Vascular endothelial cellular mechanics under hyperglycemia and its role in tissue regeneration.

Diabetes is one of the most prevalent diseases worldwide. The tissue regeneration of diabetes patients is known to be rather tricky as the result of vascular dysfunction, and this leads to various clinical complications including diabetic foot ulcers. The vascular endothelial cells, which compactly line the inner surface of blood vessels, are responsible for the growth and maintenance of blood vessels and play an essential role in tissue regeneration. Although the mechanical properties of cells are generally known to be regulated by physiological/pathological conditions, few studies have been performed to investigate vascular endothelial cellular mechanics under hyperglycemia and the biological functions related to tissue regeneration. In this study, we conduct a systematic investigation of this issue. The results suggested that the stiffness of human umbilical vein endothelial cells (HUVECs) can be significantly regulated by the glucose concentration, subsequently, leading to significant alterations in cell migration and proliferation capabilities that are closely related to tissue regeneration. The rearrangement of the cytoskeleton induced by hyperglycemia through Cdc42 was found to be one of the pathways for the alteration of the cell stiffness and the subsequent cell dysfunctions. Therefore, we suggested that the inhibition of Cdc42 might be a promising strategy to facilitate various tissue regeneration for diabetes patients.

Thermo-mechanical processing of brass components for potable-water usage increases risks of Pb leaching.

The problem of lead contamination in potable water has been a serious concern in different countries. Although the use of leaded welding solder has been banned and brass components used in potable water pipework have to be of the nominally "lead-free" grade in most jurisdictions, incidents of excessive lead leaching are still reported. The widely advocated explanation of lead leaching from brass components in terms of corrosion and the formation of electrochemical cells is inadequate since mechanical cutting is also known to cause lead segregation on brass surfaces. In this study, the effects of lead segregation on brass surfaces and subsequent leaching to contacting water resulting from thermo-mechanical processing of the brass are studied. The results indicate that mechanical milling and polishing that replicate the common processing involved in pipeline installation yield a significant increase in surface lead, and a strong correlation exists between lead leaching and the plastic deformation of the brass surface. Furthermore, flame-torch treatment that replicates the common brazing of brass also results in a significant increase in surface lead. These results indicate that the common thermo-mechanical processing of brass piping components poses a real risk of lead contamination in potable water, and revision in the common protocols for handling lead components may be necessary.

Elastic modulus and migration capability of drug treated leukemia cells K562.

Leukemia is a commonly seen disease caused by abnormal differentiation of hematopoietic stem cells and blasting in bone marrow. Despite drugs are used to treat the disease clinically, the influence of these drugs on leukemia cells' biomechanical properties, which are closely related to complications like leukostasis or infiltration, is still unclear. Due to non-adherent and viscoelastic nature of leukemia cells, accurate measurement of their elastic modulus is still a challenging issue. In this study, we adopted rate-jump method together with optical tweezers indentation to accurately measure elastic modulus of leukemia cells K562 after phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRA), Cytoxan (CTX), and Dexamethasone (DEX) treatment, respectively. We found that compared to control sample, K562 cells treated by PMA showed nearly a threefold increase in elastic modulus. Transwell experiment results suggested that the K562 cells treated with PMA have the lowest migration capability. Besides, it was shown that the cytoskeleton protein gene α-tubulin and vimentin have a significant increase in expression after PMA treatment by qPCR. The results indicate that PMA has a significant influence on protein expression, stiffness, and migration ability of the leukemia cell K562, and may also play an important role in the leukostasis in leukemia.

Actin cytoskeleton stiffness grades metastatic potential of ovarian carcinoma Hey A8 cells via nanoindentation mapping.

Recent studies have indicated that the nanoindentation measured stiffness of carcinoma adherent cells is in general lower than normal cells, thus suggesting that cell stiffness may serve as a bio-marker for carcinoma. However, the proper establishment of such a conclusion would require biophysical understanding of the underlying mechanism of the cell stiffness. In this work, we compared the elastic moduli of the actin cytoskeletons of Hey A8 ovarian carcinoma cells with and without metastasis (HM and NM), as measured by 2D atomic force microscopy (AFM) with low-depth nanoindentation via a rate-jump method. The results indicate clearly that HM cells showed lower actin cytoskeleton stiffness atop of their nucleus position and higher actin cytoskeleton stiffness at their rims, compared to NM cells, suggesting that the local stiffness on the cytoskeleton can reflect actin filament distribution. Immunofluorescence staining and scanning electron microscopy (SEM) also indicated that the difference in stiffness in Hey A8 cells with different metastasis is associated with their F-actin rearrangement. Finite-element modelling (FEM) shows that a migrating cell would have its actin filaments bundled together to form stress fibers, which would exhibit lower indentation stiffness than the less aligned arrangement of filaments in a non-migrating cell. The results here indicate that the actin cytoskeleton stiffness can serve as a reliable marker for grading the metastasis of adherent carcinoma cells due to their cytoskeleton change and potentially predicting the migration direction of the cells.

Cell-structure specific necrosis by optical-trap induced intracellular nuclear oscillation.

A drug-free procedure for killing malignant cells in a cell-type specific manner would represent a significant breakthrough for leukemia treatment. Here, we show that mechanically vibrating a cell in a specific oscillation condition can significantly promote necrosis. Specifically, oscillating the cell by a low-power laser trap at specific frequencies of a few Hz was found to result in increased death rate of 50% or above in different types of myelogenous leukemia cells, while normal leukocytes showed very little response to similar laser manipulations. The alteration of cell membrane permeability and cell volume, detected from ethidium bromide staining and measurement of intracellular sodium ion concentration, together with the observed membrane blebbing within 10min, suggest cell necrosis. Mechanics modelling reveals severe distortion of the cytoskeleton cortex at frequencies in the same range for peaked cell death. The disruption of cell membrane leading to cell death is therefore due to the cortex distortion, and the frequency at which this becomes significant is cell-type specific. Our findings lay down a new concept for treating leukemia based on vibration induced disruption of membrane in targeted malignant cells.

Direct microfabrication of oxide patterns by local electrodeposition of precisely positioned electrolyte: the case of Cu2O.

An efficient technique for writing 2D oxide patterns on conductive substrates is proposed and demonstrated in this paper. The technique concerns a novel concept for selective electrodeposition, in which a minimum quantity of liquid electrolyte, through an extrusion nozzle, is delivered and manipulated into the desired shape on the substrate, meanwhile being electrodeposited into the product by an applied voltage across the nozzle and substrate. Patterns of primarily Cu2O with 80~90% molar fraction are successfully fabricated on stainless steel substrates using this method. A key factor that allows the solid product to be primarily oxide Cu2O instead of metal Cu - the product predicted by the equilibrium Pourbaix diagram given the unusually large absolute deposition voltage used in this method, is the non-equilibrium condition involved in the process due to the short deposition time. Other factors including the motion of the extrusion nozzle relative to the substrate and the surface profile of the substrate that influence the electrodeposition performance are also discussed.

Characterizing the malignancy and drug resistance of cancer cells from their membrane resealing response.

In this report, we showed that two tumor cell characteristics, namely the malignancy and drug-resistance status can be evaluated by their membrane resealing response. Specifically, membrane pores in a number of pairs of cancer and normal cell lines originated from nasopharynx, lung and intestine were introduced by nano-mechanical puncturing. Interestingly, such nanometer-sized holes in tumor cells can reseal ~2-3 times faster than those in the corresponding normal cells. Furthermore, the membrane resealing time in cancer cell lines exhibiting resistance to several leading chemotherapeutic drugs was also found to be substantially shorter than that in their drug-sensitive counterparts, demonstrating the potential of using this quantity as a novel marker for future cancer diagnosis and drug resistance detection. Finally, a simple model was proposed to explain the observed resealing dynamics of cells which suggested that the distinct response exhibited by normal, tumor and drug resistant cells is likely due to the different tension levels in their lipid membranes, a conclusion that is also supported by direct cortical tension measurement.

Mechanical oscillations enhance gene delivery into suspended cells.

Suspended cells are difficult to be transfected by common biochemical methods which require cell attachment to a substrate. Mechanical oscillations of suspended cells at certain frequencies are found to result in significant increase in membrane permeability and potency for delivery of nano-particles and genetic materials into the cells. Nanomaterials including siRNAs are found to penetrate into suspended cells after subjecting to short-time mechanical oscillations, which would otherwise not affect the viability of the cells. Theoretical analysis indicates significant deformation of the actin-filament network in the cytoskeleton cortex during mechanical oscillations at the experimental frequency, which is likely to rupture the soft phospholipid bilayer leading to increased membrane permeability. The results here indicate a new method for enhancing cell transfection.

Volumetric deformation of live cells induced by pressure-activated cross-membrane ion transport.

In this work, we developed a method that allows precise control over changes in the size of a cell via hydrostatic pressure changes in the medium. Specifically, we show that a sudden increase, or reduction, in the surrounding pressure, in the physiologically relevant range, triggers cross-membrane fluxes of sodium and potassium ions in leukemia cell lines K562 and HL60, resulting in reversible volumetric deformation with a characteristic time of around 30 min. Interestingly, healthy leukocytes do not respond to pressure shocks, suggesting that the cancer cells may have evolved the ability to adapt to pressure changes in their microenvironment. A model is also proposed to explain the observed cell deformation, which highlights how the apparent viscoelastic response of cells is governed by the microscopic cross-membrane transport.

A mathematical model on water redistribution mechanism of the seismonastic movement of Mimosa pudica.

A theoretical model based on the water redistribution mechanism is proposed to predict the volumetric strain of motor cells in Mimosa pudica during the seismonastic movement. The model describes the water and ion movements following the opening of ion channels triggered by stimulation. The cellular strain is related to the angular velocity of the plant movement, and both their predictions are in good agreement with experimental data, thus validating the water redistribution mechanism. The results reveal that an increase in ion diffusivity across the cell membrane of <15-fold is sufficient to produce the observed seismonastic movement.

Frequency-dependent cell death by optical tweezers manipulation.

Optical tweezers were used to scan individual Chronic Myelogenous Leukemia cells to determine if the cell death depends on the scanning conditions. Although increasing the scanning frequency or amplitude means greater force applied to the cells, their effects on cell death are not a simple increasing trend, as observed in the optical microscopy. Indeed, cell death sharply increased at particular screening frequencies and amplitudes, whereas other frequencies or amplitudes were less detrimental. These results suggest that cell damage was more sensitive to certain scanning conditions, rather than simply high-applied forces.

Compression-induced alignment and elongation of human mesenchymal stem cell (hMSC) in 3D collagen constructs is collagen concentration dependent.

Controlling cell organization is important in tissue engineering. Guidance by aligned features on scaffolds or stimulation by physical signals can be used to induce cell alignment. We have previously demonstrated a preferred alignment of human MSCs (hMSCs) along the compression loading axis in 3D collagen construct. In this study, we aim to investigate the collagen concentration dependence of the compression-induced hMSC organization. Results demonstrated that the compression-induced alignment and elongation of hMSCs exhibited a biphasic dose-dependent relationship with collagen concentration, and associated well with both collagen ligand density and elastic modulus of the constructs. Moreover, collagen concentration and compression loading significantly affected the expression level of integrin beta 1 and antibody neutralization against this molecule aborted the compression-induced alignment and elongation responses.

Hepatitis B surface antigen-antibody interactions studied by optical tweezers.

The protein-protein interactions between hepatitis B surface antigen (HBsAg) and its antibodies (anti-HBs) were studied by measuring the binding force between microspheres coated with such proteins using optical tweezers. The interaction force between the protein-coated microspheres was found to be strongly influenced by the acidity of the surrounding liquid medium, as well as the experimental temperature, and it reaches a maximum value at around pH 7.5 and temperature around 37°C. By measuring the protein distribution on the surfaces of the microspheres and their contact areas using scanning electron microscopy, the specific binding force between an HBsAg and anti-HBs protein pair is estimated to be around 4.8 pN at the optimum pH value and temperature at an applied loading rate of around 1 pN/s.

Reliable measurement of elastic modulus of cells by nanoindentation in an atomic force microscope.

The elastic modulus of an oral cancer cell line UM1 is investigated by nanoindentation in an atomic force microscope with a flat-ended tip. The commonly used Hertzian method gives apparent elastic modulus which increases with the loading rate, indicating strong effects of viscoelasticity. On the contrary, a rate-jump method developed for viscoelastic materials gives elastic modulus values which are independent of the rate-jump magnitude. The results show that the rate-jump method can be used as a standard protocol for measuring elastic stiffness of living cells, since the measured values are intrinsic properties of the cells.

Nano-scale structure and mechanical properties of the human dentine-enamel junction.

Despite being an interface between two mechanically mismatched phases of the soft dentine and hard enamel, the dentine-enamel junction (DEJ) in a human tooth is in general capable of withstanding a long working life of repeated dynamic loading. The current poor understanding of the structure and properties of the DEJ has presented a major obstacle to designing better therapeutic protocols for complications concerning the DEJ. In this investigation, it was discovered that the DEJ is a thin, but gradual interface with characteristics transiting from those of dentine to those of enamel. The collagen fibres in dentine enter into the enamel side of the DEJ and terminate in a region in which the hydroxyapatite crystals begin to show enamel characteristics. Using focused ion beam machining, micro-beams were fabricated from regions within 50 µm of the DEJ and were subjected to bend tests. In spite of the similarity in the flexural strength of the DEJ and enamel, fractographs revealed cracks in the DEJ that propagated along structures with dentine characteristics. To the best of our knowledge, this is the first report on the testing of the mechanical properties of the DEJ.

Degraded prism sheaths in the transition region of hypomineralized teeth.

OBJECTIVES: Failure of the enamel adjacent to the defects in teeth with molar incisor hypomineralization (MIH) limits the success rate of the restorations placed in these teeth and this frequently leads to their ultimate extraction. To understand the cause, a state-of-the-art combination of focused ion beam (FIB) and nanoindentation techniques was used to evaluate the fracture properties and microstructure of enamel from specific regions of two MIH teeth. METHODS: Nanoindentation, bend tests on micro-cantilevers and transmission electron microscopy (TEM) were employed to compare the microstructure and mechanical properties of the unaffected, opaque and transitional region in two MIH teeth. Special attention was paid to the transitional region in all the experiments in an attempt to identify its role in affecting the overall integrity of the MIH teeth. RESULTS: The enamel in the transitional region, despite its translucent appearance under the naked eye, was found, under TEM, to have prism sheaths that were significantly less mineralized than unaffected enamel and were proved to be weaker in holding the prisms together when measured using bend tests on micro-cantilever samples machined from the region. CONCLUSION: The enamel in the transitional region adjacent to the demarcated defects in MIH has notable alterations in their prism sheaths which likely contribute to their lowered mechanical properties.

Use of focused ion beam milling for investigating the mechanical properties of biological tissues: a study of human primary molars.

In this paper, the usefulness of the specimen shaping ability of focused ion beam (FIB) milling in the micrometer scale and the high force resolution of the nanoindentation technique are demonstrated on human primary teeth. Micro-cantilevers, with a triangular cross-section <5 microm in width and 10 microm in length, were produced within 50 microm of the dentin-enamel junction (DEJ) using FIB milling, and were point-loaded at their free ends at 20 microN/s until failure using a nanoindenter. The elastic modulus and flexural strength of such micro-samples of human enamel, and their variation with respect to prism orientation, were studied and compared to data from bulk enamel measured using nanoindentation and three-point bend tests. The elastic modulus of the micro-cantilever samples was found to be comparable to that obtained by nanoindentation on bulk samples, but it demonstrated significant anisotropy commensurate with the microstructure of enamel which was not measurable using nanoindentation on bulk samples. The flexural strength of the enamel micro-cantilevers also exhibited strong anisotropy, and was about one order of magnitude higher than that of bulk specimens measured by three-point bending. Through a Weibull analysis, this size dependence of the strength was found to be similar to the normal behaviour in brittle materials. The flexural strength of the enamel samples was also found to be sensitive to changes in the degree of mineralization of the samples.

A microplate compression method for elastic modulus measurement of soft and viscoelastic collagen microspheres.

Hydrogel-based microspheres are commonly used for drug and cell delivery in regenerative medicine. Characterization of their physical and mechanical properties is important in monitoring their quality during fabrication and in predicting their performance upon injection. However, existing methods have limitations in measuring these micron-sized, soft and viscoelastic spherical structures. In this study, a protocol is developed to measure the elastic modulus of non-linear viscoelastic spheres by microplate compression, and is applied to collagen microspheres fabricated with or without cells. During the measurement, a microsphere is placed on a rigid surface and is compressed by a calibrated flexible microplate gripped to a rigid end. A step increase in the displacement rate of the rigid end of the flexible microplate is introduced and the reduced elastic modulus of the microsphere is calculated from the deformation response of the microsphere, using an equation derived in this study. The reduced elastic modulus of collagen microspheres with and without mesenchymal stem cells measured by this method was 9.1 kPa and 132 Pa, respectively.

A method to quantitatively measure the elastic modulus of materials in nanometer scale using atomic force microscopy.

A method is proposed for quantitatively measuring the elastic modulus of materials using atomic force microscopy (AFM) nanoindentation. In this method, the cantilever deformation and the tip-sample interaction during the early loading portion are treated as two springs in series, and based on Sneddon's elastic contact solution, a new cantilever-tip property α is proposed which, together with the cantilever sensitivity A, can be measured from AFM tests on two reference materials with known elastic moduli. The measured α and A values specific to the tip and machine used can then be employed to accurately measure the elastic modulus of a third sample, assuming that the tip does not get significantly plastically deformed during the calibration procedure. AFM nanoindentation tests were performed on polypropylene (PP), fused quartz and acrylic samples to verify the validity of the proposed method. The cantilever-tip property and the cantilever sensitivity measured on PP and fused quartz were 0.514 GPa and 51.99 nm nA(-1), respectively. Using these measured quantities, the elastic modulus of acrylic was measured to be 3.24 GPa, which agrees well with the value measured using conventional depth-sensing indentation in a commercial nanoindenter.

Effect of weight-bearing on bone-bonding behavior of strontium-containing hydroxyapatite bone cement.

The purpose of this study was to investigate and compare the chemical composition and nanomechanical properties at the bone-cement interface under non-weight-bearing and weight-bearing conditions, in order to understand the effect of weight-bearing on the bone-bonding behavior of strontium-containing hydroxyapatite (Sr-HA) cement. In one group, Sr-HA cement was injected into rabbit ilium (under non-weight-bearing conditions). Unilateral hip replacement was performed with Sr-HA cement (under weight-bearing conditions) in the other group. Six months later, scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX) analysis and nanoindentation tests were conducted on the interfaces between cancellous bone and the Sr-HA cement. The nanoindentation results revealed two different transitional behaviors under different conditions. nder weight-bearing conditions, both the Young modulus and hardness at the interface were considerably higher than those at either the Sr-HA cement or cancellous bone. On the contrary, under non-weight-bearing conditions, both the Young modulus and hardness values at the interface were lower than those at the cancellous bone, but were higher than the Sr-HA cement. In addition, EDX results showed that the calcium and phosphorus contents at the interface under weight-bearing conditions were considerably higher than those under non-weight-bearing conditions. The differences in chemical composition and nanomechanical properties at the cement-bone interface under two different conditions indicate that weight-bearing produces significant effects on the bone-bonding behavior of the Sr-HA cement.