Surfactant Copolymer Annealing of Chemically Permeabilized Cell Membranes.

Structural breakdown of the cell membrane is a primary mediator in trauma induced tissue necrosis. When membrane disruption exceeds intrinsic membrane sealing processes, biocompatible multi-block amphiphilic copolymer surfactants such as Poloxamer 188 (P188) have been found to be effective in catalyze or augment sealing. Although in living cells copolymer induced sealing of membrane defects has been detected by changes in membrane transport properties, it has not been directly imaged. In this project we used Atomic force microscopy (AFM) to directly image saponin permeabilized and poloxamer sealed plasma membranes of monolayer cultured MDCK and 3T3 fibroblasts. AFM image analysis resulted in the density and diameter ranges for membrane indentations per 5×5 µm area. For control, saponin lysed, and P188 treatment of saponin lysed membranes, the supra-threshold indentation density was 3.6 ± 2.8, 13.8 ± 6.7, and 4.9 ± 3.3/cell, respectively. These results indicated that P188 catalyzed reduction in size of AFM indentations which correlated with increase cell survival. This evidence confirm that biocompatible surfactant P188 augment natural cell membrane sealing capability when intrinsic processes are incapable alone.

Endothelial invasive response in a co-culture model with physically-induced osteodifferentiation.

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co-culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity-induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co-culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co-culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen-like morphology, suggesting that cell-cell interaction in the co-culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co-culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre-vascularized bone grafts.

Control of adipogenesis by ezrin, radixin and moesin-dependent biomechanics remodeling.

We have recently shown that altered stem cell biomechanics can regulate the lineage commitment through a family of the membrane-cytoskeleton linker proteins (ERM; ezrin, radixin, moesin). The ERM proteins not only modulate the cell stiffness and actin cytoskeleton organization, but also rearrange focal adhesions and therefore influence the biochemically-directed stem cell differentiation. Combining silencing RNA, atomic force microscopy, and fluorescence microscopy, the role of the ERM proteins involved in the regulation of stem cell biomechanics and adipogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi caused disassembly of actin stress fibers and focal adhesions and a decrease in the cell stiffness. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired adipogenesis in a time-dependent manner. While siRNA ERM treatment at day 0 substantially interfered with adipogenesis, the same treatment at day 3 of adipogenic differentiation significantly facilitated adipogenesis, as assessed by the expression of adipocyte-specific markers. The intact biomechanics homeostasis appears to be critical for the adipogenic induction. These findings may lead to potential biomechanical intervention techniques and methodologies to control the fate and extent of adipogenesis that would likely be involved in stem cell-based therapeutics for soft tissue repair and regeneration.

Millimeter wave-induced modulation of calcium dynamics in an engineered skin co-culture model: role of secreted ATP on calcium spiking.

We have previously designed and characterized a 94 GHz exposure system that allows real-time monitoring of subcellular interactions induced by millimeter wave (MMW) stimulation. For example, studies of the calcium dynamics in neuronal cells in response to 94 GHz irradiation suggested that MMW stimulation increased calcium spiking. In this study, we engineered a 3D co-culture model that represents the major constituents of skin. We used this experimental model along with the custom-designed MMW exposure system to investigate the effects of 94 GHz irradiation in the skin-like tissue construct. Unlike typical non-excitable cells, keratinocytes exhibited calcium spikes in their resting state. Exposure to a 94 GHz irradiation induced a statistically significant increase in the calcium spiking. When co-cultured with neuronal cells in the 3D co-culture skin model, changes in the calcium spiking in neuronal cells depended on the MMW input power. Further, the 94 GHz irradiation caused ATP secretion by keratincytes. ATP is a major factor that modulates the calcium spiking in neuronal cells. Surprisingly, while a 5-fold increase in the ATP secretion enhanced the calcium spiking in neuronal cells, a 10-fold increase significantly hindered the calcium dynamics. Computational simulation of ATP-induced calcium dynamics was in general agreement with the experimental findings, suggesting the involvement of the ATP-sensitive purinergic receptors. The engineered co-culture skin model offers a physiologically relevant environment in which the calcium dynamics is regulated both by the cell-MMW and cell-cell interactions.

Altered osteogenic commitment of human mesenchymal stem cells by ERM protein-dependent modulation of cellular biomechanics.

Cellular mechanics is known to play an important role in many cellular functions including adhesion, migration, proliferation, and differentiation. Human mesenchymal stem cells (hMSCs) demonstrate unique mechanical properties distinct from fully differentiated cells. This observation suggests that the stem cell mechanics may be modulated to regulate the hMSCs' lineage commitment. Specifically, ERM (ezrin, radixin, moesin) proteins are known to mediate the membrane-cytoskeleton adhesion, cell elasticity, actin cytoskeleton organization, and therefore could serve as potential targets for modulation of the cellular mechanics. Combining silencing RNA, atomic force microscopy, and laser optical tweezers, the role of the ERM proteins involved in the regulation of stem cell biomechanics and osteogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi causes disassembly of actin stress fibers and focal adhesions, a decrease in the cell stiffness, and membrane separation from the cytoskeleton. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired biochemically-directed osteogenic differentiation. The intact actin cytoskeleton and focal adhesions of hMSCs appear critical for the osteogenic induction. Thus, ERM knockdown modulates the dynamics of cell mechanical changes during hMSC differentiation and regulates the expression of tissue specific molecular markers. These findings are of particular interest for modulation of the cellular biomechanics to control hMSCs' activities and fate in tissue engineering, regenerative medicine, and other stem cell-based therapeutic applications.

A new perspective for stem-cell mechanobiology: biomechanical control of stem-cell behavior and fate.

Biomechanics is known to play an important role in cell metabolism. Cell phenotype, tissue-specific functions, and fate critically depend on the extracellular mechanical environment. The mechanical properties of the cell itself, such as cytoskeleton elasticity, membrane tension, and adhesion strength, may also play an important role in cell homeostasis and differentiation. Pluripotent bone marrow-derived human mesenchymal stem cells, for example, can be differentiated into many tissue-specific lineages. While cellular biomechanical properties are significantly altered during stem-cell specification to a particular phenotype, the complexity of events associated with transformation of these precursor cells leaves many questions unanswered about morphological, structural, proteomic, and functional changes in differentiating stem cells. A thorough understanding of stem-cell behavior would allow the development of more effective approaches to the expansion of stem cells in vitro and the regulation of their commitment to a specific phenotype. Control of cell behaviors might be feasible through manipulation of the cellular biomechanical properties using various external physical stimuli, including electric fields, mechanical stimuli, and genetic manipulation of the expression of particular genes. Biomechanical regulation of stem-cell differentiation can greatly minimize the number of chemicals and growth factors that would otherwise be required for composite tissue engineering. Determination and the appropriate use of the known physicochemical cues will facilitate current research effort toward designing and engineering functional tissue constructs.

Physicochemical control of adult stem cell differentiation: shedding light on potential molecular mechanisms.

Realization of the exciting potential for stem-cell-based biomedical and therapeutic applications, including tissue engineering, requires an understanding of the cell-cell and cell-environment interactions. To this end, recent efforts have been focused on the manipulation of adult stem cell differentiation using inductive soluble factors, designing suitable mechanical environments, and applying noninvasive physical forces. Although each of these different approaches has been successfully applied to regulate stem cell differentiation, it would be of great interest and importance to integrate and optimally combine a few or all of the physicochemical differentiation cues to induce synergistic stem cell differentiation. Furthermore, elucidation of molecular mechanisms that mediate the effects of multiple differentiation cues will enable the researcher to better manipulate stem cell behavior and response.

oxLDL-induced decrease in lipid order of membrane domains is inversely correlated with endothelial stiffness and network formation.

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

Controlling cellular biomechanics of human mesenchymal stem cells.

The therapeutic efficacy of human mesenchymal stem cells (hMSCs) depends on proper characterization and control of their unique biological, mechanical and physicochemical properties. For example, cellular biomechanics and environmental mechanical cues have been shown to critically influence cell commitment to a particular lineage. We characterized biomechanical properties of hMSCs including cytoskeleton elasticity and plasma membrane/cytoskeleton coupling. As expected, during osteogenic differentiation of hMSCs, the cellular biomechanics is remodeled, and such remodeling precedes up-regulation of the osteogenic markers. Further, application of an electrical stimulation modulates the cellular biomechanics and therefore may be used to facilitate stem cell differentiation for stem cell-based tissue engineering.

Regulation of cell cytoskeleton and membrane mechanics by electric field: role of linker proteins.

Cellular mechanics is known to play an important role in the cell homeostasis including proliferation, motility, and differentiation. Significant variation in the mechanical properties between different cell types suggests that control of the cell metabolism is feasible through manipulation of the cell mechanical parameters using external physical stimuli. We investigated the electrocoupling mechanisms of cellular biomechanics modulation by an electrical stimulation in two mechanically distinct cell types--human mesenchymal stem cells and osteoblasts. Application of a 2 V/cm direct current electric field resulted in approximately a twofold decrease in the cell elasticity and depleted intracellular ATP. Reduction in the ATP level led to inhibition of the linker proteins that are known to physically couple the cell membrane and cytoskeleton. The membrane separation from the cytoskeleton was confirmed by up to a twofold increase in the membrane tether length that was extracted from the cell membrane after an electrical stimulation. In comparison to human mesenchymal stem cells, the membrane-cytoskeleton attachment in osteoblasts was much stronger but, in response to the same electrical stimulation, the membrane detachment from the cytoskeleton was found to be more pronounced. The observed effects mediated by an electric field are cell type- and serum-dependent and can potentially be used for electrically assisted cell manipulation. An in-depth understanding and control of the mechanisms to regulate cell mechanics by external physical stimulus (e.g., electric field) may have great implications for stem cell-based tissue engineering and regenerative medicine.

Hybrid superporous scaffolds: an application for cornea tissue engineering.

Engineering a cell-based keratoprosthesis often requires a struggle between two essential parameters: natural 3-D biological adhesion and mechanical strength. A novel hybrid scaffold of natural and synthetic materials was engineered to achieve both cell adhesion and implantable strength. This scaffold was characterized in terms of cell adhesion, cell migration, swelling, and strength. While the study was focused on engineering a biointegrable prosthetic skirt, a clear central core with an appropriate refractive index and light transmission was also incorporated into the design for potential functionality. The hybrid scaffold was tested in rat corneas. This uniquely designed scaffold was well tolerated and encouraged host cell migration into the implant. The hybrid superporous design also enhanced cell adhesion and retention in a superporous scaffold without altering the bulk mechanical properties of the hydrogel.

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells.

Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated osteoblasts), and the average membrane tether length measured with laser optical tweezers (10.6 +/- 1.1 microm for stem cells, and 4.0 +/- 1.1 microm for osteoblasts). These differences in cell elasticity and membrane mechanics result primarily from differential actin cytoskeleton organization in these two cell types, whereas microtubules did not appear to affect the cellular mechanics. The membrane-cytoskeleton linker proteins may contribute to a stronger interaction of the plasma membrane with F-actins and shorter membrane tether length in osteoblasts than in stem cells. Actin depolymerization or ATP depletion caused a two- to threefold increase in the membrane tether length in osteoblasts, but had essentially no effect on the stem-cell membrane tethers. Actin remodeling in the course of a 10-day osteogenic differentiation of hMSC mediates the temporally correlated dynamical changes in cell elasticity and membrane mechanics. For example, after a 10-day culture in osteogenic medium, hMSC mechanical characteristics were comparable to those of mature bone cells. Based on quantitative characterization of the actin cytoskeleton remodeling during osteodifferentiation, we postulate that the actin cytoskeleton plays a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics at the early stage of stem-cell osteodifferentiation.

Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus.

Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.

Distinct membrane mechanical properties of human mesenchymal stem cells determined using laser optical tweezers.

The therapeutic efficacy of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is determined by their unique biological, mechanical, and physicochemical characteristics, which are yet to be fully explored. Cell membrane mechanics, for example, has been shown to critically influence MSC differentiation. In this study, we used laser optical tweezers to measure the membrane mechanics of human MSCs and terminally differentiated fibroblasts by extracting tethers from the outer cell membrane. The average tether lengths were 10.6+/-1.1 microm (hMSC) and 3.0+/-0.5 microm (fibroblasts). The tether extraction force did not increase during tether formation, which suggests existence of a membrane reservoir intended to buffer membrane tension fluctuations. Cytoskeleton disruption resulted in a fourfold tether length increase in fibroblasts but had no effect in hMSCs, indicating weak association between the cell membrane and hMSC actin cytoskeleton. Cholesterol depletion, known to decrease lipid bilayer stiffness, caused an increase in the tether length both in fibroblasts and hMSCs, as does the treatment of cells with DMSO. We postulate that whereas fibroblasts use both the membrane rigidity and membrane-cytoskeleton association to regulate their membrane reservoir, hMSC cytoskeleton has only a minor impact on stem cell membrane mechanics.

Mathematical model of static platelet adhesion on a solid surface.

The scheme of platelet/surface interaction and a kinetic model of platelet adhesion on a solid surface are suggested. The elaborated approach takes into account the platelet activation by the surface and accumulation of free activated cells in the bulk of the liquid phase. This effect has an especially important role in static experimental conditions. The suggested model explains three types of adhesion kinetic curves, obtained in experiments in vitro: sigmoid curves with or without saturation and an exponential curve with saturation. According to the model, the curve shape is determined by material surface properties, platelet functionality, and experimental conditions of the platelet/surface interaction. The data of static platelet adhesion from platelet rich plasma on glass, siliconized glass, hexadecyltrichlorosilane monolayers, and low-density polyethylene are described mathematically with the proposed model. Numerical parameters are calculated from approximation of experimental data by the model. These parameters allow quantitative characterization of platelet interaction with the material surface.