We acknowledge financial support from Research Growth Initiative (Award No. 101X340), National Science Foundation, Major Research Instrumentation Program (Grant No. PHY-1626450), Greater Milwaukee Foundation (Shaw Award) and University of Wisconsin System (Applied Research Grant).

1. Reagents Solution Preparation
<ol>
	<li>Prepare a starting protein solution by dissolving/diluting the protein of interest to the desired concentration, using a Tris buffer [20 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl, pH 7.4]. 
	NOTE: The smallest protein concentration for which cross-linking leads to hydrogels depends on the protein used and is typically &#62; 1 mM.</li>
	<li>Prepare stocks of ammonium persulfate (APS) (1 M) and tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]2+) (6.67 mM) solutions by dissolving APS and [Ru(bpy)3]2+ powders in the Tris buffer.</li>
</ol>
2. Protein-based Hydrogel Synthesis
<ol>
	<li>Fix a 23 G needle on a 1 mL syringe with a pressed plunger.</li>
	<li>Cut a 10 cm polytetrafluoroethylene (PTFE) tube (with an inner diameter of 0.022 in and an outer diameter of 0.044 in) using a razor blade. Attach the needle and syringe to one end of the PTFE tube.</li>
	<li>Insert the second end of the tube into a silane solution and fill the tube by retracting the syringe plunger. Leave the tube for ~30 min.</li>
	<li>Remove the silane solution and dry the tube with compressed air. 
	NOTE: Make sure that all of the silane solution is dried and that no residue is left in the tube.</li>
	<li>Mix the protein solution with APS and [Ru(bpy)3]2+ in a 1.5 mL tube using a constant volume ratio [e.g., 15:1:1 or 15:0.5:0.5 (v:v:v)].</li>
	<li>Vortex the photoactive solution until it is mixed completely.</li>
	<li>Centrifuge the mix at maximum speed (e.g., 14,000 x g) to remove any bubbles from the solution.</li>
	<li>Insert the open end of the treated PTFE tube into the photoactive mixture and draw the solution into the tube by retracting the syringe plunger.</li>
	<li>Place the loaded tube ~10 cm away from a 100 W mercury lamp to prevent heating it and keep it there for up to 30 min at room temperature (Figure 1B). 
	NOTE: In some cases, the exposure time to light can be as low as 30 s. Shorter gelation times are used here for fluorescent gels, to limit photobleaching.</li>
	<li>Remove the tube from the needle and cut the edges of the tube near the hydrogel ends with a razor blade.</li>
	<li>Use a blunted 24 G needle to extrude the hydrogel into the Tris solution (Figure 1C). 
	NOTE: Blunted needles are used to avoid any notches or damage to the hydrogel sample.</li>
	<li>Visually inspect the gels for any defects that might form during the extrusion or due to bubbles and discard the gels with defects.</li>
</ol>
3. Protein-based Hydrogel Attachment and Force-Clamp Rheometer Set-up
<ol>
	<li>Start the instrument control program. Turn on the voice-coil motor. Set the coil position to a value toward the end of the range (e.g., 7.5 mm). 
	NOTE: The voice-coil position is recommended to be toward the end of the maximum movement range, to maximize the possible extension of the hydrogel.</li>
	<li>Displace the hooks in the z-direction and align them at the bend in the x-direction (which is the pulling coordinate; see Figure 2B). Record the values of the micrometer screws for the x-direction.</li>
	<li>Cut 2 sterile sutures into strands of equal length (2 - 3 cm; see Figure 3A and B).</li>
	<li>Tie a loose double overhand knot into each of the strands and place the 2 loops on the hook connected to the force sensor (Figure 3C and 3D).</li>
	<li>Fill the experimental chamber with Tris buffer and transfer the hydrogel sample into the filled chamber using medical tweezers.</li>
	<li>Place the voice coil and force sensor hooks close to the solution surface and align the hooks in all directions using the x/y/z-positioning manipulators.</li>
	<li>Using medical tweezers, hang both sides of the protein hydrogel sample on the hooks connected to the voice coil and force sensor (Figure 3C).</li>
	<li>Tighten 1 suture loop around the hydrogel sample on the voice coil hook by holding both ends of the suture loop with medical tweezers and pulling them simultaneously (Figure 3D).</li>
	<li>Repeat step 3.8 for the loop connected to the force sensor (Figure 3D). 
	NOTE: Avoid an extreme tightening of the sutures to prevent any structural damage and transversal cutting of the hydrogel sample.</li>
	<li>Tighten the suture loops on the bends of each hook to prevent any slippage; use these bends as reference points to find the zero separation between the hooks in step 3.2. Cut the excess lengths of the sutures using medical scissors (Figure 3D).</li>
	<li>Move the attached hydrogel using z-manipulators along the z-axis toward the experimental chamber to immerse the hydrogel in the experimental solution.</li>
	<li>Align the hydrogel sample in y-z using the manipulators such that the gel is not under any stress.</li>
	<li>Zero the force sensor and separate the two hooks using the x-micrometer stages until the gel starts to experience force. Once this happens, slightly turn back the micrometer screw in the x-direction.</li>
	<li>Record the position of both manipulators for the voice coil motor and the sensor and use the difference between these values and the ones measured in step 3.2 to calculate the exact separation between the tethering hooks at the start of the experiment.</li>
	<li>Set the range for the slack curve to ~1.5 - 2 mm and measure the gel slack (Figure 4A). 
	NOTE: For each slack measurement, try to keep the start of the slack regime near the initial voice coil position, allowing for an optimal number of data points to fit the 2 regimes (Figure 4A). The gel length can be determined with a micron resolution using the separation between the hooks and the intersection between the 2 regimes in the slack curve (see also step 5.1). As the force sensor might drift with time due to variations in the experimental conditions, the part of the slack curve where the gel is not under force reports on this possible drift. The program controlling the instrument compensates automatically for this difference when sending the set-point command to the PID loop (Figure 4A inset).</li>
</ol>
4. Protein-based Hydrogel Characterization using Controlled Force-Ramp and Constant-Force Measurements
<ol>
	<li>Force-ramp experiments
	<ol>
		<li>To perform a force-ramp cycle by increasing the force at the desired loading rate (e.g., 0.01 mN/s), input the starting and final forces and the duration of the protocol as a flipped &#34;V&#34;. Then, hold the gel at 0 mN (or low force) for &#62; 200 s to allow the protein domains to refold and the gel elasticity to recover.</li>
		<li>Save the trace.</li>
	</ol>
	</li>
	<li>Constant-force experiments
	<ol>
		<li>Perform a constant-force protocol by applying a low force (e.g., 0.1 mN) for 30 s and then increase the force to a constant force (e.g., 1 mN) for a defined amount of time (e.g., 120 s), followed by quenching the force back to the same low value (e.g., 0.1 mN) for &#62; 300 s to allow the protein domains to refold and the gel elasticity to recover.</li>
		<li>Following the first pulse, adjust the PID settings to maximize the response time of the feedback loop (see Figure 2D). 
		NOTE: For stiff gels and for small changes in force, the response time of the loop is limited by the electronics of the force sensor and the response time of the coil, and can be as low as 5 ms7. For softer gels and large changes in force, the response time is dictated by the elasticity of the hydrogels (Figure 2D).</li>
		<li>Save the trace.</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>Utilizing the measured separation between the hooks and the calculated coil position, when the gel starts to experience force (&#916;x in the Figure 4A insert), compute the gel length L using the equation: 
	L = L0 + &#8710;x 
	Here, L0 is the separation between the hooks, measured from the position of the micrometer screws before the experiment (step 3.14). 
	NOTE: For gels with low protein concentrations that do not result in a complete cross-linking, the measured length will change from trace-to-trace. Also, over long periods of time, proteins inside hydrogels might experience aging effects25, which result in an overall lengthening of the gel.</li>
	<li>Normalize the measured extension to the gel length to obtain the strain.</li>
	<li>Normalize the measured force to the transversal surface area by using the inner diameter of the tube used for the polymerization.</li>
</ol>

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The authors have nothing to disclose.

Herein, we describe a force-clamp rheometry technique to investigate the biomechanical response of low-volume protein-based hydrogels. Additionally, a protocol is provided to synthesize a uniform cylindrical low-volume protein hydrogel sample. A protocol is also presented which describes how to tie different types of protein-based hydrogels with various elasticities without causing any mechanical deformation or damage to the protein-based hydrogel samples or slippage of the gel on the hooks. The analog PID system, togeth...

Apart from having unique physical properties, protein-based hydrogels hold the promise of revolutionizing force spectroscopy by enabling the measurement of several billion molecules in one &#39;pull&#39;, thus enabling the study of proteins in crowded environments, similar to those encountered in skin and other tissues. Protein domains remain folded inside hydrogels, allowing the study of their biomechanical response to force, binding partners, and chemical conditions. Additionally, the biomechanical response of protein domains inside hydrogels resembles the response seen with single-molecule force spectroscopy techniques. For example, chemical denaturants and oxidizing agents decrease the stability of the folded state, both at the single protein domain level1,2,3 and at the macroscopic level4,5,6,7. Similarly, osmolytes increase the stability of single proteins8,9, leading to a decrease in the viscoelastic response&#160;of hydrogels, for the same force conditions7,10.
Several approaches have been implemented to synthesize protein-based hydrogels, by either using physical interactions11,12 or covalent cross-linking4,13. Covalent reactions allow for fixed cross-linking locations and these hydrogels&#160;can recover the initial state upon a removal of the mechanical or chemical perturbations. A successful approach for covalent cross-linking relies on forming covalent carbon-carbon bonds between exposed tyrosine amino acids using ammonium persulfate (APS) as an oxidant and a ruthenium (II) salt as an initiator (Figure 1)14. Upon exposure to white light, a solution of concentrated proteins can be turned into a hydrogel. By controlling when the reaction starts, the protein-APS mix can be injected into any casting form, such as polytetrafluoroethylene (PFTE) tubes (Figure 1B and 1C), allowing the use of an extremely small solution volume15. Furthermore, the use of white light to trigger the cross-linking reaction results in a limited bleaching of fluorescent proteins and allows the formulation of composite hydrogels with fluorescent markers (Figure 1). Other protein-based hydrogel formation methods use cross-linking based on the SpyTag-SpyCatcher covalent interaction16, amine cross-linking via glutaraldehyde13, or biotin-streptavidin interactions17.
Dynamic mechanical analysis (DMA) is currently a technique extensively used to study polymer-based hydrogels13,18. While DMA can apply constant force protocols to biomaterials, it requires Young&#39;s moduli over 10 kPa, and large sample volumes of more than 200 &#181;L19. Due to these limitations, protein hydrogels are generally too soft to be investigated by this technique. As engineered polyproteins are harder to synthesize than polymers, since they require a living system to produce, such high volumes are inefficient, at best4,15. Furthermore, most biological tissues are softer than 10 kPa. Several approaches were developed for biological samples, especially in the study of muscle elasticity20,21. These techniques can also operate under feedback to apply constant force but are optimized for samples with small diameters (in the micron range) exposed to force for very short times (typically less than 1 s).
Protein-based hydrogels were successfully studied with modified rheometry techniques. For example, casting the hydrogel in a ring shape allows the use of extensional rheometry to measure the change in the experienced force as a function of extension4,22. Other approaches for studying the rheological properties of protein-based hydrogels use controlled shear-stress rheometry. These techniques can also achieve low sample volume and tolerate soft materials. However, these methods lack the ability to mimic the pulling forces that cause protein unfolding in vivo, and Young&#39;s modulus is calculated based on complex theories that require various assumptions and corrections23.
We have recently reported a new approach that utilizes a small volume of proteins, polymerized inside tubes with&#160;diameters &#60; 1 mm. Our first implementation of this technique was operating in length-clamp mode, where the gel was extended following the desired protocol15. In this method, the proteins experience a continuous change in both extension and force while the domains unfold, making the data interpretation cumbersome. Recently, we have reported a new force-clamp rheometry technique, where a feedback loop can expose low-volume protein hydrogels to a predefined force protocol7 (Figure 2). An analog PID system compares the force measured by the force sensor with the set point sent from the computer and adjusts the gel extension by moving the voice coil to minimize the difference between the two inputs. This &#39;clamping&#39; of the force now allows for new types of experiments to measure the biomechanics of protein hydrogels.
In the force-ramp mode, a tethered protein hydrogel experiences a constant increase and decrease of force with time. The PID compensates for any viscoelastic deformation by changing the extension in a non-linear way, depending on the type of protein and hydrogel formulation. The main advantage of force ramp is that it allows the quantification of standard parameters, such as Young&#39;s modulus and energy dissipation, due to an unfolding and refolding of protein domains.
In constant-force mode, the applied force changes in a step-like fashion. In this mode, the gel extends and contracts elastically when the force is increased or decreased, respectively, followed by a time-dependent deformation. This viscoelastic deformation, taking place while the gel experiences a constant force, is directly related to domain unfolding/refolding. In a simplified way, this extension can be seen as the equivalent of several billion single molecule traces averaged together and measured all at once. Constant-force protocols can be used to study the creep and relaxation of protein hydrogels as a function of force and time. As a function of force, for BSA-based protein hydrogels, we have recently shown that there is a linear dependency between the elastic and viscoelastic extension and recoil with the applied strain7.
Here we detail the operation of a force-clamp rheometer using composite gels made from a mixture of protein L (8 domains24, depicted as L8) and a protein L-eGFP construct (L-eGFP), which makes the overall hydrogel fluorescent and easy to demonstrate.

<table><tbody><tr><td>SI-KG4A force transducer</td><td>World Precision Instruments (WPI)</td><td>SI-KG4A</td><td></td></tr><tr><td>Linear Voice Coil Motor</td><td>Equipement Solutions</td><td>LFA2010</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Rocky Mountain Biologicals (RMBIO)</td><td>BSA-AAF-1XG / 100 G</td><td></td></tr><tr><td>Trizma</td><td>Sigma-Aldrich</td><td>T1503-1KG</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653-1KG</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>248614-100G</td><td></td></tr><tr><td>Tris(bipyridine)ruthenium(II) chloride</td><td>Sigma-Aldrich</td><td>544981-1G</td><td></td></tr><tr><td>EXPRESS MEDICAL SUPPLIES&amp;nbsp;6-0 NYLON SUTURE 12/PK</td><td>Fisher Scientific</td><td>NC0395626</td><td></td></tr><tr><td>1mL Syringe Only, Luer-Lok Tip</td><td>BD</td><td>309628</td><td></td></tr><tr><td>Silane, Sigmacote</td><td>Sigma-Aldrich</td><td>SL2-25ML</td><td></td></tr><tr><td>Microbore PTFE Tubing, 0.022&quot;ID x 0.042&quot;OD, 100 ft/roll</td><td>Cole-Parmer</td><td>EW-06417-21</td><td></td></tr><tr><td>Hypodermic Needle, 23 Gauge</td><td>Healthcare Supply Pros</td><td>305194</td><td></td></tr><tr><td>Jensen Global JG24-1.5X Red IT Dispensing Tips - 24 gauge</td><td>KIMCO</td><td>JG24-1.5X</td><td></td></tr><tr><td>USH-103D USHIO 100W Short Arc Mercury Lamp</td><td>ALB</td><td>USH-103D USHIO</td><td></td></tr><tr><td>Medical Tweezers</td><td></td><td></td><td></td></tr><tr><td>Medical scissors</td><td></td><td></td><td></td></tr><tr><td>Olympus</td><td></td><td></td><td></td></tr><tr><td>The computer code and CAD design of the custom parts can be made available on request to the corresponding author.</td><td></td><td></td><td></td></tr></tbody></table>

force-clamp rheometry for characterizing protein-based hydrogels

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels&#160;enables the tethering of extremely low-volume hydrogel samples (&#60; 5 &#181;L) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

Figure 1A shows the scheme of the photoactive reaction used to synthesize the L-EGP/L8 hydrogel. Figure 1B shows the hydrogel mixture in the PTFE tube before and after the photoactivation. Figure 1C presents the extruded L-eGFP-L8 hydrogel inside a Tris solution. The hydrogel sample has no structural defects such as notches. Hydrogels with clearly visible damage should be discar...

Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the &#39;clamping&#39; of the force experienced to the desired protocol.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog ...

force-clamp-rheometry-for-characterizing-protein-based-hydrogels

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

6.9K Views.  University of Wisconsin-Milwaukee. A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the 'clamping' of the force experienced to the desired protocol.

Video: Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengineering

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...

Chemistry

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size.
The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a &#8216;thick and sticky&#8217; state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are&#160;facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process.
The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer. &#160;
A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi ...

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 ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Neuroscience

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Summary

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Chapters in this video

Preparation of Complaint Matrices for Quantifying Cellular Contraction

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy