Name: experimental methods to study human postural control
Uploaded: 2019-09-11T15:00:00
Description: Watch this Scientific Journal Video about Experimental Methods to Study Human Postural Control at JoVE.com

This article was made possible by NPRP grant #6-463-2-189 from the Qatar National Research and MOP grant #81280 from the Canadian Institutes of Health Research.

All experimental methods have been approved by the McGill University Research Ethics Board and subjects sign informed consents before participating.
1. Experiments
NOTE: Each experiment involves the following steps.
<ol>
	<li>Pre-test
	<ol>
		<li>Prepare a definite outline of all trials to be performed and make a checklist for data collection.</li>
		<li>Provide the subject with a consent form with all the necessary information, ask them to read it thoroughly, ans...

Several steps are critical in performing these experiments to study human postural control. These steps are associated with the correct measurement of the signals and include: 1) Correct alignment of the shank ankle axis of rotation to that of the pedals, for the correct measurement of ankle torques. 2) Correct set-up of the range finders to ensure they work in their range and are not saturated during the experiments. 3) Measurement of EMG with good quality and minimal cross talk. 4) Application of appropriate perturbati...

Human postural control is realized through complex interactions between the central nervous and musculoskeletal systems1. The human body in standing is inherently unstable, subject to a variety of internal (e.g., respiration, heartbeat) and external (e.g., gravity) perturbations. Stability is achieved by a distributed controller with central, reflex, and intrinsic components (Figure 1).
Postural control is achieved by: an active controller, mediated by the central nervous system (CNS) and spinal cord, which changes muscle activation; and an intrinsic stiffness controller that resists joint movement with no change in muscle activation (Figure 1). The central controller uses sensory information to generate descending commands that produce corrective muscle forces to stabilize the body. Sensory information is transduced by the visual, vestibular, and somatosensory systems. Specifically, the somatosensory system generates information regarding the support surface and joint angles; vision provides information regarding the environment; and the vestibular system generates information regarding the head angular velocity, linear acceleration, and orientation with respect to gravity. The central, closed-loop controller operates with long delays that may be destabilizing2. The second element of the active controller is reflex stiffness, which generates muscle activity with short latency and produces torques resisting joint movement.
There is a latency associated with both components of active controller; consequently, joint intrinsic stiffness, which acts with no delay, plays an important role in postural control3. Intrinsic stiffness is generated by passive visco-elastic properties of contracting muscles, soft tissues and inertial properties of the limbs, which generates resistive torques instantaneously in response to any joint movement4. The role of the joint stiffness (intrinsic and reflex stiffness) in postural control is not clearly understood, since it changes with operating conditions, defined by muscle activation4,5,6 and joint position4,7,8, both of which change with the body sway, inherent to standing.
Identifying the roles of the central controller and joint stiffness in postural control is important, as it provides the basis for: diagnosing the etiology of balance impairments; the design of targeted interventions for patients; assessment of the risk of falls; the development of strategies for fall prevention in the elderly; and the design of assistive devices such as orthotics and prosthetics. However, it is difficult, because the different sub-systems act together and only the overall resulting body kinematics, joint torques, and muscle electromyography can be measured.
Therefore, it is essential to develop experimental and analytical methods that use the measurable postural variables to evaluate each subsystem&#8217;s contribution. A technical difficulty is that the measurement of postural variables is done in closed-loop. As a result, the inputs and outputs (cause and effect) are interrelated. Consequently, it is necessary to: a) apply external perturbations (as inputs) to evoke postural reactions in responses (as outputs), and b) employ specialized mathematical methods to identify system models and disentangle cause and effect9.
The present article focuses on postural control when an ankle strategy is used, that is, when the movements occur primarily about the ankle joint. In this condition, upper body and lower limbs move together, consequently, the body can be modeled as a single-link inverted pendulum in sagittal plane10. The ankle strategy is used when the support surface is firm and the perturbations are small1,11.
A standing apparatus capable of applying appropriate mechanical (proprioceptive) and visual sensory perturbations and recording the body kinematics, kinetics, and muscle activities has been developed in our laboratory12. The device provides the experimental environment needed to study the role of ankle stiffness, central control mechanisms, and their interactions by generating postural responses using visual or/and somatosensory stimuli. It is also possible to extend the device to study the role of vestibular system by the application of direct electrical stimulation to the mastoid processes, that can generate a sensation of head velocity and evoke postural responses12,13.
Others have also developed similar devices to study human postural control, where linear piezo electric actuators11, rotary electrical motors14,15, and linear electrical motors16,17,18 were used to apply mechanical perturbations to ankle in standing. More complex devices also have been developed to study multi-segment postural control, where it is possible to apply multiple perturbations to ankle and hip joints simultaneously19,20.
Standing apparatus
Two servo-controlled electrohydraulic rotary actuators move two pedals to apply controlled perturbations of ankle position. The actuators can generate large torques (&#62;500 Nm) needed for postural control; this is especially important in cases such as forward lean, where the body&#8217;s center of mass is far (anterior) from ankle axis of rotation, resulting in large values of ankle torque for postural control.
Each rotary actuator is controlled by a separate proportional servo valve, using pedal position feedback, measured by a high-performance potentiometer on the actuator shaft (Table of Materials). The controller is implemented using a MATLAB-based xPC real-time, digital signal processing system. The actuator/servo-valve together have a bandwidth of more than 40 Hz, much larger than bandwidth of the overall postural control system, ankle joint stiffness, and the central controller21.
Virtual reality device and environment
A virtual reality (VR) headset (Table of Materials) is used to perturb the vision. The headset contains an LCD screen (dual AMOLED 3.6&#8217;&#8217; screen with a resolution of 1080 x 1200 pixels per eye) that provides the user with a stereoscopic view of the media sent to the device, offering three-dimensional depth perception. The refresh rate is 90 Hz, sufficient to provide a solid virtual sense to the users22. The field of view of the screen is 110&#176;, enough to generate visual perturbations similar to real world situations.
The headset tracks the rotation of the user&#8217;s head and alters the virtual view accordingly so that the user is fully immersed in the virtual environment; therefore, it can provide the normal visual feedback; and it can also perturb vision by rotating the visual field in sagittal plane.
Kinetic measurements
Vertical reaction force is measured by four load cells, sandwiched between two plates beneath the foot (Table of Materials). Ankle torque is measured directly by torque transducers with a capacity of 565 Nm and a torsional stiffness of 104 kNm/rad; it also can be measured indirectly from the vertical forces transduced by the load cells, using their distances to ankle axis of rotation23, assuming that horizontal forces applied to the feet in standing are small2,24. Center of pressure (COP) is measured in sagittal plane by dividing the ankle torque by the total vertical force, measured by the load cells23.
Kinematic measurements
Foot angle is the same as pedal angle, because when an ankle strategy is used, the subject&#8217;s foot moves with the pedal. Shank angle with respect to the vertical is obtained indirectly from the linear displacement of the shank, measured by a laser range finder (Table of Materials) with a resolution of 50 &#956;m and bandwidth of 750 Hz25. Ankle angle is the sum of the foot and shank angles. Body angle with respect to the vertical is obtained indirectly from the linear displacement of the mid-point between the left and right posterior superior iliac spines (PSIS), measured using a laser range finder (Table of Materials) with a resolution of 100 &#956;m and bandwidth of 750 Hz23. Head position and rotation are measured with respect to the global coordinate system of the VR environment by the VR system base stations that emit timed infrared (IR) pulses at 60 pulses per second that are picked up by the headset IR sensors with sub-millimeter precision.
Data acquisition
All signals are filtered with an anti-aliasing filter with a corner frequency of 486.3 and then sampled at 1000 Hz with high performance 24-bit/8-channel, simultaneous-sampling, dynamic signal acquisition cards (Table of Materials) with a dynamic range of 20 V.
Safety mechanisms
Six safety mechanisms have been incorporated into the standing apparatus to prevent injuries to subjects; the pedals are controlled separately and never interfere with each other. (1) The actuator shaft has a cam, which mechanically activates a valve that disconnects hydraulic pressure if the shaft rotation exceeds &#177; 20&#176; from its horizontal position. (2) Two adjustable mechanical stops limit the range of motion of the actuator; these are set to each subject&#8217;s range of motion prior to each experiment. (3) Both the subject and the experimenter hold a panic button; pressing the button disconnects hydraulic power from the actuators and causes them to become loose, so they can be moved manually. (4) Handrails located at either side of the subject are available to provide support in case of instability. (5) The subject wears a full body harness (Table of Materials), attached to rigid crossbars in the ceiling to support them in case of a fall. The harness is slack and does not interfere with normal standing, unless the subject becomes unstable, where the harness prevents the subject from falling. In the case of fall, the pedal movements will be stopped manually either by the subject, using the panic button or by the experimenter. (6) The servo-valves stop the rotation of the actuators using fail-safe mechanisms in case of electrical supply interruption.

<table>
	<tbody>
		<tr>
			<td>5K potentiometer</td>
			<td>Maurey</td>
			<td>112P19502</td>
			<td>Measures actuator shaft angle</td>
		</tr>
		<tr>
			<td>8 channel Bagnoli surface EMG amplifiers and electrodes</td>
			<td>Delsys</td>
			<td></td>
			<td>Measures the EMG of ankle muscles</td>
		</tr>
		<tr>
			<td>AlienWare Laptop</td>
			<td>Dell Inc.</td>
			<td>P69F001-Rev. A02</td>
			<td>VR-ready PC laptop</td>
		</tr>
		<tr>
			<td>Data acquisition card</td>
			<td>National instruments</td>
			<td>4472</td>
			<td>Samples the analogue signals from the sensors</td>
		</tr>
		<tr>
			<td>Directional valve</td>
			<td>REXROTH</td>
			<td>4WMR10C3X</td>
			<td>Bypasses the flow if the angle of actuator shaft goes beyond &#177;20&#176;</td>
		</tr>
		<tr>
			<td>Full body harness</td>
			<td>Jelco</td>
			<td>740</td>
			<td>Protect the subjects from falling</td>
		</tr>
		<tr>
			<td>Laser range finder</td>
			<td>Micro-epsilon 1302-100</td>
			<td>1507307</td>
			<td>Measures shank linear displacement</td>
		</tr>
		<tr>
			<td>Laser range finder</td>
			<td>Micro-epsilon 1302-200</td>
			<td>1509074</td>
			<td>Measures body linear displacement</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>Omega</td>
			<td>LC302-100</td>
			<td>Measures vertical reaction forces</td>
		</tr>
		<tr>
			<td>Proportional servo-valve</td>
			<td>MOOG</td>
			<td>D681-4718</td>
			<td>Controls the hydraulic flow to the rotary actuators</td>
		</tr>
		<tr>
			<td>Rotary actuator</td>
			<td>Rotac</td>
			<td>26R21VDEISFTFLGMTG</td>
			<td>Applies mechanical perturbations</td>
		</tr>
		<tr>
			<td>Torque transducer</td>
			<td>Lebow</td>
			<td>2110-5k</td>
			<td>Measures ankle torque</td>
		</tr>
		<tr>
			<td>Virtual Environment Motion Trackers</td>
			<td>HTC inc.</td>
			<td>1551984681</td>
			<td>Tracks the head motion</td>
		</tr>
		<tr>
			<td>Virtual Reality Headset</td>
			<td>HTC inc.</td>
			<td>1551984681</td>
			<td>Provides visual perturbations</td>
		</tr>
	</tbody>
</table>

experimental methods to study human postural control

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied by appropriate mathematical methods are needed to understand the role of the different sub-systems involved in human postural control. This article describes a protocol for performing perturbed standing experiments, acquiring experimental data, and carrying out the subsequent mathematical analysis, with the aim of understanding the role of musculoskeletal system and central control in human upright posture. The results generated by these methods are important, because they provide insight into the healthy balance control, form the basis for understanding the etiology of impaired balance in patients and the elderly, and aid in the design of interventions to improve postural control and stability. These methods can be used to study the role of somatosensory system, intrinsic stiffness of ankle joint, and visual system in postural control, and may also be extended to investigate the role of vestibular system. The methods are to be used in the case of an ankle strategy, where the body moves primarily about the ankle joint and is considered a single-link inverted pendulum.

Pseudo random ternary sequence (PRTS) and TrapZ signals
Figure 2A shows a PRTS signal, which is generated by integrating a pseudo random velocity profile. For each sample time <img alt="Equation 76" src="/files/ftp_upload/60078/60078eq76.jpg" />, the signal velocity may be equal to zero, or acquire a pre-defined positive or negative value, <img alt="Equation 77" src="/files/ftp_uplo...

Watch this Scientific Journal Video about Experimental Methods to Study Human Postural Control at JoVE.com

Experimental Methods to Study Human Postural Control

This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control.

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

These experimental and analytical methods provide guidelines that aim to understand to role of the many components of the central nervous and musculoskeletal systems in human postural control. Postural control models with meaningful physical parameters can be used to investigate the role and interaction of the sensory systems and their changes due to disease and aging. These methods can be used to assess patient balance problems, to reveal the ideology of the impairment, and to aid in the design of interventions for improving postural control.These methods can also be used to study the interactions between sensory motor pathologies and balance control. For example, for fall prevention in the elderly. This protocol provides a means to investigate the relative contributions of sensory modalities including proprioceptive and visual systems and their interactions as well as muscle passive contributions to postural control.To prepare a subject for the measurement of electromyography from ankle muscles, use single differential electrodes with an interelectrode distance of one centimeter. Mark the medial gastrocnemius at the most prominent bulge of the muscle, the lateral gastrocnemius at one third of the line between the head of the fibula and the heel, the soleus at two thirds of the line between the medial condyles of the femur and the medial malleolus, and the tibialis anterior at one third of the line between the tip of the fibula and the tip of the medial malleolus. When all of the points have been marked, use a razor to shave each area and clean the skin with alcohol.When the skin has dried, use double sided tape to attach one electrode to each area taking care that each electrode is fixed to the skin securely. To prepare the subject for a kinematic measurement, first use a strap to attach a reflective marker as high as possible on the subject's shank and have the subject put on the body harness. Use a strap to attach a reflect marker to the subject's waist and have the subject climb on to the standing apparatus.Adjust the subject's foot position to align the lateral and medial malleoli of each leg to the pedal axis of rotation and use a marker to outline the foot positions. Instruct the subject to keep their feet in the same locations during the experiments and adjust the vertical position of the laser range finders to point to the center of the reflective markers. Then, adjust the horizontal distance between the laser range finder and the reflective markers so that the range finders work in their mid range and do not saturate during standing experiments.Before beginning the experiment, inform the subject of what to expect for each trial condition. Instruct the subject to stand quietly with hands at the side while looking forward maintaining their balance as they do so when faced with the real world perturbations. For a quiet standing trial, have the subject stand still for two minutes with no perturbations.For perturbed experiments, if the objective is to investigate the role of somatosensory system or ankle stiffness in standing, apply pedal perturbations for two to three minutes while recording the data. If the objective is to examine the role of vision in postural control, apply visual perturbations by rotating the visual field using the virtual reality headset for two to three minutes while recording the data. If the objective is to examine the interaction of the two systems in postural control, apply the visual and pedal perturbations simultaneously.For non-parametric identification of the dynamic relation of the body angle to visual perturbations after loading the visually perturbed trial data into a suitable analysis software program, use the commands as indicated to decimate the raw body angle and the visual perturbation signals and remove the means from the decimated signals. Determine the decimated sampling frequency, then select the lowest frequency of interest to determine the window length and choose the degree of overlap for the estimation of power spectra. Define the vector of frequencies at which the frequency response is to be estimated.Use the TF Estimate function to find the frequency response of the system as indicated and find the gain and phase of the estimated frequency response as demonstrated. Then, use the command as indicated to calculate the coherence function and plot the gain, phase, and coherence as a function of frequency. In this example of a typical standing trial with visual perturbations, a trapezoidal signal applied by the virtual reality headset can be observed where the field of view rotates from zero to plus or minus 0.087 rad in the sagittal plane.The ankle and body angles were very similar in this analysis since the foot angle is zero and the shank and upper body move together. The ankle torque was also correlated with the shank and body angles. Electromyographs from the ankle muscles demonstrate that the soleus and the lateral gastrocnemius are continuously active, but the medial gastrocnemius periodically generates large bursts of activity with body sway and that the tibialis anterior is silent.Here, a frequency response of the transfer function relating the visual input to the body angle for the standing trial data is shown. In this experiment, the coherence was high at low frequencies up to around one hertz and dropped significantly at higher frequencies, meaning the frequency response is meaningful up to one hertz. The gain initially increased from 0.1 to 0.2 hertz before decreasing to one hertz, demonstrating the expected low pass behavior due to the body's high inertia.The phase also started at zero and decreased almost linearly with the frequency indicating that the output was delayed with respect to the input. Take care to align the ankle axis of rotation with that of the actuator. Make sure that the subject does not generate extra movements and ensure that the appropriate mechanical and visual perturbations are used.First time users may have difficulty in establishing a consistent repeatable experimental paradigm and in using the appropriate identification methods that account for close loop, non-linear, and time varying effects in postural control. These methods have been used to investigate healthy postural control and its adaptation as well as to quantify changes in balance control under a variety of experimental and clinical conditions.

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Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...