Digital optical and scanning probe microscopy for biocells inspection and manipulation

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Nanobiotechnology and scanning probe microscopy (SPM) are new fields that are of interest to modern medical science. Nowadays atomic force microscopy (AFM) is widely used in medical-biological researches from the all variety of SPM. Without special methods of preparation AFM gives an opportunity to investigate the morphology of the surface of different biological objects with nanoresolution. Also this method allows to analyze the physical and mechanical properties at micro- and nanoscale. Our experimental complex with the functions of scanning probe and optical microscopy is intended for different materials investigation including biological cells. A special optical system makes it possible to visualize the objects position of the probe in the microscale. AFM is used for visualization and identification of the local adhesion and viscoelastic properties of biological cells. Dynamic laser speckle (DLS) is used for real-time monitoring of cells motility in living tissues. Another opportunity of this complex is some manipulation with the cell by means of the applied load variation. This technique greatly enhances the possibilities and opens a new field of experiments in cell biology. The purpose of this work is to show the possibility of AFM and DLS for studies of biological cells, namely measurement of the general cells motility in living tissues, the elastic modulus of the single cell membrane, as well as to identify the forces causing damage of the membrane.This study is partially funded by the National Academy of Sciences and Foundation for Basic Research of Belarus with grants and projects “ЭЭ 1.6.1”, T11MC-023, T10Р-029

Validation of an electrogoniometry system as a measure of knee kinematics during activities of daily living

Purpose: The increasing use of electrogoniometry (ELG) in clinical research requires the validation of different instrumentation. The purpose of this investigation was to examine the concurrent validity of an ELG system during activities of daily living. Methods: Ten asymptomatic participants gave informed consent to participate. A Biometrics SG150 electrogoniometer was directly compared to a 12 camera three dimensional motion analysis system during walking, stair ascent, stair descent, sit to stand, and stand to sit activities for the measurement of the right knee angle. Analysis of validity was undertaken by linear regression. Standard error of estimate (SEE), standardised SEE (SSEE), and Pearson’s correlation coefficient r were computed for paired trials between systems for each functional activity. Results: The 95% confidence interval of SEE was reasonable between systems across walking (LCI = 2.43 °; UCI = 2.91 °), stair ascent (LCI = 2.09 °; UCI = 2.42 °), stair descent (LCI = 1.79 °; UCI = 2.10 °), sit to stand (LCI = 1.22 °; UCI = 1.41 °), and stand to sit (LCI = 1.17 °; UCI = 1.34 °). Pearson’s correlation coefficient r across walking (LCI = 0.983; UCI = 0.990), stair ascent (LCI = 0.995; UCI = 0.997), stair descent (LCI = 0.995; UCI = 0.997), sit to stand (LCI = 0.998; UCI = 0.999), and stand to sit (LCI = 0.996; UCI = 0.997) was indicative of a strong linear relationship between systems. Conclusion: ELG is a valid method of measuring the knee angle during activities representative of daily living. The range is within that suggested to be acceptable for the clinical evaluation of patients with musculoskeletal conditions

Biomechanics

Biomechanics is a vast discipline within the field of Biomedical Engineering. It explores the underlying mechanics of how biological and physiological systems move. It encompasses important clinical applications to address questions related to medicine using engineering mechanics principles. Biomechanics includes interdisciplinary concepts from engineers, physicians, therapists, biologists, physicists, and mathematicians. Through their collaborative efforts, biomechanics research is ever changing and expanding, explaining new mechanisms and principles for dynamic human systems. Biomechanics is used to describe how the human body moves, walks, and breathes, in addition to how it responds to injury and rehabilitation. Advanced biomechanical modeling methods, such as inverse dynamics, finite element analysis, and musculoskeletal modeling are used to simulate and investigate human situations in regard to movement and injury. Biomechanical technologies are progressing to answer contemporary medical questions. The future of biomechanics is dependent on interdisciplinary research efforts and the education of tomorrow’s scientists

Intervertebral disc characterization by shear wave elastography: an in-vitro preliminary study

Patient-specific numerical simulation of the spine is a useful tool both in clinic and research. While geometrical personalization of the spine is no more an issue, thanks to recent technological advances, non-invasive personalization of soft tissue’s mechanical properties remains a challenge. Ultrasound elastography is a relatively recent measurement technique allowing the evaluation of soft tissue’s elastic modulus through the measurement of shear wave speed (SWS). The aim of this study was to determine the feasibility of elastographic measurements in intervertebral disc (IVD). An in-vitro approach was chosen to test the hypothesis that SWS can be used to evaluate IVD mechanical properties and to assess measurement repeatability. Eleven oxtail IVDs were tested in compression to determine their stiffness and apparent elastic modulus at rest and at 400 N. Elastographic measurements were performed in these two conditions and compared to these mechanical parameters. The protocol was repeated six times to determine elastographic measurement repeatability. Average SWS over all samples was 5.3 ± 1.0 m/s, with a repeatability of 7 % at rest and 4.6 % at 400 N; stiffness and apparent elastic modulus were 266.3 ± 70.5 N/mm and 5.4 ± 1.1 MPa at rest, respectively, while at 400 N they were 781.0 ± 153.8 N/mm and 13.2 ± 2.4 MPa. Correlations were found between elastographic measurements and IVD mechanical properties; these preliminary results are promising for further in-vivo application.The authors are grateful to the ParisTech BiomecAM chair program on subject-specific musculoskeletal modelling for funding (with the support of Proteor, ParisTech and Yves Cotrel Foundations)

A DIC based technique to measure the contraction of a skeletal muscle engineered tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response.The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based systemallows for an accurate and noninvasive measurement of biological tissues’ spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

elastography in primary open-angle glaucoma

Objective: The aim of this study was to compare sonoelastographic findings in the retina-choroid-sclera (RCS) complex and vitreous in glaucomatous and healthy eyes.Methods: For this cross-sectional comparative study, 20 patients with primary open-angle glaucoma and 20 healthy volunteers were recruited. Ultrasound elastography measurements were taken with a sonographic scanner of the RCS complex, anterior vitreous (AV), posterior vitreous (PV), retrobulbar fat tissue (RFT), optic disc (OD) and optic nerve (ON) in each eye.Results: The elasticity index of the RCS complex, RFT, OD, ON, AV and PV was similar in both groups (p > 0.05), although the AV/PV strain ratio in the group of patients with glaucoma was significantly higher (p = 0.04).Conclusion: Glaucoma increases the AV/PV strain ratio. In providing reproducible and consistent values, the real-time elastography (RTE) technique may be helpful in elucidating the mechanisms of glaucoma in some aspects.Advances in knowledge: This study can help to evaluate the elasticity of the RCS complex and vitreous in glaucomatous eyes with RTE

Hepatic steatosis and fibrosis: Non-invasive assessment

Chronic liver disease is a major cause of morbidity and mortality worldwide and usually develops over many years, as a result of chronic inflammation and scarring, resulting in end-stage liver disease and its complications. The progression of disease is characterised by ongoing inflammation and consequent fibrosis, although hepatic steatosis is increasingly being recognised as an important pathological feature of disease, rather than being simply an innocent bystander. However, the current gold standard method of quantifying and staging liver disease, histological analysis by liver biopsy, has several limitations and can have associated morbidity and even mortality. Therefore, there is a clear need for safe and noninvasive assessment modalities to determine hepatic steatosis, inflammation and fibrosis. This review covers key mechanisms and the importance of fibrosis and steatosis in the progression of liver disease. We address non-invasive imaging and blood biomarker assessments that can be used as an alternative to information gained on liver biopsy

Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial