An impedance-based cell contraction assay using human primary smooth muscle cells and fibroblasts.

INTRODUCTION: Many cell types (including muscle cells and fibroblasts) can contract at physiological conditions and their contractility may change during tissue injury and repair or other diseases such as allergy and asthma. The conventional gel contraction assay is commonly used to monitor the cellular contractility. It is a manual assay and the experiment usually takes hours even days to complete. As its readout is not always accurate and reliable, the gel contraction assay is often used to qualitatively (but not quantitatively) characterize cellular contractility under various conditions. METHOD: To overcome the limits of the gel contraction assay, we developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technology utilizes special 96-well E-plates with gold microelectrode arrays printed in individual wells to monitor cellular adhesion by recording the electrical impedance in real time. The impedance change (percentage vs. control) can be used as the readout for cellular contraction. RESULTS: We demonstrated that the impedance-based contraction assay can be performed within 2h. Using this new method, we quantitatively characterized the effects of several contractile stimulators and inhibitors on human primary bronchial smooth muscle cells and primary lung fibroblasts. DISCUSSION: The impedance-based contraction assay can be applied to both basic research and drug discovery for characterizing cellular contraction quantitatively. Because it has high throughput capacity and high reproducibility, the impedance-based contraction assay is useful for high throughput functional screening in drug industry.

Sensing bacterial vibrations and early response to antibiotics with phase noise of a resonant crystal.

The speed of conventional antimicrobial susceptibility testing (AST) is intrinsically limited by observation of cell colony growth, which can extend over days and allow bacterial infections to advance before effective antibiotics are identified. This report presents an approach for rapidly sensing mechanical fluctuations of bacteria and the effects of antibiotics on these fluctuations. Bacteria are adhered to a quartz crystal resonator in an electronic bridge that is driven by a high-stability frequency source. Mechanical fluctuations of cells introduce time-dependent perturbations to the crystal boundary conditions and associated resonant frequency, which translate into phase noise measured at the output of the bridge. In experiments on nonmotile E. coli exposed to polymyxin B, cell-generated frequency noise dropped close to zero with the first spectra acquired 7 minutes after introduction of the antibiotic. In experiments on the same bacterial strain exposed to ampicillin, frequency noise began decreasing within 15 minutes of antibiotic introduction and proceeded to drop more rapidly with the onset of antibiotic-induced lysis. In conjunction with cell imaging and post-experiment counting of colony-forming units, these results provide evidence that cell death can be sensed through measurements of cell-generated frequency noise, potentially providing a basis for rapid AST.

Optical assessment of changes in mechanical and chemical properties of adipose tissue in diet-induced obese rats.

Obesity is becoming a leading cause of health problems world-wide. Obesity and overweight are associated with the structural and chemical changes in tissues; however, few methods exist that allow for concurrent measurement of these changes. Using Brillouin and Raman microspectroscopy, both the mechanical and chemical differences can be assessed simultaneously. We hypothesized that Brillouin spectroscopy can measure the adipose tissues' stiffness, which increases in obesity. Samples of brown and white adipose tissues obtained from control and diet-induced obese adult rats were analyzed. The results show that both adipose tissues of the obese group exhibit a greater high-frequency longitudinal elastic modulus than the control samples, and that the brown fat is generally stiffer than white adipose. The Raman spectra indicate that the lipids' accumulation in adipose tissue outpaces the fibrosis, and that the high-fat diet has a greater effect on the brown adipose than the white fat. Overall, the powerful combination of Brillouin and Raman microspectroscopies successfully assessed both the mechanical properties and chemical composition of adipose tissue simultaneously for the first time. The results indicate that the adipose tissue experiences an obesity-induced increase in stiffness and lipid content, with the brown adipose tissue undergoing a more pronounced change compared to white adipose.

Mechanical assessment of a hip joint stem model made of a PEEK/carbon fibre composite under compression loading.

PURPOSE: The aim of the work was to manufacture a composite stem model consisting of carbon fibres (CF) and polyether ether ketone (PEEK) and to perform the surface strain and stress distributions in the stem-femoral bone model under compression loading. METHODS: Composite stems differing in elasticity were prepared. Three types of composite stems having different arrangements of carbon fibre reinforcements (carbon fibre roving, carbon fibre sleeves and their combinations) in the polymer matrix were made. The stems were cementless fixed in the femoral bone model channel or with the use of the polymer bone cement (PMMA). Mechanical behaviour of composite stems under compression loading was compared with a metallic stem by strain gauge measurements at different parts of stem/bone model systems. RESULTS: The values of stresses in the proximal part of the bone model for cemented and cementless fixations of the composite stem in the femoral bone channel were higher than those noted for the metallic stem. The increase in proximal bone stress was almost similar for both types of fixation of composite stems, i.e., cemented and cementless fixed stems. CONCLUSIONS: The optimal range of mechanical stiffness, strengths and work up to fracture was obtained for composite stem made of carbon fibre sleeves and carbon fibres in the form of roving. Depending on the elasticity of the composite stem model, an increase in the stress in the proximal part of femoral bone model of up to 40% was achieved in comparison with the metallic stem.

Capsiate supplementation reduces oxidative cost of contraction in exercising mouse skeletal muscle in vivo.

Chronic administration of capsiate is known to accelerate whole-body basal energy metabolism, but the consequences in exercising skeletal muscle remain very poorly documented. In order to clarify this issue, the effect of 2-week daily administration of either vehicle (control) or purified capsiate (at 10- or 100-mg/kg body weight) on skeletal muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in mice. Mechanical performance and energy metabolism were assessed strictly non-invasively in contracting gastrocnemius muscle using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (31P-MRS). Regardless of the dose, capsiate treatments markedly disturbed basal bioenergetics in vivo including intracellular pH alkalosis and decreased phosphocreatine content. Besides, capsiate administration did affect neither mitochondrial uncoupling protein-3 gene expression nor both basal and maximal oxygen consumption in isolated saponin-permeabilized fibers, but decreased by about twofold the Km of mitochondrial respiration for ADP. During a standardized in vivo fatiguing protocol (6-min of repeated maximal isometric contractions electrically induced at a frequency of 1.7 Hz), both capsiate treatments reduced oxidative cost of contraction by 30-40%, whereas force-generating capacity and fatigability were not changed. Moreover, the rate of phosphocreatine resynthesis during the post-electrostimulation recovery period remained unaffected by capsiate. Both capsiate treatments further promoted muscle mass gain, and the higher dose also reduced body weight gain and abdominal fat content. These findings demonstrate that, in addition to its anti-obesity effect, capsiate supplementation improves oxidative metabolism in exercising muscle, which strengthen this compound as a natural compound for improving health.

The design and development of a high-throughput magneto-mechanostimulation device for cartilage tissue engineering.

To recapitulate the in vivo environment and create neo-organoids that replace lost or damaged tissue requires the engineering of devices, which provide appropriate biophysical cues. To date, bioreactors for cartilage tissue engineering have focused primarily on biomechanical stimulation. There is a significant need for improved devices for articular cartilage tissue engineering capable of simultaneously applying multiple biophysical (electrokinetic and mechanical) stimuli. We have developed a novel high-throughput magneto-mechanostimulation bioreactor, capable of applying static and time-varying magnetic fields, as well as multiple and independently adjustable mechanical loading regimens. The device consists of an array of 18 individual stations, each of which uses contactless magnetic actuation and has an integrated Hall Effect sensing system, enabling the real-time measurements of applied field, force, and construct thickness, and hence, the indirect measurement of construct mechanical properties. Validation tests showed precise measurements of thickness, within 14 µm of gold standard calliper measurements; further, applied force was measured to be within 0.04 N of desired force over a half hour dynamic loading, which was repeatable over a 3-week test period. Finally, construct material properties measured using the bioreactor were not significantly different (p=0.97) from those measured using a standard materials testing machine. We present a new method for articular cartilage-specific bioreactor design, integrating combinatorial magneto-mechanostimulation, which is very attractive from functional and cost viewpoints.

Assessment of modified gold surfaced titanium implants on skeletal fixation.

Noncemented implants are the primary choice for younger patients undergoing total hip replacements. However, the major concern in this group of patients regarding revision is the concern from wear particles, periimplant inflammation, and subsequently aseptic implant loosening. Macrophages have been shown to liberate gold ions through the process termed dissolucytosis. Furthermore, gold ions are known to act in an anti-inflammatory manner by inhibiting cellular NF-κB-DNA binding. The present study investigated whether partial coating of titanium implants could augment early osseointegration and increase mechanical fixation. Cylindrical porous coated Ti-6Al-4V implants partially coated with metallic gold were inserted in the proximal region of the humerus in ten canines and control implants without gold were inserted in contralateral humerus. Observation time was 4 weeks. Biomechanical push out tests and stereological histomorphometrical analyses showed no statistically significant differences in the two groups. The unchanged parameters are considered an improvement of the coating properties, as a previous complete gold-coated implant showed inferior mechanical fixation and reduced osseointegration compared to control titanium implants in a similar model. Since sufficient early mechanical fixation is achieved with this new coating, it is reasonable to investigate the implant further in long-term studies.

Effect of eldecalcitol, an active vitamin D analog, on hip structure and biomechanical properties: 3D assessment by clinical CT.

The effects of an active vitamin D analog, eldecalcitol (ELD), on bone mineral density (BMD), bone geometry, and biomechanical properties of the proximal femur were investigated by using clinical CT. The subjects--a subgroup of a recent randomized, double-blind study comparing anti-fracture efficacy of ELD with alfacalcidol (ALF) - constituted 193 ambulatory patients with osteoporosis (189 postmenopausal women and 4 men aged 52-85 years, average ± SD: 70.9 ± 6.92 years) enrolled at 11 institutions. Multidetector-row CT data was acquired at baseline and at completion of 144 weeks' treatment. Cross-sectional densitometric and geometric parameters of the femoral neck were derived from three-dimensional CT data. Biomechanical properties including cross-sectional moment of inertia (CSMI), section modulus (SM) and buckling ratio (BR) of the femoral neck, and CSMI of the femoral shaft were also calculated. We found that, (1) with respect to the femoral neck cross-sectional parameters (total bone), in the ALF group, volumetric BMD (vBMD) decreased but bone mass was maintained and cross-sectional area (CSA) increased. In contrast, ELD maintained vBMD with a significant increase in bone mass and a trend toward increased CSA. (2) With respect to the femoral neck cross-sectional parameters (cortex), cortical thickness decreased in the ALF group, but was maintained in the ELD group. In the ALF group, vBMD and bone mass increased, and CSA was maintained. In the ELD group, vBMD, CSA, and bone mass increased. (3) With respect to the biomechanical properties of the femoral neck, ELD improved CSMI and SM to a greater extent than did ALF. BR increased in both the ALF and ELD groups. (4) With respect to the femoral shaft parameters, overall the results of bone geometry and CSMI of the femoral shaft were very consistent with the results for the femoral neck; however, cortical vBMD of the femoral shaft decreased significantly in both the ELD and ALF groups. In conclusion, our longitudinal analysis of hip geometry by clinical CT revealed the unexpected potential of ELD to increase cortical CSA, vBMD, and bone mass, and to maintain cortical thickness, probably through the more potent effect of ELD in mitigating endocortical bone resorption than ALF. By improving the biomechanical properties of the proximal femur, ELD may have the potential to reduce the risk of hip fractures.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering.

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-ß1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-ß1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-ß1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

Mechano-functional assessment of human mesenchymal stem cells grown in three-dimensional hyaluronan-based scaffolds for cartilage tissue engineering.

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

DNA-DNA interactions in tight supercoils are described by a small effective charge density.

DNA-DNA interactions are important for genome compaction and transcription regulation. In studies of such complex processes, DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation. Commonly, a charge adaptation factor is used to address limitations of this theoretical approach. Despite considerable theoretical and experimental efforts, a rigorous quantitative assessment of this parameter is lacking. Here, we comprehensively characterized DNA-DNA interactions in the presence of monovalent ions by analyzing the supercoiling behavior of single DNA molecules held under constant tension. Both a theoretical model and coarse-grained simulations of this process revealed a surprisingly small effective DNA charge of 40% of the nominal charge density, which was additionally supported by all-atom molecular dynamics simulations.