Shujin Sun

Differential regulation of stiffness, topography, and dimension of substrates in rat mesenchymal stem cells

The physiological microenvironment of the stem cell niche, including the three factors of stiffness, topography, and dimension, is crucial to stem cell proliferation and differentiation. Although a growing body of evidence is present to elucidate the importance of these factors individually, the interaction of the biophysical parameters of the factors remains insufficiently characterized, particularly for stem cells. To address this issue fully, we applied a micro-fabricated polyacrylamide hydrogel substrate with two elasticities, two topographies, and three dimensions to systematically test proliferation, morphology and spreading, differentiation, and cytoskeletal re-organization of rat bone marrow mesenchymal stem cells (rBMSCs) on twelve cases. An isolated but not combinatory impact of the factors was found regarding the specific functions. Substrate stiffness or dimension is predominant in regulating cell proliferation by fostering cell growth on stiff, unevenly dimensioned substrate. Topography is a key factor for manipulating cell morphology and spreading via the formation of a large spherical shape in a pillar substrate but not in a grooved substrate. Although stiffness leads to osteogenic or neuronal differentiation of rBMSCs on a stiff or soft substrate, respectively, topography or dimension also plays a lesser role in directing cell differentiation. Neither an isolated effect nor a combinatory effect was found for actin or tubulin expression, whereas a seemingly combinatory effect of topography and dimension was found in manipulating vimentin expression. These results further the understandings of stem cell proliferation, morphology, and differentiation in a physiologically mimicking microenvironment.

Spreading area and shape regulate apoptosis and differentiation of osteoblasts

The in vivo observations have indicated that at the remodeling sites of bone, the spreading area or shape of preosteoblasts is confined by the mineralized matrix. But it remains unknown whether this spreading confinement regulates the differentiation or apoptosis of osteoblasts. In the present study, osteoblast-like cells (MC3T3-E1) were seeded on micropatterned islands with different area and shape. The expression of three osteogenic differentiation markers was measured by immunofluorescence staining and apoptotic cells were detected using a terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling assay kit. The membrane fluorescence staining results showed that the actual spreading area of micropatterned osteoblasts coincided with the designed value. When the area of a micropatterned cell was confined as 314 or 615 µm(2), which was lower than that of freely spreading osteoblasts, the circular shape promoted the expression of osteogenic differentiation markers and the percentage of apoptotic osteoblasts compared with the branched shape. This shape-regulated differentiation and apoptosis of osteoblasts with confined spreading area were abolished when actin polymerization was inhibited by cytochalasin D. The present study gives an insight into the roles of spreading morphology on osteoblastic differentiation and apoptosis.

Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model

Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

Effects of oriented substrates on cell morphology, the cell cycle, and the cytoskeleton in Ros 17/2.8 cells.

Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts. The underlying mechanism, however, of cellular sensing and responding to the gravity vector is poorly understood. This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-, downward- or edge-on-oriented substrates. Cell morphology and nuclear translocation, cell proliferation and the cell cycle, and cytoskeletal reorganization were found to vary significantly in the three orientations. All of the responses were duration-dependent. These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.

Mechanical remodeling of normally-sized mammalian cells under a gravity vector

Translocation of the dense nucleus along a gravity vector initiates mechanical remodeling of a cell, but the underlying mechanisms of cytoskeletal network and focal adhesion complex (FAC) reorganization in a mammalian cell remain unclear. We quantified the remodeling of an MC3T3‐E1 cell placed in upward‐, downward‐, or edgeon‐orientated substrate. Nucleus longitudinal translocation presents a high value in downward orientation at 24 h or in edge‐on orientation at 72 h, which is consistent with orientation‐dependent distribution of perinuclear actin stress fibers and vimentin cords. Redistribution of total FAC area and fractionized super mature adhesion number coordinates this dependence at short duration. This orientation‐dependent remodeling is associated with nucleus flattering and lamin A/C phosphorylation. Actin depolymerization or Rho‐associated protein kinase signaling inhibition abolishes the orientation dependence of nucleus translocation, whereas tubulin polymerization inhibition or vimentin disruption reserves the dependence. A biomechanical model is therefore proposed for integrating the mechanosensing of nucleus translocation with cytoskeletal remodeling and FAC reorganization induced by a gravity vector.—Zhang, C., Zhou, L., Zhang, F., Lü, D., Li, N., Zheng, L., Xu, Y., Li, Z., Sun, S., Long, M. Mechanical remodeling of normally sized mammalian cells under a gravity vector. FASEB J. 31, 802–813 (2017). http://www.fasebj.org

Fluid Dynamics Analysis of a Novel Micropatterned Cell Bioreactor

Although flow-based bioreactor has been widely used to provide sufficient mass transportation and nutrient supply for cell proliferation, differentiation, and apoptosis, the underlying mechanism of cell responses to applied flow at single cell level remains unclear. This study has developed a novel bioreactor that combines flow bioreactor with microfabrication technique to isolate individual cells onto micropatterned substrate. A mechanical model has also been developed to quantify the flow field or the microenvironment around the single cell; flow dynamics has been analyzed on five geometrically different patterns of circle-, cube-, 1:2 ellipse-, 1:3 ellipse-, and rectangle-shaped “virtual cells.” The results of this study have demonstrated that the flow field is highly pattern dependent, and all the hydrodynamic development length, cell spacing, and orientation of inlet velocity vector are crucial for maintaining a fully developed flow. This study has provided a theoretical basis for optimizing the design of micropatterned flow bioreactor and a novel approach to understand the cell mechanotransduction and cell–surface interaction at single cell level.

Theoretical modeling of mechanical homeostasis of a mammalian cell under gravity-directed vector

Translocation of dense nucleus along gravity vector initiates mechanical remodeling of a eukaryotic cell. In our previous experiments, we quantified the impact of gravity vector on cell remodeling by placing an MC3T3-E1 cell onto upward (U)-, downward (D)-, or edge-on (E)- orientated substrate. Our experimental data demonstrate that orientation dependence of nucleus longitudinal translocation is positively correlated with cytoskeletal (CSK) remodeling of their expressions and structures and also is associated with rearrangement of focal adhesion complex (FAC). However, the underlying mechanism how CSK network and FACs are reorganized in a mammalian cell remains unclear. In this paper, we developed a theoretical biomechanical model to integrate the mechanosensing of nucleus translocation with CSK remodeling and FAC reorganization induced by a gravity vector. The cell was simplified as a nucleated tensegrity structure in the model. The cell and CSK filaments were considered to be symmetrical. All elements of CSK filaments and cytomembrane that support the nucleus were simplified as springs. FACs were simplified as an adhesion cluster of parallel bonds with shared force. Our model proposed that gravity vector-directed translocation of the cell nucleus is mechanically balanced by CSK remodeling and FAC reorganization induced by a gravitational force. Under gravity, dense nucleus tends to translocate and exert additional compressive or stretching force on the cytoskeleton. Finally, changes of the tension force acting on talin by microfilament alter the size of FACs. Results from our model are in qualitative agreement with those from experiments.

Microgravity-Induced Alterations of Inflammation-Related Mechanotransduction in Endothelial Cells on Board SJ-10 Satellite

Endothelial cells (ECs) are mechanosensitive cells undergoing morphological and functional changes in space. Ground-based study has provided a body of evidences about how ECs can respond to the effect of simulated microgravity, however, these results need to be confirmed by spaceflight experiments in real microgravity. In this work, we cultured EA.hy926 ECs on board the SJ-10 Recoverable Scientific Satellite for 3 and 10 days, and analyzed the effects of space microgravity on the ECs. Space microgravity suppressed the glucose metabolism, modulated the expression of cellular adhesive molecules such as ICAM-1, VCAM-1, and CD44, and depressed the pro-angiogenesis and pro-inflammation cytokine secretion. Meanwhile, it also induced the depolymerization of actin filaments and microtubules, promoted the vimentin accumulation, restrained the collagen I and fibronectin deposition, regulated the mechanotransduction through focal adhesion kinase and Rho GTPases, and enhanced the exosome-mediated mRNA transfer. Unlike the effect of simulated microgravity, neither three-dimensional growth nor enhanced nitric oxide production was observed in our experimental settings. This work furthers the understandings in the effects and mechanisms of space microgravity on ECs, and provides useful information for future spaceflight experimental design.

Selectivity of biopolymer membranes using HepG2 cells

Bioartificial liver (BAL) system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration. One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor, the core unit of BAL system. Here, we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight, commercially available biocompatible membranes made of acetyl cellulose–nitrocellulose mixed cellulose (CA–NC), acetyl cellulose (CA), nylon (JN), polypropylene (PP), nitrocellulose (NC), polyvinylidene fluoride (PVDF), polycarbonate (PC) and polytetrafluoroethylene (PTFE). Physicochemical analysis and mechanical tests indicated that CA, JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding. Cells prefer to adhere on CA, JN, PP or PTFE membranes with high proliferation rate in spheriod-like shape. Actin, albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane, whereas protein filtration is consistent among all the eight membranes. These results further the understandings of cell growth, morphology and spreading, as well as protein filtration on distinct membranes in designing a liver bioreactor.