Molecular Biomechanics - American Society of Biomechanics [PDF]

Also discussed will be a product, based on biodegradable polymers, that has been used specifically to ...... Using short

4 downloads 5 Views 5MB Size

Recommend Stories


Molecular Biomechanics Laboratory
Live as if you were to die tomorrow. Learn as if you were to live forever. Mahatma Gandhi

Biomechanics
In the end only three things matter: how much you loved, how gently you lived, and how gracefully you

Biomechanics
What you seek is seeking you. Rumi

Tendon Biomechanics
If your life's work can be accomplished in your lifetime, you're not thinking big enough. Wes Jacks

PDF Download Applied Biomechanics
Love only grows by sharing. You can only have more for yourself by giving it away to others. Brian

PDF Introductory Biomechanics
We can't help everyone, but everyone can help someone. Ronald Reagan

Biomechanics of Stair Climbing
If your life's work can be accomplished in your lifetime, you're not thinking big enough. Wes Jacks

Spine Biomechanics
What we think, what we become. Buddha

spine biomechanics
If you are irritated by every rub, how will your mirror be polished? Rumi

Physics & Biomechanics
Learning never exhausts the mind. Leonardo da Vinci

Idea Transcript


Plenary Session I .................................................................................................................................................................... 4 Plenary Session 2 ................................................................................................................................................................... 5 Plenary Session 3 ................................................................................................................................................................... 6 Plenary Session 4 ................................................................................................................................................................... 8 Plenary Session 5 ................................................................................................................................................................... 9 Plenary Session 6 ................................................................................................................................................................. 11 Plenary Session 7 ................................................................................................................................................................. 13 Plenary Session 8 ................................................................................................................................................................. 14 1-1

Protein Mechanics ........................................................................................................................................... 15

1-3

Cell response to mechanical stress .................................................................................................................. 20

2-1

Molecular and Cellular Exp Tools ..................................................................................................................... 24

2-3

Cell mechanics and cell function ...................................................................................................................... 29

3-1

Nucleic Acid Nanostructures ............................................................................................................................ 33

3-2

Molecular mechanisms of biological lubrication I ........................................................................................... 38

3-3

Mechano-sensitive signaling pathways I ......................................................................................................... 43

4-1

DNA Mechanics and Assembly......................................................................................................................... 47

4-2

Molecular mechanisms of biological lubrication II .......................................................................................... 50

4-3

Mechano-sensitive signaling pathways II ........................................................................................................ 56

5-1

Mechanics of the Nuclear Pore and Nucleocytoplasmic Transport................................................................. 61

5-2

Duling Memorial: Cancer Metastasis and the Glycocalyx II ............................................................................. 66

5-3

Cellular Mechanotransduction ........................................................................................................................ 72

6-1

Mechanics of biomolecular complexes............................................................................................................ 76

6-2

Duling Memorial: Cancer Metastasis and the Glycocalyx II ............................................................................. 80

6-3

Cell-substrate interaction I .............................................................................................................................. 85

7-1

Design, fabrication and analysis of hierarchical biomaterials ......................................................................... 90

7-2

Acto-myosin Mechanobiology I ...................................................................................................................... 95

7-3

Cell-substrate interaction II ............................................................................................................................. 99

8-1

Bio-inspired Manufacturing ........................................................................................................................... 103

8-2

Acto-myosin Mechanobiology II ................................................................................................................... 107

8-3

Cell-substrate interaction III .......................................................................................................................... 112

9-1

Bio-inspired Materials from Nanostructures I ............................................................................................... 117

9-2

Engineering Molecular Mechanics with Synthetic Biology I .......................................................................... 121

9-3

Biophysical aspects of cell/cell adhesion ....................................................................................................... 125

10-1

Bio-inspired Materials from Nanostructures II .............................................................................................. 131

10-2

Engineering Molecular Mechanics with Synthetic Biology II ......................................................................... 136

10-3

Cell/cell adhesion and cell rheology .............................................................................................................. 142

11-1

Nanomechanics of the cellular microenvironment ....................................................................................... 146

11-2

Single molecule mechanics of motor proteins and motor assemblies I ........................................................ 150

11-3

Mechanotransduction at the Focal Adhesions .............................................................................................. 153

12-1

Molecular Brushes: models and experiments ............................................................................................... 157

12-2

Single molecule mechanics of motor proteins and motor assemblies II ....................................................... 162

12-3

Molecular adhesion ....................................................................................................................................... 167

12-12

Multi-scale Modeling in Cardiovascular......................................................................................................... 169

13-1

Nanostructured biomaterials ......................................................................................................................... 173

13-2

Mechanics of weak protein-ligand interaction: experiments and modeling I ............................................... 176

13-3

Subcellular biophysics and mechanosensing ................................................................................................. 180

14-2

Mechanics of weak protein-ligand interaction: experiments and modeling II .............................................. 186

14-3

Measurements and models for cell-ECM interactions .................................................................................. 191

15-1

Enhanced Imaging and Treatment with Nanoparticles ................................................................................. 195

15-2

Implications for Flow on Cell Adhesion and Drug Delivery ............................................................................ 198

15-3

Cytoskeletal mechanics and physics of adhesion .......................................................................................... 203

16-1

Micro/Nano Technology in Cryopreservation ............................................................................................... 211

16-2

CNS transport and drug delivery: Experimental ............................................................................................ 215

16-3

Cytoskeletal mechanics and physics of adhesion .......................................................................................... 219

17-1

Novel devices and modeling for nanoparticle and cell transport in biological systems - Portonovo Ayyaswamy 70th Birthday Tribute Special Sessions I .................................................................................... 223

17-2

CNS transport and drug delivery: Modeling .................................................................................................. 227

17-3

Cytoskeletal mechanics and physics of adhesion .......................................................................................... 233

18-1

Functional micro/nanodevices for quantitative cell and tissue mechanics measurements ......................... 237

18-2

Molecular Imaging and Therapeutic Approaches .......................................................................................... 240

18-3

Prenatal Skeletal Development: Mechanobiology and Mechanotransduction ............................................. 245

19-1

Biophysical regulation of cell reprogramming and directed differentiation using micro/nanostructured surfaces ..................................................................................................................... 252

19-2

SMART BioSym Session I: Biofilm Ecomechanics ........................................................................................... 256

19-3

Computational modeling of cellular cytoskeletal mechanics 1 .................................................................... 258

20-1

Nano and Mesoscale Organization and Behavior of Biomolecular Materials I ............................................. 262

20-2

SMART BioSyM Session II: Stem Progenitor Cell Chemomechanics ............................................................. 266

20-3

Computational modeling of cellular cytoskeletal mechanics II .................................................................... 270

21-1

Nano and Mesoscale Organization and Behavior of Biomolecular Materials II ............................................ 275

21-2

SMART BioSyM Session III: Stem Progenitor Cell Chemomechanics II ......................................................... 280

21-3

Computational modeling of cellular cytoskeletal mechanics III ................................................................... 283

22-1

Molecular Design and Nanomechanics of Biomimetic Materials and Adhesives .......................................... 288

22-2

SMART BioSyM Session IV: Cancer Anti-Metastasis ...................................................................................... 293

22-3

Computational modeling of cellular cytoskeletal mechanics IV ................................................................... 297

POSTERS

303

Plenary Session I From matrix mechanics to nuclear mechanics D. E. Discher; University of Pennsylvania, Philadelphia, PA. Tissues can be soft like brain or fat, which bear little stress, or rigid like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, an unbiased proteomics approach revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

Plenary Session 2 Noninvasive Functional Assessment of Coronary Artery Disease using Cardiac CT Imaging and Computational Fluid Dynamics C. Taylor; HeartFlow, Inc., Redwood City, CA. Atherosclerosis in the coronary arteries is the cause of nearly one-third of all global deaths. The severity of coronary artery disease and the consequent effect on blood flow to the heart are difficult to measure, yet this information is critical for treating patients. Currently, the gold-standard for assessing the functional significance of coronary artery disease, fractional flow reserve (FFR) involves invasive measurement of pressure in the coronary arteries at the time of diagnostic cardiac catheterization. FFRCT is a noninvasive technology whereby patient-specific models of blood flow are constructed from coronary CT angiography (cCTA) data and used to provide data to physicians related to the functional significance of coronary artery disease. FFRCT requires an accurate segmentation of the coronary artery lumen from cCTA data, but additionally, it leverages established biologic principles relating form (anatomy) to function (physiology). Finally, FFRCT exploits recent advances in computational fluid dynamics to solve the governing equations of blood flowing in the coronary arteries. Ultimately, any theoretical model must be tested against experiment and the data must be the final arbiter of model validity. FFRCT has been evaluated against measured FFR data in three diagnostic accuracy trials to date. Most recently, the NXT trial has demonstrated accuracy against measured FFR data in 254 patients and 484 vessels not only significantly exceeding that of cCTA alone, but superior to that reported for any other noninvasive diagnostic test when compared to measured FFR as the reference standard. In a demonstration of the expected clinical use of the technology, FFRCT correctly reclassified 68% of coronary CTA false positive patients and 67% of coronary CTA false positive vessels as true negatives. The clinical use of this technology could substantially improve the identification of patients that would benefit from coronary stenting and hence should be referred to invasive cardiac catheterization. Clinical trials examining the impact of FFRCT on clinical outcomes and costs are underway. At present, FFRCT is being used for clinical decision making in parts of Europe and Asia and FDA Clearance is pending in the U.S. Ultimately, tens of millions of patients worldwide could benefit from this technology.

Plenary Session 3 Microrheology of living cells B. Fabry; University of Erlangen-Nuremberg, Erlangen, GERMANY. The elastic, viscous, or plastic behavior of living cells is important for force generation, migration, division, growth, and responses to mechanical stimulation. Cell mechanical measurements, when carried out over a frequency range between 0.01 Hz and 1 kHz, universally and regardless of cell type and measurement method show that cell elasticity (the storage modulus G’) increases weakly with frequency according to a power-law. Dissipative properties (the loss modulus G”) are smaller but also increase weakly with frequency according to the same power-law. Moreover, pharmacological interventions may change the exponent of the power-law, but generally do not change the absolute value of G’ and G” at a cell-type specific characteristic high frequency. The molecular origin of this frequency-scale invariant behavior is currently debated, although actin-myosin interactions and other metastable crosslinks and bonds within the disordered cytoskeleton can explain the absence of a characteristic frequency scale. Moreover, actin-myosin interactions link another universally observed cell behavior to cell rheology: Cell stiffness increases linearly with the contractile prestress of the cytoskeleton. When external forces that are applied to the cell lead to a further increase of the mechanical stress within the cytoskeleton, the cell stiffness increases linearly with the total cytoskeletal stress. It follows that single cells show an exponential stress-strain behavior, which is also observed in many tissues of the human body. In addition to stiffness, also the power law exponent increases with force, indicating a simultaneous fluidization of the cytoskeleton. The amount of stress stiffening and fluidization scales with the contractile prestress as the only free parameter. Therefore, by modulating the internal mechanical tension, cells can actively control their mechanical properties over a large range. This behavior is of fundamental importance for protection against damage caused by large external forces, and allows the cells to adapt to the highly variable and nonlinear mechanical properties of the extracellular matrix.

To Infinity and Beyond: Musculoskeletal Biomechanics in the Age of the Virtual Physiological Human M. Viceconti; Department of Mechanical Engineering and Insigneo Institute for in silico Medicine, University of Sheffield, UNITED KINGDOM. Reductionism is the unavoidable necessity imposed by the investigation of an infinitely complex reality with a finite cognitive capacity. But biological systems are deeply entangled and thus too frequently by focusing our attention to a specific space-time scale we loose an essential systemic determinant of the process under investigation. But if we can observe that process at multiple space-time scales, develop reductionist mechanistic theories for each of them, and capture these into computer models, we can in principle re-compose the systemic behaviour by creating hypermodels, orchestrations of multiple models each capturing a distinct reductionist perspective of the same process. The Virtual Physiological Human (VPH) is a framework of methods and technologies that enable this integrative investigation of living organisms. While the development of such framework poses huge challenges, the preliminary results produced worldwide in the last ten years confirm that this approach is having a transformational impact on biomechanics research, and on its ability to translate into the clinical practice. The musculoskeletal system primary function is biomechanical in nature; thus it is not a surprise that physics-based multiscale modelling results particularly effective in this context. From a heterogeneous collection of patient’s data that include clinical data, medical imaging, gait analysis, wearable sensing, and lab exams, it is possible to build patient-specific hypermodels that can be used for diagnosis, prognosis, or treatment planning [1]. A typical example is the technology developed by the VPHOP consortium, funded by the European Commission, where such approach is used to predict whose patients are at higher risk of low-energy bone fractures, due to an increased propensity to fall and overload, and to a progressive reduction of bone mass; in two independent clinical studies recently completed, we showed that such integrative approach can improve the predictive accuracy of at least 10% with respect to the current standard of care. Now this approach is used in our group to optimise spine surgery, to explore the role of biomechanical determinants in the onset and severity of juvenile idiopathic arthritis, or in better recognising abuserelated fractures in babies, just to name a few applications. Other applications are the Digital Mouse, where the same approach is used to individually model mice in order to reduce, refine, and partially replace animal experiments, or Population Modelling, where hypermodels are used within a stochastic framework to answer more basic research questions. We conclude that the VPH will have a transformational impact on biomechanics.

Plenary Session 4 Translating Biomechanics Research into Clinical Practice M. J. Pearcy; Queensland University of Technology, Brisbane, AUSTRALIA. There is a large amount of research conducted each year examining every aspect of the mechanics of the human body and its interaction with medical devices and the environment; from the cellular level through to the whole body. While, as researchers, we obtain great pleasure from conducting studies and creating new knowledge we need to keep in mind that while this is a good thing it is even better if this new knowledge can lead to improvement in the quality of life for individuals suffering from biomechanical disorders. There are examples of the successful translation of biomechanical research into clinical practice but if we consider the number of individuals and institutions involved in research then it is legitimate to question what the benefit really is. In reviewing outcomes of research it is important to consider not only the relatively few commercial successes but also evidence for the benefit to clinical practice more generally. It becomes clear that research often leads to incremental improvements in design and service provision as researchers work with clinicians to understand and solve their day-to-day problems. We have available many sophisticated tools to analyse biomechanical issues and these can be used to provide evidence for clinicians to change their practice to improve outcomes for individual patients and for companies to modify the design of their devices so that they are available for more diverse populations. For example: modern mechanical testing methods combined with modelling techniques can assist engineers and surgeons in designing total joint replacement components that will suit different populations; and three dimensional reconstructions from medical imaging and finite element studies can assist spinal surgeons to plan spinal deformity correction surgery. In addition, quite simple biomechanical analyses can provide answers that assist clinical practice by clarifying why devices fail the way they do and enable changes to surgical techniques to be developed to reduce the likelihood of failure. So sometimes relatively simple mechanical analyses can provide important insights into clinical problems and suggest effective solutions. In summary, when considering translating biomechanical research into clinical practice, while commercialisation is a good aim not all research leads to marketable outcomes, however, it can lead to improvements in surgical techniques and clinical practice. It is important for us to identify and promote how the outcomes of research lead to improvements in quality of care, as this is perhaps the most important outcome for individual patients.

Plenary Session 5 Establishing a Translational Pathway in Biomechanics K. A. Athanasiou; Univ of California Davis, Davis, CA. This presentation will cover two different areas in biomechanics: Articular cartilage healing and technology translation. Articular cartilage is possibly the tissue most pivotal for motion and overall function. The demanding biomechanical milieu of a joint, plus cartilage’s relative lack of cells and blood supply, renders this tissue almost unique in its inability to repair adequately. If one compares cartilage to bone, one will realize that, unlike the former, bone can mount a robust repair response that often results in successful healing (1). This presentation will describe our group's efforts toward helping joint cartilages heal via tissue engineering approaches employing biomechanical relationships established at multiple dimensional levels. The second part of the presentation will be devoted on efforts to commercialize outcomes of our academe-based research, as outlined in the schematic diagram below. Specific examples and results will be presented to illustrate a specific pathway of commercializing research outcomes. Of particular interest will be a product to deliver drugs through bone to patients with compromised peripheral blood supply. Also discussed will be a product, based on biodegradable polymers, that has been used specifically to fill small defects in musculoskeletal tissues. Throughout the presentation, lessons learned from the translational process will be discussed along with efforts to portray the entrepreneurial world from an academic's perspective. Seeing one’s technology to full commercialization is exceedingly exciting when it works and quite demoralizing when it does not. Central to translational efforts is, of course, the development of exciting, innovative, and useful work; also central are perseverance, stubbornness, and a general philosophy of eschewing traditional approaches.

Variety of Molecular and Biophysical Mechanisms Underlying Cell Mechanosensing M. Sokabe; Nagoya Unversity Graduate School of Medicine, Nagoya, JAPAN. Mechanosensing is an indispensable function to support the life of organisms from bacteria to human. Virtually every cell can respond to mechanical stimuli, by which cells can regulate their shape, proliferation, differentiation, survival and migration. In the past decade a variety of mechanosensor molecules have been identified, including mechanosensitive ion channels (MSCs) and non-channel type mecahnosensors such as talin, integrin and actin cytoskeletons. This paper deals with three topics 1) dynamic structure-function of the bacterial MSC MscL, 2) a mecahnosensing complex in eukaryotic cells that can act as a sensor for environmental stiffness, and 3) actin fibers working as a negative tension sensor to regulate cell shape and migration. MscL is the best studied MSC owing to its resolved 3 D crystal structure of the closed channel. It is activated simply by tension in the membrane, contributing to the cell volume regulation against hypoosmotic challenge. However, the biophysical mechanism underlying the channel opening process is unknown. We first identified experimentally the potential amino acids for the tension sensor and the gate of MscL, then conducted molecular dynamic simulations for channel opening, focusing on the relationship between the sensor and gate. We found a critical structural change, a breakdown of alfa-helix near the gate, which triggers channel opening in response to increased membrane tension. Eukaryotic MSCs seem to be activated mainly by tension in the actin cytoskeleton stress fiber (SF) that terminates at the focal adhesions (FAs). In the case of a Ca2+-permeable MSC in endothelial cells, forces conducting along the SF/FA complex activate the MSCs within FAs, causing intracellular Ca2+ increases with a surprisingly high sensitivity to mechanical force as low as 1 pN. The SF/FA/MSC complex has another important function called “active-touch sensing” in which contractile forces in SFs pull the cell substrate via FAs to activate MSCs. As tension and/or strain in the SF/FA depend on substrate stiffness, the MSCs can transduce substrate stiffnesses into intracellular Ca2+ levels. It is known that intra- and extracellular forces affect the dynamics of actin cytoskeletons, raising a possibility that they can be a mechanosensor. We found that tension in an actin fiber (AF) protect it from severing by ADF/cofilin. By contrast, relaxed AFs were easily severed through tension-dependent binding/unbinding of cofilin to AFs. In other words, tension in the AF regulates the apparent severing activity of cofilin, thus AFs can be called as a negative tension sensor.

Plenary Session 6 Powered Upper Limb Prostheses: State-of-the-Art and Clinical Challenges K. Englehart; University of New Brunswick, Fredericton, NB, CANADA. Artificial limbs have provided cosmetic and functional replacements for those with deficiencies due to congenital defect or traumatic injury for many years. The first electrically powered prostheses became available in the 1950’s, which was a significant advance in usability. A further advance in functionality came in the 1960’s, when the first control system using signals from remaining muscles was developed. This form of control, using the myoelectric signal, provides a user with a self-contained, autonomous means of controlling a powered prosthesis. The past decade has seen the development of powered upper limbs that have dramatically improved speed and dexterity. The impact of these devices upon usability and enhanced function has been limited by the need for a better man-machine interface to impart user intent. This has motivated intense research in novel methods of accessing motor intent from the central nervous system, including cortical and peripheral nerve interfaces. These invasive approaches hold promise, but require solving considerable medical and technical challenges before they are viable solutions. The most practical solution in the near future remains using the myoelectric signal. The adoption of targeted muscle reinnervation (TMR), a procedure in which the brachial nerves are transferred to residual muscles in an amputee, allows the restoration of absent neural pathways. Patients can then contract the reinnervated muscles by attempting to move their missing limb. TMR, when combined with advanced pattern recognition methods, can enable intuitive control of many degrees of freedom using the myoelectric signal from reinnervated sites. This is particularly advantageous in individuals with highlevel amputation. This seminar will describe the evolution of myoelectric control to its current state-of-the-art. This will be set in the context of major new initiatives in the field, including breakthroughs in medical science, signal processing, and robotics.

Design, Development and Evaluation of Innovative Fusion Augmenting Spinal Hardware V. K. Goel; University of Toledo, Toledo, OH. Introduction: Chronic lower back pain patients who don’t respond to conservative therapies may require surgical intervention. Surgery may range from procedures like discectomy, fusion with instrumentation, to application of motion preservation systems (e.g., artificial discs). At present, in the US, clinical outcomes of the motion preservation devices are suboptimal. In addition, due to the increase in the elderly population, shift to minimal invasive surgical procedures-MIS, changes in the health care laws, and growing medical-markets in developing nations, the stake holders are once again innovating fusion augmenting spinal implants. The talk presents our efforts to assess the technologies over the years, using state-of-art test protocols. Methods: Besides the bench type and other tests to obtain FDA approval and CE Mark in EU, we have developed clinically relevant in-vitro protocols to evaluate an implant’s ability to reduce/restore motion across the decompressed and adjacent segments. Finite element models were developed to predict load sharing, and stresses/strains within the device and other spinal structures (Goel et al., 2006). Cages of various shapes and sizes, including expandable cages, posterior and anterior instrumentation, dynamic systems such as artificial discs (fabricated from stainless steel, titanium, PEEK, and bioresorbable materials) were evaluated. More recent testing involved spinal implants suitable for MIS. Results: Cages alone didn’t reduce the motion across the index segment as effectively as cages supplemented with additional posterior/anterior instrumentation - 360 fusion systems. More recent expandable cage concepts were found to reduce motion to a level justifying their use without additional instrumentation. Biomechanics of posterior dynamic systems, with a few exceptions, was similar to fusion devices. Artificial discs restored the motion to normal values. Discussion: Biomechanics of instrumentation support the in vivo data that devices do facilitate fusion. However, some patients still experience pain, thus not a clinical success. Motion restoring devices, including artificial discs, were thus developed. In-vitro and in-silico data predict these devices do maintain alignment and restore motion to normal values. However, clinical follow up data, to a large extent, reveal that these devices also lead to reduction in motion. The adjacent segment degeneration rates are within the range of the fusion devices as well. Hence, the shift back to the development of fusion devices suitable for MIS. Biomechanics of these devices is similar to their counter parts used for open surgery.

Plenary Session 7 Combined Mechanical and Biologic approach to Improve Integrity of Bone-Implant interface and increase longevity of Orthopaedic Joint Replacement Implants J. E. Bechtold1, C. Goreham Voss2, P. Swider3, J. Baas4, K. Soeballe4; 1 University of Minnesota and Minneapolis Medical Research Foundation, Minneapolis, MN, 2University of Minnesota, Minneapolis, MN, 3University of Toulouse, Toulouse, FRANCE, 4Aarhus University, Aarhus, DENMARK. Total Joint Replacement (TJR) has one of the highest quality of life improvements per health care dollar spent, and currently a combined 1 million hips and knees are implanted in the United States annually. Given increased lifespans and younger age at implantation, the pool of patients with functioning implants is growing. While most implants work well (5-10% failure at 15 years), the larger pool of implants is leading to a larger number of revisions for failed implants. In some hospitals revisions comprise from 10-30% of all TJR surgeries. For over two decades, our laboratory has studied the role of mechanical loading and implant stability (lack of relative motion) in combined experimental and computational approaches, with a focus on the revision implant. This presentation will present a synopsis of our experimental findings and focus on two of our mechanical approaches. Both approaches focus on evaluating the integrity of the bone implant interface. Osseointegration, or direct bone ingrowth into an implant surface is a critical factor in the long-term function of these implants. Proper implant fixation prevents implant migration and loosening leading to failure. Static pushout tests of implants excised from animal studies are a common way to assess the fixation strength of various experimental implant coatings and surgical techniques. However, these tests are costly, timeconsuming, and destructive. In this talk we will discuss a method developed to create computational models of implant fixation, in order to complement physical pushout tests. Another approach is to evaluate the fixation of the implants with dynamic loading, to differentiate differences in early soft tissue healing that could lead either to secure bone ingrowth or to persistence of fibrous tissue. The following figure is an example of a micro-CT derived bone-implant specimen for pushout simulation.

Plenary Session 8 Unraveling large-strain structure-function relationships in the joint and spine tissues N. D. Broom; University of Auckland, Auckland, NEW ZEALAND. Most conventional engineering materials are utilized primarily for their intrinsic strength and high elastic stiffness, and over a strain range that involves minimal structural rearrangement. The strains in these solid materials over the normal limits of elastic loading (25% to 3.5x10^-4 in response to low shear stress ( 20 kPa) exhibited upregulation of Runx2, type I collagen, and alkaline phosphatase (ALP) mRNA. Additionally, ALP enzyme activity was increased with an increased matrix stiffness compared to lower stiffnesses (4.5-19 kPa). An initially high cell density (30×106 cells/mL) resulted in a decrease in the number of cells, while a low cell density (1×106 cells/mL) resulted in an increase. The decrease in cell number per bead over time in the highest seeding density is likely due to cell migration and / or leaching from the alginate beads. The increasing trend in cell number at the lowest seeding density likely suggests that there may be an optimum number of cells maintained in alginate beads in in vitro culture. Seeding of cells at a low density also resulted in an increase in the expression of mRNA osteogenic markers and ALP enzyme activity compared to the higher cell densities. Together, the results of the present study with MSC in 3D culture are in agreement with previous studies investigating the influence of these factors on MSC differentiation in 2D cultures. Specifically, a high matrix stiffness and low cell density lead to increased osteoblast differentiation through the early upregulation of osteogenic markers. It can be speculated that the physical environment influences the integrin binding and associated change in cytoskeleton, influencing the subsequent MSC differentiation. However, further investigation is necessary to elucidate the exact mechanisms involved.

2-1

Molecular and Cellular Exp Tools

Development of High Throughput Single Molecule Force Spectroscopy using DNA Nanotechnology R. Patton, E. Briggs, C. Castro; The Ohio State University, Columbus, OH. This work focuses on the development, validation, and application of a nanoscale device for fluorescence-based high throughput single molecule force spectroscopy. This device will enable investigation of mechanical properties, kinetics, and stability of bimolecular interactions and singular biomolecules in a highly parallel fashion. While it is widely understood that molecular interactions are fundamental to many biological processes, it is only in the past few decades that technologies such as optical trapping, atomic force microscopy, and magnetic tweezers have been developed to probe single molecule interaction. The instrumentation required for these experiments is often costly, cumbersome, and reliant upon user skill or expertise. Furthermore, the experiments are generally low-throughput (one at a time). Our goal is to develop a DNA origami device to allow force spectroscopy measurements in single molecular interaction assays on 100s of devices simultaneously using a basic laboratory fluorescence microscope. The nanostructure comprising this device will consist of a structurally stiff framework of doublestranded DNA bundles, functionalized attachment points for two biomolecules (a receptor and a ligand), a flexible single stranded DNA linker (which will function as a force probe exploiting entropic elasticity), and fluorescent molecules to facilitate readout of the binding interaction between the biomolecules under study. The device will rely on fluctuations of the single-stranded DNA linker to both facilitate an interaction between biomolecules and apply a force acting to rupture the interaction once it is formed. The magnitude of the force can be controlled by tuning the extension of the linker. When the biomolecules are bound together, the fluorescent molecules contained within the device will undergo Förster resonance energy transfer (FRET). This design will enable the measurement of lifetimes of single biomolecular interactions as a function of force using a FRET-based measurement. This FRET based force spectroscopy can be integrated into a single molecule fluorescence assay that is amenable to high throughput highly parallel data collection. We have constructed the device and performed proof of principle experiments probing DNA basepairing interactions. We have demonstrated that the device can facilitate and rupture these interactions with estimated forces ranging from ~1-25pN. The fraction of bound interactions decrease with increasing magnitude of force. We are currently developing the single molecule FRET assay and will initially measure the kinetics of previously characterized DNA interactions to verify the behavior of the device. After verifying the functionality, this device will be used to probe the receptor-ligand kinetics of DNA-protein interactions.

Nanomechanical Behaviors of α-Catenin under Tension as a Mechanotransduction Switch revealed by AFM Nanofishing K. Maki, S. Han, T. Adachi; Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JAPAN. Alpha-catenin, a component molecule of adherens junctions (AJ), recruits vinculin under intercellular tension for mechanotransduction. Yonemura et al. (2011) elucidated the residues 276-634, which form helix bundles E, F and G, play a core role as a force-sensor, molecular switch regulating vinculin recruitment. The residues 510-634 (helix bundles F and G) inhibits the vinculin recruitment interacting with helix bundle E that contains vinculin binding site 1 (VBS1; residues 325-402). However, it is unclear that how alpha-catenin dynamically behaves in vinculin recruitment under tension at molecular level. Therefore, we analyzed the nanomechanical behaviors of residues 276-634 of alpha-catenin, employing atomic force microscopy (AFM) nanofishing. AFM nanofishing is a method for analyzing mechanical behaviors of single biomolecules by directly loading. In order to analyze the mechanical behaviors of alpha-catenin as a mechanotransduction switch, we designed alpha-catenin fusion proteins containing wild type helix bundles EFG and its mutant without the interaction between helix bundles E and G. We also designed fusion proteins containing helix bundle E and FG to elucidate the behaviors of individual domains. These fusion proteins were Nterminus- GST-tagged and C-terminus- His-tagged for binding with GSH-modified AFM tip and NTA-Ni2+modified glass substrate. The AFM cantilever was controlled by extension of piezo-electric device and the molecules were loaded at constant speed of 50 [nm/s]. Force curves obtained in nanofishing showed multiple tension relaxation, indicating that alpha-catenin dynamically changes its conformation under tension. The contour lengths were analyzed using a wormlike chain model. Because the histogram of contour lengths for wild type EFG fragment exhibited distinct multiple peaks, EFG fragment would unfold via a specific unfolding pathway. Comparing the histogram with those of other fragments, we elucidated the order of unfolding of EFG fragment. In the initial behavior under tension, the auto inhibited of EFG fragment unfurls structure under tension into open structure, referred to “switching” in this study. After switching behavior, helix bundle E is unfolded before unfolding of helix bundles FG, depending on structural stabilities of individual helix bundles. Our results which revealed the mechanical behaviors of alpha-catenin as a molecular switch could expect to explore the multicellular dynamics that underlies in biological processes from a view point of mechanics.

Characterizing Deformability of Cancer Cells with Dielectrophoretic-based Microfluidic Chip Y. Teng1, P. Xiao2, F. Lin1, M. Chu2, H. Lin3, Y. Wang2, C. Xiong1; 1 Peking University, Beijing, CHINA, 2Peking University Health Science Center, Beijing, CHINA, 3The State University of New Jersey, Rutgers, NJ. Alterations in cell mechanical properties are closely associated with many physiological and pathological changes in cell function, which endows researchers with important implications for detection and diagnosis of disease. In this study, a micro-electrode array chip was designed and fabricated on ITO glass substrate to study the mechanical properties of cells. By applying high frequency electric field, the cells are captured by dielectrophoretic force and gradually deformed between the electrodes. Through CCD camera, real-time and dynamic process of cell deformation was achieved. The cell deformation curves can be quantified by image processing. Epithelial-mesenchymal transition (EMT), a term describing the loss of epithelium-specific function and the acquisition of migration and invasion function, has critical physical and biological significance for investigation of tumor progression and metastasis. We used this microarray chip to study the mechanical properties of breast epithelial cells MDA-MB-231, MCF-7 and lung epithelial cells A549 in EMT process. The deformation curves showed three cells lines have greater deformability after EMT. Another experiment aimed to comparative study the mechanical properties of the non-tumorigenic (MCF-10A), non-invasive malignant (MCF-7) and highly-invasive malignant (MDA-MB-231) breast epithelial cell lines, commonly used as models of cancer metastasis. Their stretching and relaxation curves were analyzed by an optimized mechanical model, which can calculate the relaxation modulus of the cell. Our data showed that cell deformability through dielectrophoresis is sensitive enough to monitor the metastatic state of cancer cell. Furthermore, optimization of layout and injection mode of microfluidic channels enables multi-channel and parallel detection, which is expected to establish a low-cost and high-throughput characterization

system for cell mechanical studies.

3D Traction Force Microscopy Combined with Computer Simulation Models for the Analysis of Filopodia Dynamics Y. R. Silberberg1, M. Kim1, H. H. Asada2; 1 Singapore-MIT Alliance for Research and Technology (SMART), SINGAPORE, SINGAPORE, 2 Massachusetts Institute of Technology,, Cambridge, Massachusetts, MA. Angiogenesis, the process in which new blood vessels are formed from pre-existing ones, is an important mechanism governing a variety of processes in health and disease, from wound healing to the study of cancer tumor invasion. During angiogenic sprouting, filopodia is extended and retracted from tip cells at the leading edge, which are sensing the micromechanical environment of the ECM through a dynamic feedback mechanism. In this study, real-time, three dimensional (3D) traction force microscopy (TFM) of collagen-embedded vascular endothelial cells was performed in order to analyze the 3D dynamics of filopodia during the process of angiogenic sprouting. Forces exerted by single filopodium were characterized, and were correlated with variations in ECM stiffness. In addition, experimental observations were compared with computer simulations, using a specially developed extensive model that comprises of three modules: cell mechanics, filopodia dynamics and ECM fiber structure and mechanics. Investigating the mechanisms that are involved in angiogenic sprouting and filopodia dynamics at near-physiological conditions and in a true 3D environment, together with the codevelopment of an extensive multi-module simulation model, may help tackle crucial questions and enable the understanding of emergent behaviors in angiogenesis and vasculogenesis.

Dynamic Alterations in Stem Cell Nuclear Architecture and Mechanobiology as a Consequence of Mechanical Perturbation S. Heo1, T. D. Driscoll1, S. D. Thorpe2, D. A. Lee2, R. L. Mauck1; 1 University of Pennsylvania, Philadelphia, PA, 2Queen Mary, University of London, London, UNITED KINGDOM. Lineage specification is accompanied by regulation of nuclear structural proteins (i.e. Lamin A/C) and chromatin reorganization in mesenchymal stem cells (MSCs). While it is clear that mechanical cues can instigate these changes, it remains unclear how such physical cues are transduced into biological response. In this study we investigated nuclear remodeling in MSCs in response to dynamic tensile loading (DL) and the time scales over which DL regulated chromatin condensation and changes in nuclear mechanics. In doing so, we identified load induced ATP release as a key signaling event in this process and further determined the duration over which these changes were “imprinted” on the nucleus to establish a mechanical “memory”. In the absence of exogenous growth factors, DL (3%, 1Hz) up-regulated expression of fibrochondrogenic markers (e.g. type I collagen, aggrecan, and TGF-beta) and induced Lamin A/C reorganization and heterochromatin condensation (HTC, Fig. 1a) increasing nuclear stiffness, and did so more rapidly than soluble differentiation factors. A quantitative measure of chromatin condensation (the chromatin condensation parameter, CCP, Fig. 1b) increased with short term loading (150 sec or 600 sec) and was dependent on extracellular release of ATP. Changes in CCP persisted over time scales that depended on both the duration of stimulation and acto-myosin contractility (Fig. 1c). DL induced ATP release was regulated through the TGF beta superfamily (Fig1. d), and short term loading increased CCP through both purinergic and TGF signaling pathways. However neither pathway was involved in increases in CCP with longer term 1hr or 3hr loading (Fig. 1 e, f). Further, we also queried whether the number of loading events regulates chromatin condensation and nuclear mechanics. Larger increases in CCP and up-regulation of genes associated with chromatin movement (SMC1A), transcriptional repression (CTCF), and ECM synthesis were observed with increasing number of loading cycles. Chromatin relaxation and changes in nuclear mechanics was also dependent on the number of loading cycles. These findings suggest that, through alterations in nuclear structure, biophysical cues may direct lineage specification in MSCs, and that MSCs might establish a mechanical memory, encoded in structural changes in the nucleus.

2-3

Cell mechanics and cell function

Mechanical Coupling of Cardiomyocytes on PDMS Film Enables Synchronization B. Williams, T. Saif; University of Illinois at Urbana-Champaign, Urbana, IL. We present the use of a freestanding PDMS film as a tool to independently probe the role of mechanical coupling in the sychronization of cardiomyocytes. Previous investigations have demonstrated induced cardiomyocyte contractility as well as variation in Connexin-43 expression by applying cyclic external strain to the substrate of a cell culture. However, previous investigations have been unable to isolate the effect of mechanical coupling between cardiomyocytes due to experimental limitations. We suspend thin, 10-20 micron thick, 15 mm diameter PDMS films from a glass ring to provide a taut, drum-like substrate with fixed boundary conditions (Fig. 1a). We separately culture dense populations of primary cardiomyocytes from neonatal rats on each side of the film, while confining cell adhesion to the membrane to ensure that no direct contact between the two populations are formed. Within 2-3 days, each side synchronizes to itself, enabled by well characterized gap junctions. Thus each side is a single oscillator. With sufficient cell density, in-plane, micron-scale displacements of the membrane are observed, which stretch the substrate and, consequentially, the group of cells on the other side of the film. We quantify the system by measuring the time dynamics of each group of cells by particle tracking and subtracting the motion of the substrate (Fig. 1b). Cells are readily identified as being on opposite sides of the film by staining with CellTracker (red and green). Displacement shapes are consistent for all cells on the same side of the film, indicating local synchronization. We map each contractile function to a phase space (Fig. 1c), assuming linear phase propagation between local displacement maxima, and utilize the relative phase between the two sides to demonstrate synchronization (Fig 1d). We find that mechanical coupling alone is sufficient to induce phase locking indicated by a constant relative phase with occasional missed contractions from the trailing waveform. We provide additional platforms with similar results and a computational model to support and explain our observations. We propose metrics to quantify the dependency of mechanical coupling strength on relative phase, which may play a role in heart function.

Nuclear Shape Prescribes the Mechanical Properties of Human Stem Cells O. A. Lozoya, 20111, C. L. Gilchrist1, E. T. O'Brien2, R. Superfine2, F. Guilak1; 1 Duke University Medical Center, Durham, NC, 2University of North Carolina at Chapel Hill, Chapel Hill, NC. Cells perform physiological processes such as differentiation, mechanotransduction and tissue development, which involve structural connections between their nucleus and cytoskeleton. However, neither the mechanical properties of these connections are fully understood, nor how those properties vary in response to biophysical stimuli or among different cell types. We hypothesize that the perinuclear region that links the nucleus with the cell’s cytoskeleton possesses an adaptable structure that exhibits distinct mechanical properties among diverse cell types, morphologies and cytokine stimuli. In this study, we used in vitro passive tracking microrheology with cytoplasmic microspheres to characterize cytoskeletal mechanical properties in human stem cells subject to different treatments (IL1α, TGFß1, Cytochalasin D), with controlled morphology (microphotopatterned substrates), or from distinct stem cell sources (hASCs, hMSCs, hiPSCs). We used an elliptical coordinate system with orthonormal basis set i={u,v} prescribed to the best-fit ellipse of the nuclear envelope (NE) in each cell, and found that rheological properties of the perinuclear cytoskeleton (pnCSK), as measured from microspheres with subdiffusive motion, were: a) described in a physiologically relevant frequency span 0.2 Hz 80%) using 2 M 1,2-propanediol and 0.5 M trehalose. This is attributed to the high cooling and thawing rates that could be achieved with the use of the tiny QMC. Moreover, by microencapsulating mesenchymal stem cells in ~100 μm hydrogel microbeads of 2% alginate, ~90% of the cells can survive after vitrification in a 400 μm thin-walled (10 μm) QMC with 1.5 M dimethyl sulfoxide (DMSO) as the CPA. This further enhancement of vitrification by the alginate hydrogel is due to the preferential vitrification of water in the hydrogel that creates a nano liter ice-free environment around the microencapsulated cells to minimize IIF in them. Research is ongoing to adapt this alginate hydrogel microencapsulation and/or QMC technologies to further improve low-CPA vitrification of embryonic stem cells and to achieve low-CPA vitrification of mammalian oocytes with promising outcome.

The Application of Nanoparticles in the Cryopreservation of Biological System" B. Liu, F. Lv, B. Hao, W. Li; University of Shanghai for Science and technology, Shanghai, CHINA. Usually there are two ways for the cryopreservation of biological materials, freezing and vitrification. In the freezing process, the uncontrolled formation of ice crystals is often the major cause of freeze damage. So vitrification is regarded as a potential method to preserve biomaterials, especially for tissues and organs with large volume. Vitrification can be defined as the solidification of a solution by an extreme increase in viscosity without crystallization, which requires high concentrations of glass-inducing solutes and ultra-fast cooling rate. Many studies had been performed to obtain the glass transition of a biological system. Nanoparticles in solution offer unique electrical, mechanical and thermal properties due to their physical presence in solution and their interaction with the state of dispersions. Some attempts have been made to incorporate nanoparticles into cryopreservation solutions that may offer new means for changing the phase behavior of solutions and for controlling nucleation, ice growth and ice crystal geometries. The purpose of the present study is to investigate the effect of hydroxyapatite (HA) nanoparticles on devitrification, recrystallization and ice crystal characteristics of aqueous glycerol and poly(ethylene glycol) (PEG-600) solutions. HA nanoparticles (20nm,40nm or 60nm) were incorporated into solutions at the content of 0.1% or 0.5% (w/w), and were studied by differential scanning calorimeter (DSC) and cryomicroscopy. The presence of nanoparticles does not change the glass transition temperatures and melting temperatures of quenched solutions, but significantly affects the behavior of devitrification and recrystallization upon warming. Cryomicroscopic investigation showed the complex interactions among solution type, nanoparticle size and nanoparticle content, which apparently influence ice crystal growth or recrystallization in the quenched dispersions. The presence of nanoparticles alters the behaviors of devitrification and recrystallization of quenched solutions upon warming. Data of the present study and earlier work demonstrate the complex interactions among solution type, nanoparticle size and the content of nanoparticles in the dispersion. The molecular nature of such interactions at subzero temperatures and the complexity of ice crystal growth kinetics in nanoparticle-containing dispersions are poorly understood, and further studies are needed to solve this problem.

Challenges facing the 21st century cryopreservation technology and our current and potential solutions with micro/nano technology X. Han, Y. Yuan, R. M. Roberts; University of Missouri, Columbia, MO. Confronting fast advancing biotechnologies in the biomedical sciences, e.g. genetic engineering, stem cell techniques, tissue engineering, traditional cryopreservation methods and devices face technical challenges in providing efficient approaches to storage and shipment of large numbers of emerging cell/tissue products. Overcoming these challenges will give birth to a new generation of cryopreservation technologies. One major task is to lower the cost and solve the safety issues associated with the extensive use of liquid nitrogen in cooling, storage, and shipping. Based on our investigations on the influence of different polymer molecules on the thermal stability of cryoprotectant solutions at deep low temperatures, we developed a series of media that enable long-term storage of germplasm, i.e. gametes, early-stage embryos, and stem cells at -80oC. These media contain an optimal concentration of Ficoll and permeating cryoprotectant, e.g. DMSO, glycerol, designed for each cell type. As one example of our current achievements, leukemia inhibitory factor (LIF)-dependent naïve type porcine induced pluripotent stem (pIPS) cells can be efficiently preserved by using such a deigned medium in a -80oC freezer for 10 weeks, with a post-thaw plating efficiency and cell viability very close to (difference

Smile Life

When life gives you a hundred reasons to cry, show life that you have a thousand reasons to smile

Get in touch

© Copyright 2015 - 2024 PDFFOX.COM - All rights reserved.