Yihong Gong

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H2O2) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H2O2 at a sublethal concentration of 200 μm, human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H2O2 exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H2O2 exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H2O2‐induced premature senescence. We also found that melatonin attenuated the H2O2‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.

Melatonin mediates protective effects on inflammatory response induced by interleukin‐1 beta in human mesenchymal stem cells

Joint diseases like osteoarthritis usually are accompanied with inflammatory processes, in which pro‐inflammatory cytokines mediate the generation of intracellular reactive oxygen species (ROS) and compromise survival of subchondral osteoblasts. Melatonin is capable of manipulating bone formation and osteogenic differentiation of mesenchymal stem cells (MSCs). The aim of this work was to investigate the anti‐inflammatory effect of melatonin on MSC proliferation and osteogenic differentiation in the absence or presence of interleukin‐1 beta (IL‐1β), which was used to induce inflammation. Our data showed that melatonin improved cell viability and reduced ROS generation in MSCs in a dose‐dependent manner. When exposed to 10 ng/mL IL‐1β, various concentrations of melatonin resulted in significant reduction of ROS by 34.9% averagely. Luzindole as a melatonin receptor antagonist reversed the anti‐oxidant effect of melatonin in MSCs with co‐exposure to IL‐1β. Real‐time RT‐PCR data suggested that melatonin treatment up‐regulated the expression of CuZnSOD and MnSOD, while down‐regulated the expression of Bax. To investigate the effect of melatonin on osteogenesis, MSCs were cultured in osteogenic differentiation medium supplemented with IL‐1β, melatonin, or luzindole. After exposed to IL‐1β for 21 days, 1 μm melatonin treatment significantly increased the levels of type I collagen, ALP, and osteocalcin, and 100 μm melatonin treatment yielded the highest level of osteopontin. Our study demonstrated that melatonin maintained MSC survival and promoted osteogenic differentiation in inflammatory environment induced by IL‐1β, suggesting melatonin treatment could be a promising method for bone regenerative engineering in future studies.

Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases

Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints.

Promotion of Hepatic Differentiation of Bone Marrow Mesenchymal Stem Cells on Decellularized Cell-Deposited Extracellular Matrix

Interactions between stem cells and extracellular matrix (ECM) are requisite for inducing lineage-specific differentiation and maintaining biological functions of mesenchymal stem cells by providing a composite set of chemical and structural signals. Here we investigated if cell-deposited ECM mimicked in vivo livers stem cell microenvironment and facilitated hepatogenic maturation. Decellularization process preserved the fibrillar microstructure and a mix of matrix proteins in cell-deposited ECM, such as type I collagen, type III collagen, fibronectin, and laminin that were identical to those found in native liver. Compared with the cells on tissue culture polystyrene (TCPS), bone marrow mesenchymal stem cells (BM-MSCs) cultured on cell-deposited ECM showed a spindle-like shape, a robust proliferative capacity, and a suppressed level of intracellular reactive oxygen species, accompanied with upregulation of two superoxide dismutases. Hepatocyte-like cells differentiated from BM-MSCs on ECM were determined with a more intensive staining of glycogen storage, an elevated level of urea biosynthesis, and higher expressions of hepatocyte-specific genes in contrast to those on TCPS. These results demonstrate that cell-deposited ECM can be an effective method to facilitate hepatic maturation of BM-MSCs and promote stem-cell-based liver regenerative medicine.

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

Both self-renewal and lineage-specific differentiation of mesenchymal stem cells (MSCs) are triggered by their in vivo microenvironment including the extracellular matrix (ECM) and secreted hormones. The ECM may modulate the physiological functions of hormones by providing binding sites and by regulating downstream signaling pathways. Thus, the purpose of this study was to evaluate the degree of adsorption of melatonin to a natural cell-deposited ECM and the effects of this interaction on the biological functions of melatonin in human bone marrow-derived MSCs (BM-MSCs). The fibrillar microstructure, matrix composition, and melatonin-binding affinity of decellularized ECM were characterized. The cell-deposited ECM improved melatonin-mediated cell proliferation by 31.4%, attenuated accumulation of intracellular reactive oxygen species accumulation, and increased superoxide dismutase (SOD) mRNA and protein expression. Interaction with ECM significantly enhanced the osteogenic effects of melatonin on BM-MSCs by increasing calcium deposition by 30.5%, up-regulating osteoblast-specific gene expression and down-regulating matrix metalloproteinase (MMP) expression. The underlying mechanisms of these changes in expression may involve intracellular antioxidant enzymes, because osteoblast-specific genes were down-regulated, whereas MMP expression was up-regulated, in the presence of SOD-specific inhibitors. Collectively, our findings indicate the importance of native ECM in modulating the osteoinductive and antioxidant effects of melatonin and provide a novel platform for studying the biological actions of growth factors or hormones in a physiologically relevant microenvironment. Moreover, a better understanding of the enhancement of MSC growth and osteogenic differentiation resulting from the combination of ECM and melatonin could improve the design of graft substitutes for skeletal tissue engineering.

Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H2O2) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100μM H2O2 dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs.

SIRT1‐dependent anti‐senescence effects of cell‐deposited matrix on human umbilical cord mesenchymal stem cells

Human umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi‐lineage differentiation. This investigation evaluated the effect of cell‐deposited decellularized ECM (DECM) on oxidative stress‐induced premature senescence in UC‐MSCs. Sublethal dosages of H2O2, ranging from 50 μm to 200 μm, were used to induce senescence in MSCs. We found that DECM protected UC‐MSCs from oxidative stress‐induced premature senescence. When treated with H2O2 at the same concentration, cell proliferation of DECM‐cultured UC‐MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H2O2, fewer senescence‐associated β‐galactosidase‐positive cells were observed on DECM than those on TCPS (17.6  ±  4.0% vs. 60.4  ±  6.2%). UC‐MSCs cultured on DECM also showed significantly lower levels of senescence‐related regulators, such as p16INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC‐MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)‐dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM‐mediated anti‐senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell‐deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright

Incorporation of well-dispersed calcium phosphate nanoparticles into PLGA electrospun nanofibers to enhance the osteogenic induction potential

Poly(lactide-co-glycolide) (PLGA) electrospun composite nanofibers were fabricated to mimic bone extra cellular matrix for applications in bone tissue engineering. Poly(acrylic acid) modified Zn-doped calcium phosphate (PAA-CaP/Zn) nanoparticles were first synthesized. PAA was selected because of the strong bonding ability with calcium and Zn was incorporated to enhance the bioactivity. PAA-CaP/Zn nanoparticles were well dispersed with a hydrodynamic size of 184 nm; the presence of Zn was confirmed and the composition was similar to hydroxyapatite. These nanoparticles were non-cytotoxic against adipose derived stem cells from rats (rADSC) at 0.1 mg ml−1 based on an extraction assay and direct contact method. The nanoparticles were further entrapped into PLGA nanofibers. All electrospun membranes were composed of smooth fibers and for composite membranes, nanoparticles were homogeneously distributed in the fiber matrix. Furthermore, the tensile strength was increased with compromised elasticity, while the degradation was retarded and the biomineralization property was enhanced due to the presence of nanoparticles. rADSC proliferated very well on composite membranes and the one with 5% nanoparticles (P-5) demonstrated the highest osteogenic induction effect as significantly more calcium was deposited after osteogenic induction for 21 days. Sample P-5 was the most suitable for applications in bone tissue engineering.

In situ transformation of casein/CaCO3 microspheres into hierarchical hydroxyapatite composite microparticles and its cytocompatibility evaluation

Biomimetic scaffold is promising for repairing bone defects that are resulted from diseases or injuries. An ideal scaffold should be similar to native bones. Thus, hydroxyapatite (HAp) composite microparticles in which HAp closely resembles the mineral component in native bones were fabricated. Casein/CaCO3 microspheres, precipitated in the presence of casein, were used as starting material and an in situ ion-exchange method was applied under mild conditions. In order to obtain hierarchical structure in which casein/CaCO3 core was wrapped with HAp layer, various conditions, including starting material (resuspended powder or mother solution), reaction temperature, concentration, and addition rate of Na2HPO4 solution, were intensively studied and optimized. The products were characterized using X-ray diffractometer (XRD), Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscope (SEM). After the in situ transformation, the presence of casein was confirmed and its amount was not significantly changed. The formed microparticles could be in only HAp phase of irregular shape or in HAp/CaCO3 composite phase of well-dispersed microspheres. The cytotoxicity of samples was investigated using direct culturing method and its bioactivity was also evaluated by seeding cells on poly(lactic-co-glycolic acid) (PLGA)/HAp films. Here, adipose-derived stem cells (ADSCs) were used as model cells. The results demonstrated that the hierarchical HAp/CaCO3 composite microparticles were cytocompatible and could be potentially used for bone tissue engineering.

Enhancing proliferation and osteogenic differentiation of HMSCs on casein/chitosan multilayer films

Creating a bioactive surface is important in tissue engineering. Inspired by the natural calcium binding property of casein (CA), multilayer films ((CA/CS)n) with chitosan (CS) as polycation were fabricated to enhance biomineralization, cell adhesion and differentiation. LBL self-assembly technique was used and the assembly process was intensively studied based on changes of UV absorbance, zeta potential and water contact angle. The increasing content of chitosan and casein with bilayers was further confirmed with XPS and TOF-SIMS analysis. To improve the biocompatibility, gelatin was surface grafted. In vitro mineralization test demonstrated that multilayer films had more hydroxyapatite crystal deposition. Human mesenchymal stem cells (HMSCs) were seeded onto these films. According to fluorescein diacetate (FDA) and cell cytoskeleton staining, MTT assay, expression of osteogenic marker genes, ALP activity, and calcium deposition quantification, it was found that these multilayer films significantly promoted HMSCs attachment, proliferation and osteogenic differentiation than TCPS control.