A rate-jump method for characterization of soft tissues using nanoindentation techniques

The biomechanical properties of soft tissues play an important role in their normal physiological and physical function, and may possibly relate to certain diseases. The advent of nanomechanical testing techniques, such as atomic force microscopy (AFM), nano-indentation and optical tweezers, enables the nano/micro-mechanical properties of soft tissues to be investigated, but in spite of the fact that biological tissues are highly viscoelastic, traditional elastic contact theory has been routinely used to analyze experimental data. In this article, a novel rate-jump protocol for treating viscoelasticity in nanomechanical data analysis is described. © 2012 The Royal Society of Chemistry.postprin

Accurate measurement of tip-sample contact size during nanoindentation of viscoelastic materials

Polypropylene (PP) and amorphous selenium (a-Se) were used as prototype materials at room temperature to explore the problems that may exist in the accurate measurement of the reduced modulus of viscoelastic materials using depth-sensing nanoindentation. As has been reported previously by others, we observed that a "nose" in the load - displacement curve may occur during unloading, indicating significant creep effects at the onset of unloading. To accurately measure the elastic modulus in viscoelastic materials like PP or a-Se, both the contact stiffness and the contact area at the onset of unloading must be determined accurately. The issue of removing the influence of creep on the measurement of the contact stiffness using the Oliver - Pharr method has been addressed in a previous paper by Feng and Ngan. In this work, the effect of creep on contact-depth measurement is considered. Removal of creep effects in both contact stiffness and contact-area measurement leads to satisfactory prediction of the reduced moduli in PP and a-Se.published_or_final_versio

Viscoelastic effects during unloading in depth-sensing indentation

With polypropylene as a prototype viscoelastic material at room temperature, it was found that a "nose" may appear in the unloading segment of the load-displacement curve during nanoindentation when the holding time at peak load is short and/or the unloading rate is small, and when the peak load is high enough. The load at which the nose appears was also found to decrease linearly with decreasing unloading rate. A linear viscoelasticity analysis was performed to interpret this effect. The analysis predicts a linear variation between the nose load and the unloading rate, and the slope of such a linear variation is also shown to be proportional to the viscosity parameter of the material. Thus, by measuring the slope of the nose-load versus unloading rate plot at a given temperature, the viscosity parameter of the specimen can be found. This is a new way of measuring the viscosity parameter of a material in addition to the existing method of force modulation and noting the frequency response of the displacement.published_or_final_versio

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. © 2011 Elsevier Ltd.postprin

Accurate measurement of stiffness of leukemia cells and leukocytes using an optical trap by rate-jump method

Accurate measurement of the elastic modulus of soft biological cells in the micro/nano scale range is still a challenging task. Tests involving constant-rate loading often yield results that are rate dependent, due to the viscous component of the deformation. In this work, a rate-jump indentation method was employed in an optical tweezers system to measure the stiffness of non-adherent blood cells, which are the softest types of cells. Compared to the traditional Hertzian method of indentation, the rate-jump method is found to be able to yield invariant elastic modulus from K562 myelogenous leukemia cells. The optical tweezers indentation method proposed can therefore serve as a standard protocol for obtaining the intrinsic elastic modulus of extremely soft cells, with applied forces in the pico-newton range. This method is also found to be effective in grading the stiffness values of myelogenous leukemia cell lines (K562 and HL60) and normal leukocytes, indicating that it can be used to identify normal cells from diseased counterparts without biochemical analysis.postprin

The biomechanics of drug-treated leukemia cells investigated using optical tweezers

Leukemia is a very common cancer worldwide, and different drugs have been applied to treat the disease. However, the influence of the drugs on the biomechanical properties of leukemia cells, which are related to the risk of leukostasis, is still unknown. Moreover, accurate measurement of biomechanical properties of leukemia cells is still a challenging task because of their non-adherent nature and high sensitivity to the surrounding physiological conditions. In this study, a protocol to measure the biomechanical properties of leukemia cells by performing indentation tests using optical tweezers is proposed. The biomechanical properties of normal leukemia cells and cells treated with various cancer drugs, including phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRa), Cytoxan (CTX), and Dexamethasone (DEX), were measured. The adhesion between the cells and certain proteins existing in the extracellular matrix, i.e., fibronetin and collagen I, was also characterized with the help of a static adhesion assay. It was found that after treatment by ATRa, CTX, and DEX, the cells became softer, and the adhesion between the cells and the proteins became weaker. PMA treatment caused no change in the stiffness of the HL60 cells, but increased the stiffness of the K562 cells, and increased the cell–protein adhesion of both K562 cells and HL60 cells.postprin

An improved method for the measurement of mechanical properties of bone by nanoindentation

Nanoindentation is widely used to measure the mechanical properties of bio-tissues. However, viscoelastic effects during the nanoindentation are seldom considered rigorously, although they are in general very significant in bio-tissues. In this study, a recently developed method for correcting the viscoelastic effects during nanoindentation is applied to mice bone samples. This method is found to yield reliable elastic modulus and hardness results from forelimb and femur cortical bone samples of C57 BL/6N and ICR mice. The creep properties of the samples are also characterized by a novel procedure using nanoindentation. The measured mechanical properties correlate well with the calcium content of the bone samples. © 2007 Springer Science+Business Media, LLC.postprin

An alternative method for continuous property measurement during depth-sensing indentation

Based on the Oliver-Pharr framework, an iteration scheme is proposed to continuously evaluate the hardness during monotonic loading in nanoindentation. The new method was applied to measure the hardness of various crystalline materials including Al, Cu, Ni3Al(Cr,B) and MgO, where an obvious Indentation Size Effect (ISE) was observed. In a-Se and PP, no ISE was observed when using exponential loading schemes at constant strain rates. The new method therefore produced the correct ISE behaviours in various materials under different loading situations.postprin

Human papillomavirus-16/18 AS04-adjuvanted cervical cancer vaccine: Immunogenicity and safety in healthy Chinese women from Hong Kong

Objective To assess the immunogenicity and safety of human papillomavirus-16/18 AS04-adjuvanted cervical cancer vaccine in Chinese women aged 18 to 35 years enrolled from Hong Kong. Design Double-blind, randomised controlled trial with vaccine and placebo groups. Setting Single-centre study in Hong Kong. Participants Three hundred women enrolled (150 per group) between March 2006 and June 2007. Interventions Subjects received three doses of human papillomavirus-16/18 vaccine or placebo (aluminium hydroxide), administered intramuscularly at 0, 1, and 6 months. Main outcome measures Human papillomavirus-16/18 seroconversion rates and geometric mean titres at month 7 (in human papillomavirus-16/18 recipients); reactogenicity and safety (in all subjects). Results A total of 294 women completed the study (148 in the vaccine group, 146 in placebo group). All initially seronegative subjects in the vaccine group had seroconverted for human papillomavirus-16/18 antibodies by month 7. Anti-human papillomavirus-16 and anti-human papillomavirus-18 antibody geometric mean titres were 10 422 (95% confidence interval, 8730-12 442) EL.U/mL and 4649 (3975-5437) EL.U/mL, respectively. High compliance (99% in both groups) was observed for the three-vaccination course. The frequencies of local injection site reactions were higher in the vaccine than placebo group; pain being the most common symptom in both groups. Regarding solicited symptoms, fatigue and myalgia were the most frequent in both groups. Five serious adverse events (four in vaccine group, one in placebo group) were reported, but all were considered unrelated to the vaccinations. Conclusion The human papillomavirus-16/18 AS04-adjuvanted vaccine was highly immunogenic, safe, and generally well tolerated in Chinese women from Hong Kong.published_or_final_versio

AFM nanoindentation detection of the elastic modulus of tongue squamous carcinoma cells with different metastatic potentials

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