figure 9

    The effect of superoxide dismutase (SOD) inhibitors on gene expression of matrix metalloproteinases (MMPs) in differentiated bone-marrow-derived mesenchymal stem cells (BM-MSCs), in the presence of extracellular matrix (ECM) and melatonin. The mRNA levels of MMP1 (A and B) and MMP2 (C and D) were measured by real-time RT-PCR. The application of diethyldithiocarbamate (DDC), an inhibitor of SOD1, reversed the suppressive effects of melatonin on MMP1 expression when BM-MSCs were cultured on decellularized ECM. Data are expressed as the mean±s.e.m. of four independent experiments (*P<0.05 and **P<0.01).

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  1. Published online before print September 10, 2014, doi: 10.1530/JOE-14-0430 J Endocrinol 223 167-180
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