Lisha Zheng

Effect of substrate stiffness on the functions of rat bone marrow and adipose tissue derived mesenchymal stem cells in vitro

Regenerative medicine treatments that combine the use of cells and materials may open new options for tissue/organ repair and regeneration. The microenvironment of mesenchymal stem cells (MSCs) strictly regulates their self-renewal and functions. In this study, when rat bone marrow derived MSCs (rBMSCs) and rat adipose tissue derived MSCs (rAMSCs) in passages 2-4 were cultured on different substrates, they presented the cellular functions to be dependent of substrate stiffness. The cells attached better on the softer substrate than on the stiffer one. The substrate stiffness had no significant influence on the proliferation of those cells. However, the substrate stiffness significantly promoted the osteogenic differentiation of the two kinds of stem cells. Furthermore, rBMSCs cultured on the same stiffness expressed more osteoblast-related markers than rAMSCs. In addition, combined biomaterials and biochemical reagents treatment yielded a stronger effect on osteogenic differentiation of MSCs than either treatment alone. These results have significant implications for further extending our capabilities in engineering functional tissue substitutes.

Effect of Cyclic Strain on Cardiomyogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Involvement of large conductance Ca 2+ -activated K + channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation

The large conductance Ca2+-activated K+ (BKCa) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BKCa channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BKCa channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BKCa protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BKCa in SS-induced inhibition of proliferation was examined by BKCa inhibition using a BKCa specific blocker, iberiotoxin (IBTX), and by BKCa transfection in BKCa non-expressing CHO cells. The changes in [Ca2+]i were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC4(3). We found that laminar SS inhibited VSMC proliferation and stimulated BKCa channel expression. Furthermore, laminar SS induced an increase in [Ca2+]i and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BKCa channel activity on cell proliferation in response to SS was also confirmed in BKCa-transfected CHO cells showing a decline in proliferation. Blocking BKCa channel reversed its inhibitory effect, providing additional support for the involvement of BKCa in SS-induced proliferation reduction. Our results suggest, for the first time, that BKCa channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: Involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways

Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12 dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.

Galanin protects against nerve injury after shear stress in primary cultured rat cortical neurons.

The neuropeptide galanin and its receptors (GalR) are found to be up-regulated in brains suffering from nerve injury, but the specific role played by galanin remains unclear. This study aimed to explore the neuroprotective role of galanin after shear stress induced nerve injury in the primary cultured cortical neurons of rats. Our results demonstrated that no significant changes in cell death and viability were found after galanin treatment when subjected to a shear stress of 5 dyn/cm2 for 12 h, after increasing magnitude of shear stress to 10 dyn/cm2 for 12 h, cell death was significantly increased, while galanin can inhibit the nerve injury induced by shear stress with 10 dyn/cm2 for 12 h. Moreover, Gal2-11 (an agonist of GalR2/3) could also effectively inhibit shear stress-induced nerve injury of primary cultured cortical neurons in rats. Although GalR2 is involved in the galanin protection mechanism, there was no GalR3 expression in this system. Moreover, galanin increased the excitatory postsynaptic currents (EPSCs), which can effectively inhibit the physiological effects of shear stress. Galanin was also found to inhibit the activation of p53 and Bax, and further reversed the down regulation of Bcl-2 induced by shear stress. Our results strongly demonstrated that galanin plays a neuroprotective role in injured cortical neurons of rats.

The effects of fluid shear stress on proliferation and osteogenesis of human periodontal ligament cells

Shear stress is one of the main stress type produced by speech, mastication or tooth movement. The mechano-response of human periodontal ligament (PDL) cells by shear stress and the mechanism are largely unknown. In our study, we investigated the effects of fluid shear stress on proliferation, migration and osteogenic potential of human PDL cells. 6dyn/cm(2) of fluid shear stress was produced in a parallel plate flow chamber. Our results demonstrated that fluid shear stress rearranged the orientation of human PDL cells. In addition, fluid shear stress inhibited human PDL cell proliferation and migration, but increased the osteogenic potential and expression of several growth factors and cytokines. Our study suggested that shear stress is involved in homeostasis regulation in human PDL cells. Inhibiting proliferation and migration potentially induce PDL cells to respond to mechanical stimuli in order to undergo osteogenic differentiation.

Static magnetic field regulates proliferation, migration, differentiation and YAP/TAZ activation of human dental pulp stem cells

The dental pulp stem cells (DPSCs) are a population of mesenchymal stem cells, which have multilineage potential and high proliferation. DPSCs are regarded as a promising tool for tissue regeneration of dentine, dental pulp, bone, cartilage, and muscle. Recently, magnetic materials have become commonly applied in dental clinics. Static magnetic field has been reported to regulate the proliferation, migration, or differentiation of stem cells. However, whether static magnetic fields affect DPSCs is still unknown. In our study, we investigated the effect of static magnetic field on the proliferation, migration, and differentiation of DPSCs. The results indicated that static magnetic field rearranged the cytoskeleton of DPSCs. A static magnetic field of 1 mT increased DPSC proliferation, as well as the gene expression of several growth factors such as FGF‐2, TGF‐β, and VEGF. Moreover, the static magnetic field promoted the migration of DPSCs by regulating MMP‐1 and MMP‐2 gene expression. Static magnetic field of 1 mT also induced osteo/odontogenesis and mineralization in DPSCs. Otherwise, the static magnetic field recruited YAP/TAZ to the nucleus, inhibited the phosphorylation of YAP/TAZ, and upregulated the two YAP/TAZ‐regulated genes, CTGF and ANKRD1. Cytoskeleton inhibitor, cytochalasin D, obviously inhibited the nuclear localization of YAP/TAZ. When YAP/TAZ were knocked‐down, the static magnetic field‐induced mineralization of DPSCs was diminished. Our findings provide an insight into the effect of static magnetic field on DPSCs and provide the foundation for the future tissue regeneration.